Arch Virol (2011) 156:2205–2213 DOI 10.1007/s00705-011-1119-4
ORIGINAL ARTICLE
High genetic variability and recombination in a begomovirus population infecting the ubiquitous weed Cleome affinis in northeastern Brazil Sarah J. C. da Silva • Gloria P. Castillo-Urquiza • Braz T. Hora Ju´nior Iraildes P. Assunc¸a˜o • Gaus S. A. Lima • Gilvan Pio-Ribeiro • Eduardo S. G. Mizubuti • F. Murilo Zerbini
•
Received: 24 April 2011 / Accepted: 14 September 2011 / Published online: 18 October 2011 Ó Springer-Verlag 2011
Abstract Diseases caused by begomoviruses are a serious constraint to crop production in many tropical and subtropical areas of the world, including Brazil. Begomoviruses are whitefly-transmitted, single-stranded DNA viruses that are often associated with weed plants, which may act as natural reservoirs of viruses that cause epidemics in crop plants. Cleome affinis (family Capparaceae) is an annual weed that is frequently associated with leguminous crops in Brazil. Samples of C. affinis were collected in four states in the northeast of Brazil. Analysis of 14 full-length DNA-A components revealed that only one begomovirus was present, with 91-96% identity to cleome leaf crumple virus (ClLCrV). In a phylogenetic tree, ClLCrV forms a basal group relative to all other Brazilian begomoviruses. Evidence of multiple recombination events was detected among the ClLCrV isolates, which also display a high degree of genetic variability. Despite ClLCrV being the only begomovirus found, its phylogenetic placement, high genetic variability and recombinant nature suggest that C. affinis may act as a source of novel viruses Electronic supplementary material The online version of this article (doi:10.1007/s00705-011-1119-4) contains supplementary material, which is available to authorized users. S. J. C. da Silva G. P. Castillo-Urquiza B. T. Hora Ju´nior E. S. G. Mizubuti F. M. Zerbini (&) Departamento de Fitopatologia/BIOAGRO, Universidade Federal de Vic¸osa, Vic¸osa, MG 36570-000, Brazil e-mail:
[email protected] S. J. C. da Silva G. S. A. Lima G. Pio-Ribeiro Departamento de Agronomia, Universidade Federal Rural de Pernambuco, Recife, PE 52171-900, Brazil I. P. Assunc¸a˜o G. S. A. Lima Departamento of Fitossanidade/CECA, Universidade Federal de Alagoas, Rio Largo, AL 57100-000, Brazil
for crop plants. Alternatively, ClLCrV could be a genetically isolated begomovirus. Further studies on the biological properties of ClLCrV should help to clarify the role of C. affinis in the epidemiological scenario of Brazilian begomoviruses.
Introduction Geminiviruses (family Geminiviridae) have circular, single-stranded (ss) DNA genomes that are packaged within twinned quasi-isometric virions. Geminiviruses are divided into four genera, Mastrevirus, Topocuvirus, Curtovirus and Begomovirus, based on genome organization and biological properties, the most important being the type of insect vector (either whitefly, leafhopper or treehopper) and host range (either mono- or dicotyledonous hosts) [9]. Begomoviruses (whitefly-transmitted geminiviruses) cause serious diseases in a number of economically important crops, mostly in tropical and subtropical regions [32]. Over the last four decades, agricultural intensification and the emergence and prevalence of a new and more aggressive biotype of the insect vector (Bemisia tabaci biotype B) have led to an increase in begomovirus populations and their expansion to new plant hosts throughout tropical and subtropical regions of the Americas [15, 16, 21, 27]. In Brazil, begomoviruses are limiting factors for tomato and common bean production [8, 44]. In beans (Phaseolus vulgaris and P. lunatus), bean golden mosaic virus (BGMV) has been an important pathogen since the 1970s, following the increase in soybean (Glycine max) cultivation [4, 5]. The emergence of novel begomoviruses in tomatoes has been attributed to the introduction and spread of the B biotype of B. tabaci in the mid-1990s [30].
