Received for publication: 15 Abril, 2013. Accepted for publication: 5 June, 2013. 1 Plantae Research Group, Department of Biology, Faculty of Basic Sciences, Universidad Francisco de Paula Santander. Cucuta (Colombia). gchavesbe@gmail.com 2 Plant Virus Laboratory, Institute of Biotechnology, Universidad Nacional de Colombia. Bogota (Colombia). 3 Department of Basic Sciences, Universidad de los Llanos. Villavicencio (Colombia). Agronomía Colombiana 31(2), 161-168, 2013 Genetic structure and evidence of putative Darwinian diversifying selection in the Potato yellow vein virus (PYVV) Estructura genética y evidencia de una posible selección darwiniana diversificadora en el virus del amarillamiento de las venas de la papa (PYVV) Giovanni Chaves-Bedoya1, Mónica Guzmán-Barney2, and Luz Yineth Ortíz-Rojas3 ABSTRACT RESUMEN The population structure and genetic variation of Potato yellow vein virus (PYVV) were estimated by analysis of the nucleo- tide and deduced amino acid sequence of the coat protein of 69 isolates, reported in GenBank, from Solanum tuberosum (ST) and Solanum phureja (SP) hosts from different regions; predominantly Cundinamarca, Antioquia and Nariño, located in central and southwestern Colombia. Bioinformatics analysis revealed that despite the wide geographic distribution of dif- ferent hosts and different collecting years, PYVV maintains a genetic similarity between 97.1 to 100.0%, indicating high spatial and temporal genetic stability of the major coat protein. No recombination events were found, but evidence was seen for the first time that this protein could be undergoing Darwinian diversifying selection. En este trabajo se estimó la estructura poblacional y variación genética del virus del amarillamiento de las venas de la papa (PYVV) infectando cultivos en Colombia por medio del análisis de 69 secuencias nucleotídicas y aminoácidos deducidos de la proteína mayor de la cápside (CP) reportados en el banco de genes (GenBank). Los aislamientos de PYVV fueron obtenidos de los hospederos Solanum tuberosum (ST) y Solanum phureja (SP) en diferentes regiones de Colombia, predominantemente los departamentos de Cundinamarca, Antioquia y Nariño localizados en la región Central y Sur Oeste del país respec- tivamente. El análisis bioinformático reveló que a pesar de la amplia distribución geográfica de los hospederos y diferentes años de colecta, PYVV mantiene un similitud genética entre 97,1 y 100,0% indicando una gran estabilidad genética espacial y temporal en la CP. En este estudio no se detectaron eventos de recombinación, pero se presenta evidencia por primera vez de que esta proteína podría estar bajo selección darwiniana diversificadora. Key words: Solanum tuberosum, v ira l proteins, genetic variability, Crinivirus. Palabras clave: Solanum tuberosum, proteinas v ira les, variabilidad genética, Crinivirus. is semi-persistent, transmitted by the greenhouse whitefly (Trialeurodes vaporariorum, Westwood) vector (Livieratos et al., 2004). PYVV is the causal agent of the potato yel- low vein disease (PYVD) which reduces the production in number of tubers by over 50% in the Solanum tuberosum group Andigena (Livieratos et al., 2004). A characteristic of RNA viruses is that they have high genetic variability, due to the ability to generate large populations and the lack of a proof reading activity of RNA polymerase (Domingo and Holland, 1997). However, there are other factors that influence genetic diversity, such as genetic recombination, genomic rearrangement, genetic drift and natural selection (Garcia-Arenal et al., 2001). Genetic diversity among different viruses varies according Introduction Worldwide, the potato is considered the fourth most important crop, after rice, wheat and maize. The potato can be infected by different viruses, including the Potato yellow vein virus (PYVV) which reduces the yield and quality of tubers (Salazar et al., 2000). PYVV has affected potato crops in Colombia for more than 50 years and has spread to neighboring countries in the Andean region. PYVV is classified as a tentative species of the Crinivirus genus in the Closteroviridae family. PYVV has a tripartite single strand and positive sense RNA genome. Virions are flexible, located in the phloem of the plant. The genome sequence indicates that the CP protein of PYVV consists of 756 nucleotides (252 aa) (Salazar et al., 2000). The virus 162 Agron. Colomb. 