Biological Forum – An International Journal 7(2): 981-992(2015)

ISSN No. (Print): 0975-1130 ISSN No. (Online): 2249-3239 The last changes , Phylogeny and Evolution of Plant-parasitic Hamid Abbasi Moghaddam* and Mohammad Salari** *M.Sc. Student, Department of Plant Protection, Faculty of Agriculture, University of Zabol, IRAN **Associate Professor, Department of Plant Protection, College of Agriculture, University of Zabol, IRAN (Corresponding author: Hamid Abbasi Moghaddam) (Received 28 August, 2015, Accepted 29 November, 2015) (Published by Research Trend, Website: www.researchtrend.net) ABSTRACT: Nematodes can be identified using several methods, including light microscopy, fatty acid analysis, and PCR analysis. The phylum Nematoda can be seen as a success story. Nematodes are speciose and are present in huge numbers in virtually all marine, freshwater and terrestrial environments. A phylogenetic framework is needed to underpin meaningful comparisons across taxa and to generate hypotheses on the evolutionary origins of important properties and processes. We will outline the backbone of phylogeny and focus on the phylogeny and evolution of plant-parasitic Tylenchomorpha. We will conclude with some recent insights into the relationships within and between two highly successful representatives of the Tylenchomorpha; the genera Pratylenchus and Meloidogyne. In this study, we present last changes taxonomy, phylogeny and evolution of plant-parasitic nematodes. Keywords: Phylogeny, taxonomy, plant-parasitic nematode, evolution comparative anatomy of existing nematodes, trophic INTRODUCTION habits, and by the comparison of nematode DNA Members of the phylum Nematoda (round worms) have sequences (Thomas et al. 1997, Powers et al. 1993). been in existence for an estimated one billion years, Based upon molecular phylogenic analyses, it appears making them one of the most ancient and diverse types that nematodes have evolved their ability to parasitize of on earth (Wang et al. 1999). They are animals and plants several times during their evolution thought to have evolved from simple animals some 400 (Blaxter et al. 1998). One point is clear; nematodes million years before the "Cambrian explosion" of have evolved to fill almost every conceivable niche on invertebrates able to be fossilized (Poinar, 1983). The earth that contains some amount of water. Nematodes two nematode classes, the Chromadorea and Enoplea, are extremely abundant and diverse animals; only have diverged so long ago, over 550 million years, that insects exceed their diversity. Most nematodes are free- it is difficult to accurately know the age of the two living and feed on bacteria, fungi, protozoans and other lineages of the phylum (Fig. 1). Nematodes are nematode (40% of the described species); many are multicellular animals in the group Ecdysozoa, or parasites of animals (invertebrates and vertebrates (44% animals that can shed their cuticle. Also included in this of the described species) and plants (15% of the group with nematodes are insects, arachnids and described species). crustaceans. In contrast to some of their relative Order Triplonchida invertebrates, nematodes are soft-bodied. Thus, very CHANGING PERSPECTIVES IN NEMATODE few nematodes have been fossilized (22 species from PHYLOGENY AND CLASSIFICATION 11 genera) and exactly what ancestral nematodes looked like remains unknown. While we do not know In the face of overwhelming diversity, a phylogenetic the morphology of the first nematodes, it is probable framework is needed to underpin meaningful that they were microbial feeders in the primordial comparisons across taxa and to generate hypotheses on oceans. The oldest known fossil nematodes are only the evolutionary origins of interesting properties and 120-135 million years old; by then nematodes had processes. Our understanding of nematode relationships diversified to feed on microbes, animals and plants has a varied and at times turbulent history, reflecting (Poinar et al. 1994, Manum et al. 1994). The oldest not only wider developments in phylogenetics, but also fossil nematodes are found in amber and are commonly the expertise and perspectives of those few systematists associated with insects. This is probably due to the fact who produce comprehensive classifications. Until that tree sap, which fossilizes to make amber, captures recently, the data for most nematode phylogenies and preserves insects and their associated nematodes consisted of relatively few morphological characters much more easily than an - or a nematode- derived primarily from light microscopy and often by infested portion of a plant. Much of what we know individual effort of the lone taxonomist. about the evolution of nematodes is inferred from the Moghaddam and Salari 982