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Weeds are considered reservoirs of begomoviruses that infect crop plants, as well as sources of novel recombinant viruses due to mixed infections [2, 13, 19]. Some of the economically important begomoviruses in crop plants are closely related to begomoviruses found in weeds [1, 19, 22]. Similar to what is observed for begomoviruses in crops, the species diversity of begomoviruses infecting weeds is very high [2, 10–12, 14, 17, 34, 35, 37]. For example, at least 15 begomoviruses are reported to infect Sida spp. [2, 9, 42]. The characterization of weed-infecting begomoviruses is therefore important in elucidating their ecological and evolutionary behavior [38]. In this report, we examine a begomovirus population present in Cleome affinis, an annual weed that belongs to the family Capparaceae and which is frequently present within or around fields of common bean (Phaseolus vulgaris) and lima bean (P. lunatus) crops in northeastern Brazil.
S. J. C. da Silva et al. Table 1 Location, year of collection and full-length begomovirus clones obtained from Cleome affinis samples collected in five northeastern Brazilian states from 2007 to 2010 Sample code
Collection site
Year of collection
Clones (DNA-A)
Accession number
Alagoas state SC201
Paripueira
2009
BR:ALPar1:09A
JN103427
SC202
Maragogi
2009
BR:ALMar1:09A
HM195184
SC203
2009
BR:ALSmc1:09A
JN103435
SC205
Sa˜o Miguel dos Campos Maceio´
2010
BR:ALMac6:10A
JN103436
SC215
Atalaia
2007
BR:ALAta1:07A
JN103433
SC216
Rio Largo
2007
BR:ALRil:07A
JN103434
2010
BR:BACds1:10A
JN103439
2010
BR:PBAlh1:10A
JN103438
2010
BR:PEGoi1:10A
JN103437
BR:SEInd1:10A BR:SENps1:09A
JN103428
Bahia state SC207
Materials and methods
Costa do Sauı´pe Paraı´ba state
SC226
Alhandra
Sample collection Samples of Cleome affinis were collected during the years 2007 to 2010 in the states of Alagoas (AL), Bahia (BA), Paraı´ba (PA), Pernambuco (PE) and Sergipe (SE), all in northeastern Brazil (Table 1). Plants displaying symptoms of mosaic, yellowing and growth reduction typical of begomovirus infection were preferentially collected. Samples were desiccated by pressing and stored at room temperature.
Pernambuco state SC218
Goiana Sergipe state
SC208
Indiaroba
2010
SC209
Neo´polis
2009
SC210
Japoata˜
2009
BR:SEJpt1:09A
JN103430
DNA amplification and cloning
SC212
Neo´polis
2009
BR:SENps2:09A
JN103431
Total DNA was extracted according to Doyle and Doyle [6]. To confirm the presence of begomoviruses, PCR was carried out using universal primers for members of the genus [31]. Full-length viral genomes were amplified from PCR-positive samples by rolling-circle amplification (RCA) [20], cloned in pBLUESCRIPT KS ? (Stratagene) after monomerization with the restriction enzymes Cla I, Hind III or Pst I, and sequenced at Macrogen Inc. (Seoul, South Korea) by primer walking. Prior to cloning, RCA products were also cleaved with the 4-base cutter enzymes Msp I and Hae III in order to verify the presence of possible satellite DNAs, as reported by Paprotka et al. [28] for C. affinis samples from the state of Mato Grosso do Sul.
SC213
Neo´polis
2009
BR:SENps3:09A
JN103432
Sequence comparisons and phylogenetic analysis DNA-A nucleotide sequences were subjected to a BLAST search for preliminary species assignment based on the
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JN103429
All clones were classified as Cleome leaf crumple virus (ClLCrV) based on the ICTV-established criteria of 89% nucleotide sequence identity for the full-length DNA-A [9]
89% threshold level established by the Geminiviridae Study Group of the ICTV [9]. Additional nucleotide pairwise comparisons were performed with DNAMan version 4.0 (Lynnon Co.) using the Optimal Alignment option with the following parameters: Ktuple = 2, Gap penalty = 7, Gap open = 10, Gap extension = 5. Nucleotide sequences of begomoviruses used in the phylogenetic and recombination analyses (see Supplementary Table S1 for the full-length virus names and their respective GenBank accession numbers) were aligned using the Muscle module in MEGA 5 [40]. Phylogenetic analysis was performed by the neighbor-joining method
Begomoviruses infecting Cleome affinis in Brazil
implemented replications.
