Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units

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Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units
Jose Vladimir SANDOVAL-SIERRA, Mar
ıa P. MART
IN, Javier DIEGUEZ-URIBEONDO*
nico-CSIC, Plaza Murillo 2, 28014 Madrid, Spain Departamento de Micolog
ıa, Real Jard
ın Bota

article info

abstract

Article history:

The lack of a robust taxonomy in the genus Saprolegnia is leading to the presence of incor-

Received 4 May 2013

rectly named isolates in culture collections and of an increasing number of misassigned se-

Received in revised form

quences in DNA databases. Accurate species delimitation is critical for most biological

9 October 2013

disciplines. A recently proposed approach to solve species delimitation (taxonomic diagno-

Accepted 16 October 2013

sis system) of difficult organisms is the definition of molecular operational taxonomic units

Corresponding Editor:

(MOTUs). We have used 961 sequences of nrDNA ITS from culture collections (461 se-

Pieter van West

quences) and GenBank (500 sequences), to perform phylogenetic and clustering optimization analyses. As result, we have identified 29 DNA-based MOTUs in agreement with

Keywords:

phylogenetic studies. The resulting molecular clusters support the validity of 18 species

ITS

of Saprolegnia and identify 11 potential new ones. We have also listed a number of incor-

Pathogens

rectly named isolates in culture collections, misassigned species names to GenBank se-

Phylogeny

quences, and reference sequences for the species. We conclude that GenBank represents

Taxonomy

the main source of errors for identifying Saprolegnia species since it possesses sequences

GenBank

with misassigned names and also sequencing errors. The presented taxonomic diagnosis system might help setting the basis for a suitable identification of species in this economically important genus. ª 2013 Published by Elsevier Ltd on behalf of The British Mycological Society.

Introduction The genus Saprolegnia belongs to the Oomycetes, which are fungal-like organisms with a zoosporic stage and are related to brown algae and diatoms (Baldauf et al. 2000). Oomycetes thrive in wet terrestrial and aquatic ecosystems (Sparrow 1960, 1976) and, in addition to saprobes, they include destructive plant and animal pathogens (Hulvey et al. 2007; Beakes et al. 2012). The genus Saprolegnia comprises species responsible for diseases on aquatic animals that are dramatically rising both in nature and in aquaculture. Investigations on Saprolegnia have become of increasing interest, especially

those focused on the identification and characterization of pathogenic species in this genus. Species delimitation in Saprolegnia is, however, complicated and troublesome mainly due to a number of problems that are listed next: (1) species description is largely based on ambiguous characters i.e., sexual structures such as oogonia, oospores, and antheridia (Coker 1923; Seymour 1970; Johnson et al. 2002) and many isolates, especially those from animals, often fail to produce these sexual structures in vitro. In addition, there are very few cultures of reference for Saprolegnia species (or type species) to compare with or to be used in molecular studies; (2) there are also limited studies


nico-CSIC, Plaza Murillo 2, 28014 Madrid, Spain. Tel.: þ34 91 420 30 * Corresponding author. Departamento de Micolog
ıa, Real Jard
ın Bota 17; fax: þ34 91 420 01 57.
guez-Uribeondo). E-mail address: [email protected] (J. Die 1878-6146/$ e see front matter ª 2013 Published by Elsevier Ltd on behalf of The British Mycological Society. http://dx.doi.org/10.1016/j.funbio.2013.10.005

Please cite this article in press as: Sandoval-Sierra JV, et al., Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.10.005

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on biodiversity; and (3) there are few molecular studies in this genus that could allow one to distinguish phylogenetic species. As a result of the complication to identify Saprolegnia spp. based on currently used taxonomic characters, DNA databases are being used as the main resource for identifying isolates as it happens with other organisms with similar taxonomic problems (Tautz et al. 2003). However, the increasing number of misassigned names of DNA sequences in databases such a GenBank negatively influences the correct identification of species (Bridge et al. 2003; Vilgalys 2003). An accurate species delimitation is critical for most biological disciplines. A recently proposed approach to solve species delimitation (taxon diagnosis system) of difficult organisms is the definition of molecular operational taxonomic units (MOTUs) using molecular data (Blaxter & Floyd 2003; Blaxter 2004; Blaxter et al. 2005). In the genus Saprolegnia, some studies attempted to solve specific taxonomic problems, e.g., the Saprolegnia diclinaeparasitica complex that encompasses the most virulent pathogens. For example, Molina et al. (1995) used restriction fragment length polymorphisms (RFLPs) in two regions of the nuclear ribosomal DNA (nrDNA), and, more
guez-Uribeondo et al. (2007) carried out phylogerecently, Die netic analysis of internal transcribed spacer (ITS) of the nrDNA. Both studies confirmed previous results suggesting that S. parasitica is a different species from S. diclina (Beakes
guez-Uribeondo et al. (2007) found & Ford 1983). In addition, Die that this complex appears to hold at least four species (Saprolegnia australis, S. diclina s. str., S. diclina named clade V, and S. parasitica). These authors also observed that several formally described species were actually synonyms of existing ones, e.g., Saprolegnia salmonis and Saprolegnia multispora. Some recent studies have also used ITS nrDNA in order to identify the species of Saprolegnia involved in amphibian decline (Johnson et al. 2008), and to compare between molecular and morphological data (Hulvey et al. 2007). They conclude that DNA barcoding might provide an insight to solve the difficulty of using morphological characters, and to identify the presence of cryptic phylogenetic species. Recent studies have also recommended the ITS region as one of the most suitable candidate markers for barcoding in the Oomycetes (Robideau et al. 2011) and even in fungi (Schoch et al. 2012). Recent molecular phylogenetic studies and application of new software for species delimitation have allowed resolving taxonomically difficult problems in fungi (Stielow et al. 2011; Druzhinina et al. 2012), fungal-like organism such as Oomy€ ker et al. 2009b) and other organisms (Pons et al. cetes (Go 2006; Birky et al. 2010; Lim et al. 2012). Research focused on solving the taxonomic boundaries in Saprolegnia are considerably delayed. This is having severe consequence in key-target areas of research such as genomics, population genetics, phytogeography, pathology, etc. Thus, the purpose of this work was to identify main source of problems when identifying Saprolegnia species using ITS nrDNA, and to assess a taxon diagnosis system for this genus. Thus, we have first searched for potential misidentifications in main culture collections and GenBank database. Finally, we have delimited DNA-based molecular operational units MOTUs, in Saprolegnia, by applying a clustering optimization analysis.

J. V. Sandoval-Sierra et al.

Materials and methods Culture collection, pure cultures, and genus identification A total of 461 isolates of the genus Saprolegnia and closely related genera were maintained as pure cultures and used in our analyses (Appendix A). A total of 438 cultures were from
nico-CSIC (RJB-CSIC, Oomycete culture the Real Jard
ın Bota collection, Madrid, Spain), 20 from the Fungal Biodiversity Culture Collection (CBS, The Netherlands), and three from American Type Culture Collection (ATCC, USA). The isolates originated from a worldwide distribution (Appendix A). For the genus identification, first, pieces of agar with isolated mycelium were grown in 0.5 ml of peptone glucose agar (PGA) for 2 d at 20  C. To trigger sporulation, the mycelia were washed three times in sterile mineral water, and then incubated in petri dishes containing 25 ml sterile mineral water, during 14 h at 15  C. Then, the genus of the isolates was identified according to sporangium morphology and zoospore discard by microscopic observations (Seymour 1970; Johnson et al. 2002). Microscopic examinations were performed using an Olympus CKX41SF inverted microscope (Olympus Optical, Tokyo, Japan). Because there are no isolates in culture of the strains used for the original descriptions of the majority of Saprolegnia species, we selected the following isolates as reference for species (Appendix A): Saprolegnia anisospora CBS 178.44 (SAP0364), Saprolegnia asterophora CBS 531.67 (SAP1296), Saprolegnia crustosa CBS 281.38 (SAP1295), Saprolegnia delica CBS 343.62 (SAP353), Saprolegnia diclina ATCC 56851 (SAP0229), Saprolegnia eccentrica CBS 551.67 (SAP1288), Saprolegnia ferax ATCC 26116 (SAP0157), Saprolegnia furcata CBS 542.67 (SAP1294), Saprolegnia hypogyna CBS 869.72 (SAP1289), Saprolegnia lapponica CBS 284.38 (SAP1475), Saprolegnia litoralis CBS 535.67 (SAP1486), Saprolegnia megasperma CBS 532.67 (SAP1287), Saprolegnia mixta CBS 149.65 (SAP1292), Saprolegnia monilifera CBS 558.67 (SAP1470), Saprolegnia monoica CBS 539.67 (SAP1279), Saprolegnia parasitica CBS 223.65 (SAP1458), and ATCC 42062 (SAP0208), Saprolegnia semihypogyna CBS 109568 (SAP1483), Saprolegnia subterranea CBS 278.52 (SAP1293), Saprolegnia terrestris CBS 533.67 (SAP1285), Saprolegnia turfosa CBS 313.81 (SAP1291), and Saprolegnia unispora CBS 213.35 (SAP1290) (Appendix A).

