Phylogeography of Rice yellow mottle virus in Africa

Zakia Abubakar1, Fadhila Ali1, Agnes Pinel2, Oumar Traoré3, Placide N'Guessan4,{dagger}, Jean-Loup Notteghem5, Frances Kimmins6, Gnissa Konaté3 and Denis Fargette2

1 ZARC, Zanzibar, PO Box 1062, Tanzania
2 IRD, BP 64501, 34394 Montpellier cedex 5, France
3 INERA, 01 BP 476 Ouagadougou, Burkina Faso
4 CNRA, Man, Côte d'Ivoire
5 ENSAM, 34060 Montpellier cedex 1, France
6 NRI, Chatham, Kent ME4 4TB, UK

Correspondence
Denis Fargette
Denis.Fargette{at}mpl.ird.fr


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The sequences of the coat protein gene of a representative sample of 40 isolates of Rice yellow mottle virus (RYMV) from 11 African countries were analysed. The overall level of nucleotide diversity was high ( ~14 %). Great geographical distances between the sites where isolates were collected were consistently associated with high genetic distances. In contrast, a wide range of genetic distances occurred among isolates spread over short geographical distances. There was no evidence of long-range dispersal. RYMV diversity in relation to land area was eight times greater in East Africa than in West/Central Africa. West/Central African isolates with up to 9 % divergence belonged to a monophyletic group, whereas the East African isolates with up to 13 % divergence fell into distantly related groups. In East Africa, each Tanzanian strain had a specific and restricted geographical range, whereas West/Central African strains had large and partially overlapping geographical distributions. Overall, our results suggest an earlier RYMV diversification in East Africa and a later radiation in West/Central Africa. The West African situation was consistent with virus adaptation to savanna, forest and other ecological conditions. In contrast East Africa, as exemplified by the Tanzanian situation, with numerous physical barriers (mountain chains, sea channel, lakes), suggested that RYMV strains resulted from divergence under isolated conditions. For RYMV and for two other viruses, phylogenetic relationships were established between isolates from Madagascar and isolates from the Lake Victoria region.

This paper is dedicated to the memory of our respected colleague, friend and co-author Placide N'Guessan, who was killed while attempting to escape from fighting between rival forces during the current unrest in Côte d'lvoire.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Studies of several plant virus species have shown genetic differences among isolates that are correlated with their geographical origins (Roossinck et al., 1999; Bousalem et al., 2000; García-Arenal et al., 2001; Krause-Sakate et al., 2002; Ohshima et al., 2002). However, the relationship is often difficult to discern because of virus dissemination by man and vectors. Rice yellow mottle virus (RYMV) offers a convenient model for evolutionary studies of a plant virus that is present on a continental scale, because the lack of efficient biotic or abiotic means of long-distance spread has conserved the original genetic structure of the virus populations. Furthermore, the range of diversity within the coat protein gene in RYMV populations ( ~14 %) is appropriate for phylogenetic studies. Due to the coordinated efforts of African scientists and their collaborators, representative isolates from most of the countries where RYMV disease causes economic losses have now been characterized.

Rice cultivation throughout the African continent is severely affected by RYMV. First reported in Kenya in 1966 (Bakker, 1974), RYMV has since been detected in most rice-growing countries of Africa and in Madagascar, but not elsewhere. The virus is considered to be among the most important rice pathogens within sub-Saharan Africa (Abo et al., 1998). It is a member of the genus Sobemovirus (van Regenmortel et al., 2000). The host range of RYMV is limited to grasses of the Oryzeae and Eragrostidae tribes (Abo et al., 1998). RYMV is transmitted by several species of beetles (Coleoptera), most of which belong to the Chrysomelidae, and is not seed-borne (Fauquet & Thouvenel, 1977; Konaté et al., 2001). There are conflicting observations on the role and significance of abiotic transmission by mechanical means during cultural practices, through soil residues or by irrigation water (Abo et al., 2000).