in
MEGA
5,
with
2,000
2207
bootstrap
Recombination analysis Phylogenetic network analysis for evidence of recombination was performed using the neighbour-net method implemented in SplitsTree4 [18]. Additional analysis of potential recombination events and identification of putative parental sequences was carried out using the Recombination Detection Program (RDP) ver. 3.0 [26]. Recombination events detected by at least four of the analysis methods available in the program were considered reliable. Alignments were scanned using default settings for each analysis method using a Bonferroni-corrected p value cutoff of 0.05. Genetic variability of the viral population The partition of genetic variability and inferences about population structure were based on Wright’s F fixation index [41]. The UST parameter was calculated with the program Arlequin 3.11 [7], using the Kimura 2-parameter distance and estimating statistical significance by permutation analysis with 1,000 replications. The main descriptors of genetic variability were quantified using the program DnaSP version 5 [36]: number of polymorphic sites, total number of mutations (g), average number of nucleotide differences (k), nucleotide diversity (p), number of haplotypes, haplotype diversity (Hd), number of segregating sites, Watterson’s estimate of the population mutation rate based on the total number of segregating sites (Theta-W) and on the total number of mutations (Theta-Eta). Four types of neutrality tests were used to test the hypothesis of occurrence of selection in the population: Tajima’s D, Fu and Li’s D* and F*, and the test based on the number of synonymous (Ds) and non-synonymous (Dns) substitutions with the Pamilo-Bianchi-Li (PBL) model.
Results Fourteen samples of Cleome affinis showing mosaic, yellowing and growth reduction were collected: six from the state of Alagoas, one each from the states of Bahia, Paraı´ba and Pernambuco, and five from the state of Sergipe (Table 1). All 14 samples tested positive for the presence of a begomovirus by PCR with universal primers (data not shown). No evidence of the presence of alphasatellites, or of any other kind of DNA satellite, was obtained after digestion with Msp I or Hae III. Fourteen full-length DNAA viral genomes were cloned (Table 1). Pairwise sequence
comparisons showed that all fourteen cloned viral genomes corresponded to isolates of cleome leaf crumple virus (ClLCrV), with their nucleotide sequences displaying 91-96% identity with a recently described ClLCrV isolate from the state of Mato Grosso do Sul, Brazil (FN435999) (Supplementary Table S2). In fact, clone BR:AL-Ata1:07 is the only one whose sequence shows 91% identity to ClLCrV, with the sequences of the remaining 13 clones displaying 95-96% identity (Supplementary Table S2). Besides, sequence identity between BR:AL-Ata1:07 and the other 13 isolates ranges from 93-94% (Supplementary Table S2), with most differences in the Rep gene and in the common region (data not shown). This indicates that BR:AL-Ata1:07 represents a distinct strain of ClLCrV, for which we propose the denomination ClLCrV-Ata. A neighbor-joining tree was constructed based on the complete DNA-A nucleotide sequences of the 14 ClLCrV isolates from C. affinis plus 22 Brazilian begomoviruses and additional sequences of begomoviruses from the Americas (Figure 1). The viruses clustered into five major groups. Groups I and V comprise only non-Brazilian viruses. Group II includes, in addition to viruses from other countries in the Americas, four begomoviruses from Brazil (abutilon Brazil virus, AbBV; euphorbia yellow mosaic virus, EuYMV; sida yellow leaf curl virus, SiYLCV; and tomato common mosaic virus, ToCmMV). Group III includes Brazilian begomoviruses and one virus from neighboring Argentina (soybean blistering mosaic virus, SoBlMV). All ClLCrV isolates grouped with the original ClLCrV isolate from Mato Grosso do Sul in group IV, which is placed at a basal position relative to clusters I-III. Recombination analysis Neighbor-net analysis indicated the