J. Eukaryot. Microbiol., 48(2), 2001 pp. 161±169 q 2001 by the Society of Protozoologists Diversity and Phylogeny of Insect Trypanosomatids Based on Small Subunit rRNA Genes: Polyphyly of Leptomonas and Blastocrithidia EKATERINA MERZLYAK,a VYACHESLAV YURCHENKO,a,1 ALEXANDER A. KOLESNIKOV,a KIRILL ALEXANDROV,b SERGEI A. PODLIPAEVc and DMITRI A. MASLOVd aDepartment of Molecular Biology, Moscow State University, 119899 Moscow, Russia, and bDepartment of Physical Biochemistry, Max-Planck Institute for Molecular Physiology, Otto-Hahn Strasse 11, 44227 Dortmund, Germany, and cZoological Institute, Russian Academy of Sciences, 199034 St. Petersburg, Russia, and dDepartment of Biology, University of California, Riverside, California 92521, USA ABSTRACT. With the aim of further investigating phylogenetic relationships in insect trypanosomatids, we have determined the sequences of small subunit rRNA genes from ten isolates, which were originally classi®ed as Leptomonas, Blastocrithidia, and Walla- ceina based on their morphology in the hosts. The inferred maximum likelihood, parsimony, and distance trees indicate that the Leptomonas and Blastocrithidia are polyphyletic, and con®rm the polyphyly of Herpetomonas and Crithidia. Blastocrithidia triatoma and Leptomonas collosoma were among the earliest branching lineages among the insect trypanosomatids, while most other isolates were found within a closely related terminal clade, which also included Crithidia fasciculata. This analysis has clearly demonstrated that the morphological classi®cation system of insect trypanosomatids does not always re¯ect their genetic af®nities warranting its revision in the future. Key Words. Ribosomal RNA, taxonomy, Trypanosomatidae, Wallaceina. RYPANOSOMATIDS (family Trypanosomatidae Do¯ein 2000). Trypanosoma, which appeared paraphyletic in earlier T 1901, order Kinetoplastida Honigberg 1963, suborder Try- works due to unequal rate effects (Fernandes, Nelson, and Bev- panosomatina Kent 1880) are de®ned as a group of kinetoplas- erley 1993; Maslov et al. 1996), was shown to be monophyletic tid protozoa with a single ¯agellum and a relatively small ki- when the fast evolving lineages were subdivided by the inclu- netoplast containing densely packed DNA (Vickerman 1976). sion of additional taxa of salivarian trypanosomes (Haag, The family includes four genera of digenetic organisms that O'hUigin, and Overath 1998; LukesÏ et al. 1997; Stevens et al. parasitize vertebrate (Trypanosoma, Leishmania, and Endotry- 1999; Wright et al. 1999). Another monophyletic group was panum) or plant (Phytomonas) hosts, with insects or leeches obtained for Phytomonas (Hollar and Maslov 1997; Marche et serving as vectors, and the monogenetic genera (Leptomonas, al. 1995), although this result did not mean that all promastigote Crithidia, Blastocrithidia, Herpetomonas, Rhynchoidomonas, isolates from plants would necessarily belong to this group. A and Wallaceina) that are found largely in hemipteran and dip- strong monophyletic assembly was found for the symbiont- teran insect hosts with a limited distribution in seven other or- bearing trypanosomatids, which included species from Blasto- ders of insects, as well as in ciliates. crithidia, Crithidia, and Herpetomonas (Du, Maslov, and So far, only light microscopy has been used to establish the Chang 1994; Hollar, LukesÏ, and Maslov 1998). The endosym- trypanosomatid taxonomy. Eight genera were described before biont-free species of Herpetomonas formed a separate mono- 1910, one genus in 1959 and the last one in 1990 (see Podlipaev phyletic group, and a single endosymbiont-free crithidia (Cri- 1990). The assignment of isolates or species of trypanosomatids thidia fasciculata) grouped elsewhere in the tree together with to a certain genus is determined by the morphological charac- the only analyzed species of Leptomonas sp. and several Leish- ters and the host range (Hoare and Wallace 1966; Molyneux mania and Endotrypanum species. Thus, at least two genera, and Ashford 1983). The relative positions of the nucleus and Crithidia and Herpetomonas, were found to be polyphyletic pri- the kinetoplast and the shape of cells are the important mor- or to this study. No conclusions could be drawn for Blastocrith- phological features. However, it has become increasingly clear idia or Leptomonas, with a single isolate analyzed for each that this system does not always re¯ect the genetic af®nities of genus. The dearth of data on isolates from insects represents trypanosomatids on one hand, and their real diversity on the the major gap in our knowledge of the trypanosomatid phylog- other. The discrepancies between the groups established by bio- eny. chemical, molecular or phylogenetic analyses and the tradition- Currently there are