The diversity among RYMV isolates was first studied immunologically with polyclonal and monoclonal antibodies (mAbs) (Fauquet & Thouvenel, 1977; Mansour & Baillis, 1994; Konaté et al., 1997; N'Guessan et al., 2000). Later, analyses of sequences of the coat protein gene were made (Pinel et al., 2000; Traoré et al., 2001; Fargette et al., 2002). The coat protein gene was found to be a reliable part of the genome for assessing diversity, as congruent conclusions were obtained by sequencing other genes (Pinel et al., 2000; Fargette et al., 2002) or the full genome (A. Pinel & D. Fargette, unpublished results) of representative RYMV isolates. Both serological and molecular analyses showed a large virus diversity and a geographical distribution of the strains, with a split between isolates from East and West Africa, and with different strains in the savanna and forested regions of West Africa. Isolates from Central Africa were related to isolates of the savanna strain from West Africa (Traoré et al., 2001).

However, the studies in East Africa have been done with few isolates, so the diversity has not been assessed in this region. Furthermore, analyses with an unbalanced number of isolates from different regions may lead to biased conclusions on the phylogenetic relationships between strains (Graur & Li, 2001). Consequently, additional work is needed in East Africa to allow a comprehensive phylogeographic assessment of RYMV in Africa. Accordingly, to complement earlier studies in West and Central Africa (N'Guessan et al., 2000, 2001; Pinel et al., 2000; Traoré et al., 2001; Fargette et al., 2002), we have assessed the diversity of RYMV in Tanzania, which is one of the largest East African countries and one of the main African rice producers where RYMV has been known for several years (Rossel et al., 1982; Ali & Abubakar, 1995). Isolates were collected from the main rice-producing zones of Tanzania, their serological profile was determined using a range of mAbs and their coat protein gene was sequenced.

Subsequently, a balanced and representative sample of isolates from West, Central and East Africa was subjected to phylogeographical analyses. The results suggested that: (i) there was no evidence of long-distance spread; (ii) an earlier diversification occurred in East Africa, and a later strain radiation took place in West/Central Africa; (iii) the present genetic population structure in West/Central Africa reflected adaptation to different ecosystems, whereas divergence under isolated conditions was the main evolutionary factor in East Africa; and (iv) there were phylogenetic relationships between isolates from Madgascar and isolates from the Lake Victoria region for RYMV and for two other viruses.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isolate collection.
RYMV samples were collected from four major rice-growing areas of Tanzania: Mwanza, Mbeya, Morogoro and Pemba (see Fig. 2). Tanzania, covering an area of 950 000 km2, consists of a plateau at an altitude of 1200 m covering two-thirds of the country in the centre and north. It is bordered by Lake Victoria at the north (Mwanza) and Lake Malawi at the West (Mbeya). A 2000 m high mountain chain spreads south–north across the country and separates this plateau from the low-altitude coastal valley at the east of the country (Morogoro). Pemba island in the Indian ocean is 35 km east of mainland Tanzania. Isolates were collected between 1997 and 1999 in these four regions from diseased plants showing characteristic leaf mottling symptoms (Ali, 1999). RYMV was recovered by mechanical inoculation of leaf extracts to the susceptible Oryza sativa indica cultivar IR64. The virus induced the characteristic yellow discoloration and mottling of leaves. The 15 representative isolates detailed in Table 1 were selected for immunological and molecular analyses.



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Fig. 2. Top: geographical origin of the isolates, and latitude and longitude spanned in the surveys in West/Central Africa and Tanzania: Mwanza (a), Mbeya (b), Morogoro (c) and Pemba (d). Middle: surfaces surveyed, nucleotide diversity index and diversity index per unit area of land. Bottom: unrooted distance tree using Kimura two parameters from nucleotide sequences of ORF4 of 40 RYMV isolates:15 from Tanzania and 25 from different countries of West, Central and East Africa. Isolate denomination as in Table 1.