Fig. 1. Order Rhabditida. Moghaddam and Salari 983 Molecular phylogenetics, bioinformatics and digital suspicions held by several proponents of earlier communication technologies have substantially altered morphological systems, i.e., that many important the dynamics of nematode systematics, creating anatomical features have arisen repeatedly during conditions where collaborative strategies are much evolution, and that one of the two traditional classes more productive than individual effort. This approach () is deeply phylogenetically embedded was exemplified by the analyses of Blaxter et al. (1998) within the other (Adenophorea). In an effort to translate based on small subunit (SSU) rDNA sequences of 53 the implications of SSU rDNA sequences into nematode species. Just seven years later, SSU rDNA classification, De Ley & Blaxter (2002, 2004) proposed sequences are available in public databases for more a system based primarily on the molecular backbone of than 600 nematode species. The basic topology SSU phylogenies (Fig. 2), but also incorporating other obtained by Blaxter et al. (1998) appears to remain characters. The result combines elements from many quite robust, although a number of important groups previous systems, and introduces some new features remain to be included. Also, it is increasingly clear that with respect to ranking (use of infraorders; Fig. 3). For some important aspects of nematode phylogeny cannot the sake of convenience, we will follow the be resolved by SSU data alone. Two of the major nomenclature of this system here, in order to outline the features of SSU analyses have independently confirmed major features of SSU-based nematode phylogenies.

Fig. 2. Schematic overview of the evolution of the phylum Nematoda derived from SSU rDNA sequence data (based on Holterman et al. 2006). Major lineages of plant-parasites are indicated by dotted boxes (Tylenchomorpha, Dorylaimida and Triplonchida). It is noted that the infra order Tylenchomorpha is possible a polyphyletic group; it includes the members of Clade 12 and the Aphelenchoididae, a family within Clade 10. Moghaddam and Salari 984 BACKBONE OF NEMATODE PHYLOGENY the first to exploit the potential of small subunit ribosomal DNA (SSU rDNA) sequence data to resolve Analysis of large EST data sets recently reconfirmed phylogenetic relationships among nematodes. the placement of the phylum Nematoda within the Holterman et al. (2006) presented a subdivision of the superphylum Ecdysozoa (Dunn et al. 2008), a major phylum Nematoda into 12 clades based on a series of animal clade proposed by Aguinaldo et al. (1997) that mostly well-supported bifurcations in the backbone of unites all moulting animals. Blaxter et al. (1998) (53 the tree (339 taxa) (Fig. 2). taxa) and Aleshin et al. (1998) (19 taxa) were among

Fig. 3. Summarized SSU phylogeny of Nematoda with example taxa, ecological range and higher classification (adapted from De Ley & Blaxter, 2002). P = phtypoarasitic, Z = zooparasitic. The under-representation of marine nematodes in these phylogenetic tree of nematodes-it is hard to find a phylogenetic overviews was, to some extent, lifted by morphological, ecological or biological characteristic SSU rDNA-based papers from Meldal et al. (2007) that has not arisen at least twice during nematode and Holterman et al. (2008a). Recently, a phylogenetic evolution. Convergent evolution appears to be an tree based on 1,215 small subunit ribosomal DNA important additional explanation for the seemingly sequences covering a wide range of nematode taxa persistent volatility of nematode systematics. One of was presented by van Megen et al. (2009). The overall the peculiarities of the phylum Nematoda is the topology of this phylogenetic tree resembles that of multitude of times that (animal- or plant-) parasitic Holterman et al. (2006). However, the support values lifestyles have arisen. Understanding the phylogenetic for the backbone tend to be lower. The deep history of the acquisition of particular phenotypes subdivision of the phylum Nematoda should be associated with successful parasitism permits fuller regarded as a 'work in progress', and a multi loci appreciation of the evolutionary constraints approach will be required for a more definitive experienced by organisms adapting to new hosts. framework. The extensiveness of convergent evolution Plant-parasitism has evolved independently in each of is one of the most striking phenomena observed in the three major clades in the phylum Nematoda (Fig. 2). Moghaddam and Salari 985