more than three hundred descriptions of ally de®ned taxa have been observed in a number of works insect trypanosomatids in the literature (Podlipaev 1990). Many (Camargo et al. 1982; Dollet 1994; Hollar, LukesÏ, and Maslov of them may be synonyms or descriptions of organisms of du- 1998; Kolesnikov, Maslov, and Podlipaev 1990; McGhee and bious origin. At the same time, only a limited number of iso- Cosgrove 1980; Podlipaev, Malysheva, and Kolesnikov 1991; lates have been investigated in culture. Fifteen years ago, 10± Podlipaev and Lobanov 1996; Vickerman 1994; Wallace et al. 15 isolates had been studied in various laboratories (Wallace et 1983; Wright et al. 1999) demonstrating the necessity to estab- al. 1983). Today this number still does not exceed 20±30 iso- lish new genera and revise the existing ones. lates obtained from a limited range of hosts and locations. This Recently the small subunit (SSU) rRNA gene-based phylog- situation is inadequate for characterization of the diversity of enies have been used to establish the major natural groups with- insect trypanosomatids, nor does it allow full investigation of in the family (reviewed in Philippe 1998; Vickerman 1994). the phylogenetic relationships within the family. In this work, Several monophyletic clades have emerged with only a few of as a step toward solving these problems, we present the SSU them agreeing with the current taxonomy. The family Trypano- rRNA phylogenetic analysis of ten isolates that have been pre- somatidae itself is monophyletic in contrast to the other kineto- viously assigned to the genera Leptomonas, Blastocrithidia, and plastid families Bodonidae and Cryptobiidae (DolezÏel et al. Wallaceina. In order make our study more comprehensive, we combined molecular methodology with examining cell morpho- types in both the hosts and in culture. Corresponding Author: S. Podlipaev. Telephone number: 1 7 812 114 6651; FAX number: 1 7 812 114 0444; E-mail: sergei@weed. MATERIALS AND METHODS zin.ras.spb.ru 1 Current address: The Picower Institute for Medical Research, 350 Cultures. Origins of the isolates used in the study are listed Community Drive, Manhasset, NY 11030, USA in Table 1. Methods of isolation and cultivation have been de- 161 162 J. EUKARYOT. MICROBIOL., VOL. 48, NO. 2, MARCH±APRIL 2001 Table 1. Origin of trypanosomatid strains. Name Reference Insect host Place of isolation Comments Blastocrithidia gerricola Podlipaev 1985 Gerris lacustris Hemiptera: North-West Rus- Isolated by S. Podli- Gerridae sia paev in 1981 Blastocrithidia triatoma Cerisola et al. 1971 Triatoma infestans Hemip- Argentina Provided by G. tera: Reduviidae Shaub Leptomonas collosoma Wallace et al. 1960 Gerris dissortis Hemiptera: USA ATCC30261 Gerridae Leptomonas peterhof® Podlipaev 1985 Nabicula ¯avomarginata He- North-West Rus- Isolated by S. Podli- miptera: Nabidae sia paev in 1982 Leptomonas seymouri Wallace 1977 Dysdercus suturellus Hemip- USA ATCC30220 tera: Pyrrhocoridae Leptomonas sp. Cfm this work Nabicula ¯avomarginata He- North-West Rus- Isolated by A. Fro- miptera: Nabidae sia lov in 1983 Leptomonas sp. F6 Kolesnikov, Maslov, and Nabicula ¯avomarginata He- North Russia Isolated by S. Podli- Podlipaev 1990 miptera: Nabidae paev in 1986 Leptomonas sp. Nfm this work Nabicula ¯avomarginata He- North-West Rus- Isolated by A. Fro- miptera: Nabidae sia lov in 1991 Wallaceina inconstansa ZK Kolesnikov, Maslov, and Calocoris sexguttatus He- North-West Rus- Isolated by A. Fro- Podlipaev 1990; Podlipaev, miptera: Miridae sia lov in 1986 Frolov, and Kolesnikov, 1990 Wallaceina brevicula Nbr Kolesnikov, Maslov, and Nabis brevis Hemiptera: Na- North-West Rus- Isolated by A. Fro- Podlipaev 1990; Podlipaev, bidae sia lov in 1986 Frolov, and Kolesnikov 1990 a We use the generic name Wallaceina Podlipaev, Frolov et Kolesnikov, 1999 as a replacement name for Proteomonas Podlipaev, Frolov et Kolesnikov, 1990 because the name Proteomonas is preoccupied by a cryptomonad ¯agellate (Proteomonas Hill et Wetherbee 1986). The replacement name is given after the late Professor F. G. Wallace (Bulat, Mokrousov, and Podlipaev 1999). scribed previously (Podlipaev 1985; Podlipaev, Malysheva, and (L18872), Phytomonas serpens (AF016323), Phytomonas sp. Kolesnikov 1991). The cultures are stored in the Zoological EM1 (AF016322), Phytomonas sp. E.hi.Se (L35077), Phytom- Institute, St. Petersburg, Russia, and in the Max-Planck Institute onas sp. Hart1 (L35077), Blastocrithidia culicis (U05679), Cri- for Molecular Physiology, Dortmund, Germany. thidia oncopelti (AF038025), Herpetomonas cf. roitmani Trypanosomatids were cultivated at 26 8C in Brain Heart In- (AF267738), Herpetomonas roitmani (AF038023), Herpetom- fusion medium (Difco) with 10 mg/ml hemin. For Blastocrith- onas sp. TCC263