occurrence of several recombination events among ClLCrV isolates and other Brazilian begomoviruses (Fig. 2A). Strong evidence for recombination was found in cluster I, represented by the 14 ClLCrV isolates and the isolate from Mato Grosso do Sul. Recombination events were also evident in other clusters (II, III and VI). These results were corroborated when the analysis was restricted to begomoviruses from C. affinis (Fig. 2B). The same sets of sequences were analyzed using the RDP3 package with the aim of investigating these putative recombination signals. A recombination event was detected for all 14 ClLCrV isolates, with breakpoints within the Rep coding region and tomato yellow spot virus (ToYSV) identified as one of the putative parents (Table 2). An additional recombination event was observed within the Rep gene for BR:AL-Ata1:07, with BR:AL-Ril1:07 identified as one of the parents (Table 2). A strongly supported recombination event was detected involving BR:AL-Ata1:07
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S. J. C. da Silva et al. b Fig. 1 Neighbor-joining tree based on the complete DNA-A nucleotide sequences of begomoviruses from the Americas, including the cleome leaf crumple virus (ClLCrV) isolates infecting Cleome affinis in northeastern Brazil (see Supplementary Table S1 for full virus names). Group IV includes all 14 ClLCrV isolates obtained in this study, plus the isolate from Mato Grosso do Sul (FN435999)
BDMV M88179
92
SGMCRV X99550 SGMHV Y11097 98
SiYVV
Y11099
SiYMYuV
DQ875872
OYMMV DQ022611
61
ToLCSV AJ608286 TLCCUV AM050143 96
WGMV DQ395343 ToMHV Y14874
59
CdTV AF101476
98
I
OYMoIV AY751753 ToMoTV AF012300 SiGYVV HQ009519
and BR:PB-Alh1:10, with breakpoints at the common region (CR) and the Rep coding region, and BR:ALPar1:09 identified as one of the putative parents (Table 2).
ToYDLV FJ174698
84
ToMoV L14460
72
SGMV AF049336 81 91
RhRGMV HM236370
73
AbMV X15983 MaGMV EU158096 CoYSV DQ875868
51
Genetic variability of the ClLCrV population
PYMPV Y15034 PYMV D00940
100
CLCrV AF480940
66
DesLDV DQ875870 ToCmMV EU710754
96 100
84
SiYLCV EU710750 AbBV FN434438 ToSLCV AF130415
58
RhGMSV DQ406672
93 100
CaLCuV U65529
58
PepGMV
U57457
BCaMV AF110189
96
EuMV_YP DQ318937
93 96
ToMYLCAV AY927277
75
EuYMV FJ619507
51
CuLCrV AF224760
54
100
SqMLCV AF421552 100
MCLCuV AY064391 100
SqLCV M38183
SiMoV AY090555
63 92
SiYMV AY090558 ToYSV DQ336350
88
OMoV EU914817 SimMV AJ557451
58
SiCmMV EU710751 62
ToLDV
83 100
EU710749
NDNV n.a.
79
ToMIMV EU710752
III
BGMV M88686
68 68 51
SiBV
FN436001
BlYSV
EU710756
SoBlMV EF016486
55 71
ToYVSV EF417915 TGMV K02029
52
PSLDV FJ972767
64
ToCMoV AF490004 ToRMV AF291705 99
ToSRV DQ207749 BR:AL-Ata1:07 ClLCrV FN435999 100
BR:PB-Alh1:10 99
BR:AL-Ril1:07 BR:AL-Par1:09
99
BR:AL-Smc1:09 99
BR:AL-Mac6:10 BR:SE-Ind1:10
78
II
Population subdivision analysis indicated that the 14 ClLCrV isolates comprised a single population, with no structuration based on geographical location or year of collection. The analysis of genetic descriptors demonstrated that the ClLCrV population has a high degree of genetic variability, which is considerably higher than those observed for two populations of tomato infecting begomoviruses from southeastern Brazil, and similar to that observed for a population of macroptilium yellow spot virus (MaYSV), a novel begomovirus described in the weed Macroptilium lathyroides, also in northeastern Brazil (Table 3). Evidence of selection or demographic forces acting on the ClLCrV population was assessed by four different neutrality tests. The four ORFs encoded by the DNA-A (Rep, Trap, Ren and CP) varied in this regard. Significant probability for rejecting the hypothesis of neutrality was found for the Rep ORF (Table 4), indicating that this genomic region is potentially under purifying selection. Negative values were obtained, but were not statistically supported, for Tajima’s D, Fu and Li’s D* and Fu and Li’s F* for Ren, Trap and CP (Table 4). However, a dN/dS \1 for all ORFs is indicative of purifying selection acting on this population.
