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1994 Molecular Phylogenetic Studies on Filarial Parasites Based on 5S Ribosomal Spacer Sequences Hong Xie Yale Medical School

O. Bain Biologie Parasitaire, Protistologie, Helminthologie, Museum d’Histoire Naturelle

Steven A. Williams Smith College, [email protected]

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Recommended Citation Xie, Hong; Bain, O.; and Williams, Steven A., "Molecular Phylogenetic Studies on Filarial Parasites Based on 5S Ribosomal Spacer Sequences" (1994). Biological Sciences: Faculty Publications, Smith College, Northampton, MA. https://scholarworks.smith.edu/bio_facpubs/38

This Article has been accepted for inclusion in Biological Sciences: Faculty Publications by an authorized administrator of Smith ScholarWorks. For more information, please contact [email protected] Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/1994012141

M o l e c u l a r phylogenetic s t u d i e s o n f i l a r i a l p a r a s i t e s BASED ON 5S RIBOSOMAL SPACER SEQUENCES

X IE H .*, BA IN O .** and W ILLIAM S S.A.*,***

S u m m a ry : R é s u m é : É t u d e s phylogénétiques moléculaires d e s fila ires à pa r­ This paper is the first large-scale molecular phylogenetic study on t ir DE SÉQUENCES DU « SPACER- DU 5S RIBOSOMAL filarial parasites (family Onchocercidae) which includes 16 spe­ Cette première étude sur la phylogénie moléculaire des filaires cies of 6 genera : Brugia beaveri Ash et Little, 1964 ; B. buckleyi (famille des Onchocercidae) - Nématodes chez lesquels les phéno­ Dissanaike et Paramananthan, 1961 ; B. malayi (Brug, 1927) mènes de convergence sont particulièrement importants en raison de Buckley, 1 9 6 0 ; B. pahangi (Buckley et Edeson, 1956) Buckley, leur vie tissulaire - inclut 16 espèces appartenant à 6 genres diffé­ 1 9 6 0 ; B. pa tei (Buckley, Nelson et Heisch, 1958) Buckley, rents : Brugia beaveri Ash et Little, 19 64 ; B. buckleyi Dissanaike et 1 9 6 0 ; B. timori Partono e t al, 1 9 7 7 ; Paramananthan, 19 6 1 ; B. malayi (Brug, 1927) Buckley, I9 6 0 ; B. (Cobbold, 1877) Seurat, 1921; W. kalimantani Palmieri , pahangi (Buckley et Edeson, 1956) Buckley, 1960; B. patei Purnomo, Dennis and Marwoto, 1 9 8 0 ; (Buckley, Nelson et Heisch, 195 8) Buckley, 19 6 0 ; B. timori (Manson, 1891 ) Eberhard et Orihel, 1984; , Stiles, Partono et al, 19 7 7 ; Wuchereria bancrofti (Cobbold, 1877) 1 9 0 5 ; (Leuckart, 1983) Railliet et Henry, Seurat, 1921 ; W . kalimantani Palmieri, Purnomo, Dennis et 1 9 1 0 ; O. ochengi Bwangamoi, 1969; O. gutturosa Neumann, Marwoto, 1980; Mansonella perstans (Manson, 1891) Eberhard 1 9 1 0 ; Dirofilaria immitis (Leidy, 1856) Railliet et Henry, 1911 ; et Orihel, 1 9 8 4 ; Loa loa Stiles, 1 9 0 5 ; Onchocerca volvulus Acanthocheilonema viteae (Krepkogorskaya, 1933) Bain, Baker (Leuckart, 1983) Railliet et Henry, 19 10 ; O. ochengi Bwangamoi, et Chabaud, 1982 and Litomosoides sigmodontis Chandler, 19 69 ; O . gutturosa Neumann, 1 9 1 0 ; Dirofilaria immitis (Leidy, 1931. 5S rRNA gene spacer region sequence data were collec­ 1856) Railliet et Henry, 1 9 1 1 ; Aconthocheilonema viteae ted by PCR, cloning and dideoxy sequencing. The 5S rRNA gene (Krepkogorskaya, 1933) Bain, Baker, Chabaud, 1982 et spacer region sequences were aligned and analyzed by maxi­ Litomosoides sigmodontis Chandler, 1931. Le gène «spacer» du 5 S mum parsimony algorithms, distance methods and maximum likeli­ rRNA a été collecté, cloné et séquencé par PCR. Les séquences des hood methods to construct phylogenetic trees. Bootstrap analysis différentes espèces ont ensuite été alignées puis leur phylogénie w as used to test the robustness of the different phylogenetic reconstruite par les méthodes de parcimonie, de distance et de vrai­ reconstructions. The data indicated that 5S spacer region semblance. Des analyses de bootstrap ont été utilisées pour tester la sequences are highly conserved within species yet differ signifi­ robustesse des différentes reconstructions phylogénétiques. Les résul­ cantly between species. Spliced leader sequences were observed tats indiquent que cette séquence est fortement conservée dans une in all of the 5S rDNA spacers with no sequence variation, même espèce alors qu'elle diffère significativement d'une espèce à although flanking region sequence and length heterogeneity was l'autre. La séquence d'épissure est présente sans variation dans observed even within species. All of the various tree-building toutes les espèces tandis que les régions flanquantes présentent une methods gave very similar results. This study identified four clades hétérogénéité même au niveau intra-spécifique. Les différentes which are strongly supported by bootstrap analysis: the Brugia méthodes de reconstruction d'arbre présentent quelques contradic­ clade; the Wuchereria clade; the Brugia-Wuchereria clade and tions mais elles sont semblables sur plusieurs points et l'étude, très the Onchocerca clade. The analyses indicated that L. sigmodontis partielle il est vrai comparée à l'abondance des genres et espèces and A. viteae may be the most primitive among the 16 species chez les filaires, permet quelques conclusions. 1- le bootstrap sou­ studied. The data did not show any close relationship between tient fortement quatre clades: le clade Brugia, le clade Wuchereria, Loa loa and D. immitis presently classified in the same subfamily, le clade Brugia-Wuchereria, le clade Onchocerca ; ces clades cor­ and the constitution of the Dirofilariinae subfamily is questionable. respondent aux genres, ou à des genres très proches, définis par la morphologie classique. 2- Les analyses suggèrent que L. sigmodon­ tis et A. viteae pourraient être les plus primitives des 16 espèces étu­ diées; cette notion n'est pas en contradiction avec les hypothèses KEY W ORDS : phylogenetics, ribosomal genes. 5 S rDNA. spacers. PCR. faites antérieurement. 3- Les analyses ne rapprochent pas L. loa et molecular cloning, spliced leader sequence. . B. pahangi. B. D. immitis, actuellement placés dans la même sous-famille, et la timori. B. patei. B. beaveri. B. buckleyi. Wuchereria bancrofti. W . kaliman­ question de la composition des Dirofilariinae se pose. tani. Mansonella perstans. Loa loa. Onchocerca volvulus. O. ochengi. O. gutturosa. Dirofilaria immitis. Acanthocheilonema viteae. Litomosoides sigmo­ dontis. MOTS CLES : phylogénèse. 5 S rDNA. «spacers». PCR. clonage moléculaire, * Program in Molecular and Cellular Biology, University of séquence d 'épissure. Brugia malayi. B. pahangi. B. timori. B. patei. B. bea­ Massachusetts at Amherst, Amherst, MA 01003, U. S. A. veri. B. buckleyi. Wuchereria bancrofti. W . kalimantani. Mansonella pers­ ** Biologie Parasitaire, Protistologie, Helminthologie, CNRS-URA tans. Loa loa. Onchocerca volvulus. O. ochengi. O. gutturosa. Dirofilaria 114, Museum d’Histoire Naturelle, 61 rue Buffon, 75231 Paris immitis. Aconthocheilonema viteae. Litomosoides sigmodontis. Cedex 05, France *** Department of Biological Sciences, Smith College, Northampton, MA 01063, U . S. A. Correspondence : Dr. Hong Xie, Yale Medical School, 100 York Street, # 4A, New Haven, CT 06511, USA. Parasite. 1994, 1, 141-151 Mémoire 141 XIE H., BAIN O. a n d WILLIAMS S.A.