DNA extraction, amplification, and sequencing DNA extraction was carried out using a DNeasy Plant Mini Kit (QIAGEN, Valencia, California, USA). First, the isolates were
guezgrown in PGA liquid for 2 d as described in Die Uribeondo et al. (2007). The ITS nrDNA region was amplified using universal primers for eukaryotes (White et al. 1990) nuSSU-1766 (ITS5) and nu-LSU-0041 (ITS4). PCR was performed in a 25 ml volume containing 1 ml of 1–10 ng ml1 DNA of isolates, 2.5 ml of 10 RB Taq buffer (Bioline Ltd., London, UK), 1.4 ml of 50 mM MgCl2 (BioLine), 0.3 ml of 5 units ml1 Taq Polymerase (BioLine), 1.6 ml of 25 mM dNTPs (BioLine), 0.5 ml of 10 mM primers (Eurogentec Ltd., Seraing, Belgium), and 1 ml of bovine serum albumine (BSA) at 1 mg ml1. The PCR was carried out using an Eppendorf Mastercycler Epgradient S (Westbury, NY) or an MJ Research PTC-200 (Massachusetts) thermal

Please cite this article in press as: Sandoval-Sierra JV, et al., Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.10.005

Molecular taxonomy of the genus Saprolegnia

cycler under the following conditions: initiation of 2 min at 95  C, followed by 35 cycles of 1 min at 95  C, 30 s at 60  C, and 1 min at 72  C, and finished with an elongation cycle of 10 min at 72  C. Amplified products were sequenced with an automated sequencer (Applied Biosystems 3730xl DNA, Macrogen, The Netherlands). The consensus sequences of each isolate for ITS region were assembled and edited using the program Geneious v6.04 (Kearse et al. 2012).

Sequence alignment and analyses In order to prevent the incorporation into the analyses of sequences from misidentified species or ambiguous sequences, from affecting the study, we carried out a first round of analyses using only sequences obtained from pure cultures and from reliable sources such as the culture collections of ATCC, CBS, and RJB-CSIC. Four methods of alignment were tested for sequence analyses always using the default settings. These were Mafft v7.0 with G-INS-i algorithm (Katoh & Standley 2013), ClustalW v2.0 (Larkin et al. 2007), Muscle v3.6 (Edgar 2004), and Kalign v2.03 (Lassmann & Sonnhammer 2005). In order to carry out cluster optimization analyses, the genetic distances between all the sequences included in that alignment were first calculated. For this purpose, we used the software PAUP* according to model substitution Kimura 2 parameter, taking into account all the possible sequence alignments described above. The resulting matrices of pairwise comparison of genetic distances were subjected to clus€ ker ter optimization analysis using the software OPTSIL (Go et al. 2009b) (www.goeker.org/mg/clustering). We obtained the optimal threshold value for species separation taking into account the following settings for this software: linkage fraction (F ) values that ranged from 0.0 to 1.0, using a step width of 0.1. For each F value, a threshold (T ) value was calculated with a step width of 0.001. This value generated a modified Rand index (MRI), which is a rescaled version of the Rank index (Legendre & Legendre 1998), and represents a measure € ker et al. 2009b). The of similarity between two clusters (Go clustering parameters that yield the globally highest MRI were applied to the complete sequence set from pure cultures to establish the MOTUs. In addition, sequence alignments were also analyzed by applying both Maximum Parsimony (MP) and Bayesian inference methods as implemented in PAUP* v4.0b10 (Swofford 2002) and MrBayes v3.2.1 (Ronquist et al. 2012), respectively. In these analyses, when sequences associated to a particular species name were repeated, only two of them were maintained in the alignment. For the MP analysis, a heuristic search was implemented using nonweighted characters with 1000 replicates of random taxon addition sequences and ten trees were held at each step by using tree bisectionereconnection (TRB) branch swapping. The support for each clade was calculated in PAUP* by nonparametric bootstrap (bs) support (Felsenstein 1985) based on 1000 replicates under MP analysis using TRB branch swapping and saving multiple trees. For the Bayesian inference analysis, the best model-fit of nucleotide substitution for each of the alignments was selected using the Bayesian information criterion (BIC), which

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was implemented in jModelTest 2 (Darriba et al. 2012). The BIC parameters were applied for each alignment methods (Appendix B). Three simultaneous runs with eight Monte Carlo Markov Chains (MCMC) with incremental heating of 0.2, were computed for 20 million generations and were sampled every 1000 generations. Convergence of the MCMC was assessed using the effective sampling size criterion for each parameter as implemented in Tracer v. 1.5 (Rambaut & Drummond 2009). The first 2500 samplings (reflecting 250 000 generations) were discarded as ‘burn-in’, after checking for stability on the log-likelihood curves, and the remaining trees from the independent runs were combined to build a 50 % majority-rule consensus tree.

GenBank sequence analysis A total of 500 ITS nrDNA sequences were downloaded from GenBank in December of 2012 (Appendix C) including 469 isolates assigned to the Saprolegnia genus. Additionally, we also downloaded sequences ascribed to the closely related genera Achlya, Aplanopsis, Leptolegnia, Protoachlya, and Pythiopsis (Appendix C). These sequences were also from a wide range of hosts and habitats and with a worldwide distribution (Appendix C). The original assignments of these sequences were to the following species: Saprolegnia anisospora, Saprolegnia asterophora, Saprolegnia australis, Saprolegnia delica, Saprolegnia diclina, Saprolegnia eccentrica, Saprolegnia ferax, Saprolegnia hypogyna, Saprolegnia itoana, Saprolegnia lapponica, Saprolegnia litoralis, Saprolegnia longicaulis, Saprolegnia luxurians, Saprolegnia megasperma, Saprolegnia mixta, Saprolegnia monilifera, Saprolegnia monoica, Saprolegnia multispora, Saprolegnia parasitica, Saprolegnia richteri, Saprolegnia subterranean, Saprolegnia terrestris, Saprolegnia torulosa, Saprolegnia turfosa, Saprolegnia unispora, Saprolegnia sp. nuchiae, Saprolegnia sp. rodrigueziana, S. cf. ferax, Saprolegnia cf. kauffmanniana, Saprolegnia sp., Protoachlya polysporus, Pythiopsis humphreyana, and Pythiopsis terrestris. Some of these sequences were used as source of species reference when a particular culture of Saprolegnia was not available (Appendix C); these were: Saprolegnia anomala (DQ322632), Saprolegnia brachydanionis (EU292729), Saprolegnia bulbosa (AY267011), S. longicaulis (AY270032), Saprolegnia oliviae (AY270031), Saprolegnia polymorpha (DQ393560), Saprolegnia salmonis (AB219399), and Saprolegnia semihypogyna (AB219395). Both the RJB-CSIC and the whole set of GenBank sequences were aligned as mentioned above and analyzed with MP and Bayesian inference (for selected parameters see Appendix B) and cluster optimization analyses as mentioned above. The cluster optimization analysis was based on selected parameters obtained from the previous optimization analyses using sequences from pure cultures of reliable sources. The final MOTUs were then defined based on a cluster optimization analysis of sequences from both pure cultures and GenBank.

Contrasting cluster optimization groups to phylogenetic analyses Threshold specificity for delimiting species or taxonomic units was verified by comparison of the resulting MOTUs with the name assigned to each sequence of the GenBank. Based on the MOTUs delimitations and names assigned to

Please cite this article in press as: Sandoval-Sierra JV, et al., Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.10.005

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each sequence of the GenBank, the genetic distances within (intraspecific) and among (interspecific) sequences were calculated. The genetic distances were estimated using the substitution model of Kimura 2 parameter with the program MEGA v.5.1 (Tamura et al. 2011), and the standard deviation was obtained with the bs analysis based on 1000 replications. The pairwise comparisons obtained were classified as intraspecific based on the argument that two comparisons belong to same species and as interspecific when belonging to different species. The histograms analyses were generated with the program STATA v.12.0. The overlapping analyses were computed using MorisitaeHorn index (CH) (Jost 2007). Genetic distances were considered as separation criterion, and intra and interspecific pairwise comparisons were counted inside of each genetic distance (abundance of case). The MorisitaeHorn index was conducted using the Vegetarian package (Charney & Record 2013) for the statistical program, R (R Development Core Team 2010) and the standard deviation was estimated using 1000 replications with bs analysis (Chao et al. 2008). The differences between intraspecific and interspecific levels were statistically analyzed by applying a one-way analysis of the variance (ANOVA) and a receiver operating characteristics analysis (ROC) using the program STATA. The ROC analysis is a technique for visualizing, organizing, and selecting classifiers based on their performance of a binary classifier system and a discrimination threshold. This analysis has long been used in detection to show the tradeoff between plotting the fraction of true positives out of the positives (true positive rate) versus the fraction of false positives out of the negatives (false positive rate). The true positive rate is also known as sensitivity, and the false positive rate equals one minus the specificity or true negative rate (Fawcett 2006). The ROC analysis determines an area under the curve (AUC). An AUC of 0.5 indicates a random classifier, i.e., the fraction of true positives increases proportional to fraction of false positives, while an AUC of 1 indicates a perfect classifier i.e., all true positives and negatives are correctly classified. The resulting MOTU values were compared to the corresponding bs and posterior probability (pp) from MP and Bayesian inference analyses. Thus, when MOTUs included sequences in clades with high values of bs (>75) and pp (>0.90), they were validated and compared with the species defined based on traditional taxonomy. For broad phylogenetic considerations regarding genera such as Achlya, Leptolegnia, Protoachlya, Pythiopsis, and Saprolegnia isolates, higher values of bs (>75) and pp (>0.95) were taken into account.