IV
BR:SE-Nps3:09 BR:AL-Mar1:09 BR:SE-Nps1:09
54 66
Discussion
BR:SE-Nps2:09 BR:SE:Jpt1:09
98 BR:BA-Cds1:10 98 BR:PE-Goi1:10 100 99
TYCV FJ213931 CaLCuJV DQ178614 ToGMoV DQ520943
89
ToChLPV AY339618 MeMV AF068636 ToYMLCV AY508993
78 100
DiYMCUV AJ549960 DiYMV AF139168
V
BGYMV D00201
59
MaYMFV AY044135
100 98
MaYMV EF585290 MaMPRV AY044133 PHYVV
100 100
X70418
RhGMV EU339936 ToLCNDV U15015
0.05
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Cleome affinis is classified in the family Capparaceae and is often present within and near fields of lima bean (Phaseolus lunatus), common bean (Phaseolus vulgaris) and other leguminous crops in northeastern Brazil. Recently, a new begomovirus, cleome leaf crumple virus (ClLCrV) was found infecting this weed in the state of Mato Grosso do Sul [28]. Sequence analysis of the fourteen isolates obtained from C. affinis indicated 91-96% identity to the ClLCrV isolate from Mato Grosso do Sul. The ICTV guidelines propose a demarcation threshold of 89%
Begomoviruses infecting Cleome affinis in Brazil
Fig. 2 Phylogenetic evidence for recombination among: (A) all Brazilian begomoviruses, including the ones describes in this work, and (B) a population of cleome leaf crumple virus (ClLCrV) obtained from samples of Cleome affinis collected in five different states of
2209
northeastern Brazil. Neighbor-net analysis was performed using SplitsTree4. Formation of a reticular network rather than a single bifurcated tree is indicative of recombination
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S. J. C. da Silva et al.
Table 2 Putative recombination events detected among Brazilian begomoviruses, including the cleome leaf crumple virus (ClLCrV) isolates infecting Cleome affinis in northeastern Brazil Isolate
Parents
Breakpoints
P-value
Initial
Final
Rc
G
B
M
C
S
3S
BR:ALPar1:09
ToYSVa
2486b
2601
3.9 9 10-03
–d
3.3 9 10-02
1.3 9 10-05
2.1 9 10-04
–
–
BR:ALMar1:09
ToYSV
2190
2661
3.9 9 10-03
–
3.3 9 10-02
1.3 9 10-05
2.1 9 10-04
–
–
BR:ALSmc1:09
ToYSV
2201
2659
3.9 9 10-03
–
3.3 9 10-02
1.3 9 10-05
2.1 9 10-04
–
–
BR:ALMac6:10
ToYSV
2211
2674
3.9 9 10-03
–
3.3 9 10-02
1.3 9 10-05
2.1 9 10-04
–
–
BR:BACds1:10
ToYSV
2290
2652
3.9 9 10-03
–
3.3 9 10-02
1.3 9 10-05
2.1 9 10-04
–
–
BR:SEInd1:10
ToYSV
2207
2664
3.9 9 10-03
–
3.3 9 10-02
1.3 9 10-05
2.1 9 10-04
–
–
BR:SENps1:09
ToYSV
2189
2660
3.9 9 10-03
–
3.3 9 10-02
1.3 9 10-05
2.1 9 10-04
–
–
BR:SEJpt1:09
ToYSV
2201
2663
3.9 9 10-03
–
3.3 9 10-02
1.3 9 10-05
2.1 9 10-04
–
–
BR:SENps2:09
ToYSV
2212
2660
3.9 9 10-03
–
3.3 9 10-02
1.3 9 10-05
2.1 9 10-04
–
–
BR:SENps3:09
ToYSV
2201
2663
3.9 9 10-03
–
3.3 9 10-02
1.3 9 10-05
2.1 9 10-04
–
–
BR:ALAta1:07
BR:ALPar1:09
18
1620
2.1 9 10-22
6.1 9 10-12
2.6 9 10-13
1.7 9 10-19
2.1 9 10-12
4.6 9 10-25
6.1 9 10-24
BR:ALRil1:07
1658
2007
4.6 9 10-08
2.7 9 10-08
2.0 9 10-11
2.6 9 10-03
3.1 9 10-05
1.6 9 10-16
–
ToYSV
2205
2663
3.9 9 10-03
–
3.3 9 10-02
1.3 9 10-05
2.1 9 10-04
–
–
-03
–
-02
3.3 9 10
1.3 9 10
-05
2.1 9 10-04
–
–
BR:ALRil1:07
ToYSV
2201
2661
3.9 9 10
BR:PEGoi1:10
ToYSV
2201
2661
3.9 9 10-03
–
3.3 9 10-02
1.3 9 10-05
2.1 9 10-04
–
–
BR:PBAlh1:10
BR:ALPar1:09
17
1584
2.1 9 10-22
6.1 9 10-12
2.6 9 10-13
1.7 9 10-19
2.1 9 10-12
4.6 9 10-25
6.1 9 10-24
2112
2660
3.9 9 10-03
3.3 9 10-02
1.3 9 10-05
2.1 9 10-04
–
-
ToYSV a
When only the major parent is indicated, the minor parent has not been identified. ToYSV, tomato yellow spot virus
b
Numbering starts at the first nucleotide after the cleavage site at the origin of replication and increases clockwise
c
R, RDP; G, GeneConv; B, Bootscan; M, MaxChi; C, CHIMAERA; S, SisScan; 3S, 3SEQ
d
–, no recombination event detected