INTRODUCTION 5S rDNA genes were basically identical across all of the filarial species whereas the 5S rDNA spacer region sequences were very well conserved within species, ilarioid are a large group of parasites but showed significant variation between species. with species of medical or veterinary impor­ tance. Numerous detailed morphological and biologicalF studies, pioneered by Wehr (1935), have MATERIALS AND METHODS been conducted in an attempt to clarify the phylogene­ tic relationships of these parasites (Chabaud and Choquet, 1953; Anderson and Bain, 1976 ; Chabaud F i l a r i a l p a r a s i t e m a t e r i a l s and Bain, 1976). Since convergence phenomena are aboratory strains of B. pahangi a n d B. m alayi w e re common in these tissue dwelling parasites, phylogenies obtained from Dr. J. McCall (TRS Laboratory, Athens, derived solely from morphological data may be mislea­ LGA, U.S.A.). B. patei adults were kindly provided by ding. DNA sequence data collected by molecular biolo­ Drs. U.R. Rao and A.C. Vickery (University of Southern gical methods provide a new approach in phylogenetic Florida, Gainsville, FL, U.S.A.). B. beaveri adults preserved studies. This paper will focus on these molecular data in 100mM EDTA were kindly provided by Dr. T.C. Orihel and the results of this study will be compared to those (Tulane University Medical Center, New Orleans, LA, obtained by the classical methods. There were sixteen U .S .A .). B. buckleyi adults preserved in 70% ethanol were kindly provided by Dr. A.S. Dissanaike (Colombo, Sri species of six genera in the Onchocercidae family L a n k a ). B. timori microfilariae and W. kalimantani a d u lts included in this study : subfamily Onchocercinae : preserved in 100mM EDTA were kindly provided by Dr. F. Brugia beaveri Ash et Little, 1964 ; B. buckleyi Partono (University of Indonesia, Jakarta). Genomic DNA Dissanaike et Paramananthan, 1961 ; B. m alayi (Brug, samples of M. perstans preserved in ethanol were kindly 1927) Buckley, 1960; B. pahan gi (Buckley et Edeson, provided by Dr. S.E.O. Meredith (Amsterdam, the Nether­ 1956) Buckley, 1960 ; B. p a tei (Buckley, Nelson et lands). Genomic DNA samples of Loa loa, D. immitis, O. Heisch. 1958) Buckley, 1960 ; B. timori Partono et al., volvulus, A. viteae, a n d L. sigmodontis as well as Loa loa 1977 ; Wuchereria bancrofti (Cobbold. 1877) Seurat, microfilariae were kindly provided by Drs. T.B. Nutman 1921 ; W. kalimantani Palmieri, Purnomo, Dennis and and E.A. Ottesen (National Institute of Health, Bethesda, Marwoto, 1980 ; Mansonella perstans (Manson, 1891) MD, U.S.A.). Genomic DNA samples of O. volvulus. O. ochengi a n d O. gutturosa were kindly provided by Dr. T.R. Eberhard and Orihel, 1984 ; Onchocerca volvulus Unnasch (University of Alabama at Birmingham, (Leuckart, 1983) Railliet et Henry, 1910 ; O. ochengi Birmingham, AL, U.S.A). Morphological analyses were Bwangamoi, 1969 ; O. gutturosa Neumann, 1910 ; conducted to verify the identity of some specimens. Acanthocheilonema viteae (Krepkogorskaya, 1933)