Results Delimitation of MOTUs using sequences from culture collections When the 461 sequences obtained from pure cultures of culture collections were used for cluster optimization, the different methods of alignment generated a matrix that ranged from 727 to 758 characters, i.e., ClustalW, ca 727; Kaling, ca 758; Mafft, ca 742; and Muscle, ca 737. The pairwise identities were of 89.6 % for ClustalW, 89.7 % for Kaling, 90.2 % for Mafft, and 90.3 % for Muscle. The cluster optimization analysis of these sequences

J. V. Sandoval-Sierra et al.

showed an average MRI value for the alignment methods of 0.996 (0.000) and an average threshold value of 0.010 (0.001) (Appendix D), and grouped these sequences in a total of 23 clusters that were alignment method-independent (Fig 1, Appendix E, Appendix F). These clusters were named according to the species of reference that contained. These were the following: Saprolegnia diclina (cluster 1), Saprolegnia sp. 1 (cluster 2), Saprolegnia parasitica (cluster 3), Saprolegnia ferax (cluster 4), Saprolegnia australis (cluster 5), Saprolegnia delica (cluster 6), Saprolegnia litoralis (cluster 7), Saprolegnia sp. 2 (cluster 8), Saprolegnia sp. 3 (cluster 9), Saprolegnia subterranea (cluster 10), Saprolegnia torulosa (cluster 11), Saprolegnia monilifera (cluster 12), Saprolegnia terrestris (cluster 13), Saprolegnia eccentrica (cluster 14), Saprolegnia sp. 4 (cluster 15), Saprolegnia furcata (cluster 16), Saprolegnia sp. 5 (cluster 17), Saprolegnia sp. 6 (cluster 18), Saprolegnia asterophora (cluster 19), Saprolegnia sp. 7 (cluster 20), Saprolegnia megasperma (cluster 21), Saprolegnia turfosa (cluster 22), and Saprolegnia anisospora (cluster 23) (Fig 1, Appendix E). The isolates that belonged to Achlya caroliniana, Aphanomyces astaci, Leptolegnia sp., and Protoachlya paradoxa were included in separated groups (Fig 1, Appendix E). Of these groups, a number of 16 clusters had high bs and pp values, one cluster, i.e., Saprolegnia sp. 1, was only supported by MP, and nine clusters contained only one sequence, i.e., Saprolegnia sp. 4 (cluster 15), S. litoralis (cluster 7), S. subterranea (cluster 10), S. furcata (cluster 16), S. asterophora (cluster 19). Finally, two clusters, S. delica and S. monilifera, had a low support by either MP or Bayesian inference analyses (Fig 1, Appendix E). In these cases, terminal nodes did not coincide with resulting clusters. Thus, terminal nodes based on phylogenetic analyses did not separate S. australis from S. delica, nor S. monilifera from S. torulosa. However, these four species could be resolved based on genetic distance analysis, which showed that they represent distinct molecular taxonomic units.

Delimitation of MOTUs based on culture collection and GenBank sequences When the GenBank sequences were included in the alignments, a matrix of 961 sequences was generated. The number of characters ranged from 782 to 874, depending on the method of alignment (i.e., ClustalW, ca 782; Kaling, ca 874; Mafft, ca 797; and Muscle, ca 798). The pairwise identities were 88.4 % for ClustalW, 89.5 % for Kaling, 89.7 % for Mafft, and 89.5 % for Muscle. The cluster optimization analysis of the resulting alignments generated a total of 29 clusters of Saprolegnia species using the average MRI and threshold values obtained in the previous analysis (Fig 2, Appendix F, Appendix G). A total of 23 clusters coincided to those generated from sequences from pure cultures, i.e., Saprolegnia diclina (cluster 1), Saprolegnia sp. 1 (cluster 2), Saprolegnia parasitica (cluster 3), Saprolegnia ferax (cluster 4), Saprolegnia australis (cluster 5), Saprolegnia delica (cluster 6), Saprolegnia litoralis (cluster 7), Saprolegnia sp. 2 (cluster 8), Saprolegnia subterranea (cluster 10), Saprolegnia torulosa (cluster 11), Saprolegnia monilifera (cluster 12), Saprolegnia terrestris (cluster 13), Saprolegnia eccentrica (cluster 14), Saprolegnia furcata (cluster 16), Saprolegnia sp. 5 (cluster 17), Saprolegnia sp. 6 (cluster 18), Saprolegnia asterophora (cluster 19), Saprolegnia sp. 7 (cluster 20), Saprolegnia megasperma (cluster 21),

Please cite this article in press as: Sandoval-Sierra JV, et al., Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.10.005

Molecular taxonomy of the genus Saprolegnia

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Fig 1 e Schematic tree of Bayesian inference analysis based on ITS nrDNA sequences obtained from pure cultures of Saprolegnia species and related genera of culture collections (ATCC, CBS, and RJB-CSIC). The cluster numbers represent the Saprolegnia species based on MOTUs obtained from clustering optimization analysis. The numbers above the branches represent the pp (>0.90) values and bs support (> 75) based on Bayesian inference and MP analyses respectively and performed under Mafft alignment. The numbers include in brackets represent the pp (>0.95) values and bs support (> 75) for phylogenetic considerations. Species names below each cluster name indicate sequences obtained from pure culture of the culture collections CBS and ATCC with misassigned names, i.e., names that do not correspond to the MOTU, i.e., species, found with our analyses.

Please cite this article in press as: Sandoval-Sierra JV, et al., Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.10.005

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J. V. Sandoval-Sierra et al.

Fig 2 e Schematic tree of Bayesian inference analysis based on ITS nrDNA sequences obtained from pure cultures of Saprolegnia species and related genera of culture collections (ATCC, CBS, and RJB-CSIC) and combined with GenBank sequences. The cluster numbers represent the Saprolegnia species based on MOTUs obtained from clustering optimization analysis. The numbers above the branches represent the pp (>0.90) values and bs support > 75) based on Bayesian inference and MP Please cite this article in press as: Sandoval-Sierra JV, et al., Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.10.005

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B

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Intraspecific Interspecific

Percent pairwise 50

50

A

100 0

Molecular taxonomy of the genus Saprolegnia

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Intraspecific and interspecific genetic distances between MOTUs

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C

G). Three clusters had high bp and pp values, i.e., Saprolegnia sp. 9 (cluster 26), Saprolegnia sp. 10 (cluster 27), and Saprolegnia monoica (cluster 29) (Fig 2, Appendix G). The other three clusters contained just a single sequence. These were: Saprolegnia brachydanionis (cluster 24), Saprolegnia sp. 8 (cluster 25), Saprolegnia sp. 11 (cluster 28). On the other hand, we identified that sequencing errors generated three subclusters (4-1 to 4-3, Appendix G) within S. ferax (cluster 4), and one subcluster (8-1, Appendix G) within Saprolegnia sp. 2 (cluster 8). Also, we found that one sequence assigned to Saprolegnia genus most likely belongs to other genus such as Achlya, Aphanomyces, Leptolegnia, Protoachlya or Pythiopsis (Fig 2).

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.1 .2 Genetic distance

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Fig 3 e Pairwise distribution for the intra/interspecific genetic distances (Kimura 2 parameter) between each pair of ITS nrDNA sequences from: (A) GenBank with original assigned names; (B) Pure cultures of Saprolegnia species and related genera of culture collections (ATCC, CBS, and RJBCSIC) named based on MOTUs, and (C) Combined sequences from pure cultures and GenBank named based on MOTUs.

Saprolegnia turfosa (cluster 22), and Saprolegnia anisospora (cluster 23), all these clusters had high bs and pp values in MP and Bayesian inference analyses (Fig 2, Appendix G). The incorporation of GenBank sequences to the matrix made it possible to test the validity of previously identified cluster containing single sequences (four clusters), that were the following: S. litoralis (cluster 7), S. subterranea (cluster 10), S. furcata (cluster 16), and S. asterophora (cluster 19). These clusters also had high bs and pp values in MP and Bayesian inference analyses (Fig 2). However, the cluster Saprolegnia sp. 1 (cluster 2) was only supported by MP analysis (Fig 2, Appendix G) and the cluster corresponding to Saprolegnia sp. 4 (cluster 15) remained as cluster containing only a single sequence (Figs 1 and 2). The incorporation of GenBank sequences to the matrix also generated six new clusters (Fig 2, Appendix

When we performed cluster optimization analysis using GenBank sequences taking into account the originally assigned names, the intraspecific genetic distances (66 492 pairwise comparisons) ranged from 0 to 0.261 (mean ¼ 0.057  0.080) (Fig 3A). The interspecific genetic distances (394 788 pairwise comparisons) ranged from 0 to 0.264 (mean ¼ 0.074  0.065) (Fig 3A). If using only sequences from pure cultures with assigned names based on MOTUs, the intraspecific genetic distances (12 691 pairwise comparisons) ranged from 0 to 0.010 (mean ¼ 0.002  0.003) (Fig 3B). The interspecific genetic distances (93 339 pairwise comparisons) ranged from o.010 to 0.304 (mean ¼ 0.098  0.075) (Fig 3B). When combined sequences from pure cultures and GenBank with assigned names based on MOTUs (comprising calculations from 57 477 pairwise comparisons) were used, the intraspecific genetic distances ranged from 0 to 0.072 (mean ¼ 0.001  0.003) (Fig 3C), while the intraspecific genetic distances (403 803 pairwise comparisons) ranged from 0.006 to 0.271 (mean ¼ 0.083  0.069) (Fig 3C). For the analysis from pure cultures and the correctly named from the GenBank sequences the variation of the genetic distances within MOTUs ranged from 0.000 to 0.006 (Table 1). When calculating the overlapping between intra and interspecific genetic distances, we found significant overlaps between them if original names assigned in GenBank were used. The percentage of overlapping in genetic distance values was 59.05 % (CH ¼ 0.591  0.002; f1,327 ¼ 0.260; p ¼ 0.612) (Fig 3A). However, if names were corrected based on MOTUs results this overlapping was not significant for both pure culture and pure culture and combined sequences. Thus, when using sequences from pure cultures the percentage of overlapping in genetic distance values was 0.030 % (CH ¼ 0.001  0.001; f1,216 ¼ 127.650; p ¼ 0.000) (Fig 3B), and 1.97 % (CH ¼ 0.020  0.001; f1,218 ¼ 21.280; p ¼ 0.000) when using combined sequences from pure cultures and GenBank sequences (Fig 3C). Thus ROC analysis used for testing the validity of the intra and interspecific genetic distances based on MOTUs

analyses respectively and performed under Mafft alignment. The numbers include in brackets represent the pp (>0.95) values and bs support (> 75) for phylogenetic considerations. Species names below each cluster name indicate sequences obtained from pure culture of the culture collections CBS and ATCC, or from GenBank with misassigned names, i.e., names that do not correspond to the MOTU, i.e., species, found with our analyses. Please cite this article in press as: Sandoval-Sierra JV, et al., Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.10.005

