M
o l e c u l a r p h y l o g e n e t i c 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 sB A S E D O N 5 S R IB O S O M A L S P A C E R S E Q U E N C E S
X I E H . * , B A I N O . * * and W I L L I A M S S . A . * , * * *
S u m m a r y :
This paper is the first large-scale molecular phylogenetic study on filarial parasites (family Onchocercidae) which includes 16 spe
cies of 6 genera : Brugia beaveri Ash et Little, 1 9 6 4 ; B. buckleyi Dissanaike et Paramananthan, 1961 ; B. malayi (Brug, 1927) Buckley, 1 9 6 0 ; B . pahangi (Buckley et Edeson, 1956) Buckley, 1 9 6 0 ; B . p a tei (Buckley, Nelson et Heisch, 1958) Buckley, 1 9 6 0 ; B. timori Partono e t al, 1 9 7 7 ; W uchereria bancrofti (Cobbold, 1 8 77 ) Seurat, 1 9 2 1 ; W . kalimantani Palmieri , Purnomo, Dennis and Marwoto, 1 9 8 0 ; Mansonella perstans (Manson, 1891 ) Eberhard et Orihel, 1 9 8 4 ; Loa loa, Stiles,
1 9 0 5 ; O nchocerca volvulus (Leuckart, 1983) Railliet et Henry, 1 9 1 0 ; O . ochengi Bwangamoi, 1 9 6 9 ; O . gutturosa Neumann, 1 9 1 0 ; Dirofilaria immitis (Leidy, 1856) Railliet et Henry, 1911 ; A c a nthocheilonema viteae (Krepkogorskaya, 19 33 ) Bain, Baker et Chabaud, 1 9 8 2 and Litomosoides sigmodontis Chandler,
1 9 3 1 . 5 S rRNA gene spacer region sequence data were collec
ted by PCR, cloning and dideoxy sequencing. The 5 S rRNA gene spacer region sequences were aligned and analyzed by maxi
mum parsimony algorithms, distance methods and maximum likeli
hood methods to construct phylogenetic trees. Bootstrap analysis w as used to test the robustness of the different phylogenetic reconstructions. The data indicated that 5 S spacer region sequences are highly conserved within species yet differ signifi
cantly between species. Spliced leader sequences were observed in all of the 5 S rDNA spacers with no sequence variation, although flanking region sequence and length heterogeneity w as observed even within species. All of the various tree-building methods gave very similar results. This study identified four clades which are strongly supported by bootstrap analysis: the Brugia clade; the W uchereria clade; the Brugia-Wuchereria clade and the O nchocerca clade. The analyses indicated that L. sigmodontis and A . viteae may be the most primitive among the 16 species studied. The data did not show any close relationship between Loa loa and D. immitis presently classified in the same subfamily, and the constitution of the Dirofilariinae subfamily is questionable.
KEY W O R D S : phylogenetics, ribosomal genes. 5 S rD N A . spacers. PCR.
molecular cloning, spliced leader sequence. Brugia malayi. B. pahangi. B.
timori. B. patei. B. beaveri. B. buckleyi. Wuchereria bancrofti. W . kaliman
tani. Mansonella perstans. Loa loa. Onchocerca volvulus. O . ochengi. O.
gutturosa. Dirofilaria immitis. Aca nthocheilonema viteae. Litomosoides sigmo
dontis.
* P ro g ra m in M o le c u la r an d C e llu la r B io lo g y , U n iv e rsity o f M assachusetts at Am herst, Am herst, MA 0 1 003, U. S. A.
** B io lo g ie P arasitaire, P rotistologie, H elm in th olog ie, CNRS-URA 114, M useum d ’H istoire N aturelle, 61 rue B u ffo n , 75 2 3 1 Paris C ed ex 05, France
*** D e p a r t m e n t o f B i o l o g i c a l S c i e n c e s , S m ith C o l l e g e , N ortham pton, MA 0 1 063, U . S. A.
C o rresp o n d en ce : Dr. H ong X ie, Y a le M edical Sch ool, 100 Y ork Street, # 4A, New H aven, CT 0 6 5 1 1 , USA.
R é s u m é : Ét u d e sp h y l o g é n é t iq u e sm o l é c u l a ir e sd e sf il a ir e sàpa r t ir DE SÉQUENCES DU « SPACER- DU 5S RIBOSOMAL
Cette première étude sur la phylogénie moléculaire des filaires (famille des O nchocercidae) - Ném atodes ch ez lesquels les phéno
mènes de convergence sont particulièrement importants en raison de leur vie tissulaire - inclut 16 espèces appartenant à 6 genres diffé
rents : Brugia beaveri Ash et Little, 1 9 6 4 ; B. buckleyi Dissanaike et Paramananthan, 19 6 1 ; B. malayi (Brug, 1927) Buckley, I 9 6 0 ; B.