31(2) 2013 to factors such as the virus–vector relationship, host range or geographic incidence. Characterization studies of ge- netic variability are of practical interest for the control of viruses; and strategies based on monogenic resistance are influenced by genetic variation of the pathogen (Vives et al., 2002). Sequence analyses show that, in most instances, the selec- tion acting on virus genes is negative. The degree of selec- tion can be determinated from the ratio between nucleotide diversities at non-synonymous and synonymous positions (dN/dS). This ratio indicates the amount of variation in the nucleotides that results in variation in the encoded protein. Virus encoded proteins are not less constrained than those of their eukaryotic hosts and vectors, which suggests that the need to establish functional interactions with host and vector encoded factors is constraining the variability of virus encoded proteins (Garcia-Arenal et al., 2003). In virus genes, negative and positive selection may be acting in particular domains of the viral proteins, and are evidenced by detailed analyses of the encoding sequence. Poitive selection acts with resistance-breaking isolates (Garcia-Arenal et al., 2003). Sequence analysis, in silico, of nucleotide or amino acids al- lowsfor the determination of possible phylogenetic relation- ships and similarities or differences among viral isolates, as has been reported for various species of viruses that infect plants using public sequences reported in the GenBank (Ge et al., 2007; Marco and Aranda, 2005; Martin et al., 2006; Rangel et al., 2011). Since there is currently no genetic variability or Darwinian selection analysis of PYVV using the CP sequences reported from different potato producing regions in Colombia, the aim of this study was to identify genetic variation in PYVV isolates infecting the potato in producing regions and to determine the presence of posi- tive selection as an evolutionary force in PYVV based on 69 nucleotide sequences reported from different regions of Colombia. The results obtained for a region of 586 nucleotides within the coat protein gene covering a region encoding 195 aminoacids indicates low genetic variability, confirming previous studies (Guzmán et al., 2006; Offei et al., 2004; Rodriguez et al., 2010). Nevertheless, for the first time, we present evidence of positive Darwinian selection in two amino acids of the CP, suggesting this virus could be looking for change or speciation strategies. Materials and methods Origin of sequences The nucleotide analysis was carried out with a total of 69 sequences of the coat protein (CP) of PYVV, which were obtained from the public database GenBank. The 68 sequences were obtained from major potato producing departments in Colombia; namely Antioquia, Boyaca, Cauca, Cundinamarca and Nariño. One sequence repor- ted in Cajamarca, Peru (Livieratos et al., 2004) (GenBank AJ557129) was also used. Origin, year of collection and hosts are listed in Tab.1. Sequence edition for analysis Since the nucleotide sequences of PYVV ś CP reported in GenBank have different lengths, for the analysis, all sequences were aligned using the ClustalW program im- plemented in the program MegAlign™ (DNAStar, Madi- son, WI) package for sequence analysis, version 7.2.2 and were adjusted to a length of 586 nucleotides flanked by the highly conserved amino acid sequences KDDSYNLDL and DLTANYLFK (Fig. 2). The CP sequences of PYVV starting TABLE 1. Identification of sampled Colombian PYVV isolates. Accesion number Location Place Host Colection year 1 HQ620554 Nariño Ipiales - Suras NA 2010? 2 HQ620553 Nariño Ipiales - Saguaran NA 2010? 3 HQ620552 Nariño Pasto - Obonuco NA 2010? 