8

J. V. Sandoval-Sierra et al.

Table 1 e MOTUs in the genus Saprolegnia based on ITS nr DNA sequences obtained from pure cultures of culture collections (ATCC, CBS, and RJB-CSIC) and GenBank. The clusters were established by using clustering optimization, Bayesian inference, and MP analyses. Intraspecific genetic distances were implemented with Kimura 2 parameter. MOTUs are listed in same order as in Figs 1 and 2. MOTU

Cluster

Number of sequences

Genetic distance

Pure culture

GenBank

Pure culture

Pure culture and GenBank

S. diclina Saprolegnia sp. 1 S. parasitica

1 2 3

83 12 44

30 9 163

0.003 (0.001) 0.001 (0.001) 0.001 (0.001)

0.000 (0.000) 0.001 (0.001) 0.001 (0.000)

S. ferax

4 4-1 4-2 4-3

77

111 1 1 1

0.001 (0.001)

0.001 (0.000)

S. australis

5

61

28

0.001 (0.001)

0.000 (0.000)

S. delica S. litoralis Saprolegnia sp. 2

6 7 8 8-1

27 1 73

9 3 33 1

0.002 (0.001) n/c 0.004 (0.001)

0.001 (0.000) 0.000 (0.000) 0.004 (0.002)

Saprolegnia sp. 3 S. subterranea S. torulosa S. monilifera S. terrestris

9 10 11 12 13

2 1 5 4 6

0 8 3 8 6

0.000 (0.000) n/c 0.000 (0.000) 0.001 (0.001) 0.001 (0.001)

0.000 0.000 0.003 0.000 0.002

S. eccentrica

14

2

7

0.002 (0.001)

0.001 (0.001)

15 16 17 18 19 20 21

1 1 30 4 1 6 4

0 2 1 3 2 0 5

n/c n/c 0.004 (0.002) 0.000 (0.000) n/c 0.001 (0.001) 0.001 (0.001)

n/c 0.000 0.001 0.000 0.000 n/c 0.002

22 23

2 10

4 10

0.000 (0.000) 0.000 (0.000)

0.000 (0.000) 0.000 (0.000)

Saprolegnia sp. S. furcata Saprolegnia sp. Saprolegnia sp. S. asterophora Saprolegnia sp. S. megasperma S. turfosa S. anisospora

4 5 6 7

(0.000) (0.000) (0.002) (0.000) (0.001)

Previously assigned names (for more information see the Appendix B, F and G) S. australis, S. megasperma S. cf. ferax S. delica, S. diclina, S. hypogyna, S. litoralis*, S. salmonis Protoachlya polysporus, S. anomala, S, bulbosa, S. diclina, S. lapponica, S. litoralis, S. longicaulis, S. megasperma, S. mixta, S. oliviae, S. parasitica, S. sp. nuchiae, S. terrestris, S. unispora S. megasperma, S. multispora, S. parasitica, S. polymorpha S. australis, S. diclina Achlya sp., Leptolegnia sp., S. cf. kauffmanninana, S. diclina, S. parasitica S. itoana, S. rodrigueziana, S. unispora S. asterophora S. megasperma, S. unispora Leptolegnia sp., S. luxurians, S. rechteri S. luxurians, S. rechteri, S. semyhypogyna

(0.000) (0.000) (0.000) (0.000) (0.002)

Aplanopsis terrestris, Pythiopsis terrestris S. monoica Pythiopsis humphreyana, S. eccentrica, S. crustosa

S. brachydanionis 24 0 1 n/c n/c Saprolegnia sp. 8 25 0 1 n/c n/c S. monoica Saprolegnia sp. 9 26 0 7 n/c 0.000 (0.000) S. litoralis Saprolegnia sp. 10 27 0 2 n/c 0.000 (0.000) S. litoralis Saprolegnia sp. 11 28 0 1 n/c n/c Saprolegniaceae 29 0 2 n/c 0.000 (0.000) S. monoica n/c not applicable. The presence of n/c in the results denotes cases in which it was not possible to estimate genetic distances. * Sequences that have incongruent name of isolate with respect to GenBank name (see Table 2 and Appendix C).

showed high sensitivity and specificity for both sequences from pure cultures or combined with GenBank sequences (AUC ¼ 1.000 and AUC ¼ 0.996, respectively) (Fig 4), but was of low sensitivity and specificity when sequences with originally assigned names were used (AUC ¼ 0.675) (Fig 4).

number of misassigned sequences represents a total of 44 % of the whole Saprolegnia sequences available in the GenBank. In addition, we generated a list of 59 ITS nrDNA reference sequences for each MOTU and included up to seven ribotype sequences for each MOTU (Table 3).

GenBank sequences with misassigned species names and sequence types for Saprolegnia species

Phylogenetic considerations

According to our taxonomic molecular diagnosis system, we have identified 225 GenBank ITS nrDNA sequences that have misassigned species names (Table 2, Appendix C). This

MP and Bayesian inference phylogenetic analyses showed that all Saprolegnia species were engulfed into one clade with high support (pp ¼ 1 and bp ¼ 100). However, this cluster also comprised sequences of genus Achlya, Leptolegnia,

Please cite this article in press as: Sandoval-Sierra JV, et al., Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.10.005

9

1.00

Molecular taxonomy of the genus Saprolegnia

Pure cultures

Originally designated name in the GenBank sequences

0.00

0.25

Sensitivity 0.50

0.75

New designated name to GenBank sequences

0.00

0.25

0.50 1 - Specificity

0.75

1.00

Fig 4 e Predicted classification based on interaction between intra/inter-specific genetic distances based on ROC analysis and AUC. The ROC analysis used for testing the validity of the intra/inter-specific genetic distances based on MOTUs shows high sensitivity and specificity for sequences from pure cultures, and new designated GenBank sequences and pure cultures (AUC [ 1.000 and AUC [ 0.996, respectively), but was of low sensitivity and specificity when sequences with originally assigned names were used (AUC [ 0.675).

Pythiopsis, and Protoachlya (Fig 2, Appendix G). Sequences of Saprolegnia spp. formed polytomies with high bs and pp values (Figs 1 and 3). The resulting polytomy comprises six clusters: (1) one that we named Saprolegnia s. str. (pp ¼ 1 and bs ¼ 97, comprising 20 species, i.e., Saprolegnia australis, Saprolegnia brachydanionis, Saprolegnia delica, Saprolegnia diclina, Saprolegnia eccentrica, Saprolegnia ferax, Saprolegnia furcata, Saprolegnia litoralis, Saprolegnia parasitica, Saprolegnia monilifera, Saprolegnia subterranea, Saprolegnia terrestris, Saprolegnia torulosa, and seven potential new species); (2) Saprolegnia asterophora and three potential new species (pp ¼ 1 and bs ¼ 100); (3) Saprolegnia megasperma (pp ¼ 1 and bs ¼ 100); (4) S. torulosa (pp ¼ 1 and bs ¼ 100); (5) Saprolegnia anisospora (pp ¼ 1, bs ¼ 100); (6) Saprolegnia monoica and Protoachlya paradoxa (pp ¼ 1 and bs ¼ 100). The sequences of the genus Leptolegnia, that according to present study included four MOTUs showed high support (pp ¼ 1 and bs ¼ 97) (Fig 2, Appendix G).