DNA-A sequence identity for begomovirus species, and 94% for their strains [9]. The sequence of clone BR:ALAta1:07 from Atalaia (AL) showed 91-94% identity to those of other ClLCrV isolates, indicating that this isolate represents a distinct strain, hereby named ClLCrV-Ata. A striking feature of the ClLCrV isolate from Mato Grosso do Sul was its association with an alphasatellite molecule (cleome leaf crumple virus-associated DNA1), one of two recent reports of DNA satellites in association with begomoviruses in the New World [28, 33]. A careful examination of the RCA products obtained from our C. affinis samples after digestion with 4-base cutter restriction
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enzymes failed to indicate the presence of alphasatellites or of any other kind of DNA satellite. Phylogenetic analysis based on Brazilian and American begomoviruses placed ClLCrV in a basal group relative to all Brazilian begomoviruses, suggestive of an ancestral origin for this virus. It is noteworthy that phylogenetic inconsistency among the ClLCrV DNA-A and DNA-B components lead Paprotka et al. [28] to suggest that an ancient pseudorecombination event could be involved in the origin of this virus. We found evidence of multiple recombination events among the ClLCrV isolates. Recombination signals were
Begomoviruses infecting Cleome affinis in Brazil
2211
Table 3 Genetic variability of the cleome leaf crumple virus (ClLCrV) population obtained from C. affinis samples collected in five states of northeastern Brazil Population
N
Genome size
sc
Etad
ClLCrV
14
2756
253
267
MaYSV
a
ke
pf
hg
Hdh
hwi
h-Etaj
51.758
0.0191
14
1.0
0.0294
0.0311 0.0542
12
2658
402
419
150.177
0.0572
12
1.0
0.0537
ToCmMVb
22
2560
103
104
36.645
0.0143
20
0.987
0.0110
0.0111
ToYVSVb
26
2562
49
49
5.381
0.0021
25
0.997
0.0050
0.0050
a
Silva et al. [39]
b
Castillo-Urquiza et al. [3]
c
Total number of segregating sites
d
Total number of mutations
e
Average number of nucleotide differences between sequences (Tajima’s estimate of the population mutation rate, h)
f
Nucleotide diversity
g
Haplotype number
h
Haplotype diversity
i
Watterson’s estimate of the population mutation rate based on the total number of segregating sites
j
Watterson’s estimate of the population mutation rate based on the total number of mutations
Table 4 Results of the different neutrality tests for each open reading frame (ORF) in the DNA-A of isolates of cleome leaf crumple virus (ClLCrV) obtained from Cleome affinis samples collected in five states of northeastern Brazil ORFa
Tajima’s D
Fu and Li’s D*
Fu and Li’s F*
dN/dS 0.0228
Rep
-1.8653*
-2. 5503**
-2.71489*
Trap
-0.4477 (ns)b
-1.4769 (ns)
-1.3752 (ns)
0.0887
Ren
-1.3826 (ns)
-1.8803 (ns)
-1.9997 (ns)
0.3171
CP
-0.0972 (ns)
-1.0095 (ns)
-0.8731 (ns)
0.2124
* Significant at p \ 0.05 ** Significant at p \ 0.02 a
Rep, Replication-associated protein; Trap, Tans-activating protein; Ren, Replication enhancer protein; CP, Coat protein
b
ns, not significant at p [ 0.10
particularly strong for clones BR:AL-Ata1:07 and BR:PBAlh1:10, which always clustered separately of the other ClLCrV isolates in phylogenetic trees. Recombination breakpoints were identified primarily in the Rep coding region, a known hot spot for recombination among geminiviruses [24, 25]. It is interesting, though, that ClLCrV seems to be restricted to C. affinis, and also seems to be the only begomovirus associated with this host. Furthermore, it is not known if ToYSV, identified as one of the parents in the recombination event, is capable of infecting C. affinis. Parent identification in recombination analysis is obviously limited by the dataset used, and it is possible that the true viruses involved in these recombination events are either ancestral viruses that no longer exist or unknown viruses infecting distinct, unidentified hosts. Therefore, despite recombination frequently resulting in local adaptation, it is tempting to conclude that, at least in this specific virus-host system, it seems to be acting on the viral population without an obvious effect on its evolution. However, additional studies such as the biological characterization of