 

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Table 1. Isolates of RYMV used for phylogenetic analysis

 
Additional sequences of isolates were used to set a balanced and representative sample of the geographical and molecular diversity of RYMV in Africa (Table 1): four were from Madagascar, one from Kenya and 20 from West and Central Africa (Pinel et al., 2000; Traoré et al., 2001). These isolates were selected from a set of 120 coat protein gene sequences (D. Fargette & A. Pinel, unpublished results). Altogether 40 sequences were studied. This number is considered appropriate with regard to the number of informative sites for the phylogenetic analysis (Ridley, 1996). The geographical distances between the isolates were recorded. Cocksfoot mottle virus (CfMV) (Makinen et al., 1995), a virus of the genus Sobemovirus, which infects monocotyledonous plants, was added to be used as an outgroup in the phylogenetic analyses as it had the sequence the most closely related to RYMV (van Regenmortel et al., 2000). Finally, the sequences of the coat protein gene of isolates of Sweet potato feathery mottle virus (SPFMV) (Kreuze et al., 2000; Fenby et al., 2002) and of the heat-shock protein 70 homologous gene of Sweet potato chlorotic stunt virus (SPCSV) (Alicai et al., 1999) were re-analysed to investigate, using these viruses, relationships between isolates from Madagascar and isolates from the Lake Victoria region.

Immunological typing.
A panel of mAbs has been previously prepared against an isolate of RYMV from Madagascar (N'Guessan et al., 2000). Six mAbs (A, B, C, D, E and G) directed against different epitopes reacted differently against the RYMV isolates in ELISA. These mAbs were used in a triple-antibody-sandwich (TAS)-ELISA to assess the immunological profile of the RYMV isolates, as described previously (N'Guessan et al., 2000). Polyclonal antiserum against a Madagascan isolate was used as the primary antibody in the TAS-ELISA as it consistently detected all RYMV isolates. Monoclonal antibodies in culture supernatant fluids were used as secondary antibodies to discriminate among serotypes.

Molecular typing.
Genome fragments with the coat protein gene were transcribed and amplified by RT-PCR after extraction of total RNA from leaves. RT-PCR could not be performed directly on field isolates, probably because the virus concentration was too low, and virus multiplication on the susceptible cv. IR64 was necessary (see above). However, no modification of sequences was observed after passage on this host (A. Pinel & D. Fargette, unpublished results). The protocol was that of Pinel et al. (2000). Sequencing was carried out with the Taq terminator sequencing kit (Applied Biosystems) and analysed on an Applied Biosystems 373A sequencer. Two readings per base (3'->5' and 5'->3' directions) led to sequence accuracy >99·9 %. Sequences were assembled by Seqman (DNASTAR).

Phylogenetic analyses.
The following analyses were carried out. (i) The 720 bp nucleotide sequences of the coat protein gene of the 40 isolates were aligned using CLUSTAL W (Thompson et al., 1994) with default parameters, the alignment being checked by visual inspection. (ii) The genetic distances between the isolates were expressed in matrices of pairwise nucleotide divergence percentages (Nei & Kumar, 2000). (iii) Relationships between genetic and geographical distances were assessed with the Mantel test implemented in Genetix 4.02 (Mantel, 1967). (iv) The diversity index ({pi}) was calculated as described in Nei & Kumar (2000) and related to the surface areas of land from which the isolates had been collected, assuming that the surveys roughly covered the natural distribution of the virus. (v) The phylogenetic relationships between the 40 isolates were determined by the maximum-parsimony method and a distance method where nucleotide pairwise distances were corrected using the Kimura two-parameter method and trees reconstructed by the neighbour-joining method. Both methods were implemented in the PAUP program (Swofford, 2000). One thousand bootstrap replicates were carried out for each data set to assess the robustness of the nodes. (vi) Relationships between East African isolates were further studied using amino acid pairwise sequence distances to reconstruct the phylogenetic trees. (vii) Sequences of SPFMV and SPCSV isolates were compared to assess relationships between isolates from Lake Victoria region and from Madagascar.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Two serological profiles for the Tanzanian isolates
The 15 isolates from Tanzania showed two serological profiles (Table 2). The first was shared by all isolates from Mwanza and Mbeya and characterized by a full reaction against all mAbs, except the weakly reacting mAb B. This serological profile (designated Ser 4) was also shared by Madagascan isolates with which the mAbs had been prepared. The second serotype (Ser 5) was shared by all isolates from Morogoro and Pemba and was characterized by reactions against all mAbs except E. Overall, all the Tanzanian isolates differed from serotypes 1–3 found in West and Central Africa (Table 2).