pahangi (Buckley et Edeson, 1956) Buckley, 19 6 0 ; B. patei (Buckley, Nelson et Heisch, 19 5 8 ) Buckley, 19 6 0 ; B. timori Partono et al, 19 7 7 ; W uchereria bancrofti (Cobbold, 1877) Seurat, 1921 ; W . kalimantani Palmieri, Purnomo, Dennis et M arw oto, 19 8 0 ; Mansonella perstans (M anson, 1891) Eberhard et Orihel, 1 9 8 4 ; Loa loa Stiles, 1 9 0 5 ; Onchocerca volvulus (Leuckart, 1983) Railliet et Henry, 1 9 1 0 ; O . ochengi Bwangamoi,
1 9 6 9 ; O . gutturosa Neumann, 1 9 1 0 ; Dirofilaria immitis (Leidy, 1856) Railliet et Henry, 1 9 1 1 ; Aconthocheilonema viteae (Krepkogorskaya, 1933) Bain, Baker, Chabaud, 1 9 82 et Litomosoides sigmodontis Chandler, 1931. Le gène «spacer» du 5 S rRNA a été collecté, cloné et séquencé par PCR. Les séquences des différentes espèces ont ensuite été alignées puis leur phylogénie reconstruite par les méthodes d e parcimonie, d e distance et de vrai
semblance. Des analyses d e bootstrap ont été utilisées pour tester la robustesse des différentes reconstructions phylogénétiques. Les résul
tats indiquent que cette séquence est fortement conservée dans une même esp èce alors qu'elle diffère significativement d'une espèce à l'autre. La séquence d'épissure est présente sans variation dans toutes les espèces tandis que les régions flanquantes présentent une hétérogénéité même au niveau intra-spécifique. Les différentes méthodes d e reconstruction d'arbre présentent quelques contradic
tions mais elles sont semblables sur plusieurs points et l'étude, très partielle il est vrai com parée à l'abondance des genres et espèces ch ez les filaires, permet quelques conclusions. 1- le bootstrap sou
tient fortement quatre clades: le clade Brugia, le clade W uchereria, le clad e Brugia-Wuchereria, le clad e O nchocerca ; ces clades cor
respondent aux genres, ou à des genres très proches, définis p ar 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 a vec les hypothèses faites antérieurement. 3- Les analyses ne rapprochent pas L. loa et D. immitis, actuellement p lacés dans la même sous-famille, et la question d e la composition des Dirofilariinae se pose.
MOTS CLES : phylogénèse. 5 S rDNA. «spacers». PCR. clonage moléculaire, séquence d 'épissure. Brugia malayi. B. pahangi. B. timori. B. patei. B. bea
veri. B. buckleyi. Wuchereria bancrofti. W . kalimantani. Mansonella pers
tans. Loa loa. Onchocerca volvulus. O . ochengi. O . gutturosa. Dirofilaria immitis. Aconthocheilonema viteae. Litomosoides sigmodontis.
Parasite. 1994, 1, 141-151 Mémoire 141
XIE H ., BAIN O. a n d WILLIAMS S.A.
INTRODUCTION
F
ilarioid nematodes are a large group of parasites with species o f medical or veterinary importance. Numerous detailed morphological and biological studies, pioneered by Wehr (1935), have been conducted in an attempt to clarify the phylogene
tic relationships of these parasites (Chabaud and Choquet, 1953; Anderson and Bain, 1976 ; Chabaud and Bain, 1976). Since convergence phenomena are common in these tissue dwelling parasites, phylogenies derived solely from morphological data may be mislea
ding. DNA sequence data collected by molecular biolo
gical methods provide a new approach in phylogenetic studies. This paper will focus on these molecular data and the results of this study will be compared to those obtained by the classical methods. There were sixteen species of six genera in the Onchocercidae family included in this study : subfamily Onchocercinae : B r u g ia b e a v e r i Ash et Little, 1964 ; B. b u c k le y i Dissanaike et Paramananthan, 1961 ; B. m alayi (Brug, 1927) Buckley, 19 60; B. p a h a n g i (Buckley et Edeson, 1956) Buckley, 1960 ; B. p a t e i (Buckley, Nelson et Heisch. 1958) Buckley, 1960 ; B. timori Partono et al., 1977 ; W uchereria ban crofti (Cobbold. 1877) Seurat, 1921 ; W. kalim an tan i Palmieri, Purnomo, Dennis and Marwoto, 1980 ; M ansonella perstans (Manson, 1891) Eberhard and Orihel, 1984 ; O n c h o c e r c a volvulus (Leuckart, 1983) Railliet et Henry, 1910 ; O. ochen gi Bwangam oi, 1969 ; O. g u ttu rosa Neumann, 1910 ; A c a n th o c h e ilo n e m a v itea e (Krepkogorskaya, 1933) Bain, Baker et Chabaud, 1982 and Litomosoides sigm o
dontis Chandler, 1931 ; subfamily Dirofilariinae : Loa loa, Stiles, 1905 and D irofilaria immitis (Leidy, 1856) Railliet et Henry, 1911.