Bain, Baker et Chabaud, 1982 and Litomosoides sigmo­ E xperim ental p r o c e d u r e s dontis Chandler, 1931 ; subfamily Dirofilariinae : Loa loa, Stiles, 1905 and Dirofilaria immitis (Leidy, 1856) E xtraction of parasite DNA Railliet et Henry, 1911. The ribosomal RNA gene spacer regions can be used to Individual adult filarial parasites were picked from 70% infer phylogeny among closely related taxa (<50 mil­ ethanol solution and evaporated to dryness. The worms were resuspended in 50 µl H 2 O and boiled for 10 minutes. lion years old) (Verbeet et al., 1984 ; McIntyre et al., These preparations were used as the PCR templates. Similar 1988 ; Yokota et al., 1989 ; Gonzalez et al., 1990 ; treatment was performed for B. timori a n d Loa loa m ic ro fi­ Kjems and Garrett, 1990). The 5S rDNA gene spacer lariae, and for W. kalimantani a n d B. beaveri adult worms region was chosen for this study based on the follo­ which were preserved in lOOmM EDTA. This treatment was wing three observations : 1. Filarial parasites may be not necessary for those species whose genomic DNA was recently derived (less than 65 million years old, already available (except the DNA sample of M. perstans Maggenti, 1983). Therefore, the 5S rDNA spacer region which was preserved in ethanol). These genomic DNA sequences were likely to possess more phylogenetic samples were used directly in the PCR reactions. The DNA information than the ribosomal DNA coding region s a m p le o f M. perstans was evaporated dry from an original volume of 1 ml, resuspended in 100 µl of dd H 2 O and dia- sequences. 2. The 5S rDNA gene organization was lyzed against 0.05x TE buffer for four hours before it was already established for B. m alayi. Genomic titration used for PCR reactions. experiments indicated that the Brugia malayi nuclear

genome contains about 250 copies of the 5S rRNA Am plification of 5S rDNA sequences from parasite genes (Ransohofff et al., 1989). In addition, the detailed DNA organization of 5S genes in nematodes has been stu­ C . died in eleg a n s (Nelson and Honda, 1985). 3. The polymerase chain reactions (PCR) were conducted Preliminary data suggested that the coding regions of using Perkin-Elmer-Cetus 480 and System 9600 thermal

1 4 2 Parasite, 1994, 1. 141-151 MOLECULAR PHYLOGENETIC STUDIES ON FILARIAL PARASITES

cycl ers. Two primers matching the coding region of the B. ng of plasmid DNA and 2 pmol of primer were used for m alay i 5S rDNA gene were custom synthesized by the each sequencing reaction. All of the other reagents were DNA oligo nucleotide service of the Program in Molecular provided in the kit. The Perkin Elmer Cetus thermal cycler and Cellular Biology, University of Massachusetts (Amherst, 480 was used with the following program : 95°C, 20 MA, U.S.A.). The sequences of the two primers were : seconds ; 55°C, 20 seconds ; 72°C, 20 seconds for a total of 20 cycles. Primer S2 : 5’-GTTAAGCAACGTTGGGCCTGG-3’ ; Primer S16 : 5’-TTGACAGATCGGACGAGATG-3’. All PCR reagents except primers, template and double distil- S equence D ata A nalysis led water (ddH2O) were from the GeneAmp PCR kit purcha- sed from Perkin-Elmer Cetus (Norwalk, CT). The PCR Sequence alignment reaction contents included : 5 µl of 10x reaction buffer, 8 µl of 1.25mM dNTP mixture, 20 pmoles of each primer, 1-2 µl The P ileu p computer program from the GCG package template DNA solution, 1 unit of T aq polymerase and (Genetics Computer Group, Inc. Madison, WI, USA) was ddH2O added to a total volume of 50 µl. The PCR cycling used to align all of the 5S rDNA sequences. Because more programs used were: 93°C, 1 minute ; 55°C, 1 minute ; 72°C, than one 5S rDNA spacer region sequence was obtained for 1 minute for a total of 30 cycles (Model 480 thermal cycler) ; each species, p ileu p was used first to align the multiple and 93°C, 30 seconds ; 55°C, 30 seconds ; 72°C, 30 seconds sequences from the same species. The gap penalty (penalty for a total of 30 cycles (System 9600 thermal cycler). for opening a gap) was set to two and gap length penalty (penalty for each character space in the gap) was set to Cloning of the PCR amplified 5S rDNA spacer region zero. Another program in the GCG package, Pretty, was used to print out consensus sequences for each species. sequences Any site that had more than 40% of a nucleotide other than the major nucleotide was considered polymorphic and The TA cloning system from Invitrogen Corporation (San IUPAC-IUB nucleotide notations were used. The final Diego, CA) was used to clone the 5S rDNA PCR products. consensus sequences of all the sixteen species were then All of the reagents needed for ligation (except the amplified aligned by the p ileu p program. Only the portion of the 5S rDNA products) were provided in the TA cloning kit. sequences that were well aligned were selected for further The manufacturer’s protocols were strictly followed. analysis. Recombinant Eschericbia coli colonies (white colonies) were picked with a sterilized platinum wire loop and scree- ned by PCR using the same primer pairs as in the original Phylogenetic reconstruction from sequence data PCR amplification. The loop of E. coli cells was placed PAUP 3.1 (Phylogenetic Analysis Using Parsimony, directly into the PCR reaction tube. The PCR program was Swofford, 1993) was used for phylogenetic tree reconstruc- the same as for the original 5S rDNA amplification except tion (Swofford and Olsen, 1990). Characters were treated as the reactions were incubated in the thermal cycler at 95°C unordered (Fitch, 1971) and were equally weighted. Gaps for five minutes to burst the cells before the initiation of the were treated as missing data. The exhaustive search option 30 cycles. The PCR products were run on 1.5% agarose gels was used on ten of the sixteen species. The branch-and- and stained in ethidium bromide to identify positive clones. bound algorithm was used for the full set of 16 species. Bootstrap analysis was used with PAUP 3.1. Plasmid DNA preparation and purification PHYLIP 3.5c (Phylogeny Inference Package, Felsenstein, 1993) was also used for data analysis. Maximum likelihood Individual E. coli clones with 5S rDNA inserts were inocula­ algorithms (Felsenstein, 1981) and distance methods ted into 20 ml of LB medium containing 50pg/ml of kana- (Neighbor-joining algorithm, Saitou and Nei, 1987) were mycin and incubated at 37°C with vigorous shaking used in the analyses. Bootstrapping analysis was also used overnight. The cells were harvested the next morning by with the PHYLIP package to estimate confidence intervais centrifugation at 5,000g for ten minutes. The cell pellets in the phylogenetic trees. were resuspended in P1 buffer from the Plasmid Midi Kit and plasmid DNA was isolated following the manufacturer’s All of the PAUP computation and some of the PHYLIP dis­ protocol [Qiagen Inc. (Chatsworth. CA. U.S.A.)] tance method analyses were done using a Macintosh SE-30 and a Macintosh Ilci computer. Sequence alignment, maxi­ DNA sequencing mum likelihood analyses, and bootstrap analyses (except bootstrap with PAUP) were done on a Sun Sparc worksta- The DNA sequencing primers used were primers S2 and tion running Sun OS 4.1.1 located in the University of S16 as described before and the M13 universal sequencing Massachusetts at Amherst. primers (primer 1201 and 1211 from New England Biolabs, Beverly, MA, U.S.A.). The CircumVent Thermal Cycle Sequencing Kit from New England Biolabs was used to sequence the plasmid DNA. The manufacturer’s protocols for thermal cycle sequencing with labeled 35s dATP incor­ poration were followed. 35S dATP was purchased from Amersham Corp. (Arlington Heights, IL, USA). About 200