4 HQ620551 Nariño Pasto - La Victoria NA 2010? 5 HQ620550 Antioquia La Union - Buena Vista NA 2010? 6 HQ620549 Antioquia La Union -El Vergel NA 2010? 7 HQ620548 Cundinamarca Facatativa NA 2010? 8 HQ620547 Antioquia Carmen del Viboral NA 2010? 9 HQ620546 Antioquia Santuario - El Carmen NA 2010? 10 HQ620545 Antioquia Sonson NA 2010? 11 JF718318 Cundinamarca Chipaque S. tuberosum 2008 12 JF718317 Cundinamarca Chipaque S. tuberosum 2008 13 JF718316 Cundinamarca Sibate S. tuberosum 2008 14 JF718315 Cundinamarca Sibate S. tuberosum 2008 Continues 163Chaves-Bedoya, Guzmán-Barney, and Ortíz-Rojas: Genetic structure and evidence of putative Darwinian diversifying selection in the Potato yellow vein virus (PYVV) TABLE 1. Identification of sampled Colombian PYVV isolates. Location, place, host and collection date are indicated (continued). Accesion number Location Place Host Colection year 15 FJ718314 Cundinamarca Sibate S. tuberosum 2008 16 JF718313 Cundinamarca Sibate S. tuberosum 2008 17 JF718312 Cundinamarca Sibate S. tuberosum 2008 18 JF718311 Cundinamarca Sibate S. tuberosum 2008 19 JF718310 Cundinamarca Chipaque S. phureja 2008 20 JF718309 Cundinamarca ? S. tuberosum 2008 21 JF718308 Cundinamarca ? S. tuberosum 2008 22 JF718307 Cundinamarca ? S. tuberosum 2008 23 JF718306 Cundinamarca ? S. tuberosum 2008 24 JF718305 Antioquia Marinilla S. phureja 2008 25 JF718304 Antioquia Marinilla S. phureja 2008 26 JF718303 Antioquia Marinilla S. phureja 2008 27 JF718302 Antioquia Marinilla S. phureja 2008 28 JF718301 Antioquia Marinilla S. phureja 2008 29 JF718300 Cundinamarca ? S. tuberosum 2008 30 JF718299 Cundinamarca ? S. tuberosum 2008 31 JF718298 Cundinamarca ? S. tuberosum 2008 32 JF718297 Cundinamarca ? S. tuberosum 2008 33 JF718296 Cundinamarca ? S. tuberosum 2008 34 JF718295 Nariño Puerres S. phureja 2008 35 JF718294 Nariño Puerres S. phureja 2008 36 JF718293 Nariño Puerres S. phureja 2008 37 JF718292 Nariño Puerres S. phureja 2008 38 JF718291 Nariño Puerres S. phureja 2008 39 JF718290 Cundinamarca ? S. tuberosum 2008 40 JF718289 Cundinamarca ? S. tuberosum 2008 41 JF718288 Cundinamarca ? S. tuberosum 2008 42 JF718287 Cundinamarca Chipaque S. phureja 2008 43 JF718286 Cundinamarca Chipaque S. phureja 2008 44 JF718285 Cundinamarca Chipaque S. phureja 2008 45 JF718284 Cundinamarca Chipaque S. phureja 2008 46 GQ344830 Cauca San Sebastian S. phureja 2008 47 GQ397987 Antioquia Sonson S. phureja 2008 48 GQ397986 Antioquia La Union S. tuberosum 2008 49 GQ397985 Antioquia ? S. tuberosum 2008 50 GQ397984 Nariño Pupiales S. phureja 2008 51 GQ397983 Nariño Pupiales S. tuberosum 2008 52 GQ397982 Antioquia Marinilla S. phureja 2008 53 GQ397981 Antioquia Marinilla S. phureja 2008 54 GQ397980 Antioquia Sonson S. phureja 2008 55 GQ397979 Antioquia Sonson S. phureja 2008 56 GQ397978 Boyaca Tunja S. tuberosum 2008 57 GQ397977 Boyaca Tunja S. tuberosum 2008 58 GQ397976 Cundinamarca ? S. phureja 2008 59 GQ397975 Cundinamarca ? S. chaucha 2008 60 GQ397974 Cundinamarca ? Solanum. sp 2008 61 GQ397973 Cauca ? S. phureja 2008 62 GQ397972 Cauca ? S. phureja 2008 63 AJ560291 Cundinamarca ? S. tuberosum 2003? 64 AJ586117 Cundinamarca ? S. phureja 2003? 65 AJ586116 Cundinamarca ? S. tuberosum 2003? 66 AJ586115 Cundinamarca ? S. tuberosum 2003? 67 AJ586114 Cundinamarca ? S. phureja 2003? 68 AJ586113 Cundinamarca ? S. tuberosum 2003? 164 Agron. Colomb. 31(2) 2013 with GenBank code HQ (i.e., HQ620554) were adjusted from nucleotide position 86 to 668. Sequences starting with code JF (i.e., JF718316) were adjusted from nucleotide position 109 to 696. Sequences starting with code GQ (i.e., GQ397987) were adjusted from nucleotide position 1 to 588 and sequences starting with code AJ (i.e., 586113) were adjusted from nucleotide position 76 to 661. We assumeda high qualityfor the sequences deposited in GenBank, which were generated by third parties. Phylogenetic analysis The phylogenetic relationship of the nucleotide sequences was inferred by the Neighbor-Joining method (Saitou and Nei, 1987). Evolutionary distances were calculated using the Kimura 