Fig. 4. Summarized SSU phylogeny of Rhabditida with example taxa, ecological range and higher classification (adapted from De Ley & Blaxter, 2002). Note the use of infraorder names (ending in -omorpha). The plant-parasitic Tylenchomorpha, Dorylaimida, However, the evolution of this diversity of complex Triplonchida have acquired their ability to parasitize feeding traits is not yet fully understood. In recent years plants independently. The infra order Tylenchomorpha LSU and SSU rDNA sequences have been used to infer comprises manifestly the economically most relevant relationships among Tylenchomorpha (Subbotin et al. plant-parasites and within this chapter we will mainly 2006; Bert et al. 2008; Holterman et al. 2009). A focus on this particular group. Apart from the scientific multiple gene approach derived from an EST mining merits of studying of the phylogeny of the Nematoda, strategy has been used to characterize the relationships the underlying molecular framework can be used for between the plant-parasitic genera Meloidogyne, DNA barcode-based nematode detection and Heterodera and Globodera (Scholl and Bird 2005). community analysis. It is (in most cases) possible to Other phylogenetic studies within Tylenchomorpha, define species specific sequence signatures and to mainly based on rDNA sequences or the internal design simple and cheap PCR primers that allow real- transcribed spacers, have been restricted to individual time PCR-based detection and quantification of (super) families or genera. Recent studies include pathogenic nematodes in complex DNA backgrounds. analyses of (Subbotin et al. 2001), At the same time, the SSU rDNA alignment has been Anguinidae (Subbotin et al. 2004), Criconematoidea used to design many family-specific PCR primers (see (Subbotin et al. 2005), Hoplolaimidae (Subbotin et al. for example Holterman et al. 2008b) and quantitative 2007), Meloidogyne (Tandingan De Ley et al. 2002; DNA barcode-based nematode community analyses Tenente et al. 2003; Tigano et al. 2005) and under field conditions are currently being tested. Pratylenchus (Subbotin et al. 2008). In Fig. 5, a schematic phylogenetic framework of the PHYLOGENY OF TYLENCHOMORPHA Tylenchomorpha based on Bert et al. (2008) and A. Overview Holterman et al. (2009) is shown. The families The Tylenchomorpha, the most intensively investigated Hoplolaimididae (including Heteroderinae), infra order within the Tylenchina, comprises the largest Pratylenchidae (except Pratylenchoides) and and most economically important group of Meloidogynidae, which comprises the economically plantparasitic nematodes. Although there are examples most important plant-parasites, plus the genera of nematodes that exploit all plant organs including Tylenchorhynchus and Macrotrophurus form a well flowers and seeds, they mostly attack roots. The supported clade. A robust sister relationship between evolution of plant-parasitic Tylenchomorpha is of Meloidogyne (root-knot nematodes) and representatives particular interest because associations range from of the migratory endoparasitic Pratylenchidae transitory grazing by root-hair feeders to the highly (Pratylenchus, Zygotylenchus and Hirschmanniella) complex host-pathogen interactions of gall-inducing can be observed. The Hoplolaimidae, which include the nematodes and their hosts. Non plant-parasitic Heteroderinae, Hoplolaiminae, Rotylenchoidinae and Tylenchomorpha feed on fungi, algae, lichens, mosses, Rotylenchulinae according to the classification of de insects, mites, leeches or frogs (Siddiqi 2000). Ley and Blaxter (2002), appear as a monophyletic group. Moghaddam and Salari 986 Remarkably, the migratory endoparasitic Radopholus, a the Tylenchomorpha (without Aphelenchoididae) is still notorious pest in banana and citrus, has a well- ambiguous; depending on the reconstruction method supported sister relationship with the Hoplolaimidae. this is fungal-feeding, lower plant/root-hair feeding or Thus, the cyst forming (Heteroderinae) and root-knot bacteriovore feeding. Conclusions on this hypothesis nematodes (Meloidogyne) are