isolates from the two strains of ClLCrV (including their host ranges), should be carried out to test this hypothesis. In contrast to the low diversity of viruses found infecting C. affinis, the ClLCrV population displays a high degree of genetic variability, which is highlighted by the presence of unique haplotypes and high rates of nucleotide diversity and mutation (besides the aforementioned recombination events). These values were considerably higher than those observed for two populations of tomato-infecting begomoviruses from southeastern Brazil [3] and were similar to those observed for a MaYSV population obtained from Macroptilium lathyroides samples collected in northeastern Brazil [39]. Although additional studies are necessary to confirm this relationship, this presumptive higher genetic variability of weed-infecting begomoviruses compared to crop-infecting viruses suggests either that a longer co-evolution time leads to greater variability, or that viruses infecting hosts with a wider genetic base are allowed to diversify to a greater extent. Either way, it is reasonable to propose that weeds can act not only as virus reservoirs, but
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also as reservoirs of genetic variability, from which novel viruses can emerge following a host switch. On the other hand, the recombination analysis shows mostly inward recombination (other viruses contributing sequences to ClLCrV) instead of outward recombination (ClLCrV contributing sequences to other viruses). This could actually be constructed as a counterargument to C. affinis being a reservoir of crop viruses or contributing to crop virus diversity. Instead, it suggests that these viruses may be genetically isolated, as it has been reported for legume begomoviruses in Asia [29]. As mentioned above, ClLCrV seems to be restricted to C. affinis, which is further indication of a genetic isolation. However, a larger number of samples must be analyzed to confirm this observation. Results of several neutrality tests indicated that the Rep ORF is potentially under purifying selection. As Rep encodes the only protein essential for begomovirus replication, purifying selection may be acting to preserve its function. Purifying selection and population expansion were concluded to be the major evolutionary forces acting on a begomovirus in Eupatorium makinoi [43], on ToYVSV and ToCmMV in tomato [3], and on the tospovirus tomato spotted wilt virus (TSWV) in peanut [23]. In summary, our results indicate a low species diversity of begomoviruses infecting C. affinis, but the single species detected seems to be ancestral to Brazilian begomoviruses, has a recombinant nature, and displays a high degree of genetic variability. Whether C. affinis is a source of novel (recombinant) viruses for crop plants or a host of a genetically isolated begomovirus remains to be determined. Further studies on the biological properties of ClLCrV (including its host range and vector transmission properties) as well as its ability to form viable pseudorecombinants with other begomoviruses should help to elucidate the role of this pathosystem in the ever increasingly complex epidemiological scenario of Brazilian begomoviruses.
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14. Acknowledgments The authors wish to thank Poliane AlfenasZerbini for critical review of the manuscript. This work was carried out under the framework of a CAPES PROCAD-NF (no. 93-2008) collaborative project among UFAL, UFRPE and UFV, and was additionally funded by FAPEMIG grants CAG-666-08 and CAG-949-09 to FMZ. GPCU was the recipient of a CAPES-PNPD postdoctoral fellowship.
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