Discussion This study describes for the first time a taxon diagnosis system for the genus Saprolegnia. The described MOTUs included sequences from main public culture collections and all ITS nrDNA sequences available in GenBank. For this classification, we have followed a clustering optimization approach that determines the distance function and clustering setting for se€ ker et al. quences resulting in an optimal agreement (Go 2009b, 2010) that includes selected cultures and sequences as reference for species. The optimal agreement or threshold value of the genetic distance for species delimitation in Saprolegnia is similar to those obtained in studies on other

€ ker et al. 2009b), Oomycetes such as the Peronosporales (Go € ker et al. 2009a; Stielow et al. 2011; Setaro et al. true fungi (Go 2012), and also other organisms such as planktonic Foraminif€ ker et al. 2010). The validity of the generated groups in era (Go this taxon diagnosis system for Saprolegnia was in agreement with two phylogenetic approaches, and the statistically significance of the overlaps between the intraspecific and interspecific genetic distances of the proposed MOTUs (molecular species) of this taxonomy. Thus, based on our approach we identified the following MOTUs that coincided with previously described species (Seymour 1970; Johnson et al. 2002; Ke et al. 2009a): Saprolegnia anisospora, Saprolegnia asterophora, Saprolegnia australis, Saprolegnia brachydanionis, Saprolegnia delica, Saprolegnia diclina, Saprolegnia eccentrica, Saprolegnia ferax, Saprolegnia furcata, Saprolegnia litoralis, Saprolegnia megasperma, Saprolegnia monilifera, Saprolegnia monoica, Saprolegnia parasitica, Saprolegnia subterranean, Saprolegnia terrestris, Saprolegnia torulosa, Saprolegnia turfosa. The cluster S. delica engulfed previous isolates named as S. diclina clade V, which possesses similar characters to S.
guez-Uribeondo et al. 2007). Thus, the characters diclina (Die of S. diclina seem to be homoplasic, resulting in several species being often confused, e.g., S. australis, S. delica, and S. diclina, which have made these species occasionally be considered as a single species or species complex (Neish 1976). A similar case appears to occur for the species S. litoralis since there is an important number of isolates that have been assigned to this species that falls in different MOTUs. The existence of homoplasic characters and/or false species assignments may be the reasons for this observed clustering. On the other hand, some MOTUs included sequences that have been assigned to diverse names. The best example is the MOTUs for S. ferax. This species is the most frequently misidentified taxon and the one given the higher number of names (e.g., Protoachlya polysporus, Saprolegnia anomala, Saprolegnia bulbosa, S. diclina, Saprolegnia lapponica, S. litoralis, Saprolegnia longicaulis, S. megasperma, Saprolegnia mixta, Saprolegnia nuchiae, Saprolegnia oliviae, S. parasitica, S. terrestris, and Saprolegnia unispora). Thus, the morphological characters of S. ferax seem to have a high plasticity resulting in definition of several synonymic species. The fact that the majority of these synonym species does not have a backup culture makes it complicate to quantify this plasticity. Furthermore, our result supports the separation of the species that are part of the Saprolegnia diclinaeparasitica complex (S. australis, S. diclina, and S. parasitica) (Neish 1976). The results confirm previous reports providing evidence that S. parasitica has a species level and that this taxon is different from S.
guez-Uribeondo diclina (Willoughby 1978; Beakes 1983; Die et al. 2007). This species still formally considered as S. diclina but the genetic distances of S. diclina MOTU from S. parasitica MOTU clearly separated them as different taxa. The recently genome sequenced strain of S. parasitica (Jiang et al. 2013) grouped into the S. parasitica MOTU. Moreover, other described species such as Saprolegnia hypogyna or Saprolegnia salmonis, do not show any genetic difference from isolates of S. parasitica and, therefore, they seem to represent specimens
guezof the same taxon as previously pointed out by Die Uribeondo et al. (2007). The case of S. hypogyna represents a conflicting case of a pragmatic definition of species versus

Please cite this article in press as: Sandoval-Sierra JV, et al., Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.10.005

Genbank Accession No.

Designated name in GenBank

New designated name by MOTUs

AM228818 AY647195 DQ393507 DQ393535 DQ393541

S. australis S. australis Saprolegnia sp. S. megasperma Saprolegnia sp.

S. diclina S. diclina S. diclina S. diclina S. diclina

AM228782 AM947036 FN186016 FN186023 AB219389 AB219399

S. cf. ferax S. cf. ferax S. cf. ferax S. cf. ferax S. hypogyna S. salmonis

Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. S. parasitica S. parasitica

AM228724 AM228728 AM228729 AM228814 AM228815 AM228846

S. hypogyna S. hypogyna S. hypogyna S. hypogyna S. hypogyna S. hypogyna

AY647188

Cluster

Strain

Origin

Publications generating the sequences

SAP203 NJM 9852 UNCW114 UNCW283 ATCC 36144

Spain Japan USA USA United Kingdom


guez-Uribeondo et al. 2007 Die Phadee et al. Unpublished Hulvey et al. 2007 Hulvey et al. 2007 Hulvey et al. 2007

2 2 2 2 3 3

SAP151 VI04022 VI02389 VI04808 NBRC 36711 NJM 9851

Spain Norway Norway Norway Japan


guez-Uribeondo et al. 2007 Die Vr alstad et al. 2009 Stueland et al. Unpublished Stueland et al. Unpublished Inaba & Tokumasu Unpublished Inaba & Tokumasu Unpublished

S. parasitica S. parasitica S. parasitica S. parasitica S. parasitica S. parasitica

3 3 3 3 3 3

SAP24 SAP33 SAP37 SAP194 SAP196 SAP231

Spain Spain Spain Spain Spain Spain


guez-Uribeondo Die
guez-Uribeondo Die
guez-Uribeondo Die
guez-Uribeondo Die
guez-Uribeondo Die
guez-Uribeondo Die

S. hypogyna

S. parasitica

3

ATCC 52721

United Kingdom

Phadee et al. Unpublished

AY647193

S. salmonis

S. parasitica

3

NJM 9851

Japan

Phadee et al. Unpublished

DQ393525 DQ393527 DQ393531

Saprolegnia sp. S. litoralis Saprolegnia sp.

S. parasitica S. parasitica S. parasitica

3 3 3

UNCW278 ATCC 26116 ATCC 90213

USA USA Japan

Hulvey et al. 2007 Hulvey et al. 2007 Hulvey et al. 2007

DQ393532

Saprolegnia sp.

S. parasitica

3

ATCC 200013

Japan

Hulvey et al. 2007

DQ393537 DQ393544 DQ393545 DQ393548 DQ393549 DQ393551 DQ393557 EF175573 EU551152 EU551153 FJ236033 FN186034 FN186049

Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. S. diclina Saprolegnia sp. S. salmonis S. salmonis Saprolegnia sp. S. hypogyna S. salmonis

S. parasitica S. parasitica S. parasitica S. parasitica S. parasitica S. parasitica S. parasitica S. parasitica S. parasitica S. parasitica S. parasitica S. parasitica S. parasitica

3 3 3 3 3 3 3 3 3 3 3 3 3

UNCW288 UNCW312 UNCW314 UNCW328 UNCW329 UNCW300 UNCW373 MCA 3030 Argentina 3.12 Argentina 3.13 DC3U9_2007 VI04814 NJM 9851

USA USA USA USA USA Italy USA USA Argentina Argentina USA United Kingdom Japan

Hulvey et al. 2007 Hulvey et al. 2007 Hulvey et al. 2007 Hulvey et al. 2007 Hulvey et al. 2007 Hulvey et al. 2007 Hulvey et al. 2007 Straus et al. Unpublished Paul & Steciow Unpublished Paul & Steciow Unpublished Straus et al. 2009 Stueland et al. Unpublished Stueland et al. Unpublished

1 1 1 1

et et et et et et

al. al. al. al. al. al.

2007 2007 2007 2007 2007 2007

Isolate and named assigned by

Willoughby et al. 1983, S. diclina

Hussein & Hatai 1999, S. salmonis

Fregeneda-Grandes et al. 2000, S. diclina Willoughby et al. 1983, S. hypogyna Hussein & Hatai 1999, S. salmonis R Emerson, S. ferax Hatai et al. 1990, S. parasitica Hatai et al. 1990, S. parasitica

Hussein & Hatai 1999, S. salmonis

J. V. Sandoval-Sierra et al.

1 1 1 1 1

10

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Table 2 e Misassigned species names of Saprolegnia to nrDNA ITS GenBank sequences, and new designated species names according to MOTUs. The MOTUs are based on clustering optimization, Bayesian inference, and MP analyses.

S. delica Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. S. mixta S. diclina S. bulbosa

S. parasitica S. parasitica S. parasitica S. parasitica S. ferax S. ferax S. ferax

3 3 3 3 4 4 4

CBS 344.62 JE1 HZL1 CY1 CBS 307.37 SAP132 LPS 45800/745

USA China China China France Australia Argentina

Robideau et al. 2011 Zhang et al. Unpublished Zhang et al. Unpublished Zhang et al. Unpublished Inaba & Tokumasu Unpublished
guez-Uribeondo et al. 2007 Die Steciow et al. 2007

AY270031 AY270032

S. oliviae S. longicaulis

S. ferax S. ferax

4 4

LPS 45828/746 Arg 004

Argentina Argentina

Steciow 2003 Steciow & Paul Unpublished

AY310503 AY666084 DQ322632

S. litoralis Saprolegnia sp. S. anomalies

S. ferax S. ferax S. ferax

4 4 4

BP-S1

Germany Moroccan India

Oidtmann et al. 2004 Paul et al. Unpublished Gandhe & Kurne 2003

DQ393506 DQ393508 DQ393509 DQ393510 DQ393511 DQ393518 DQ393519 DQ393520 DQ393526 DQ393529

S. megasperma S. megasperma Saprolegnia sp. Saprolegnia sp. S. diclina Saprolegnia sp. S. terrestris S. megasperma Saprolegnia sp. Saprolegnia sp.

S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax

4 4 4 4 4 4 4 4 4 4

UNCW105 UNCW162 UNCW170 UNCW172 UNCW176 UNCW251 UNCW253 UNCW254 UNCW262 ATCC 10396, CBS 283.38

USA USA USA USA USA USA USA USA USA

Hulvey Hulvey Hulvey Hulvey Hulvey Hulvey Hulvey Hulvey Hulvey Hulvey

DQ393530

Saprolegnia sp.