likely to have arisen await a better resolution in the basal part of the independently and the migratory endoparasitic Tylenchomorpha tree and additional information on the Pratylenchidae appears to be polyphyletic. feeding behavior of the basal Tylenchomorpha such as Between the earliest divergences within the the Tylenchidae and Anguinidae. Within the tylenchid Tylenchomorpha (Aphelenchoidea) and the top parts of nematodes, migratory ectoparasitic feeding is ancestral the tree, a number of branching points remain for all major clades of nematodes that exclusively unresolved. Although we cannot define the relationship parasitize higher plants (Fig. 5). Migratory between the suborder Criconematoidea and other endoparasitism has evolved independently several times Tylenchomorpha, its members clearly constitute a within Anguinidae and four times, always from separate and well supported clade. Also, the tylenchid migratory ectoparasitic ancestors, in the polyphyletic nematodes with supposedly ancestral morphological Pratylenchidae. Sedentary endoparasitism has also characters, including Tylenchidae and Sphaerularioidea, evolved three times independently; Nacobbus (false do not have an established phylogenetic relationship. root-knot nematodes) and the cyst-forming nematodes Nevertheless, the tylenchid nematodes-those most likely evolved from migratory ectoparasitic Tylenchomorpha that are characterized by a tylenchid nematodes, while root-knot nematodes appear to have stylet (Tylenchomorpha without Aphelenchoididae)- evolved from migratory endoparasitic nematodes. The appear to be clearly monophyletic. The Aphelenchoidea number of independent developments is higher than or "aphelenchs" comprising the mainly fungal-feeding expected mainly due to the polyphyly of the Aphelenchidae and Aphelenchoididae are appointed as Pratylenchidae. Although the development of plant- polyphyletic in all molecular analyses to date. parasitism is usually gradual, endoparasitism seems to However, the morphology based hypothesis of their have developed directly from several simple forms of monophyly could not be significantly rejected based on plant-parasitism including ectoparasites (giving rise to statistical analysis of molecular data (Bert et al. 2008). Heteroderinae, Pratylenchidae, Pratylenchoides and Several studies have confirmed the sister relationship of Tylenchulus) and epidermal and root hair feeders (from the predominantly plant-parasitic Tylenchomorpha which the Anguinidae evolved). (without Aphelenchoidea) with the bacteriovorous Although the parasitic biology of certain plant-parasitic Cephalobidae (Blaxter et al. 1998). However, this was Tylenchomorpha is now relatively well documented, we not unequivocally supported by van Megen et al. should achieve a broader understanding of the evolution (2009). of the mode and direction of plant-parasitism from intermediate groups, including economically less B. Evolution of Plant-Parasitism in Tylenchomorpha important plant-parasites. Within Clade 12 the family The development of plant-parasitism in the Aphelenchidae (fungivores) appears in a sister position Tylenchomorpha has traditionally beenseen as a gradual to all "tylenchs" (Holterman et al. 2006; Bert et al. evolution from fungal feeding to facultative parasitism 2008). However, it should be noted that in a more of root hairs and epidermal cells into more complex recent, maximum likelihood analysis this positioning forms of plant-parasitism, culminating in the could not be robustly confirmed (van Megen et al. development of sedentary endoparasitism (Luc et al. 2009). For the plant-parasitic Tylenchomorpha families 1987). To investigate this hypothesis, ancestral feeding positioned at the base of clade 12 such as the types were reconstructed among Tylenchomorpha using Tylenchidae, the Psilenchidae and the Belonolaimidae three different methods: unordered parsimony, (the 'economically less important plant-parasites'), the parsimony using a stepmatrix, and likelihood (Bert et number of (ribosomal) DNA sequences available is al. 2008; Holterman et al. 2009). Here we present only very limited. It is expected that molecular a schematic overview of these results (Fig. 5). The characterization of specimens from these families and feeding type analysis supported the classical hypothesis improved insight in their feeding habits will give us of the gradual evolution of simple forms of plant- more insight in the transition from fungivorous parasitism, such as root hair and epidermal feeding and lifestyles, via facultative plant-parasitism towards ectoparasitism towards more complex forms of obligatory plant-parasites. endoparasitism. However, the ancestral feeding state of Moghaddam and Salari 987

