Fungal Genetics and Biology xxx (2014) xxx–xxx Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi Regular Articles A combined mitochondrial and nuclear multilocus phylogeny of the genus Phytophthora a, b c Frank N. Martin ⇑, Jaime E. Blair , Michael D. Coffey a USDA-ARS, Salinas, CA, United States b Department of Biology, Franklin & Marshall College, Lancaster, PA, United States c Department of Plant Pathology & Microbiology, University of California, Riverside, CA, United States article info abstract Article history: The most recent phylogenetic analysis of the genus Phytophthora was completed in 2008 (Blair et al., Received 23 September 2013 2008) and utilized 8.1 kb of sequence data from seven nuclear loci. Given the large number of species that Accepted 24 February 2014 have recently been described, this study was undertaken to broaden the available information on the Available online xxxx phylogeny of the genus. A total of 166 isolates representing 92 recognized species and 17 provisional spe- cies were analyzed, including many of the same isolates used in the nuclear multilocus study of Blair et al. Keywords: (2008). Four mitochondrial genes (cox2, nad9, rps10 and secY) were sequenced with a total of 2373 bp Oomycetes used in the analysis; the species relationships recovered with mitochondrial data were largely consistent Phytophthora with those observed previously in the nuclear analysis. Combining the new mitochondrial data with the Phylogeny Multispecies coalescent nuclear data from Blair et al. (2008) generated a dataset of 10,828 bp representing 11 loci, however res- olution of basal clade relationships was still low. We therefore implemented a modified multispecies coa- lescent approach with a subset of the data, and recovered increased resolution and moderate to high support for clade relationships. A more detailed analysis of species from clades 2 and 8 identified an addi- tional seven phylogenetic lineages that warrant further investigation to determine if they represent dis- tinct species. As has been reported in other phylogenetic studies of the genus, there was no consistent correlation between phylogenetic relatedness and morphological features or ecology. Published by Elsevier Inc. 1. Introduction and was recently reported to be approximately 117 species (Martin et al., 2012). With the recent descriptions of P. pluvialis (Reeser Species in the genus Phytophthora (Oomycetes) are capable of et al., 2013), P. mississippiae (Yang et al., 2013), P. cichorii, P. dauci infecting a wide range of plant species and can cause significant and P. lactucae (Bertier et al., 2013) and the hybrid species P.x damage to economically important crop plants worldwide. While serendipita and P.xpelgrandis (Man in‘t Veld et al., 2012) there some species have a narrow host range (e.g., P. infestans), others are at least 124 described species. Given the number of provisional are capable of infecting host species reflecting a wide range of species names used in the literature, this number will continue to plant genera (e.g., P. cinnamomi)(Erwin and Ribeiro, 1996). While increase in the future. There are several factors driving this resembling Eumycotan fungi with the production of hyphae, the expansion of species descriptions. The availability of DNA sequence genus is allied with the stramenopiles, a group more closely related data for multiple loci at websites like the Phytophthora to chromophyte algae and plants than to true fungi (Förster et al., Database (www.phytophthoradb.org, Park et al., 2008) and others 2000; Knoll, 1992; Baldauf and Palmer, 1993; Wainright et al., (www.phytophthora-id.org, www.q-bank.eu, www.boldsystems.org) 1993; Bhattacharya and Stickel, 1994; Weerakoon et al., 1998). In and the reduction in cost for generating such data has made it contrast to most Eumycotan fungi, Oomycetes are diploid through- easier for researchers to differentiate isolates from previously out their life cycle. described species, facilitating the identification of new lineages. Over the past 15 years there has been a significant expansion in Due to concerns with invasive species, there has also been an the description of species within the genus. In 1999 the number of increase in the number of surveys from previously under repre- species stood at approximately 55, but from 2000 to 2007 the sented ecosystems (such as streams and forests); in the process of number of valid species nearly doubled to 105 (Brasier, 2007) conducting these surveys a number of new species have been iso- lated and described. Given the increasing problems with invasive species such as P. ramorum, P. kernoviae and P. alni impacting com- Corresponding author. ⇑ E-mail address: [email protected] (F.N. Martin). mercial agriculture and natural ecosystems (Hansen et al., 2012), http://dx.doi.org/10.1016/j.fgb.2014.02.006 1087-1845/Published by Elsevier Inc. Please cite this article in press as: Martin, F.N., et al. A