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Table 2. Serological profiles of RYMV isolates assessed with mAbs in TAS-ELISA tests

Absorbance values in ELISA were coded as follows: ‘0’<0·30, 0·30<=‘1’<0·60, ‘0·60<=‘2’<1·20, 1·20<=‘3’<1·80, ‘4’>=1·80.

 
High molecular variability of Tanzanian isolates
RYMV diversity in Tanzania was high, with the sequence divergence of the coat protein gene reaching 12·5 % for nucleotides and 10·8 % for amino acids (data not shown). This diversity had a specific geographical pattern. All the isolates collected in Mwanza, near Lake Victoria, were closely related to each other, with a divergence of <2 % for nucleotides and <1 % for amino acids. In contrast, they differed from the Mbeya isolate by 5·8–6·9 %, from the Pemba island isolates by 9·3–10·6 % and from the Morogoro isolates by 9·4–12·1 %. The limited number of isolates from these last three regions preclude firm conclusions on regional diversity. Nevertheless, a comparatively high intra-region diversity, although lower than the inter-region diversity, was suggested, as the three Morogoro isolates differed by 3·0 % between each other and the two Pemba isolates by 3·1 %. This was higher than the intra-group diversity observed in Mwanza ({pi}=1·5 %) and Madagascar ({pi}=1·4 %). Although Morogoro is less than 500 km away from Pemba and Mbeya, Morogoro isolates differed by 11·8–12·5 % from the Pemba isolates and by 9–10 % from the Mbeya isolate.

Relationships between genetic and geographical distances
There was a highly significant relationship (P<0·001 after the Mantel test; see Methods) between genetic and geographical distances of the 780 pairwise comparisons from the 40 isolates (Fig. 1). On average, the greater the geographical distance, the higher the genetic distance. The best regression fit between genetic and geographical distances was after logarithmic transformation of the geographical distances (data not shown). Geographical distances between 4000 and 8000 km were associated with genetic distances between 7 and 14 % in comparisons between East and West African isolates, and between East and Central African isolates. Geographical distances between 1000 and 4000 km corresponded to genetic distances between 4 and 14 %. In no instance was great distance (>1000 km) associated with low divergence (<2 %), so there was no evidence of long-distance dispersal of isolates. Geographical distances <1000 km were associated with divergence ranging from 0·1 to 13 %. Divergences up to 9 % resulted from comparisons between West, Central and East African isolates. Divergence above 9 % was found for East African isolate comparisons.



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Fig. 1. Pairwise comparisons of geographical distance (km) and genetic distance (% divergence in nucleotides) between each of the 40 African isolates studied: East vs East ({circ}), East vs Central ({lozenge}), Central vs Central (x), East vs West ({square}), Central vs West ({triangleup}), West vs West (+).

 
Higher variability in Tanzania despite the smaller area surveyed
The nucleotide diversity index was 5·4 % for the 20 West and Central African isolates compared with 7·7 % for the 20 East African isolates and 7·5 % for the 15 Tanzanian isolates (Fig. 2). Greater variation occurred in Tanzania although the surveys in West/Central Africa were conducted over a larger geographical area. Tanzania spans a total area of 950 000 km2, whereas the samples in West/Central Africa were collected across an area 3500 km in longitude by 1500 km in latitude, i.e. a total area of ~5 250 000 km2 (Fig. 2). Subsequently, the diversity index per surface area (% nucleotide diversity per million square kilometres) surveyed was 8·0 in Tanzania and 1·0 in West/Central Africa. Overall, the diversity index per surface unit was eight times greater in Tanzania than in West/Central Africa.