Fig. 5. The evolution of feeding among Tylenchomorpha super posed on a ribosomal DNA based phylogenetic backbone. (Based on Bert et al. 2008; Holterman et al. 2009). TYLENCHOMORPHA-TOP END PLANT- A2 quarantine status such as M. chitwoodi and M. fallax PARASITES (EPPO = European and Mediterranean Plant Protection Organisation) (Fig. 6). Analysis of a larger molecular A. Root-Knot Nematodes-Towards the Origin(s) of this data set and more Meloidogyne species resulted in a Highly Successful Genus confirmation of these distal clades. Ribosomal DNA Root-knot nematodes-members of the genus sequence data point at Meloidogyne artiellia-a Meloidogyne can be said without too much polyphagous root-knot nematode typically inducing exaggeration to represent the ultimate success story very small galls (host plant range includes both mono among plant-parasitic nematodes. The more distal and dicotyledons)-as being sister to the distal members of this genus have host ranges encompassing Meloidogyne clades I, II and III (III being basal to more than a thousand plant species, including numerous Clades I and II) (Holterman et al. 2009). major crops, and are spread all over the temperate and One of the elements that could explain the success of (sub)tropical regions of the world. On the basis of SSU this genus is the ability of these endoparasites to rDNA sequences Tandingan de Ley et al. (2002) migrate between plant cells. Cell wall-degrading defined three distal clades within the genus enzymes and expansions are likely to be widespread Meloidogyne. Clade I comprises a number of species throughout Meloidogyne, although they have so far only often referred to as the tropical root-knot nematodes been characterized for some of the more derived (e.g. M. incognita, M. javanica and M. arenaria), the members of this genus. most well known representative of Clade II is M. hapla, while Clade III harbors a number of species with EPPO Moghaddam and Salari 988

Fig. 6. Schematic overview of the phylogeny of the Meloidogynidae derived from SSU rDNA sequence data. (Based on Holterman et al. 2009). These enzymes and proteins make it possible for these It is notable that Clade I is not homogenous in its mode plant-parasites to move towards the most nutritional of replication. Although mitotic parthenogenesis part of the plant root, namely the vascular cylinder dominates this clade, one of its members (M. (stele). Migrating between cells, rather than through floridensis) multiplies by meiotic parthenogenesis. cells as is the case for cyst nematodes, may help the The same holds true for Clades II and III; although pre-parasitic second-stage juveniles to outpace and/ or meiotic parthenogenesis is most frequently found there avoid the host plant defense response. The question are a number of exceptions. Most remarkable is the case why lesion (Pratylenchus spp.) and cyst nematodes of Meloidogyne hapla for which two races are defined. (Globodera spp. and Heterodera spp.) do not migrate Race A reproduces by facultative meiotic intercellularly while infective life stages are producing parthenogenesis, whereas the polyploid Race B and secreting various cell wall degrading enzymes and multiplies by mitotic parthenogenesis. It would be proteins remains to be answered. useful to examine more basal root-knot nematode species when seeking further insight into the extreme Modes of Reproduction Among Root-Knot Nematodes diversity in modes of reproduction of root-knot Another fascinating aspect of root-knot nematode nematodes. According to McCarter (2008), most root- biology is the wide range of modes of reproduction knot species are "sexually reproducing diploids, a status present with the group. Meloidogyne incognita thought to reflect the root-knot nematode ancestral multiplies by mitotic parthenogenesis. Mothers that state". A careful examination of M. artiellia, which is produce genetically identical daughters could be sister to the distal Meloidogyne Clades I-III, suggests considered as a dead end road in evolutionary terms- this could be correct; the frequent occurrence of males such a genetic constitution would make it impossible suggests this species could multiply sexually. However, for