New Trends in Entomopathogenic Nematode Systematics: Impact of Molecular Biology and Phylogenetic Reconstruction

New Trends in Entomopathogenic Nematode Systematics: Impact of Molecular Biology and Phylogenetic Reconstruction S.P. Stock Department of Plant Pathology, University of Arizona, Tucson, Arizona, U.S.A. Summary Assimilation of molecular approaches into entomopathogenic nematode (EPN) systematics has escalated dramatically in the last few years. Various molecular methods and markers have been used not only for diagnostic purposes, sorting out of cryptic species, populations and strains, but also to assess evolutionary relationships among these nematodes. In this presentation, the current state of affairs in the taxonomy of the EPN is reviewed, with particular emphasis on the application of molecular methods. A combined approach using traditional (morphology) and molecular methods is considered as an example to demonstrate the value of this integrated perspective to address key questions in the systematics of this group of nematodes. Introduction Entomopathogenic nematodes (EPN) are a ubiquitous group of ob- ligate and lethal parasites of insects. They are excellent and widely used biological control agents of many insect pests (Kaya & Gaugler, 1993). These nematodes are characterized by their ability to carry specific pathogenic bacteria, Xenorhabus for Steinernematidae and Photorhabdus with Heterorhabditidae, which are released into the hemocoel after penetration of the host has been attained by the infective stage of the nematode. Two EPN families are currently known: Steinernematidae Chitwood and Chitwood, 1937 and Heterorhabditidae Poinar, 1976. These fami- ©2002 by Monduzzi Editore S.p.A. MEDIMOND Inc. C804R9051 1 2 The Tenth International Congress of Parasitology lies are not closely related phylogenetically, but share life histories and morphological and ecological similarities throughout convergent evolution (Poinar, 1993; Blaxter et al., 1998). Presently, Steinernematidae comprises two genera: Steinernema (the type genus) with 30 described species, and Neosteinernema, with only one species, N. longicurvicauda. The family Heterorhabditidae comprises only one genus, Heterorhabditis, with H. bacteriophora as the type species and 9 other species described. Accurate identification of these nematodes has important implica- tions in many areas including systematics, population genetics, ecology, and is also central to diagnosis for selection of the appropriate species and/or strains in biological control applications. EPN species have been described through the implicit application of phenetic and biological species concepts, with morphological data and cross-breeding tests most frequently used for differential diagnosis and identification (Dix et al., 1992; Hominick et al., 1997; Kaya and Stock, 1997). However, with the increasing number of described species, traditional approaches, such as comparative morphology, have become of limited utility in EPN taxonomy. The paucity of diagnostic morphological traits in members of both families hinders successful identification of species even for trained experts in the group. In addition, the application of the biological species concept via cross-breeding tests has recently been questioned. The discovery of hermaphroditism in steinernematids by Griffin and Callaghan (2001) has set a caution signal for the consideration of hybridizations assays to test validity of biological species in this group. To overcome these difficulties, many biochemical and molecular methods have been devel- oped as alternative tools (Akhurst, 1987; Joyce et al., 1994; Grenier et al., 1996; Reid and Hominick, 1992). In this presentation, I provide an overview of the most commonly used molecular techniques and markers applied in EPN systematics. The value of combined traditional and morphological approaches is discussed and demonstrated. EPN and the Molecular Revolution The advent of the polymerase chain reaction (PCR) and DNA sequencing has revolutionized nematode taxonomy and genetics not only because of their usefulness for diagnostic purposes, but also because they are powerful tools to interpret phylogenetic relationships at different taxonomic levels. A variety of molecular methods and loci are currently available for interpretation of genetic diversity in EPN. The utility of some common molecular methods and genetic markers used to identify, diagnose and assess evolutionary relationships of EPN is discussed below. Vancouver, Canada, August 4-9, 2002 3 - Molecular Methods Several molecular approaches have been used to identify, diagnose, delimit species and assess phylogenetic relationships of EPN (Reid and Hominick, 1992; Gardner et al., 1994; Liu and Berry, 1995; Liu et al., 1997; Reid et al., 1997; Nguyen et al., 2001; Stock et al., 2001). Among