Parasite, 1994, 1. 141-151 Mémoire 143 XIE H., BAIN O. a n d WILLIAMS S.A.

Table I. - 5S rDNA sequence data matrix used for phylogenetic tree reconstruction. There are 161 nucleotide positions in this data matrix. Forty-eight of the 161 sites (30%) are informative. (“-” = gap; Constant = invariable site; Uninf. = uninformative site as calculated by PAUP 3.1. Underlinecl nucleotides are the spliceci leader sequence).

144 Mémoire Parasite, 1994, 1, 141-151 MOLECULAR PHYLOGENETIC STUDIES ON FILARIAL PARASITES

RESULTS are the following : consistency index (CI) = 0.758 ; CI excluding uninformative characters = 0.624 ; CI expected = 0.588; rétention index (RI) = 0.727 ; resca- DNA SEQUENCE COLLECTION AND ALIGNMENT led consistency index (RC) = 0.551. t least two 5S rDNA sequences were obtai- ned for each species. Consensus sequences 2. Analyses by the neighbor-joining method (Saitou from each species were constructed and ali- and Nei, 1987) and by the maximum likelihood Agned to one another. The sequence data matrix method (Felsenstein, 1981) which is the input for ail of the tree reconstruction was used as the outgroup spe­ algorithms is shown in Table I. The Ascaris lumbri- cies in these analyses. The branching pattern of the coides 5S rDNA spacer région sequence (Nelsen et shortest trees obtained was very similar to that of al., 1989) was included as the outgroup. Figure 3, except that Loa loa and M. perstans form a group which switches position with the O nchocerca P hylogenetic t r e e reconstruction and D. immitis group in these analyses.

1. Analysis by maximum parsimony methods 3. Analysis by bootstrap methods (Felsenstein, 1985) (Swofford, 1993) Bootstrapping with parsimony analysis Exhaustive search A total of 500 bootstrap replicates were analyzed Due to the relatively large size of this data set, ten using the parsimony algorithm. The strongly suppor- species representing ail six généra were selected to ted clades are the O n ch ocerca clade (100%), the undergo exhaustive search in PAUP 3.1. Brugia-Wuchereria clade (97%), the A. viteae and L. Exhaustive search settings : sigmodontis clade (97%) and the Brugia clade (94%). Branches having a maximum length of zéro collapsed This tree is unable to resolve the branching order of to yield polytomies; topological constraints were not the Brugia-Wuchereria clade, Loa loa, M. perstans, D. enforced ; trees were unrooted ; multi-state taxa were immitis and the O nchocerca clade (Fig. 4 and Table interpreted as polymorphism ; character-state optimi- II). zation was by accelerated transformation (reversais preferred) Bootstrapping with neighbor-joining analysis Search results : A total of 485 bootstrap replicates were analyzed. One shortest tree of length 121was found (Fig. 1) The This analysis demonstrated that the O nchocerca clade statistics of the exhaustive search tree are the follo- (100%), Brugia clade (99%), W uchereria clade (98%) wing (Kluge and Farris, 1969; Farris, 1989; Sanderson and Brugia-Wuchereria clade (99%) were strongly and Donoghue, 1989) : consistency index (CI) = 0.777; supported by the data set (Fig. 4 and Table II). CI excluding uninformative characters = 0.710; CI expected = 0.684; rétention index (RI) = 0.690; resca- Bootstrapping with Maximum likelihood analysis led consistency index (RC) = 0.536. The frequency dis­ A total of 100 replicates were examined. The tribution of tree lengths is shown in Figure 2. O nchocerca clade (100%) and the Brugia clade (94%) were strongly supported (Fig. 4 and Table II). Branch-and-bound search Search settings : DISCUSSION An analysis of ail sixteen ingroup species was under- taken with Ascaris lumbricoides as the outgroup spe­ cies. Initial upper bound (upper limit of tree length) DNA SEQUENCE DATA COLLECTION AND SEQUENCE was unknown (to be computed via stepwise) ; addi­ ALIGNMENT tion sequence was set to furthest ; branches having a ost of the parasite materials were available maximum length of zéro were collapsed to yield in only limited quantities for this study. It polytomies ; topological constraints were not was not practical to use traditional methods enforced ; multi-state taxa were interpreted as poly­ M for extracting genomic DNA, endonuclease digestion, morphism ; character-state optimization was by acce­ and cloning. A very effective method was developed lerated transformation. using PCR to amplify 5S rDNA spacers and then to Search results : directly clone the PCR products. This method proved One shortest tree with length 198 was found in the very efficient and reliable since the B. m alay i 5S branch-and-bound search (Fig. 3)- The tree statistics

Parasite, 1994, 1, 141-151 Mémoire 145 XIE H.. BAIN O. a n d WILLIAMS S.A.