an individual to cope with changing environmental based on the analysis of rDNA sequence data at least conditions. Nevertheless, in terms of distribution and four Meloidogyne species are placed in more basal host plant range this is probably the most successful positions, viz. M. ulmi, M. mali, an undescribed member of the genus Meloidogyne. M. incognita is one Meloidogyne species found on Sansevieria, and M. of the members of Clade I (as defined above). ichinohei (Holterman et al. 2009). Moghaddam and Salari 989 Meloidogyne ichinohei is an unusual root-knot rare (Siddiqi and Booth 1991). The morphology of M. nematode species both in its morphology and in its mersa resembles that of M. spartinae, another root-knot extreme host specificity. Unlike most RKN females, nematode species living in marine or brackish habitats. adult females of M. ichinohei show a prominent 4. Thorne (1969) described Meloidogyne ottersoni as a posterior protuberance, a laterally located neck and a parasite of canary grass ( Phalaris arundinacea). As for perineal pattern showing remarkably faint and broken M. graminis, M. ottersoni was reproduced by meiotic striae (Araki 1992). parthenogenesis when males are absent but in the Previously the first two characters were considered to presence of males reproduction was by amphimixis be rather atypical, and they had been the characteristics (Triantaphyllou 1973). Based on cytological data, defining the genus Hypsoperine, a genus synonymized Triantaphyllou suggested for a close phylogenetic with Meloidogyne by Jepson (1987). M. ichinohei relationship of M. ottersoni, M. graminis, M. males are very rare according to the original description graminicola (Clade III) and M. naasi (Clade III). by Araki (1992). This observation has been confirmed 5. Meloidogyne propora was first described by Spaull by the Dutch National Plant Protection Organisation (1977) as a parasite on the roots of Cyperus obtusiflorus (NPPO); not a single male was found in M. ichinohei and Solanum nigrum from an atoll in the Indian Ocean population C2312 (G. Karssen, unpublished data). (Aldabra). Males were reported to be common in soil around infested roots and near ovigerous females. On the Former Genus Hypsoperine 6. Meloidogyne spartinae is a root root-knot nematode Araki remarked that M. ichinohei would have been a producing galls on cordgrass (Spartina alterniflora) typical member of the genus Hypsoperine if it still which is found in intertidal wetlands, especially existed (Araki 1992). Sledge and Golden (1964) estuarine salt marshes. On the basis of SSU rDNA suggested that the genus Hypsoperine actually occupied sequences Plantard et al. (2007) clearly showed this a position in between Heterodera and Meloidogyne species to be related to M. maritima, and as such (though showing closer resemblance to the root-knot residing in Meloidogyne Clade II. nematodes). This information prompted us to check a 7. Meloidogyne megriensis-rather incomplete number of other Hypsoperine-like rootknot nematodes: description published in Russian by Poghossian (1971)- (1) Hypsoperine acronea (now Meloidogyne acronea); was collected from roots of Mentha longifolia. Only (2) H. graminis (M. graminis); (3) H. mersa (M. known from the type locality, an orchard in Megri, and mersa); (4) H. ottersoni ( M. ottersoni); (5) H. propora a nearby village named Vagravar, Armenia Karssen and (M. propora); (6) H. spartinae (M. spartinae) and (7) van Hoenselaar 1998. H. megriensis (M. megriensis). As very little is This overview, based on various data, indicates that published about the members of this genus, information members of the former genus Hypsoperine are scattered about host range and mode of reproduction (if known) all over the Meloidogyne phylogenetic tree, and-other is summarized here. than the observation that their morphology is different 1. Meloidogyne acronea is a root-knot nematode-like from what is considered to be typical for root-knot parasite found on roots of sorghum (Sorghum vulgare) nematodes-they have little in common. Another by Coetzee in 1956. The author reported that this Meloidogyne species sharing a number of particular isolate could also parasitize and multiply on morphological characteristics with M. ichinohei (but beans and tomatoes. In the original description never classified as member of the genus Hypsoperine) morphological characteristics of both males and is Meloidogyne kralli. M. kralli was first found in 1968 females