This ratio is likely to be an underestimate because of sampling differences. Only 20 isolates from East Africa, including the 15 from Tanzania, were sequenced, and additional sampling is likely to reveal greater diversity. In contrast, the selected sample of 20 West/Central African isolates was from a set of ~100 isolates, which was itself selected from among ~200 isolates tested serologically using strain-specific mAbs (D. Fargette & A. Pinel, unpublished results). It was representative of the current diversity, as new isolates from West/Central Africa invariably fell within the groups already known (D. Fargette & A. Pinel, unpublished results).

Topology of the RYMV tree
The molecular diversity of RYMV was high and reached up to 14·3 % between an isolate from Pemba island in the Indian Ocean and one from Mali 5500 km westward. Phylogenetic trees derived from parsimony and distance analyses of sequences were congruent and only distance trees are presented (Fig. 3). The topology of the phylogenetic tree was quite unbalanced. West/Central African isolates belonged to the same phylogenetic group and different clusters could be distinguished clearly following the bifurcation pattern of the internal nodes. The group could first be divided into two clusters, one consisting mostly of isolates from the savanna regions, the other mostly from forest regions, termed ‘savanna’ and ‘forest’ clusters, respectively. The ‘savanna’ cluster could be further divided into two groups, later regarded as strains. One included isolates from West Africa ({pi}=2·6), the other isolates from Central Africa ({pi}=4·1). The ‘forest’ cluster was made up of one major strain ({pi}=1·5) and a numerically minor strain ({pi}=1·0) consisting of isolates from Sierra Leone at the far west of Africa and originating from mangrove habitats (Fomba, 1984).



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Fig. 3. Neighbour-joining tree calculated from the pairwise nucleotide sequence distances (with the Kimura two-parameter model) between the aligned coat protein genes of 40 RYMV isolates: 15 isolates originated from Tanzania (Tz), and 25 from 10 other countries of East, Central and West Africa. Isolate denomination as in Table 1. An isolate of CfMV was used as an outgroup. The scale bar indicates a genetic distance of 0·01. Bootstrap supports of the nodes are indicated as percentages (from 1000 replicates). Branches with bootstrap values less than 70 % were collapsed. The geographical and ecological origins of the isolates are indicated by vertical bars to the right of the figure. Different letters indicate the different regions of Tanzania surveyed: Mwanza (a), Mbeya (b), Morogoro (c) and Pemba (d). The diversity index of each group is indicated at the right of the figure.

 
Phylogenetic relationships between the East African isolates showed a different pattern (Fig. 3). East African isolates did not belong to a resolved monophyletic group. Uncertainties about the phylogenetic relationships of the East African isolates were reflected by the basal polytomy. Five clusters with high bootstrap support were apparent. They showed the longest and most basal branches, suggesting an earlier diversification in East Africa (see Discussion). They had a 6–13 % nucleotide divergence and were designated as strains. Each of the four main rice-growing regions of Tanzania harboured a different strain. Tanzanian strains not only differed between each other, but also differed from other strains including the Madagascan one. There was a notable exception in that the original Kenyan isolate collected in 1966 at Kimusu in Kenya at the east of Lake Victoria (Bakker, 1974) was close genetically to isolates from Mwanza in Tanzania at the south of Lake Victoria, suggesting they belonged to a ‘Lake Victoria’ strain. Altogether we distinguished nine strains in Africa, four in Tanzania, one in Madagascar, one in Central Africa and three in West Africa.

In Tanzania, there was a significant link between genetic and geographical distances (P<0·001). In contrast, there was no relationship between genetic and geographical distances in West Africa (P=0·14). This reflected the specific and restricted geographical range of each Tanzanian strain, and the large and partially overlapping geographical distributions of West African strains.