Fig. 1. - The tree found by exhaustive search. Tree length: 121. Fig. 3. - The branch-and-bound tree rooted by A. lumbricoides. T ree The numbers above each branch dénoté the branch length calcula- length: 198. This analysis showed that A. lu m bricoides joins the tree at the ted by PAUP 3-1 L. sigm odontis branch.

Fig. 2. - The frequency distribution of tree lengths of the 5S rDNA spacer sequences. A total of 34,459,425 trees were evaluated. The mean tree length is 167 steps.

Fig. 4. - The basic bootstrapping pattern of the species studied in this paper. Small case letters dénoté the clades identified by the various bootstrap analyses. Clade names noted with an * indicate low bootstrap values for those clades.

* PA : Parsimony; NJ: Neighbor-joining; ML: Maximum likelihood

T able II Summary of the bootstrap values*

146 Mémoire Parasite, 1994, 1, 141-151 MOLECULAR PHYLOGENETIC STUDIES ON FILARIAL PARASITES

rDNA gene sequences obtained from PCR and clo­ perstans, D. immitis and the O n c h o c erc a clade ning were identical to the sequences in the literature (Table II). When IJ. immitis, which belongs to the which were collected by traditional genomic DNA Dirofilariinae subfamily and has a long branch length cloning and sequencing methods. (this can cause problems in parsimony analysis), was The coding région sequence is virtually identical excluded from the analysis, M. perstans and Loa loa across the 16 species studied here (data not shown). appeared more closely related to the B r u g ia - On the contrary, the spacer région of the 5S rDNA W uchereria clade (data not shown). Parsimony analy­ exhibited great length variations among the différent sis is very sensitive to unequal évolution rates in the species. They were as short as 400 nucleotides in branches due to parallelisms and reversais. This is O nchocerca species or as long as 600 nucleotides in especially a problem with long branches (Felsenstein, some Brugia species. Even within the same species, 1978 ; Lake, 1987). For closely related sequences, there were sometimes différent size classes of 5S spa­ such as those studied in this paper, parallelisms and cers. The spacer length heterogeneity is probably due reversais should be rare. Therefore, the parsimony to deletion and insertion events that have occurred method can be considered reliable. during the évolution of these parasites. This pheno- menon was confirmed by sequence comparison ana­ Distance analysis : lysis. Sequence alignment is the first step in sequence Distance methods may not be accurate for distantly comparison and phylogenetic reconstruction analysis. related sequences since the observed pairwise dis­ The 22-nucleotide spliced leader région sequences tances usually underestimate the true evolutionary dis­ were found in ail of the filarial species studied tances due to homoplasy. Proper correction is needed (Table I). This conserved région was used as a focus in many cases to compensate for multiple-hits at a for the sequence alignment. single site (Gojobori et a l., 1990). Nei’s (1991) compu­ ter simulation studies have shown that the neighbor- T r e e reconstruction a l g o r i t h m s joining method is superior to most of the other methods in retrieving the true tree under a variety of Three main catégories of methods of analysis were circumstances. Différent distance methods were used used in this study: parsimony, distance and maximum in this study and they ail gave similar branching pat­ likelihood. terns (data not shown). The neighbor-joining method also gave very high bootstrap values at some branches Parsimony analysis : (Table II). It is interesting to note that D. immitis and Branch-and-bound and exhaustive searches were per- the O nchocerca clade appeared in close proximity in formed in this analysis. Because the sequence région most of the trees generated by différent distance studied was the non-coding 5S rDNA spacer région, methods (data not show'n). However, the bootstrap ail of the characters were equally weighted. Différent value for such groupings are low (Table II). The same sequence addition methods were tried and ail gave is true for Loa loa, M. perstans and the Wuchereria- the same topologies. The consistency indices were B m gia clade. This phenomenon was observed in par­ high (except in the exhaustive search, where some simony and maximum likelihood analysis as well. taxa were excluded) for the 5S spacer région data set, indicating a low level of homoplasy in the spacer Maximum likelihood analysis : région. The 5S spacer région data set showed a fre- Maximum likelihood methods are not used as fre- quency distribution of tree length that is heavily ske- quently as parsimony or distance methods because of wed towards the end of long trees (Fig. 2), which is the computational complexities of the former. The an indication of the phylogenetic information content PHYLIP 3 -5c program used on a SUN Sparc worksta- in the data sets (Hillis, 1991). Ail of the parsimony tion greatly facilitatecl the computations. Maximum analyses (some not shown) indicate that the Brugia likelihood methods are the only ones that assume clade and the W uchereria clade are close neighbors. stochastic models of sequence évolution in which the A. viteae and L. sigmodontis also form a clade, while user can specify rates of évolution at individual sites the O n c h o c e r c a species form their own clade. of the sequences. These methods are considered Although the two shortest trees (Fig.l and Fig. 3) potentially the most reliable because of their statisti- indicate that D. immitis and the O nchocerca clade are cal properties (Sidow and Wilson, 199D- In this doser to the Brugia-Wuchereria clade than are M. study, the maximum likelihood method gave very perstans and Loa loa, this relationship can be rever- similar branching patterns to those seen with other sed with a cost of less than five steps (data not methods. Différent transition/transversion ratios were shown). In other words, parsimony analysis was used in various tree reconstructions. Ail ratios yielded unable to resolve the branching order of Loa loa, M. very similar results (data not showTi).

Parasite, 1994, 1, 141-151 Mémoire 147 XIE H.. BAIN O. AND WILLIAMS SA.