many others, three molecular methods, random amplified polymorphic DNA (RAPD), restriction fragment length polymorphisms (RFLP) and more recently DNA sequencing, have been the most extensively applied. For instance, RAPDs have been used to complement description of new species (Gardner et al., 1994; Stock et al., 1996), explore genetic diversity among EPN species and strains (Hashmi et al., 1996) and interpret their phylogenetic relationships (Liu and Berry, 1996). How- ever, despite of these efforts, the use of RAPDs has been discouraged, mainly because of the recognition that reproducibility of results can be affected by many factors such as the quality and concentration of DNA, PCR cycling conditions (including type of PCR machine used), etc. RFLPs have extensively been used for diagnoses and identification of Steinernema spp (Hominick et al., 1997; Stock et al., 1998; Phan et al., 2001). Moreover, RFLP of the ITS region have been used to interpret their evolutionary relationships (Reid et al., 1997). DNA sequence analysis has recently been incorporated into nematode systematics. This molecular approach has been demonstrated to yield more information about variation within and among nematode species than the RFLP approach (Powers et al., 1997; Szalanski et al., 2000). In addition, sequence analysis has shown to be a more suitable tactic in assessing phylogenetic relationships at different taxonomic levels and as well as for species delimitation (Adams et al., 1998; Blaxter et al., 1998; Szalanski et al., 2000; Stock et al., 2001). - Target Markers The choice of a target region for amplification by PCR depends on the questions to be addressed and the purpose it should serve. All regions of nuclear and mitochondrial genomes of parasites accumulate mutations over time, and some regions are more accessible to nucle- otide changes than others. For instance, non-coding regions and introns usually evolve more rapidly that coding regions, as they are unlikely to be constrained by functions. If the purpose of a study is identify species, then the level of within-species variation in the sequence should be substantially lower that the degree of variation between or among species. However, if the goal is to provide markers for identification of strains, then a significant level of sequence variation should exit within the species under investigation. Nuclear rDNA has been a useful source for providing markers in- volved in delimitation of EPN at different taxonomic levels. For exam- 4 The Tenth International Congress of Parasitology ple, analysis of the 18S or small subunit (SSU) of rDNA has revealed that the two EPN families represent distinct phylogenetically unrelated lineages (Blaxter et al., 1998). However, this region has pr be conserved to resolve relationships among Heterorhabditis and Steinernema species (Liu et al., 1997; Stock et al., 2001). The internal transcribed spacer region (ITS) of rDNA has revealed sufficient genetic variation for differentiating Heterorhabditis species and has proved useful for delimitation and interpretation of evolutionary relationships among species of this genus. The ITS region (span- ning ITS-1, 5.8s rDNA, and ITS-2) has also been evaluated to assess phylogenetic relationships among Steinernema species and to determine its utility for taxonomic purposes (Reid et al., 1997; Nguyen et al., 2001). This highly variable region has revealed numerous markers useful for diagnostics. However, the level of variation is such that phylogenetic interpretation based on this region has proved to be difficult. Nguyen et al. (2001) suggested that the examination of other regions may be necessary to provide characters more suitable for a robust phylogenetic analysis. More recently, the 28S or large subunit (LSU) of rDNA has been considered to assess phylogenetic relationships among Steinernema species. This region has been valuable not only for interpreting evolutionary relationships, but also to test hypothesis of historical lineage independ- ence of Steinernema species and for diagnostic purposes (Stock et al., 2001). A few mitochondrial genes have been considered for analyzing genetic variation within and among EPN populations. For instance, Blouin et al (1999) and Liu et al. (1999) studied the genetic variation among several Heterorhabditis marelatus populations using the ND4 gene of mtDNA, and found limited infra-specific variation. Other mtDNA genes explored have been COX II and 16S rDNA (Szalanski et al., 2000). These loci proved useful for discrimination among Steinernema species, but did not show intraspecific variation among several S. feltiae strains considered in their study. The Future of EPN Systematics: Integrating Molecules and Morphology Molecular techniques have made EPN systematics a lot more

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