combined mitochondrial and nuclear multilocus phylogeny of the genus Phytophthora. Fungal Genet. Biol. (2014), http://dx.doi.org/10.1016/j.fgb.2014.02.006 2 F.N. Martin et al. / Fungal Genetics and Biology xxx (2014) xxx–xxx and the quarantine restrictions put in place to prevent their spread, (http://phytophthora.ucr.edu). Many of these isolates were also it is imperative that molecular techniques be available to accu- used in the prior analysis of the genus by Blair et al. (2008). A total rately identify isolates to the species level as well as provide an of 92 recognized species (including 30 type cultures), 17 provi- easy means for identification of isolates representing new species. sional species, and seven distinct phylogenetic taxa are repre- Establishing a robust phylogenetic framework is therefore crucial sented in the analysis. Rather than give provisional names for for isolate identification and also for delimiting species boundaries. these phylogenetic taxa without having conducted a careful mor- A well resolved phylogeny will also assist in the analysis of geno- phological analysis, the following naming convention was used: mic sequencing studies as well as allow us to predict the behavior Phytophthora sp. affinis followed by the name of the most closely and ecological functions of new species based on the characteris- related species. In cases where more than one distinct entity is tics of their close relatives, which may be particularly important encountered a number is added sequentially to the name. when new invasive species are identified. To be consistent with Mostowfizadeh-Ghalamfarsa et al. (2010) Early studies on the phylogeny of the genus utilized a limited the naming of P. cryptogea isolates in this study followed a similar number of species and relied mainly on portions of the ribosomal convention. Isolate P1739 was in GI of Mostowfizadeh-Ghalamf- DNA (rDNA), primarily the internal transcribed spacer (ITS) region arsa et al. (2010) and was labeled as P. cryptogea in this study. In (Förster et al., 2000; Cooke and Duncan, 1997; Crawford et al., addition, the ITS sequences of isolates from Mostowfizadeh-Gha- 1996) or mitochondrial genes (Martin and Tooley, 2003a, 2003b). lamfarsa et al. (2010) were compared to isolates used in this cur- The first comprehensive examination of a wide range of species rent study; our P. cryptogea isolates (P1088, FJ801524) in the genus was reported by Cooke et al. (2000) using the ITS re- correspond to the P. cryptogea GI isolates (SCRP230, AY659441) gion to examine the phylogenetic relationship among 50 species. A of Mostowfizadeh-Ghalamfarsa et al. (2010). The P. sp. aff. crypto- total of 8 primary clades were identified with two additional gea GII isolates (P1380, FJ802061) corresponded to P. cryptogea clades, 9 and 10, consisting of P. macrochlamydospora and P. richar- GII (SCRP204, AY659422) while the P. sp. kelmania isolates diae as well as P. insolita, respectively. While there was some cor- (P10367, FJ801461) corresponded to P. cryptogea GIII (SCRP209, relation between phylogenetic grouping and morphological AY659426); this latter grouping was also supported by elongation features like sporangial shape, this was not absolute and many factor 1 a data (P10613, EU079607 vs. SCRP209, AY659519). The clades show more than one sporangial type. Using a similar num- grouping of P. cryptogea GI and GII isolates used in this study were ber of species, Kroon et al. (2004) expanded the analysis of the also supported by cox1 sequence data (data not shown; GenBank genus by using two nuclear (translation elongation factor 1a, accession numbers in Supplementary Table S1). b-tubulin) and two mitochondrial (cox1 and nad1) genes. While in A number of the cultures included in this analysis were part of general the results were congruent with those reported by Cooke other phylogenetic studies as well. For example, representatives of et al. (2000), there were some notable exceptions. For example, clade 2 species that were part of the P. citricola species complex in- the species in clade 9 and 10 of Cooke et al. (2000) were included clude isolate P10204, which is isolate P53 of Hong et al. (2011) that as part of clade 8 rather than as separate clades. Likewise, some was used in their resurrection of P. pini as a species. Isolate P10338 species were placed in different clades (P. tentaculata was placed in P. citricola clade E is IMI031372 in Jung and Burgess (2009) in clade 2a with P. multivesiculata rather than in clade 1 as reported where this phylogenetic species is described and isolates P6624 by Cooke et al., 2000). The most recent analysis of the genus was and P1321 were used as representatives of this entity in reported by Blair et al. (2008) using seven nuclear genes represent- Bezuidenhout et al. (2010). Isolates P7902, P10458 and P1817 were ing 8.1 kb of sequence data for 82 Phytophthora spp. This larger used as representatives of P.