S. ferax

4

ATCC 36146

United Kingdom

Hulvey et al. 2007

DQ393536 DQ393554

S. megasperma Pr. polysporus

S. ferax S. ferax

4 4

UNCW286 ATCC 28092

USA USA

Hulvey et al. 2007 Hulvey et al. 2007

DQ393565 DQ393566 EF064134 EF126324 EF126335 EF126339 EF152546 EF175574 EF460351 EU071707 EU152130 EU163405 EU163406 EU240041 EU240070 EU240097 EU240103

Saprolegnia sp. Saprolegnia sp. S. anomala S. bulbosa Saprolegnia sp. S. mixta Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. S. sp. nuchiae Saprolegnia sp. Saprolegnia sp. S. longicaulis S. diclina S. longicaulis Saprolegnia sp.

S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

CBS 305.37 CBS 543.67 WD1D WD20A WD4C WD8F W805b MCA 3070 HRACA0702

France United Kingdom Poland Poland Poland Poland Poland USA Germany USA Korea China China Poland Poland Poland Poland

Hulvey et al. 2007 Hulvey et al. 2007 Belbahri et al. Unpublished Belbahri et al. Unpublished Belbahri et al. Unpublished Belbahri et al. Unpublished Belbahri et al. Unpublished Straus et al. Unpublished Hirsch et al. 2008 Ruthig 2009 Lee et al. Unpublished Ke et al. 2009b Ke et al. 2009b Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished

JY BMY WD1D GD18A K07 WD18C

et al. et al. et al. et al. et al. et al. et al. et al. et al. et al.

2007 2007 2007 2007 2007 2007 2007 2007 2007 2007

R.L. Seymour, S. delica

F. Moreau, S. mixta Steciow et al. 2007, S. bulbosa Steciow 2003, S. oliviae Steciow 2001, S. longicaulis

Gandhe & Kurne 2003, S. anomala

Molecular taxonomy of the genus Saprolegnia

W.R. Ivimey Cook, S. ferax Willoughby et al. 1983, S. ferax Ritchie & Slovin 1974, Pr. polysporus F. Moreau, S. ferax

(continued on next page)

11

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HQ643977 JX535247 JX535253 JX535254 AB219390 AM228850 AY267011

12

Genbank Accession No.

Designated name in GenBank

New designated name by MOTUs

Cluster

Strain

Origin

Publications generating the sequences

EU240106 EU240107 EU240108 EU240110 EU240111 EU240112 EU240113 EU240114 EU240116 EU240120 EU240121 EU240122 EU240128 EU240129 EU240136 EU240146 EU240148 FJ236032 FJ794908 FN186037 GU014282 GU014283 HM637287 HQ643980 HQ643990

Saprolegnia sp. S. diclina Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. S. longicaulis S. longicaulis Saprolegnia sp. S. bulbosa Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. S. litoralis S. litoralis Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. S. diclina S. lapponica

S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

WD1E WD1F WD1G WD1I WD1J WD1K WD1L WD1M WD20C WD3D WD3E WD3G WD3A WD3B WD34A WD4D WD7C DC3U2_2007 SAP2_MAR01 VI04818 AESB SPE JL CBS 326.35 CBS 284.38

Poland Poland Poland Poland Poland Poland Poland Poland Poland Poland Poland Poland Poland Poland Poland Poland Poland USA Germany United Kingdom USA USA China Netherlands United Kingdom

Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Cordier et al. Unpublished Straus et al. 2009 Wolinska et al. 2009 Stueland et al. Unpublished Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Cao et al. Unpublished Robideau et al. 2011 Robideau et al. 2011

HQ643994 HQ643995 HQ644001 HQ644016 HQ660706 JX857688 EU240109 GQ165533 AB219394 AY197329

S. mixta S. mixta S. parasitica S. unispora Saprolegnia sp. Saprolegnia sp. S. bulbosa Saprolegnia sp. S. polymorpha S. multispora

S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. ferax S. australis S. australis

4 4 4 4 4 4 4-2 4-3 5 5

CBS 149.65 ATCC 66855, CBS 307.37 CBS 397.34 CBS 110066 197907 PB-2012 WD1H F-1561.8 CBS 618.97 F-1247

Denmark France Netherlands Japan United Kingdom India Poland France France

Robideau et al. 2011 Robideau et al. 2011 Robideau et al. 2011 Robideau et al. 2011 Tuffs & Oidtmann 2011 Prabha et al. Unpublished Cordier et al. Unpublished Khatibi et al. Unpublished Inaba & Tokumasu Unpublished Paul & Steciow 2004

DQ393528

Saprolegnia sp.

S. australis

5

ATCC 22284

USA

Hulvey et al. 2007

DQ393546 DQ393547 DQ393560

Saprolegnia sp. S. megasperma Saprolegnia sp.

S. australis S. australis S. australis

5 5 5

UNCW315 UNCW320 CBS 618.97

USA USA United Kingdom

Hulvey et al. 2007 Hulvey et al. 2007 Hulvey et al. 2007

EF460350

Saprolegnia sp.

S. australis

5

LB

Germany

Hirsch et al. 2008

Isolate and named assigned by

A. Meurs, S. diclina W.R. Ivimey Cook, S. lapponica H. Holter, S. mixta F. Moreau, S. mixta A. Meurs, S. parasitica S. Inaba, S. unispora

L.G. Willoughby Paul & Steciow 2004, S. multispora Powell Jr. et al. 1972, S. parasitica

Willoughby 1998, S. polymorpha

J. V. Sandoval-Sierra et al.

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Table 2 e (continued )

S. parasitica

S. australis

5

CBS 302.56

HQ644007 JX535255 AM228811 AM228812 AM228813 AM228816 AM228817 AM228819 EU544190 HM988957 DQ393558 AY310502 DQ393513 EU071706 EU124766 EU240098 FN186033 GU014261 GU014262 GU014263 GU014264 GU014265 GU014266 GU014267 GU014268 GU014269 GU014270 GU014271 GU014272 GU014273 GU014274 GU014275 GU014276 HQ644000 AB219380 AY270033 DQ393534 DQ393564

Saprolegnia sp. Saprolegnia sp. S. diclina S. australis S. australis S. australis S. australis S. australis Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Leptolegnia sp. S. diclina Leptolegnia sp. S. cf. kauffmanniana Leptolegnia sp. Leptolegnia sp. Achlya sp. Achlya sp. Achlya sp. Achlya sp. Achlya sp. Achlya sp. Achlya sp. Achlya sp. Achlya sp. Achlya sp. Achlya sp. Achlya sp. Achlya sp. Achlya sp. Achlya sp. Achlya sp. S. parasitica S. unispora Saprolegnia sp. S. itoana S. itoana

S. australis S. australis S. delica S. delica S. delica S. delica S. delica S. delica S. delica S. delica S. litoralis Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. S. subterranea S. subterranea S. subterranea S. subterranea

5 5 6 6 6 6 6 6 6 6 7 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 10 10 10 10

CBS 632.85 JY2 SAP175 SAP179 SAP183 SAP200 SAP202 SAP204 WM_1 MF-2010 CBS 535.67 CBS 177.86 UNCW217 PSCR0503 H1E2 K08 VI04813 SCAAD CORA1 EM31B EM31E EM26B EM7 EM25 DG PCRTB2 MOTAD3 O3EG1 EM23 EM32C EM47A MOT2 02RCT CBS 540.67 CBS 110066 Arg 009 UNCW280 CBS 278.52

USA USA USA Poland United Kingdom USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA United Kingdom Japan Argentina USA USA

Robideau et al. 2011 Zhang et al. Unpublished
guez-Uribeondo et al. 2007 Die
guez-Uribeondo et al. 2007 Die
guez-Uribeondo et al. 2007 Die
guez-Uribeondo et al. 2007 Die
guez-Uribeondo et al. 2007 Die
guez-Uribeondo et al. 2007 Die Wolinska et al. 2008 Caruana et al. 2012 Hulvey et al. 2007 Oidtmann et al. 2004 Hulvey et al. 2007 Ruthig 2009 Johnson et al. 2008 Cordier et al. Unpublished Stueland et al. Unpublished Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Ruthig & Provost-Javier 2012 Robideau et al. 2011 Inaba & Tokumasu Unpublished Steciow & Paul Unpublished Hulvey et al. 2007 Hulvey et al. 2007

HQ644006 DQ393567 DQ393521 DQ393522 DQ393559

S. rodrigueziana S. asterophora S. megasperma S. megasperma Saprolegnia sp.

S. subterranea S. torulosa S. monilifera S. monilifera S. monilifera

10 11 12 12 12

CBS 119354 CBS 110064 UNCW257 UNCW258 ATCC 66859, CBS 213.35

Argentina Japan USA USA United Kingdom

Robideau et al. 2011 Hulvey et al. 2007 Hulvey et al. 2007 Hulvey et al. 2007 Hulvey et al. 2007

DQ393562

Saprolegnia sp.

S. monilifera

12

CBS 386.52 ,UNCW381

USA

Hulvey et al. 2007

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

Netherlands

China Spain Spain Spain Spain Spain Spain USA United Kingdom

Robideau et al. 2011

A.J. van der PlaatsNiterink, S. parasitica

M.W. Dick, S. litoralis

Molecular taxonomy of the genus Saprolegnia

M.W. Dick, S. parasitica S. Inaba, S. unispora

H.S. Vishnia, S. subterranea S. Inaba, S. torulosa

W.R. Ivimey Cook, S. unispora (continued on next page)

13

Please cite this article in press as: Sandoval-Sierra JV, et al., Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.10.005

HQ644002

14

Genbank Accession No.