Phylogenetic relationships between East African strains
The proportion of observed synonymous (Ds) and non-synonymous (Dn) substitutions after the Jukes–Cantor correction for multiple hits was calculated on nine isolates representing each of the nine strains. The average of all pairwise comparisons indicated that Ds was 0·29 and Dn 0·04. The ratio Ds/Dn was 7·3, indicating a high conservative selection pressure on the coat protein gene. Then, in an attempt to resolve uncertainties over the phylogenetic relationships among East African strains, phylogenetic trees were reconstructed from amino acid distances, since amino acids sequences are more conserved than nucleotide sequences and thus provide useful information on the long-term evolution of genes (Nei & Kumar, 2000). Actually, the amino acid distance tree suggested phylogenetic relationships between strains from Mwanza and Mbeya (Fig. 4). Madagascan and Kenyan isolates fell within the East African phylogenetic group shared with Mwanza and Mbeya isolates, but Madagascan isolates belonged to a subcluster within this group (Fig. 4). Similar relationships were found with non-synonymous substitution distances (data not shown) and were consistent with the serological relationships, as isolates from Mwenza and Mbeya regions and from Madagascar all shared the Ser 4 serological profile (Table 2). In contrast, these phylogenetic relationships were not apparent using distances based on synonymous nucleotide differences (data not shown) or all differences (Fig. 3). Similar phylogenetic relationships between virus isolates from Madagascar and from the Lake Victoria region were found with SPFMV and SPCSV. SPFMV and SPCSV isolates from Uganda at the north of Lake Victoria and isolates from Madagascar belonged to the same phylogenetic group, suggesting a common ancestor not shared with isolates outside East Africa (Fig. 5).



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Fig. 4. Neighbour-joining tree calculated from the pairwise amino acid sequence distances between the aligned coat protein genes of 20 RYMV isolates from East Africa. The isolate Ca30 from Cameroon in Central Africa was used as an outgroup. Bootstrap supports of the nodes are indicated as percentages (from 1000 replicates). Branches with bootstrap values less than 70 % were collapsed. The geographical origin of the isolates is indicated by vertical bars to the right of the figure.

 


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Fig. 5. Phylogenetic trees of SPFMV (top) and SPCSV (bottom) isolates from Uganda at the north of Lake Victoria and from Madagascar, using isolates from Central Africa as an outgroup. SPFMV and SPCSV trees were reconstructed from amino acid and nucleotide sequence distances, respectively. Bootstrap supports of the nodes separating East African isolates (including Madagascar) from other isolates, and of the nodes separating isolates from Lake Victoria region and from Madagascar, are given.

 
Isolates from Morogoro and Pemba at the east of the major mountain chain that spreads across Tanzania (see Methods) differed from Mwenza and Mbeya isolates at the west. Moreover, isolates from Morogoro and Pemba belonged to different strains taking either nucleotide (Fig. 3) or amino acid (Fig. 4) characters for the phylogenetic analysis. However, Morogoro and Pemba strains shared the same serological profile, Ser 5 (Table 2). Moreover, there were only two events of deletion/insertion of one amino acid in the set of 40 coat proteins (data not shown). Both events also distinguished the Morogoro and Pemba strains from all other strains. Morogoro and Pemba strains had an extra amino acid at position 60. Five isolates had an Arg60 (CGC) and one (Tz10) had a Gly60 (GGC). The Pemba strain differed from the Morogoro strain and from any other strains by a Lys19 deletion. The common serotype and the unique extra amino acid at position 60 suggested a phylogenetic relationship between these two strains, although remote since it was undetectable by coat protein gene sequence analyses.