are given. M. acronea seems to have a very by Dr. E. Krall on the roots of sedge (Carex acuta) restricted geographical distribution (Jepson 1983). Adult females share a number of as it has been reported from southern Africa only. characteristics with M. ichinohei; they have a distinct 2. Meloidogyne graminis was found in 1964 by Sledge neck (contrary to M. ichinohei, the neck was not set off) and Golden as a parasite on St. Augustine grass and the vulva was positioned on a posterior (Stenotaphrum secundatum). When few males were protuberance. Although males have been described for present, M. graminis reproduces by meiotic M. kralli (Jepson 1983), they are seldom found under parthenogenesis. However, in the presence of males natural conditions (Karssen 2002). Meloidogyne kralli reproduction was by amphimixis (Triantaphyllou 1973). seems to have a relatively small host range as it is Based on cytological data, Triantaphyllou (1973) found only on four Carex species, namely C. acuta, C. suggested a close phylogenetic relationship of M. vesicaria, C. riparia and C. pseudocyperus, and on graminis, M. ottersoni, M. graminicola (Clade III) and Scirpus sylvaticus. Under laboratory conditions M. M. naasi (Clade III). kralli populations were reported to reproduce well on 3. Meloidogyne mersa is an unusual root-knot nematode barley (Jepson 1983). It is noted that both M. ichinohei found in a marine habitat (mangrove swamps in Brunei) and M. kralli exclusively parasitize a small number of parasitizing roots of the mangrove apple (Sonneratia monocotyledons. alba). The original paper describes both males (100 paratypes) and females (50 paratypes) and males are not Moghaddam and Salari 990 However, SSU rDNA sequence analysis revealed that alignments revealed a subdivision of the genus M. ichinohei and M. kralli are rather distinct, with M. Pratylenchus into six groups (Subbotin et al. 2008). kralli robustly positioned in Meloidogyne Clade III Members of group 1 sensu Corbett and Clarke (1983) close to M. oryzae (Helder and Karssen, unpublished are scattered over major clades a, b and d. Group 2 results). Based on the information discussed above, M. (Corbett and Clarke 1983) corresponds to group c acronea would be worthwhile investigating in more (Subbotin et al. 2008), and Group 3 (Corbett and Clarke detail, as it resembles M. ichinohei. Cytological and 1983) members all reside in group a according to scattered molecular data place most of the former Subbotin et al. (2008) except for P. scribneri which members of the genus Hypsoperine in the distal resides in clade b. No representatives of the genus Meloidogyne Clades II or III, except for M. acronea Meloidogyne were included in this analysis. Holterman and M. propora. It is concluded et al. (2009) presented a phylogenetic tree based on a that molecular data from particularly these two species full length SSU rDNA data set including nine could possibly contribute to a better understanding of Pratylenchus species. However, poor backbone support the origin of the genus Meloidogyne. However, both in this particular part of the tree did not allow for the species seem to have a restricted distribution (southern identification of a Pratylenchus (or at least Africa, and an atoll in the Indian Ocean) and this Pratylenchidae) candidate that could be a likely living complicates the collection of DNA from these highly representative close to the common ancestor interesting root-knot nematode species. Currently, M. of all root-knot nematodes. ichinohei-together with a so far non-described Phylogenetic Relationship Between the Genera Meloidogyne species from Kenya found on Sansevieria Meloidogyne and Pratylenchus (Holterman et al. 2009)-seem to reside genuinely at the On the basis of shared morphological characteristics of very base of the Meloidogyne tree of life. Both species the labial region and pharyngeal structures Ryss (1988) have a very limited host range, and for both species and Geraert (1997) proposed a common ancestry males are very rare. In our eyes, this justifies a between the genera Pratylenchus and Meloidogyne. hypothesis stating that (at least facultative) asexual Analysis of SSU and LSU rDNA sequences from a reproduction is the root-knot nematode ancestral state. considerable range of lesion and root-knot nematodes LESION NEMATODES-A STENOMORPHIC supports a close phylogenetic relationship between the GENUS CLOSELY two genera. Subbotin et al. (2006) found "evidence for a