The ribosomal RNA gene spacer regions can be used to infer phylogeny among closely related taxa (<50 mil
lion years old) (Verbeet et al., 1984 ; McIntyre et al., 1988 ; Yokota et al., 1989 ; Gonzalez et al., 1990 ; Kjems and Garrett, 1990). The 5S rDNA gene spacer region was chosen for this study based on the follo
wing three observations : 1. Filarial parasites may be recently derived (less than 65 m illion years old, Maggenti, 1983). Therefore, the 5S rDNA spacer region sequences were likely to possess more phylogenetic information than the ribosomal DNA coding region sequences. 2. The 5S rDNA gene organization was already established for B. m alayi. Genomic titration experiments indicated that the Brugia m alayi nuclear genome contains about 250 copies o f the 5S rRNA genes (Ransohofff et al., 1989). In addition, the detailed organization of 5S genes in nematodes has been stu
died in C . e le g a n s (N elson and Honda, 1985). 3.
Preliminary data suggested that the coding regions of
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, but showed significant variation between species.
MATERIALS A N D METHODS
Fi l a r i a l p a r a s i t e m a t e r i a l s
L
a b o r a t o r y s t r a in s o f B. p a h a n g i a n d B. m alayi w e r e o b t a in e d f r o m D r . J . M c C a ll ( T R S L a b o r a t o r y , A t h e n s , G A , U .S .A .) . B. p a tei a d u lt s w e r e k i n d l y p r o v id e d b y D r s . U .R . R a o a n d A . C . V i c k e r y ( U n i v e r s i t y o f S o u t h e r n F lo r id a , G a i n s v il le , F L , U .S .A .) . B. beaveri a d u lt s p r e s e r v e d in 1 0 0 m M E D T A w e r e k in d l y p r o v id e d b y D r . T . C . O r i h e l ( T u l a n e U n i v e r s i t y M e d i c a l C e n t e r , N e w O r l e a n s , L A , U .S .A .) . B. buckleyi a d u lt s p r e s e r v e d in 7 0 % e t h a n o l w e r e k i n d l y p r o v i d e d b y D r . A . S . D i s s a n a i k e ( C o l o m b o , S r i L a n k a ) . B. timori m ic r o f ila r ia e a n d W. kalim an tan i a d u lt s p r e s e r v e d in 1 0 0 m M E D T A w e r e k i n d l y p r o v id e d b y D r . F . P a r t o n o ( U n i v e r s i t y o f I n d o n e s ia , J a k a r t a ) . G e n o m ic D N A s a m p le s o f M. perstans p r e s e r v e d in e t h a n o l w e r e k i n d l y p r o v id e d b y D r . S .E .O . M e r e d it h ( A m s t e r d a m , t h e N e t h e r la n d s ) . G e n o m ic D N A s a m p le s o f Loa loa, D. immitis, O.volvulus, A. viteae, a n d L. sigm odontis a s w e l l a s Loa loa
m ic r o f il a r ia e w e r e k i n d l y p r o v i d e d b y D r s . T . B . N u t m a n a n d E . A . O t t e s e n ( N a t io n a l I n s t it u t e o f H e a l t h , B e t h e s d a , M D , U . S . A . ) . G e n o m i c D N A s a m p le s o f O . volvulus. O.
ochengi a n d O. gutturosa w e r e k i n d l y p r o v id e d b y D r . T .R . U n n a s c h ( U n i v e r s i t y o f A l a b a m a a t B i r m i n g h a m , B i r m i n g h a m , A L , U . S . A ) . M o r p h o l o g i c a l a n a l y s e s w e r e c o n d u c t e d to v e r i f y t h e id e n t it y o f s o m e s p e c im e n s .
Ex p e r i m e n t a l p r o c e d u r e s
E x t r a c t i o n o f p a r a s i t e D N A
I n d i v i d u a l a d u lt f i l a r i a l p a r a s i t e s w e r e p i c k e d f r o m 7 0 % e t h a n o l s o l u t i o n a n d e v a p o r a t e d to d r y n e s s . T h e w o r m s w e r e r e s u s p e n d e d in 5 0 µ l H2O a n d b o ile d f o r 10 m in u t e s . T h e s e p r e p a r a t io n s w e r e u s e d a s th e P C R t e m p la t e s . S im ila r t r e a tm e n t w a s p e r f o r m e d f o r B. timori a n d Loa loa m ic r o f i
la r ia e , a n d f o r W. kalim an tan i a n d B. beaveri a d u lt w o r m s w h ic h w e r e p r e s e r v e d in lO O m M E D T A . T h i s tr e a tm e n t w a s n o t n e c e s s a r y f o r th o s e s p e c ie s w h o s e g e n o m ic D N A w a s a l r e a d y a v a ila b le ( e x c e p t t h e D N A s a m p le o f M. perstans
w h i c h w a s p r e s e r v e d in e t h a n o l ) . T h e s e g e n o m i c D N A s a m p le s w e r e u s e d d ir e c t ly in th e P C R r e a c t io n s . T h e D N A s a m p le o f M. perstans w a s e v a p o r a t e d d r y f r o m a n o r ig in a l v o lu m e o f 1 m l, r e s u s p e n d e d in 1 0 0 µ l o f d d H2O a n d d ia - ly z e d a g a in s t 0 .0 5 x T E b u f f e r f o r f o u r h o u r s b e f o r e it w a s u s e d f o r P C R r e a c t io n s .