C o n f id e n c e lim it s o n phylogenies two subfamilies (Dirofilariinae and Onchocercinae) of the Onchocercidae family form a single natural group One of the statistical approaches used to assess confi­ which does not exclude the suggestion of Bain dence levels in phylogenetic hypotheses is the (1981a). bootstrap analysis (Felsenstein, 1985). Bootstrap ana­ In the O n c h o c e r c a clade, the 5S rDNA spacer lysis is a method for the estimation of statistical error sequence of O. volvulus is indistinguishable from that in situations where the underlying sampling distribu­ tion is difficult to assess (Efron, 1982 ; Efron and of O. ochengi. It was suggested that O. volvulus Gong, 1983). Instead of repeatedly sampling from the belongs to a small line of O n ch ocerca in African underlying distribution of taxonomie characters itself, Bovidae of the Savanna which is morphologically which is often impractical, the bootstrap resamples highly evolved (musculature atrophied and hypoder- the original data set to approximate the distribution. mis hypertrophied in the females, Bain, 1981b). In this study, algorithms from ail three major catégo­ Chabaud (1981) recognized the capture phenomenon ries were applied to the bootstrapped data sets in parasite évolution. According to Chabaud, a captu- (Table II). Taking ail of the bootstrap results together, red parasite is defined as a parasite which, after beco- it is clear that the Brugia clade, the W uchereria clade, ming isolated in a new host, undergoes spéciation the Brugia-Wuchereria clade and the O n chocerca and becomes morphologically distinct from the origi­ clade are strongly supported by the original data set. nal species. O. volvulus only infects humans while O. It was shown that confidence levels estimated by ochengi infects cattle. Morphological evolutionary stu- bootstrap analysis usually underestimates the real dies indicated that O. volvulus and O. ochengi are the relationships among taxa (Sanderson, 1989). Some of most closely related species in the genus the groups in this study did not reach a 95% confi­ Onchocerca. It is possible that O. volvulus evolved dence level by bootstrap analysis, but that does not from the captured O. ochengi of the herbivore reser- necessarily mean these groupings should be discar- voir too recently for O. volvulus to show genetic dis­ ded (Sanderson, 1989). The A. viteae-L. sigm odontis tinctions from O. ochengi. group hacl confidence levels ranging from 85% to The two species in the W uchereria clade are the only 97% in the différent bootstrap analyses , indicating a species that have been described for this genus. They close relationship. The D.immitis-Oncbocerca group form a natural group and are very closely related appeared in the majority of trees generated by based on the DNA sequence data in this study. W. various methods in this study, although it had only a bancrofti has a world-wide distribution and its only confidence level of 69% in the bootstrap analysis with host is humans, while W. kalimantani is limited to the neighbor-joining method (data not shown, see South Kalimantan in Indonesia and infects monkeys. previous discussion). There is no corrélation between morphological cha­ racters and geographical origin in W. bancrofti except T h e h y p o t h e s is at the microfilarial level (Schacher and Geddawi, It is very clear from analysis of the 5S rDNA spacer 1969 ; Bain et a !, 1985). 5S rDNA spacer région sequence data and the above discussion, that there sequences of W. b a n c r o ft i from India, Egypt, are at least four strongly supported clades: the Indonesia and Tahiti were analyzed and were identi- B ru g ia clade, the W u chereria clade, the B ru gia- cal to one another in this study (data not shown). W uchereria clade and the O nchocerca clade. The A. Laurence (1989) placed the early infection of humans viteae and L. sigmodontis group is also strongly sup­ by W. bancrofti in Southeast Asia about 3,000 years ported (97%) in the bootstrap analysis using parsi- ago based on historical records. He suggested that mony. Litomosoides has a long buccal capsule in the only through mass migration of human beings did L3 and adult stages which is considered a very primi­ Bancroftian become widely spread. It is pos­ tive character state (Anderson and Bain, 1976). The sible that both species of W uchereria originated from genus Acanthocheilonema has a developed buccal the same place in Indonesia and the spéciation was cavity and was considered the most primitive genus completed long enough ago to allow the observation of the Dipetalonema line of the Onchocercinae subfa- of différences at the DNA level as revealed in this mily (Chabaud and Bain, 1976). The outgroup Ascaris study. lumbricoides rooted most of the 5S rDNA trees at the The bootstrap analyses of the 5S rDNA spacer région L. sigmodontis branch (except those trees derived by sequence data demonstrated that B r u g ia and algorithms which assume constant rates of évolution, W uchereria are very closely related. These results are data not shown). These observations are examples of in very good agreement with morphological data congruence of molecular data with morphological (Buckley, 1958). For the six species studied in the data. The outgroup analyses also suggest that at least Brugia clade, the 5S rDNA spacer sequences alone

148 Mémoire Parasite, 1994, 1, 141-151 MOLECULAR PHYLOGENETIC STUDIES ON FILARIAL PARASITES

were not sufficient to resolve the branching order. these DNA sequence data, Loa loa appeared to be This fact, combined with the observation that H ba I closest to M. perstan s and the Brugia-Wuchereria repeats are only present in Brugia, clearly indicates clade in most of the trees generated by various that Brugia is a natural group and that the species in methods (data not shown). The bootstrap value is this genus are very closely related (Xie, 1993). low, however, and morphologically the L3 of Loa loa The hypothesis for the phylogenetic relationships in and M. perstans are totally différent. The the filarial parasite Onchocercidae family is illustrated Dirofilariinae subfamily is characterized by highly in Figure 5. The use of 5S rDNA spacer région developed caudal alae and papillae while the sequences seems to confirm that the filarial parasites Onchocercinae subfamily is characterized by a long, in this study ( filarial parasites of ) are non-alate tail. Although some trees were rooted at the recently derived, perhaps after the Spiruria went D. immitis branch (data not shown) which might sup­ through a bottleneck in the Cretaceous disaster about port D. immitis as a différent subfamily, the position 60 million years ago (Maggenti, 1983). In fact, the ori- of Loa loa in ail of the trees generated by ail of the gin and spéciation events of the O nchocerca genus in différent methods suggest its close connection with Africa may ail be within the past 2.5 million years the Onchocercinae subfamily. These molecular data (during the Pleistocene epoch, Bain, 1981b). question the constitution of the Dirofilariinae subfa­ However, the filariae in the subfamily Oswaldofilarii- mily. nae have a Gondwanian distribution which suggests their existence possibly as early as the late Jurassic period (about 120 million years ago, Bain, 1981). ACKNOWLEDGMENTS

he authors wish to thank J. McCall, U.R. Rao, A.C. Vickery, T.C. Orihel, A.S. Dissanaike, L.A. McReynolds, F. Partono, S.E.O. Meredith, T.B. T Nutman, E.A. Ottesen and T.R. Unnasch for providing the parasite materials used in this study; J. Felsenstein and D.L. Swofford for technical assistance with their computer programs ; Dr. Pierre Darlu for his construc­ tive suggestions in the préparation of this manuscript and his help in writing the French translation of the abstract. Support for this study was provided by the Blakeslee fund for genetics research at Smith College.