Designated name in GenBank

HQ644015

S. unispora

AM228851 DQ393523 DQ393524 AB219395

New designated name by MOTUs

Cluster

Strain

Origin

S. monilifera

12

ATCC 66859, CBS 213.35

United Kingdom

Robideau et al. 2011

Leptolegnia sp. S. richteri S. luxurians S. semihypogyna

S. terrestris S. terrestris S. terrestris S. eccentrica

13 13 13 14

SAP248 UNCW259 UNCW260 CBS 109568

Norway USA USA Japan


guez-Uribeondo et al. 2007 Die Hulvey et al. 2007 Hulvey et al. 2007 Inaba & Tokumasu Unpublished

AY647194 DQ393556 DQ393563

S. semihypogyna S. richteri S. luxurians

S. eccentrica S. eccentrica S. eccentrica

14 14 14

IA1424 CBS 551.62 CBS 109568

Japan United Kingdom Japan

Phadee et al. Unpublished Hulvey et al. 2007 Hulvey et al. 2007

DQ393561 AB219375 AB219383 HQ643406 HQ643407 HQ644012 AB219376 AB219382

Saprolegnia sp. Ap. terrestris Py. terrestris Py. terrestris Py. terrestris S. monoica S. eccentrica Py. humphreyana

S. furcata S. megasperma S. megasperma S. megasperma S. megasperma S. turfosa S. anisospora S. anisospora

16 21 21 21 21 22 23 23

CBS 542.67 IA 1339 CBS 110058 CBS 110059 CBS 110058 CBS 559.67 CBS 110061 CBS 110056

United Kingdom Japan Japan Japan Japan United Kingdom Japan Japan

Hulvey et al. 2007 Inaba & Tokumasu Unpublished Inaba & Tokumasu Unpublished Robideau et al. 2011 Robideau et al. 2011 Robideau et al. 2011 Inaba & Tokumasu Unpublished Inaba & Tokumasu Unpublished

EU292729

S. brachydanis

S. brachydanionis

24

China

Ke et al. 2009a

AB219392 AM228847 AB219391 HQ643992 HQ438029 EU124749 EU124750 EU124751 EU124752 EU124753 EU124754 EU124755 EU124756 EU124757 EU124758 EU124759 EU124760 FJ801989 GU258930 GU259207 JQ974995 JQ974996 JQ974997

S. monoica S. litoralis S. litoralis S. litoralis Saprolegniaceae Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp.

Saprolegnia sp. 8 Saprolegnia sp. 9 Saprolegnia sp. 10 Saprolegnia sp. 10 Saprolegnia sp. 11 Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp.

25 26 27 27 28

Japan Sweden Japan Japan Antarctica USA USA USA USA USA USA USA USA USA USA USA USA

Inaba & Tokumasu Unpublished
guez-Uribeondo et al. 2007 Die Inaba & Tokumasu Unpublished Robideau et al. 2011 Gonc¸alves et al. 2012 Johnson et al. 2008 Johnson et al. 2008 Johnson et al. 2008 Johnson et al. 2008 Johnson et al. 2008 Johnson et al. 2008 Johnson et al. 2008 Johnson et al. 2008 Johnson et al. 2008 Johnson et al. 2008 Johnson et al. 2008 Johnson et al. 2008 Coffey et al. Unpublished Coffey et al. Unpublished Coffey et al. Unpublished Ault et al. 2012 Ault et al. 2012 Ault et al. 2012

IA 1456 SAP232 CBS 110062 CBS 110062 UFMGCB 3678 BBE15 H2E1 H2E3 AE1 BBE20 H1E1 H3E2 H3E1 H3E5 H1E4 BBE08 H3E3 P8203 P8203 P8205 LR2 MB12 LB46

USA USA USA

Publications generating the sequences

Isolate and named assigned by W.R. Ivimey Cook, S. unispora

Inaba & Tokumasu 2002, S. semiphypogyna

Inaba & Tokumasu 2002, S. semiphypogyna M.W. Dick, S. furcata S. Inaba, Ap. terrestris S. Inaba, Py. terrestris S. Inaba, Py. terrestris S. Inaba, Py. terrestris M.W. Dick, S. monoica S. Inaba, S. eccentrica S. Inaba, Py. humphreyana Ke et al. 2009a, S. brachydanionis

S. Inaba, S. litoralis S. Inaba, S. litoralis

J. V. Sandoval-Sierra et al.

Please cite this article in press as: Sandoval-Sierra JV, et al., Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.10.005

Table 2 e (continued )

Czech Republic Norway USA Czech Republic Czech Republic Czech Republic Czech Republic Czech Republic Czech Republic Czech Republic Czech Republic Czech Republic Czech Republic USA Czech Republic Czech Republic Czech Republic Czech Republic

VI03659 VI03660 CBS 540.67 SAP3_LAB01 SAP3_ZLU01 VI03839 WM_3 SAP1_ZEL03+04 Clone 1 SAP1_ZEL03+04 Clone 2 SAP1_ZEL03+04 Clone 3 SAP1_RIM01 SAP1_SEC01 SAP1_TRN01 SAP1_VIR01 SAP1_ZEL01 SAP1_ZLU01 SAP1_BRN01 UNCW244 SAP1_VIR01 SAP1_ZEL01 SAP1_ZLU01 SAP1_BRN01

Norway Norway United Kingdom

Vr alstad et al. 2009 Vr alstad et al. 2009 Oidtmann et al. 2004 Wolinska et al. 2009 Wolinska et al. 2009 Vr alstad et al. 2009 Wolinska et al. 2008 Wolinska et al. 2009 Wolinska et al. 2009 Wolinska et al. 2009 Wolinska et al. 2009 Wolinska et al. 2009 Wolinska et al. 2009 Wolinska et al. 2009 Wolinska et al. 2009 Wolinska et al. 2009 Wolinska et al. 2009 Hulvey et al. 2007 Wolinska et al. 2009 Wolinska et al. 2009 Wolinska et al. 2009 Wolinska et al. 2009

M.W. Dick, S. parasitica

Molecular taxonomy of the genus Saprolegnia

15

the formal definition since the formal description of S. hypogyna was prior to that of S. parasitica. Although the first name should be used (McNeill et al. 2012) the name of S. parasitica is widely accepted and a proposal to conserve this name might be desirable. Similar problems for other MOTUs are summarized in Table 1. We have also found a number of MOTUs that either correspond to new species or to already described species with no ITS sequence. These were named Saprolegnia sp. 1, Saprolegnia sp. 2, Saprolegnia sp. 3, Saprolegnia sp. 4, Saprolegnia sp. 5, Saprolegnia sp. 6, Saprolegnia sp. 7, Saprolegnia sp. 8, Saprolegnia sp. 9, Saprolegnia sp. 10, and Saprolegnia sp. 11. Unfortunately, the isolates for the species Saprolegnia sp. 1 to sp. 7 did not produce sexual structures allowing to determine whether these were new species or not. These MOTUs did not contain any sequence of reference for any Saprolegnia species. However, some of them comprised sequences assigned to some taxa (e.g., the MOTU for Saprolegnia sp. 2 comprised sequences assigned to S. delica, S. diclina, Saprolegnia kauffmanniana, S. parasitica, Achlya sp., and Leptolegnia sp.; the MOTU for Saprolegnia sp. 8 engulfed sequences assigned to S. monoica; or the MOTU for Saprolegnia sp. 9 and Saprolegnia sp. 10 had sequences assigned to S. litoralis). Again this fact highlights the ambiguity of characters used in classic taxonomy, their plasticity, and/or presence of misidentified sequences in the GenBank. This work shows that Saprolegnia appears to be polyphyletic group. Thus, some species, e.g., S. anisospora, S. asterophora, S. megasperma, S. monoica, S. turfosa, and some Saprolegnia spp, seem to closely related to other genus such as Achlya, Leptolegnia, Protoachlya or Pythiopsis, as previously pointed (Daugherty et al. 1998; Leclerc et al. 2000). Moreover, our phylogenetic analyses seem to indicate that the genus Leptolegnia is more likely to be member of the family Saprolegniaceae than a genus in separated family, the Leptolegniaceae, with the genus Aphanomyces, as proposed by Dick (2001).

AM947030 AM947031 AY310504 FJ794909 FJ794910 AM947032 EU544192 FJ794898 FJ794899 FJ794900 FJ794901 FJ794902 FJ794903 FJ794904 FJ794905 FJ794906 FJ794907 DQ393516 FJ794904 FJ794905 FJ794906 FJ794907

Saprolegniaceae Saprolegniaceae S. parasitica Saprolegnia sp. Saprolegnia sp. Saprolegniaceae Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp.

Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Pr. paradoxa Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp. Leptolegnia. sp.

Problems encountered when developing a molecular diagnosis taxonomy system in Saprolegnia GenBank types of errors GenBank has led to a rapid increase in DNA sequence information in recent years. However, reliable and verified information on their taxonomic level is still lacking. For example, in fungi, recent work on unreliability of published DNA sequences showed that about 20 % of them appear to be misidentified, dubious or chimeric (Bridge et al. 2003; Vilgalys 2003; Nilsson et al. 2006; Bidartondo 2008). Thus, we found that GenBank represents the main source of misidentification of species (45 % of the GenBank sequences of Saprolegnia have incorrect names according to this new MOTU system, see Table 2 and Appendix C). We show that, if sequences of GenBank are analyzed based on their originally assigned names, this results in a high overlapping of intra and interspecific genetic distances. This is because GenBank contains a lot of incorrectly named sequences. One reason is that GenBank lacks suitable and active curation of sequences preventing incorrect annotations. This has resulted in a chain of misannotation uprise, a process called error percolation (Gilks et al. 2002). This type of error has been found for all MOTUs but the most

Please cite this article in press as: Sandoval-Sierra JV, et al., Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.10.005

Accession No.