A m p l i f i c a t i o n o f 5S r D N A s e q u e n c e s f r o m p a r a s i t e D N A
T h e p o ly m e r a s e c h a i n r e a c t i o n s ( P C R ) w e r e c o n d u c t e d u s in g P e r k i n - E l m e r - C e t u s 4 8 0 a n d S y s t e m 9 6 0 0 t h e r m a l
1 4 2 Parasite, 1994, 1. 141-151
cycl ers. Two primers matching the coding region of the B.
m a la y i 5S rDNA gene w ere custom synthesized by the DNA oligo nucleotide service of the Program in Molecular and Cellular Biology, University o f Massachusetts (Amherst, MA, U.S.A.). The sequences o f the two primers were : Primer S2 : 5’-GTTAAGCAACGTTGGGCCTGG-3’ ; Primer S 16 : 5’-TTGACAGATCGGACGAGATG-3’.
All PCR reagents except primers, template and double distil- led water (ddH2O) were from the GeneAmp PCR kit purcha- sed from Perkin-Elm er Cetus (N orw alk, CT). The PCR reaction contents included : 5 µl of 10x reaction buffer, 8 µl of 1.25mM dNTP mixture, 20 pmoles of each primer, 1-2 µl tem plate DNA solution, 1 unit o f T aq polym erase and ddH2O added to a total volume of 50 µl. The PCR cycling programs used were: 93°C, 1 minute ; 55°C, 1 minute ; 72°C, 1 minute for a total of 30 cycles (Model 480 thermal cycler) ; and 93°C, 30 seconds ; 55°C, 30 seconds ; 72°C, 30 seconds for a total of 30 cycles (System 9600 thermal cycler).
Cloning of the PCR amplified 5S rDNA spacer region sequences
The TA cloning system from Invitrogen Corporation (San Diego, CA) was used to clone the 5S rDNA PCR products.
All of the reagents needed for ligation (except the amplified 5S rDNA products) were provided in the TA cloning kit.
T h e m an u factu rer’s p ro to co ls w ere strictly fo llow ed . Recom binant E s c h e r ic b ia c o li colonies (w hite colonies) were picked with a sterilized platinum wire loop and scree- ned by PCR using the same primer pairs as in the original PCR amplification. The loop o f E. c o li cells was placed directly into the PCR reaction tube. The PCR program was the same as for the original 5S rDNA amplification except the reactions were incubated in the thermal cycler at 95°C for five minutes to burst the cells before the initiation o f the 30 cycles. The PCR products were run on 1.5% agarose gels and stained in ethidium bromide to identify positive clones.
Plasmid DNA preparation and purification
Individual E. coli clones with 5S rDNA inserts were inocula
ted into 20 ml of LB medium containing 50pg/ml of kana- m ycin and in cu b ate d at 37°C w ith vig oro u s shakin g overnight. The cells were harvested the next morning by centrifugation at 5,000g for ten minutes. The cell pellets were resuspended in P 1 buffer from the Plasmid Midi Kit and plasmid DNA was isolated following the manufacturer’s protocol [Qiagen Inc. (Chatsworth. CA. U.S.A.)]
DNA sequencing
The DNA sequencing primers used were primers S2 and S16 as described before and the M13 universal sequencing primers (primer 1201 and 1211 from New England Biolabs, Beverly, MA, U .S.A .). The C ircum V ent Therm al 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 follow ed. 35S dATP was purchased from Amersham Corp. (Arlington Heights, IL, USA). About 200
ng of plasmid DNA and 2 pmol of primer were used for each sequencing reaction. All o f the other reagents were provided in the kit. The Perkin Elmer Cetus thermal cycler 480 w as used w ith the follow ing program : 95°C, 20 seconds ; 55°C, 20 seconds ; 72°C, 20 seconds for a total of 20 cycles.
Sequence Data Analysis
Sequence alignment
The P ileu p com puter program from the GCG package (Genetics Computer Group, Inc. Madison, WI, USA) was used to align all o f the 5S rDNA sequences. Because more than one 5S rDNA spacer region sequence was obtained for each species, p ile u p was used first to align the multiple sequences from the same species. The gap penalty (penalty for opening a gap) was set to two and gap length penalty (penalty for each character space in the gap) was set to zero. Another program in the GCG package, Pretty, was used to print out consensus sequences for each species.
Any site that had more than 40% of a nucleotide other than the m ajor nucleotide was considered polymorphic and IUPAC-IUB n u cleotid e notations w ere used. The final consensus sequences o f all the sixteen species were then aligned by the p ile u p program. Only the portion of the sequences that were well aligned were selected for further analysis.