REFERENCES

Fig. 5. - The proposée! phylogenetic tree for the species studied in A n d f r s o n R.C. and B a in O. : Keys to généra of the Order this paper. The six species in B rugia were lumped together in a . Part 3- Diplotriaenoidea, Aproctoidea and single branch because their branching order is ambiguous based on . “CIH Keys to the Parasites of 5S rRNA gene spacer région sequences alone. Vertebrates” No. 3 (R. C. Anderson, A. G. Chabaud and S. M. Willmott, Eds). Publisher : Commonwealth Agricultural Bureaux, Farnham Royal, Bucks, England, The only serious conflict between the results of this 1976. molecular phylogenetic study and the traditional mor- A sh L.R. and L it t l e M.D. : Brugia beaveri sp. n. (Nema- phology-based classifications is in the positions of toda : Filarioidea) from the raccoon (Procyon lotor) in two species : Loa loa and D. immitis. Based on mor- Louisiana. J. Parasit., 1964, 50, 119-123 phological studies, they have been placed in the sub­ B ain O. : Filariids and their évolution. Parasitology, 1981a. family Dirofilariinae, yet they are not very close to 8 2 ( part 4), 167-168 each other in any of the DNA sequence-based phylo­ B a in O. : Le genre Onchocerca : Hypothèses sur son évolu­ genetic trees. Morphologically, the L3 (third-stage lar- tion et clé dichotomique des espèces (The genus vae) of Loa loa does not resemble that of Dirofilaria. Onchocerca : hypothesis on its évolution and a key to the species). Ann. Parasitai. Hum. Comp., 1981b, 56, In this study, although the bootstrap value is low 503-526 (Table II), D. immitis appeared in most of the trees B ain O. and C h a b a u d A.G. : Atlas des larves infestantes de generated by différent methods to be closest to the Filaires. Trop. Med. Parasit., 1986, 37, 301-340. O n chocerca clade (data not shown). These results B a in O., D is s a n a ik e A.S ., C r o s s J . H . , H a r in a s u t a C . and correspond to certain aspects of the morphology of S u c h a r it S . : Morphologie de Wuchereria bancrofti their L3 larvae (Bain and Chabaud, 1986). Based on