Pratylenchus, Hirschmanniella and Meloidogyne Related to Root-Knot Nematodes clade" (based on D2-D3 sequence data). Two years Lesion nematodes-members of the genus Pratylenchus- later Bert et al. (2008) concluded "…root-knot can be recognized easily. Lesion nematodes are nematodes are most closely related to Pratylenchus spp. relatively small (usually around 500 m), have a short μ and appear to have evolved from migratory and stout stylet (11-22 m) with strong basal knobs, a μ endoparasitic nematodes" (based on SSU rDNA data). low lip region with a well developed sclerotized In 2009 Holterman et al. concluded "our data suggest framework, and glands forming a rather short lobe that root-knot nematodes have evolved from an which ventrally overlaps the intestine (e.g. Castillo and ancestral member of the genus Pratylenchus, but it Vovlas 2007). However, recognizing individual species remains unclear which species is closest to this is difficult as the number of diagnostic characters at this branching point" (based on more extensive SSU rDNA particular taxonomic level is small. The identification data). All in all the following can be concluded: of lesion nematodes is further complicated by intra- 1. Ribosomal DNA sequences suggest that the genus specific variation in some of these characters. Pratylenchus is paraphyletic as all Meloidogyne species Pratylenchus identification to species level is usually are nested in it. done on the basis of the morphology of adult females. 2. If this (1.) is correct, the ultimate consequence would They have more informative characters than males, be the abolishment of the genus Pratylenchus and-at least equally important-for a number of species (Meloidogyne has priority following the rules of the males are extremely rare or even non-existent. Despite International Commission on Zoological these difficulties (or possibly as a consequence of Nomenclature). This could be undesirable for numerous them), this genus comprises approximately 70 nominal practical reasons, but in scientific research and species (Castillo and Vovlas 2007). Using the labial especially in our thinking about root-knot nematode region as a distinguishing character, Corbett and Clark evolution this could be useful. (1983) distinguished three groups of Pratylenchus 3. The discussion about ancient (a) sexuality as the species. Group I includes (among others) P. ancestral state of root-knot nematodes should be brachyurus, P. coffeae, P. crenatus, P. loofi, and P. replaced by a discussion about the ancestral mode of zeae; group 2 includes P. neglectus and P. thornei; and reproduction of a clade encompassing (at least) all P. penetrans, P. pratensis, P. scribneri and P. members of the genera Pratylenchus and Meloidogyne. vulnusbelong to group 3. Combined analysis of D2-D3 of 28S (= LSU rDNA) and partial 18S (= SSU rDNA) Moghaddam and Salari 991 CONCLUSION monophyly of chromadorian and secernentian nematodes. Russ J Nematol 6: 175-184. The steep increase in the amount of molecular data Araki M (1992). Description of Meloidogyne ichinohei n. sp. over the last decade has allowed for the establishment (Nematoda: Meloidogynidae) from Iris laevigata of more and more robust and versatile phylogenetic in Japan. Japanese J Nematol 22: 11-19. frameworks for the phylum Nematoda (Blaxter et al. Bert W, Leliaert F, Vierstraete AR, Vanfleteren JR, 1998; Holterman et al. 2006, Van Megen et al. 2009). Borgonie G (2008). Molecular phylogeny of the It is noted that current frameworks are based on a Tylenchina and evolution of the female gonoduct single cistron (SSU and/or LSU ribosomal DNA (Nematoda: Rhabditida). Mol Phylogenet Evol 48: 728-744. sequences), and marine taxa (including numerous Blaxter ML, De Ley P, Garey JR, Liu LX, Scheldeman P, plant-parasites) are strongly underrepresented. Current Vierstraete A, Vanfleteren JR, Mackey LY, Dorris data suggest at least three independent lineages of M, Frisse LM, Vida JT, Thomas WK (1998). A plant-parasites. This number will probably increase molecular evolutionary framework for the phylum over time; analysis of a 5? region of LSU rDNA Nematoda. Nature 392: 71-75. pointed at multiple plant-parasite lineages among the Castillo P, Vovlas N (2007). Pratylenchus (Nematoda: Dorylaimida (Holterman et al. 2008b). The infraorder Pratylenchidae): diagnosis, biology, pathogenicity Tylenchomorpha (equivalent to Clade 12 (Fig. 3.1) and management. 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