Cluster

KF717744 KF717796 KF717815 KF717820 KF717822 KF717823 KF717835 KF717837 KF717839

S. diclina S. diclina S. diclina S. diclina S. diclina S. diclina Saprolegnia sp. 1 Saprolegnia sp. 1 S. parasitica

1 1 1 1 1 1 2 2 3

SAP0229 SAP1023 SAP1281 SAP2008 SAP2045 SAP2046 SAP1124 SAP1169 SAP0208

KF717864 KF717870 KF717872 KF717876 KF717883 KF717954 KF718010 KF718015 KF718021 KF718043 KF718048 KF718083 KF718102 KF718105 KF718115 KF718117 KF718121 KF718122 KF718124 KF718125 KF718131 KF718133 KF718134 KF718135 KF718140 KF718142 KF718143 KF718150 KF718156 KF718159 KF718164 KF718166 KF718170 KF718173 KF718176

S. parasitica S. parasitica S. parasitica S. parasitica S. ferax S. ferax S. australis S. australis S. delica S. delica S. litoralis Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. S. subterranea S. torulosa S. monilifera S. monilifera S. terrestris S. terrestris S. eccentrica Saprolegnia sp. S. furcata Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp. Saprolegnia sp.

3 3 3 3 4 4 5 5 6 6 7 8 8 8 8 8 8 9 10 11 12 12 13 13 14 15 16 17 17 17 17 17 17 17 18

SAP1091 SAP1203 SAP1230 SAP1381 SAP0157 SAP1234 SAP1105 SAP1225 SAP0353 SAP1524 SAP1486 SAP1006 SAP1315 SAP1326 SAP1456 SAP1823 SAP1979 SAP1209 SAP1293 SAP0856 SAP1450 SAP1470 SAP1285 SAP1320 SAP1288 SAP0477 SAP1294 SAP0958 SAP1272 SAP1560 SAP1642 SAP1698 SAP1872 SAP1997 SAP1536

2 2 2 2 2 2 3

4 5 5 5 5 5 5 5 6

Isolate

Host/Habitat

Source

Origin

Collection

Previously named by*

Lake River River River River River River River Salmo trutta River River River River Lake River River River Salmo clarki River River River Lake River River River River River Drainage muck River River River River River River River River River River River River River River River River

Water Water Water Water Water Water Water Water Fish adult

United Kingdom Spain Spain Chile Chile Chile Greenland Argentina United Kingdom

ATCC 56851 RJB-CSIC RJB-CSIC RJB-CSIC RJB-CSIC RJB-CSIC RJB-CSIC RJB-CSIC ATCC 42062

Willoughby et al. 1983

Water Water Water Water Water Water Water Water Fish adult Water Water Water Water Water Water Water Water Water Water Water Water Water Water Water Water Water Water Water Water Water Water Water Water Water Water

Argentina Spain Ecuador Ecuador USA Ecuador Spain Ecuador USA Argentina United Kingdom Ecuador Ecuador Ecuador Ecuador Spain Spain Spain USA Argentina Ecuador United Kingdom USA Ecuador United Kingdom South Africa United Kingdom Ecuador Portugal Spain Spain Spain Spain Spain Argentina

RJB-CSIC RJB-CSIC RJB-CSIC RJB-CSIC ATCC 26116 RJB-CSIC RJB-CSIC RJB-CSIC CBS 343.62 RJB-CSIC CBS 535.67 RJB-CSIC RJB-CSIC RJB-CSIC RJB-CSIC RJB-CSIC RJB-CSIC RJB-CSIC CBS 278.52 RJB-CSIC RJB-CSIC CBS 558.67 CBS 533.67 RJB-CSIC CBS 551.67 RJB-CSIC CBS 542.67 RJB-CSIC RJB-CSIC RJB-CSIC RJB-CSIC RJB-CSIC RJB-CSIC RJB-CSIC RJB-CSIC

Willoughby et al. 1983

R Emerson

R.L. Seymour M.W. Dick

H.S. Vishniac

M.W. Dick M.W. Dick M.W. Dick M.W. Dick

J. V. Sandoval-Sierra et al.

MOTU

16

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Table 3 e GenBank accession numbers of ITS nrDNA reference sequences for Saprolegnia species according to MOTUs including all ribotypes sequences found for each MOTU. MOTUs were generated applying clustering optimization, Bayesian inference, and MP analyses.

CBS 599.67

CBS 110062

Japan Sweden USA USA Japan Antarctica United Kingdom Water Water Water Water Water Water Water

17

eloquent is the incorrectly assignments of sequences to other genera (e.g., Achlya, Leptolegnia, and Protoachlya) and vice versa. Another source of problems for molecular taxonomy is sequencing errors present in sequences of public databases (Nilsson et al. 2006). When adding GenBank sequences to our alignment based on controlled sequences from pure cultures, we detected a number of GenBank sequences containing sequencing errors. Since the genetic distance separation among MOTUs is short, these errors sometimes lead to separation of sequences from their true MOTUs. Examples of this were the single sequence clusters 4-1, 4-2, and 4-3 (which are in fact Saprolegnia ferax, Appendix E), and 8-1 (which are in fact Saprolegnia sp. 2, Appendix G). Culture collections may also contribute to taxonomic problems if cultures are not correctly identified or they have been overgrown by other Oomycetes. This was found for a number of cultures that need to be rechecked from ATCC and CBS (see Table 2 and Appendix C). Unfortunately, the nomenclature code does not require having a reference culture, which makes it difficult to carry out proper taxonomic studies. The characters of taxonomic value can only be preserved in pure cultures but not in isolates metabolically inactive as demanded by the code (McNeill et al. 2012). Thus, it urges to change these requirements for enabling to make useful and efficient taxonomies.

M.W. Dick

R.P.W.M. Jacobs A.L. van Beverwijk Ke et al. 2009a

M.W. Dick

United Kindon Ecuador Spain United Kingdom Ecuador Germany Netherlands China Water Water Water Water Water Water Water Fish adult

CBS 531.67 RJB-CSIC RJB-CSIC CBS 532.67 RJB-CSIC CBS 313.81 CBS 178.44

M.W. Dick

Molecular taxonomy of the genus Saprolegnia

* When not indicated the species assignment was done by authors of these study.

UFMGCB3678

IA1456 SAP232 UNCW292 UNCW291

25 26 26 26 27 28 29 8 9 9 9 10 11 Saprolegnia Saprolegnia Saprolegnia Saprolegnia Saprolegnia Saprolegnia S. monoica AB219392 AM228847 DQ393540 DQ393550 HQ643992 HQ438029 HQ643998

sp. sp. sp. sp. sp. sp.

S. asterophora Saprolegnia sp. 7 Saprolegnia sp. 7 S. megasperma S. megasperma S. turfosa S. anisospora S. brachydanionis KF718178 KF718183 KF718184 KF718185 KF718186 KF718190 KF718193 EU292729

19 20 20 21 21 22 23 24

SAP1296 SAP1360 SAP1996 SAP1287 SAP1373 SAP1291 SAP0364

River River River River River Peatbog Pond Brachydanio rerio

Advantages of a molecular taxonomy in Saprolegnia This method allows us now for a more rapid and precise identification of species of this taxonomically complicated genus of Saprolegnia. The fact that these taxonomic units are based on easy-to-sequence and to-compare DNA region, i.e., ITS nrDNA, makes this MOTU system a useful approach to identify species in this genus. In addition, and for the first time in the Saprolegniales, this classification is supported by a culture and DNA collection. The collection holds each taxonomic unit described in this study and comprises 461 pure cultures from worldwide distribution. This is of key importance as it enhances the possibility of further investigations with other DNA regions as well as the study of specific characters and properties for a particular species using the same material (Tautz et al. 2003). This work also represents the most extensive ITS nrDNA sequence analysis in the Saprolegniales. This region has been recently proposed as universal region for barcoding in fungi (Schoch et al. 2012). In a recent study based on Oomycetes species, the regions ITS nrDNA and cytochrome c oxidase subunit 1 were suggested as suitable regions for barcoding (Robideau et al. 2011). Here, we show that using this taxon diagnosis system the resulting intra and intergenetic distances based on MOTUs criteria for species limits may allow the use of ITS nrDNA as a potential region for barcoding in Saprolegnia. Finally, this generated taxon diagnosis system for Saprolegnia makes it possible now to carry out precise studies on a number of key scientific disciplines such as biodiversity, systematics, genomics, ecology, biogeography, pathology, etc., as well as to develop accurate methods for detecting these economically important species.

Please cite this article in press as: Sandoval-Sierra JV, et al., Species identification in the genus Saprolegnia (Oomycetes): Defining DNA-based molecular operational taxonomic units, Fungal Biology (2013), http://dx.doi.org/10.1016/j.funbio.2013.10.005

18

Acknowledgements This work was supported by grants from the European Union
n, (ITN-SAPRO-238550), and Ministerio de Ciencia e Innovacio
n San Ignacio Spain (CGL2009-10032), and project, Fundacio
V. Sandoval-Sierra was supdel Huinay, Endesa, Chile. Jose ported by ITN-SAPRO-238550. We would like to thank Dr Mar€ ker from University of Tubingen for assistance with kus Go OPTSIL program.

Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.funbio.2013.10.005.

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