Phylogenetic reconstruction from sequence data PAUP 3.1 (P h y lo g e n e tic A n alysis U sing P arsim o n y, Swofford, 1993) was used for phylogenetic tree reconstruc- tion (Swofford and Olsen, 1990). Characters were treated as unordered (Fitch, 1971) and were equally weighted. Gaps were treated as missing data. The exhaustive search option was used on ten of the sixteen species. The branch-and- bound algorithm was used for the full set o f 16 species.
Bootstrap analysis was used with PAUP 3.1.
PHYLIP 3.5c (Phylogeny Inference Package, Felsenstein, 1993) was also used for data analysis. Maximum likelihood algorithm s (F e lse n ste in , 1 9 8 1 ) and d istan ce m ethods (Neighbor-joining algorithm, Saitou and Nei, 1987) were used in the analyses. Bootstrapping analysis was also used with the PHYLIP package to estimate confidence intervais in the phylogenetic trees.
All o f the PAUP computation and some of the PHYLIP dis
tance method analyses were done using a Macintosh SE-30 and a Macintosh Ilci computer. Sequence alignment, maxi
mum likelihood analyses, and bootstrap analyses (except bootstrap with PAUP) were done on a Sun Sparc worksta- tion running Sun OS 4.1.1 located in the University of Massachusetts at Amherst.
Parasite, 1994, 1. 141-151 Mémoire 143
XIE H., BAIN O. a n d WILLIAMS S.A.
T a b le I. - 5S rDNA se q u e n ce data m atrix used fo r ph ylogen etic tree recon stru ction . T h e re are 161 n u cleotid e p osition s in this data matrix.
Forty-eight o f th e 161 sites (3 0 % ) are inform ative. ( “- ” = g a p ; Constant = invariable s ite ; Uninf. = uninform ative site as calcu lated b y PAUP 3.1. Underlinecl n u cleotid es are th e spliceci lead er seq u en ce).
144 Mémoire Parasite, 1994, 1, 141-151
RESULTS
D N A SEQUENCE COLLECTION AND ALIGNMENT
A
t least two 5S rDNA sequences were obtai- ned for each species. Consensus sequences from each species were constructed and ali- gned to one another. The seq u en ce data matrix which is the input for ail of the tree reconstruction algorithms is shown in Table I. The A scaris lum bri- coid es 5S rDNA spacer région sequence (Nelsen et al., 1989) was included as the outgroup.Ph y l o g e n e t ic t r e e r e c o n s t r u c t io n
1. Analysis by maximum parsimony methods (Swofford, 1993)
Exhaustive search
Due to the relatively large size of this data set, ten species representing ail six généra were selected to undergo exhaustive search in PAUP 3.1.
Exhaustive search settings :
Branches having a maximum length of zéro collapsed to yield polytomies; topological constraints were not enforced ; trees were unrooted ; multi-state taxa were interpreted as polymorphism ; character-state optimi- zation was by accelerated transformation (reversais preferred)
Search results :
One shortest tree of length 121was found (Fig. 1) The statistics of the exhaustive search tree are the follo- wing (Kluge and Farris, 1969; Farris, 1989; Sanderson and Donoghue, 1989) : consistency index (CI) = 0.777;
CI excluding uninformative characters = 0.710; CI expected = 0.684; rétention index (RI) = 0.690; resca- led consistency index (RC) = 0.536. The frequency dis
tribution of tree lengths is shown in Figure 2.
B ra n ch -a n d -b o u n d search Search settings :
An analysis of ail sixteen ingroup species was under- taken with A scaris lu m bricoides as the outgroup spe
cies. Initial upper bound (upper limit of tree length) was unknown (to be computed via stepwise) ; addi
tion sequence was set to furthest ; branches having a maximum length of zéro were collapsed to yield p o ly to m ies ; to p o lo g ica l co n stra in ts w ere not enforced ; multi-state taxa were interpreted as poly
morphism ; character-state optimization was by acce
lerated transformation.
Search results :
One shortest tree with length 198 was found in the branch-and-bound search (Fig. 3)- The tree statistics
are the following : consistency index (CI) = 0.758 ; CI exclu d in g u ninform ative ch aracters = 0.6 2 4 ; CI expected = 0.588; rétention index (RI) = 0.727 ; resca- led consistency index (RC) = 0.551.
2. Analyses by the neighbor-joining method (Saitou and Nei, 1987) and by the maximum likelihood method (Felsenstein, 1981)
A scaris lu m bricoides was used as the outgroup spe
cies in these analyses. The branching pattern of the shortest trees obtained was very similar to that of Figure 3, except that L oa lo a and M. perstan s form a group which switches position with the O nchocerca and D. imm itis group in these analyses.
3. Analysis by bootstrap methods (Felsenstein, 1985) Bootstrapping with p arsim on y analysis
A total of 500 bootstrap replicates were analyzed using the parsimony algorithm. The strongly suppor- ted clades are the O n c h o c e r c a clade (100% ), the B ru gia-W u chereria clade (97%), the A. viteae and L.
sigm odontis clade (97%) and the B ru gia clade (94%).