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
RYMV was first observed in 1966 in Kenya (Bakker, 1974) and has since been found almost everywhere in Africa (Abo et al., 1998), including Madagascar and Tanzania in the 1980s (Reckhaus & Andriamasintseheno, 1997; Ali, 1999). Indeed, other viruses such as Zucchini yellow mosaic virus (ZYMV) (Pfosser & Baumann, 2002) have also reached a worldwide distribution within a few decades. ZYMV spread was attributed to the efficient long-range dispersal by infected seeds and aphids (Pfosser & Baumann, 2002). This was apparent as genetically highly divergent ZYMV isolates were found within the same geographical region, whereas isolates from remote countries were sometimes similar. The genetic structure of RYMV populations offered a different picture and suggested an alternative scenario. Great geographical distance was consistently associated with high genetic distance, and closely related isolates were never found far apart, evidence of the absence of long-distance dispersal. This is consistent with the lack of efficient biotic and abiotic long-range means of dispersal for RYMV: no seed transmission (Konaté et al., 2001), short virus retention time and low flight ability of the beetle vector (Bakker, 1974). Taken together, this explains why a consistent geographical basis of the genetic structure of RYMV populations has been preserved, which would otherwise have been blurred if long-distance and barrier-crossing dispersal had occurred. Therefore, a recent and massive long-range virus dissemination by vectors or man from Kenya since 1966 to the rest of Africa is most unlikely. More probably, the virus was present in the wild grasses before spreading recently to cultivated rice when conditions became favourable, especially after intensive cultivation of susceptible cultivars in Africa concomitant with RYMV spread (Bakker, 1974; Abo et al., 1998). Thus, the observed genetic structure of RYMV populations in rice more likely reflects those that occurred in wild grasses. Accordingly, the serological profiles and the sequences of RYMV isolates from wild grasses have been found to match those of cultivated rice of the same region (N'Guessan et al., 2000; Pinel et al., 2000).

The genetic structure of the RYMV population in Tanzania was characterized by an exceptionally high diversity, which was apparent through the number of strains, the level of divergence between the strains, the diversity index and the diversity index per area sampled. This greater diversity and the topology of the phylogenetic tree suggested an earlier virus diversification in East Africa. The monophyletic origin of West/Central African isolates suggested a distinct and later strain radiation in this region. The close relationship between RYMV strains and their geographical origins has been progressively elucidated. In West/Central Africa, a link has been established between specific strain and specific ecological conditions (forest, savanna, mangrove) (N'Guessan et al., 2001).

Tanzania offered a contrasting picture with highly divergent strains of RYMV in each of the different agro-ecologies, all of which differed from those in West/Central Africa. Furthermore, each Tanzanian strain had a specific and restricted geographical range, whereas West African strains had large and partially overlapping geographical distributions. Actually, this could reflect strain adaptation to different regional ecosystems: mid-altitude plateau, lake regions (Mwanza, Mbeya), valleys (Morogoro) and island (Pemba). However, a more ‘parsimonious' scenario based on divergence, reinforced by isolated conditions, can also explain the RYMV situation in Tanzania. The high ratio of synonymous to non-synonymous substitution reflects the strong conservation pressure on the genome. It indicates that RYMV evolution has resulted from genetic drift under conservative selection, as for most animal and plant viruses (Domingo et al., 2001; García-Arenal et al., 2001). Physical barriers such as mountain chains, lakes and sea arms in Tanzania have created isolations that have allowed divergence between strains.

Evidence is presented that isolates of RYMV from the Lake Victoria region and from Madagascar form sister groups within an East African monophyletic group. Similar relationships were found with isolates of SPFMV and SPCSV. Overall, for each of the three viruses – whatever their genus or ecology – isolates from Madagascar and the Lake Victoria region had a common ancestor not shared with isolates from other geographical origins in Africa. Continental islands such as Madagascar support flora and fauna closely related to the nearby mainland East Africa, which have diverged since separation between the island and the continent in the Cretaceous period ~100 million years ago (Brown & Lomolino, 1998). Accordingly, vicariance events may be responsible for the disjunction between the Tanzanian and the Madagascan strains of each of the three plant viruses, RYMV, SPFMV and SPCSV. This hypothesis applies preferentially to RYMV for which no evidence of long-distance spread has been detected. ‘Palaeovirological’ speculations on co-evolution between viruses and their hosts over geological time have been made previously for Turnip yellow mosaic virus (Blok et al., 1987), potyviruses (Ward et al., 1994), geminiviruses (Rybicki, 1994) and tobamoviruses (Gibbs, 1999).


   ACKNOWLEDGEMENTS
 
We thank C. Brugidou, T. Candresse, C. Fauquet, A. Ghesquière, J.-F. Guegan, B. D. Harrison, J. Maley, J.-C. Pintaud and S. Seal for helpful discussions, J. M. Thresh for careful reading of the manuscript and J. Aribi for technical assistance.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 7 August 2002; accepted 17 October 2002.