2-parameter method (Kimura, 1980), using 1000 replications to estimate the confidence of the taxon grouping in tree branches (Felsenstein, 1985). All posi- tions containing gaps and missing data were eliminated. The evolutionary analysis was performed in the program MEGA 5 (Tamura et al., 2011). Recombination and selection The search for putative recombination events was done using the genetic algorithm for recombination detection (GARD) (Kosakovsky Pond et al., 2006). The nucleotide- substitution model was selected automatically before being applied to the site-recombination analysis. The search for amino acids undergoing selection was performed using the algorithms FEL (fixed effects likelihood), REL (random effects likelihood), (Kosakovsky Pond and Frost, 2005) and MEME (mixed effects model of evolution) implemented in the HyPhy program (hypothesis testing using phylogenies) (Pond and Frost, 2005) in the datamonkey server (Delport et al., 2010). This method allows for the identification of codons undergoing positive selection and removes the as- sumptions about the demographics associated with other statistical selection tests (Cavatorta et al., 2008). Results and discussion Nucleotide similarity Unlike previous reports (Guzmán et al., 2006; Offei et al., 2004), this is the first study that analyzed the genetic variability of the CP of PYVV using 68 sequences from different geographical regions of Colombia, hosts and different years of sampling. All sequences are available in GenBank (Tab. 1). Nucleotide similarity among the CP ranged from 97.1 to 100.0%, with 97.3% being the most frequent value, as indicated by the graph of frequency obtained from 2,278 nucleotide paired comparisons (data not shown). The results indicate that Colombian PYVV isolates exhibit high genetic stability over time and among different departments and years. Several collected PYVV isolates, either in different years or departments, had 100% nucleotide similarity (Tab. 2). In an attempt to discriminate potential PYVV genetic groups circulating in Colombia, we built a phylogenetic tree using 32 PYVV haplotypes that were deducted in the pro- gram SNAP (Price and Carbone, 2005) . The phylogenetic tree of PYVV haplotypes (Fig. 1) does not show evidence of genetic groups, indicating that PYVV in Colombia is TABLE 2. Identification of PYVV isolates reported in different years and/or locations with 100% of similarity. NA, not available. Accesion Department Location Year Accesion Department Location Year HQ620548 Cundinamarca Facatativa 2010 = HQ620550 Antioquia La Union - Buena Vista 2010 JF718311 Cundinamarca Sibate 2008 = HQ620549 Antioquia La Union - El Vergel 2010 JF718296 Cundinamarca NA 2008 = HQ620553 Nariño Saguaran 2010 JF718289 Cundinamarca NA 2008 = HQ620549 Antioquia La Union - El Vergel 2011 JF718288 Cundinamarca NA 2008 = HQ620549 Antioquia La Union - El Vergel 2011 GQ397986 Antioquia La Union 2006 = HQ620550 Antioquia La Union 2011 GQ397985 Antioquia NA 2006 = HQ620547 Antioquia Carmen del Viboral 2011 GQ397982 Antioquia Marinilla 2006 = HQ620547 Antioquia Carmen del Viboral 2011 GQ397981 Antioquia Marinilla 2006 = JF718298 Cundinamarca NA 2008 GQ397980 Antioquia Sonson 2006 = GQ397987 Antioquia Sonson 2006 GQ397978 Boyaca Tunja 2006 = JF718304 Antioquia Marinilla 2008 GQ397977 Boyaca Tunja 2006 = HQ620553 Nariño Ipiales 2011 GQ397975 Cundinamarca NA 2006 = GQ397976 Cundinamarca NA 2006 GQ397974 Cundinamarca NA 2006 = GQ397987 Antioquia Sonson 2006 GQ397972 Cauca NA 2006 = HQ620549 Antioquia La Union - El Vergel 2011 AJ586117 Cundinamarca NA 2004 = HQ620550 Antioquia La Union - Buena Vista 2011 AJ586113 Cundinamarca NA 2004 = HQ620549 Antioquia La Union - El Vergel 2010 AJ586115 Cundinamarca NA 2004 = AJ560291 Cundinamarca NA 2010 AJ586116 Cundinamarca NA 2004 = AJ560291 Cundinamarca NA 2010 165Chaves-Bedoya, Guzmán-Barney, and Ortíz-Rojas: Genetic structure and evidence of putative Darwinian diversifying selection in the Potato yellow vein virus (PYVV) FIGURE 1. Phylogenetic tree with 32 deduced haplotypes of PYVV’s