This tree is unable to resolve the branching order of the B rugia-W uchereria clade, Loa loa, M. perstans, D.
immitis and the O n chocerca clade (Fig. 4 and Table II).
Bootstrapping with neighbor-joinin g analysis
A total of 485 bootstrap replicates were analyzed.
This analysis demonstrated that the O n chocerca clade (100%), B ru gia clade (99%), W uchereria clade (98%) and B ru g ia -W u ch ereria clade (99%) were strongly supported by the data set (Fig. 4 and Table II).
Bootstrapping with M axim um likelih ood analysis A total o f 100 re p lic a te s w ere ex am in ed . T he
O n chocerca clade (100%) and the B rugia clade (94%) were strongly supported (Fig. 4 and Table II).
DISCUSSION
D N A SEQUENCE DATA COLLECTION AND SEQUENCE ALIGNMENT
M
ost of the parasite materials were available in only limited quantities for this study. It was not practical to use traditional methods for extracting genomic DNA, endonuclease digestion, and cloning. A very effective method was developed using PCR to amplify 5S rDNA spacers and then to directly clone the PCR products. This method proved very efficient and reliable since the B. m a la y i 5SParasite, 1994, 1, 141-151 Mémoire 145
XIE H.. BAIN O. a n d WILLIAMS S.A.
Fig. 1. - T h e tree fou nd by exh au stiv e search . T re e length: 121. Fig. 3. - T h e b ra n c h -a n d -b o u n d tree ro o te d b y A. lu m b r ic o id e s . T re e T h e n um bers a b o v e e a ch b ran ch d én o té th e b ran ch length calcu la- length: 198. T his analysis sh o w ed that A. lu m b rico id es jo in s th e tree at the
ted b y PAUP 3-1 L. sigm odon tis b ranch.
Fig. 2. - T h e freq u en cy distribution o f tree lengths o f th e 5S rDNA sp acer seq u en ces. A total o f 3 4 ,4 5 9 ,4 2 5 trees w ere evaluated. T he m ean tree length is 167 steps.
Fig. 4. - T h e b asic boo tstrap p in g pattern o f th e sp e cies studied in this pap er. Sm all c a se letters d é n o té th e clad es id entified b y the various b ootstrap analyses. C lade n am es n o ted w ith an * indicate low b ootstrap valu es for th o se clades.
* PA : Parsimony; NJ: Neighbor-joining; ML: Maximum likelihood
T ab le II
Sum m ary o f th e bootstrap values*
1 4 6 Mémoire Parasite, 1994, 1, 141-151
rDNA gene sequences obtained from PCR and clo
ning were identical to the sequences in the literature which were collected by traditional genomic DNA cloning and sequencing methods.
The coding région sequ ence is virtually identical across the 16 species studied here (data not shown).
On the contrary, the spacer région of the 5S rDNA exhibited great length variations among the différent species. They were as short as 400 nucleotides in O n chocerca species or as long as 600 nucleotides in some B rugia species. Even within the same species, there were sometimes différent size classes of 5S spa
cers. The spacer length heterogeneity is probably due to deletion and insertion events that have occurred during the évolution of these parasites. This pheno- menon was confirmed by sequence comparison ana
lysis. Sequence alignment is the first step in sequence comparison and phylogenetic reconstruction analysis.
The 22-nucleotide spliced leader région sequences w ere found in ail o f the filarial sp ecies studied (Table I). This conserved région was used as a focus for the sequence alignment.
T r e e r e c o n s t r u c t i o n a l g o r i t h m s
Three main catégories of methods of analysis were used in this study: parsimony, distance and maximum likelihood.
Parsimony analysis :
Branch-and-bound and exhaustive searches were per- formed in this analysis. Because the sequence région studied was the non-coding 5S rDNA spacer région, ail of the characters were equally weighted. Différent sequence addition methods were tried and ail gave the same topologies. The consistency indices were high (except in the exhaustive search, where some taxa were excluded) for the 5S spacer région data set, indicating a low level of homoplasy in the spacer région. The 5S spacer région data set showed a fre- quency distribution of tree length that is heavily ske- wed towards the end of long trees (Fig. 2), which is an indication of the phylogenetic information content in the data sets (Hillis, 1991). Ail of the parsimony analyses (some not shown) indicate that the B rugia clade and the W uchereria clade are close neighbors.