1 Parasite, 1994, , 141-151 Mémoire 149 XIE H., BAIN O. a n d WILLIAMS S.A.

adulte et sub-adulte. Recherche de caractères différentiels New York, 1991. entre les souches. Ann. Parasitol. Hum. Comp., 1985, 60, Kjem s J. and G a r r e t t R.A. : Secondary structural elements 613-630. exclusive to the sequences flanking ribosomal RNAs lend B u c k le y J.J.C. and E d eso n J.F.B. : On the adult morphology support to the monophyletic nature of the archaebacte- of W uchereria sp. (m alayi?) from a Monkey (M acaca ria. J. Mol. Evol., 1990, 3 1 25-32. iru s) and from cats in Malaya, and on W u ch ereria K lu g e A.G. and F a r ris J.S. : Quantitative phyletics and the p a h a n g i n.sp. from a dog and a cat. J. Helminthology, évolution of the anurans. Syst. Zool., 1969, 18, 1-32. 1956, XXX, 1-20 L ake J.A. : A rate-independent technique for analysis of B u c k le y J.J.C., Nelson G.S. and Heisch R.B. : On W uche­ nucleic acid sequences: evolutionary parsimony. Mol. reria patei n.sp. from the lymphatics of cats, dogs and Biol. Evol., 1987, 4, 167-191. genet cats on Pate island, Kenya. J. Helminthology, 1958, L au ren ce B.R. : The global dispersai of Bancroftian filariasis. XXXII, 73-80 Parasitology Today, 1989, 5, 260-264. C h a b a u d A.G. and B a in O. : La lignée Dipetalonema. Ann. M a g g e n ti A.R. : Nematode higher classification as influen- Parasitol. Hum. Comp.. 1976, 51, 365-397. ced by species and family concepts in Concepts in nema­ C h a b a u d A.G. and C h o q u e t. M.T. : Nouvel essai de classifi­ tode systematics (Edited by Stone, A. R.; H . M . Platt and cation des filaires (super-famille des Filarioidea). Ann. L. F. Khalil) pp. 25-40. Academic Press, New York, 1983. Parasit., 1953, 28, 172-192. M c In ty re C.L., Clarke B.C. and Appels R. : DNA sequence C h a b a u d A.G. : Host range and évolution of nematode analyses of the ribosomal spacer régions in the Triticeae. parasites of vertebrates. Parasitology, 1981, 8 2 (part 4), Plant Syst. Evol. 1988, 160, 91-104. 169-174. N ei M. : Relative efficiencies of différent tree-making E f r o n B. : Nonparametric standard errors and confidence methods for molecular data in Phylogenetic Analysis of intervais. C an .J. Stat., 1981, 9, 139-172. DNA Sequences (edited by Miyamoto, M.M. and Cracraft, E f r o n B. : The jackknife, the bootstrap, and other resam- J.) pp. 90-128. Oxford University Press, New York. 1991. pling plans. CBMS-NSF Régional Conférence Sériés in N e lso n D.W. and H o n d a B.M. : Genes coding for 5S riboso­ Applied Mathematics, Monograph 38. Society of Industry mal RNA of the nem atode Caenorhabditis elegans. and Applied Mathematics, Philadelphia, 1982. Gene., 1985, 38, 245-251. E f r o n B. and Gong G. : A leisure look at the bootstrap, N elsen T.W., Sh am baugh J., D en k er J., Chubb G., Faser C., jackknife, and cross validation. Am. Stat., 1983. 37, 36-48 P utn am L. and B e n n e t t K. : Characterization and expres­ F a r ris J.S. : The rétention index and the rescaled consis- sion of a spliced leader RNA in the parasitic nematode tency index. Cladistics, 1989, 5, 417-419. Ascaris lumbricoides var. suum. Mol. Cell. Biol., 1989, 9, Felsenstein J. : Cases in which parsimony or compatibility 3543-3547. methods will be positively misleading. Syst. Zool., 1978, P alm ieri J. R., Purnomo D. T. D en nis, and H . A. M a rw o to . 27, 401-410. Filarid parasites of South Kalimantan (Bornéo) Indonesia. Wuchereria kalimantani sp. n. (Nematoda: Filarioidea) Felsenstein J. : Evolutionary trees from DNA sequences : a from the silvered leaf monkey, Presbytis cristatus maximum likelihood approach. J. Mol. Evol., 1981, 17, 368-376. Eschscholtz 1921./. Parasit., 1980, 66, 645-651 P a r t o n o F. , Purnomo, Dennis D.T., Atmosoedjono S., Felsenstein J. : Confidence limits on phylogenies : An O e m ija t i S., and Cross J . H . : sp. n. approach using the bootstrap. Evolution, 1985, 39, 783-791. (Nematoda: Filarioidea) from Flores island, Indonesia. J. Felsenstein J. : Phylogenetic Inference Programs (PHYLIP), Parasit., 1977, 63, 540-546. Manual 3.5c. University of Washington, Seattle, 1993- Ransohoff R.M., Denker J.A ., Takacs A.M., Hannon G.J. and F itc h W. M. : Towards defining the course of évolution : Nilsen T.W. : Organization and expression of 5S rRN A Minimal change for a specific tree topology. Syst. Zool., genes in the parasitic nematode, Brugia malayi. Nucleic 1971, 20, 406-416. Acids Research, 1989, 17, 3773-3782. F reed m an D.J. : Molecular evolutionary studies of filarial Sanderson M.J. : Confidence limits on phylogenies : the parasites of the genus Brugia. Ph. D Thesis. University of bootstrap revisited. Cladistics, 1989, 5, 113-129. Massachusetts at Amherst, 1991. S a ito u N. and Nei M . : The neighbor-joining method : a G o jo b o r i T., M o riy a m a E.N. and K im u ra M. : Statistical new method for reconstructing phylogenetic trees. Mol. methods for estimating sequence divergence in Biol. Evol, 1987, 4, 406-425. Molecular Evolution : computer analysis of protein and nucleic acid sequences. (edited by R. F. Doolittle). pp. S a n d e rso n M.J. and D o n o g h u e M.J. : Patterns of variation in 531-550. Methods in Enzymology, vol. 183. Academic levels of homoplasy. Evolution, 1989, 43, 1781-1795. Press, New York, 1990. S c h a c h e r J.F. and G ed d aw i M.K. : An analysis of spéciation and évolution in Wuchereria bancrofti by the study of G o n z a le z I.L., S y lv e s te r J.E., Sm ith T.F., Stambolian D. and nuclear constancy (eutely) in microfilariae. Ann. Trop. S c h m ic k e l R.D. : Ribosomal RNA gene sequences and Med. Parasitol., 1969, 63, 67-82. hominoid phylogeny. Mol. Biol. Evol.. 1990, 7, 203-219. Sid ow A. and W ilso n A.C. : Compositional statistics evaluated H illis D.M. : Discriminating between phylogenetic signal by computer simulations in Phylogenetic Analysis of DNA and random noise in DNA sequences in Phylogenetic Sequences (edited by Miyamoto, M. M. and Cracraft, J.) Analysis of DNA Sequences (edited by Miyamoto, M. M. pp. 129-146. Oxford University Press, New York, 1991. and Cracraft, J.) pp 278-294. Oxford University Press,

150 Parasite, 1994, 1, 141-151 MOLECULAR PHYLOGENETIC STUDIES ON FILARIAL PARASITES

S w o f f o r d , D.L. and Olsen G.J. : Phylogeny reconstruction in Molecular Systematics (edited by Hillis, D. M. and C. Moritz) pp. 411-501. Sinauer Associates, Massachusetts, 1990.

S w o f f o r d D.L. : Phylogenetic Analysis Using Parsimony, version 3-1 Computer program distributed by the Illinois Natural History Survey, Champaign, Illinois, 1993.

V e r b e e t M.P., van Heerikhuizen H., Klootwijk J., Fontijn R.D. and Planta R.J. : Evolution of yeast ribosomal DNA : molecular cloning of the rDNA units of Kluyveromyces lactis and Hansenula wingei and their comparison with the rDNA units of other Saccharomycetoideae. Mol. Gen. Genet.. 1984, 195, 116-125.

W e h r E.E. : A revised classification of the nematode super- family Filarioidea. Proc. Helm. Soc., Wash., 1935, 2, 84- 88.

X ie II. : Molecular phylogenetic studies on filarial parasites. Ph. D Thesis. University of Massachusetts at Amherst, 1993.

Y o k o t a Y ., K a w a t a T ., I id a Y ., K a t o A. and T a n if u ji S. : Nucleotide sequences of the 5.8S rRNA gene and internai transcribed spacer régions in carrot and broad bean ribo­ somal DNA./. Mol. Evol, 1989, 29, 294-301.

Accepté le 17 mars 1994

Parasite, 1994, 1, 141-151 Mémoire 151