A. viteae and L. sigm odontis also form a clade, while the O n c h o c e r c a sp e c ie s form th eir ow n clad e.
Although the two shortest trees (F ig .l and Fig. 3) indicate that D. immitis and the O n chocerca clade are doser to the B ru g ia-W u ch ereria clade than are M.
p erstan s and L oa lo a , this relationship can be rever- sed with a cost of less than five steps (data not show n). In other words, parsimony analysis was unable to resolve the branching order of Loa lo a , M.
p e r s t a n s , D. im m itis and the O n c h o c e r c a clade (Table II). When IJ. im m itis, which belongs to the Dirofilariinae subfamily and has a long branch length (this can cause problems in parsimony analysis), was excluded from the analysis, M. p erstan s and Loa loa ap p eared m ore c lo se ly related to the B r u g ia - W uchereria clade (data not shown). Parsimony analy
sis is very sensitive to unequal évolution rates in the branches due to parallelisms and reversais. This is especially a problem with long branches (Felsenstein, 1978 ; Lake, 1987). For closely related sequences, such as those studied in this paper, parallelisms and reversais should be rare. Therefore, the parsimony method can be considered reliable.
Distance analysis :
Distance methods may not be accurate for distantly related sequences since the observed pairwise dis
tances usually underestimate the true evolutionary dis
tances due to homoplasy. Proper correction is needed in many cases to compensate for multiple-hits at a single site (Gojobori et a l., 1990). Nei’s (1991) compu
ter simulation studies have shown that the neighbor- joining m ethod is superior to most of the other methods in retrieving the true tree under a variety of circumstances. Différent distance methods were used in this study and they ail gave similar branching pat
terns (data not shown). The neighbor-joining method also gave very high bootstrap values at some branches (Table II). It is interesting to note that D. immitis and the O nchocerca clade appeared in close proximity in most of the trees generated by différent distance methods (data not show'n). However, the bootstrap value for such groupings are low (Table II). The same is true for Loa loa, M. perstan s and the W uchereria- B m g ia clade. This phenomenon was observed in par
simony and maximum likelihood analysis as well.
Maximum likelihood analysis :
Maximum likelihood methods are not used as fre- quently as parsimony or distance methods because of the computational complexities of the former. The PHYLIP 3 -5c program used on a SUN Sparc worksta- tion greatly facilitatecl the computations. Maximum likelihood methods are the only ones that assume stochastic models of sequence évolution in which the user can specify rates of évolution at individual sites of the sequences. These methods are considered potentially the most reliable because of their statisti- cal properties (Sidow and W ilson, 199D - In this study, the maximum likelihood method gave very similar branching patterns to those seen with other methods. Différent transition/transversion ratios were used in various tree reconstructions. Ail ratios yielded very similar results (data not showTi).
Parasite, 1994, 1, 141-151
Mémoire 147
XIE H.. BAIN O. AND WILLIAMS SA.
Co n f id e n c e l im it s o n p h y l o g e n ie s
One of the statistical approaches used to assess confi
d en ce levels in p h y lo g en etic h y p o th eses is the bootstrap analysis (Felsenstein, 1985). Bootstrap ana
lysis is a method for the estimation of statistical error in situations where the underlying sampling distribu
tion is difficult to assess (Efron, 1982 ; Efron and Gong, 1983). Instead of repeatedly sampling from the underlying distribution of taxonomie characters itself, which is often impractical, the bootstrap resamples the original data set to approximate the distribution.
In this study, algorithms from ail three major catégo
ries w ere applied to the bootstrapped data sets (Table II). Taking ail of the bootstrap results together, it is clear that the B ru gia clade, the W uchereria clade, the B ru g ia -W u ch ereria clade and the O n ch o c erca clade are strongly supported by the original data set.
It was shown that confidence levels estimated by bootstrap analysis usually underestimates the real relationships among taxa (Sanderson, 1989). Some of the groups in this study did not reach a 95% confi
dence level by bootstrap analysis, but that does not necessarily mean these groupings should be discar- ded (Sanderson, 1989). The A. viteae-L. sigm odontis group hacl confidence levels ranging from 85% to 97% in the différent bootstrap analyses , indicating a close relationship. The D .im m itis-O n cbocerca group appeared in the m ajority o f trees g en erated by various methods in this study, although it had only a confidence level of 69% in the bootstrap analysis with the neighbor-joining method (data not shown, see previous discussion).
Th e h y p o t h e s is
It is very clear from analysis of the 5S rDNA spacer sequence data and the above discussion, that there are at least four strongly supported clad es: the B r u g ia clade, the W u ch ereria clade, the B ru g ia - W uchereria clade and the O n chocerca clade. The A.
viteae and L. sigm odontis group is also strongly sup
ported (97%) in the bootstrap analysis using parsi- mony. Litom osoides has a long buccal capsule in the L3 and adult stages which is considered a very primi
tive character state (Anderson and Bain, 1976). The genus A c a n th o c h eilo n e m a has a developed buccal cavity and was considered the most primitive genus of the D ipetalon em a line of the Onchocercinae subfa- mily (Chabaud and Bain, 1976). The outgroup A scaris lu m bricoides rooted most of the 5S rDNA trees at the L. sigm odontis branch (except those trees derived by algorithms which assume constant rates of évolution, data not shown). These observations are examples of congruence of molecular data with m orphological data. The outgroup analyses also suggest that at least
two subfamilies (Dirofilariinae and Onchocercinae) of the Onchocercidae family form a single natural group w hich does not exclu d e the suggestion o f Bain (1981a).
In the O n c h o c e r c a cla d e, the 5S rDNA sp a ce r sequence of O. volvulus is indistinguishable from that o f O. o c h en g i. It was suggested that O. v olvu lu s belongs to a small line of O n c h o c e r c a in African Bovidae o f the Savanna which is m orphologically highly evolved (musculature atrophied and hypoder- mis hypertrophied in the fem ales, Bain, 1981b).
Chabaud (1981) recognized the capture phenomenon in parasite évolution. According to Chabaud, a captu- red parasite is defined as a parasite which, after beco- ming isolated in a new host, undergoes spéciation and becomes morphologically distinct from the origi
nal species. O. volvulus only infects humans while O.
och en g i infects cattle. Morphological evolutionary stu- dies indicated that O. volvulus and O. och en g i are the m ost c lo se ly relate d sp e c ie s in the gen u s O n chocerca. It is possible that O. volvulus evolved from the captured O. och en g i of the herbivore reser- voir too recently for O. volvulus to show genetic dis
tinctions from O. ochengi.
The two species in the W uchereria clade are the only species that have been described for this genus. They form a natural group and are very closely related based on the DNA sequence data in this study. W.
ban crofti has a world-wide distribution and its only host is humans, while W. k a lim a n ta n i is limited to South Kalimantan in Indonesia and infects monkeys.
There is no corrélation between morphological cha
racters and geographical origin in W. ban crofti except at the microfilarial level (Schacher and Geddawi, 1969 ; Bain et a ! , 1985). 5S rDNA sp acer région se q u e n c e s o f W. b a n c r o f t i from In d ia, Egypt, Indonesia and Tahiti were analyzed and were identi- cal to one another in this study (data not shown).
Laurence (1989) placed the early infection of humans by W. ban crofti in Southeast Asia about 3,000 years ago based on historical records. He suggested that only through mass migration of human beings did Bancroftian filariasis become widely spread. It is pos
sible that both species of W uchereria originated from the same place in Indonesia and the spéciation was completed long enough ago to allow the observation of différences at the DNA level as revealed in this study.
The bootstrap analyses of the 5S rDNA spacer région se q u e n c e data d em o n strated that B r u g ia and W uchereria are very closely related. These results are in very good agreem ent with m orphological data (Buckley, 1958). For the six species studied in the B ru gia clade, the 5S rDNA spacer sequences alone
148 Mémoire Parasite, 1994, 1, 141-151
were not sufficient to resolve the branching order.
This fact, combined with the observation that H ba I repeats are only present in B rugia, clearly indicates that B rugia is a natural group and that the species in this genus are very closely related (Xie, 1993).
The hypothesis for the phylogenetic relationships in the filarial parasite Onchocercidae family is illustrated in Figure 5. The use o f 5S rDNA sp acer région sequences seems to confirm that the filarial parasites in this study ( filarial parasites o f mammals) are recently derived, perhaps after the Spiruria went through a bottleneck in the Cretaceous disaster about 60 million years ago (Maggenti, 1983). In fact, the ori- gin and spéciation events of the O n chocerca genus in Africa may ail be within the past 2.5 million years (d u rin g the P le isto c e n e e p o c h , B ain , 1 9 8 1 b ).
However, the filariae in the subfamily Oswaldofilarii- nae have a Gondwanian distribution which suggests their existence possibly as early as the late Jurassic period (about 120 million years ago, Bain, 1981).
The only serious conflict between the results of this molecular phylogenetic study and the traditional mor- phology-based classifications is in the positions of two species : Loa lo a and D. immitis. Based on mor- phological studies, they have been placed in the sub
family Dirofilariinae, yet they are not very close to each other in any of the DNA sequence-based phylo
genetic trees. Morphologically, the L3 (third-stage lar- vae) of L oa lo a does not resemble that of D irofilaria.
In this study, although the bootstrap value is low (Table II), D. imm itis appeared in most of the trees generated by différent methods to be closest to the O n ch o c erca clade (data not shown). These results correspond to certain aspects of the morphology of their L3 larvae (Bain and Chabaud, 1986). Based on
these DNA sequence data, L oa lo a appeared to be closest to M. p ersta n s and the B ru g ia -W u ch ereria clade in most o f the trees generated by various methods (data not shown). The bootstrap value is low, however, and morphologically the L3 of Loa loa and M. p e r s t a n s are to ta lly d ifféren t. The Dirofilariinae subfamily is characterized by highly d e v elo p ed cau d al alae and p a p illa e w h ile the Onchocercinae subfamily is characterized by a long, non-alate tail. Although some trees were rooted at the D. immitis branch (data not shown) which might sup
port D. imm itis as a différent subfamily, the position of Loa loa in ail of the trees generated by ail of the différent methods suggest its close connection with the Onchocercinae subfamily. These molecular data question the constitution of the Dirofilariinae subfa
mily.
ACKNOWLEDGMENTS
T
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.
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.
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Accepté le 17 mars 1994
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