CPs. Solanum tuberosum (ST), Solanum phureja (SP), NA not available. Cucur- bit chlorotic yellows virus (CCYV) was used as an out-group. Collection year and precedence are indicated. JF718314_ST_08 JF718312_ST_08 JF718310_SP_08 JF718316_ST_08 JF718290_ST_08 JF718289_ST_08 CUND JF718301_SP_08 JF718303_SP_08 JF718305_SP_08 JF718304_SP_08 ANT ANTJF718302_SP_08 CCAGQ344830_SP_08 ANTHQ620550_NA_10 PERUAJ557129 JF718298_ST_08 JF718300_ST_08 JF718299_ST_08 CUND CUNDAJ560291_ST_03 NARHQ620553_NA_10 CUNDJF718297_ST_08 JF718295_SP_08 JF718294_SP_08 HQ620554_NA_10 JF718292_SP_08 HQ620551_NA_10 GQ397984_SP_08 NAR CUNDJF718309_ST_08 CUNDJF718317_ST_08 CUNDJF718318_ST_08 CUNDJF718307_ST_08 ANTGQ397987_SP_08 CUNDJF718308_ST_08 CCYV 34 21 47 36 23 85 80 29 63 54 19 43 26 30 42 36 40 34 36 0.05 very homogeneous without clear genetic groups according to geographical precedence, host or year. More Colombian PYVV sequences are needed in order to better estimate phylogenetic inferences. Haplotypes and related isolates are listed in Tab. 3. The most common PYVV haplotype in Colombia is identified as H28, which includes isolates obtained from different po- tato species sampled in the departments of Cundinamarca, Antioquia and Cauca. Geographically, the departments of Antioquia and Cundinamarca are adjacent to one another in the central region of Colombia and the Cauca depart- ment is located in the southwest of the country (Fig. 1). High nucleotide similarity values between PYVV isolates suggest: first, a high spatial and temporal genetic stability and second, the possible movement between departments of tubers infected with the virus. Indeed, the presence of 166 Agron. Colomb. 31(2) 2013 identical PYVV haplotypes infecting potato crops in dif- ferent departments of Colombia indicates that there is an urgent need to improve the quality of potato tubers with a certified seed production program. It is also important to regulate and limit the transport of seeds between de- partments and borders, because PYVV is a virus that can be transmitted through tubers (Salazar et al., 2000). The transfer of potato tubers between departments is a practice that is often used among Colombian farmers, but the detec- tion of the virus by visual analysis is not possible because the infected tubers have no apparent symptoms, making it difficult to predict whether or not they have the virus. Control of viral spread remains the most efficient method to reduce viral diseases in potato seed production. Within the family Closteroviridae, other viruses have been reported with low CP genetic diversity from geographically distant isolates (Alicai et al., 1999; Rubio et al., 2001a; Rubio et al., 2001b; Rubio et al., 1999). Several references point- ing to similar results indicate that low heterogeneity is the norm for viruses in the genus Crinivirus. Selection analysis Nucleotide or amino acid selection can be exercised to maintain the primary, secondary or tertiary structural characteristics in the viral genome that are important for replication, such as the 3 ńon coding genomic regions of the single strand RNA virus. Another group of selection factors is associated with the host plant. The differentiation of natural populations according to the host plant can also be taken as evidence of host-associated selection (Garcia- Arenal et al., 2001). Most positive-strand plant RNA viruses are adaptedtoinfection of plant hosts. The comparison of genetic maps of representative viruses has revealed genes in plant viral genomes that appear to be essential adaptations needed for success fulinvasion and dispersal throughout their plant hosts. (Goldbach et al., 1994). A non-synonymous substitution rate (dN) significantly higher than the percentage of synonymous substitution (dS), or ω = dN/dS greater than 1, points to positive Darwin- ian selection (Delport et al., 2009). Since the selection acting on viral genes is negative in most cases, (Garcia-Arenal et al., 2003), and positive selection is less frequent (Gojobori et al., 1990), we focused on positively selected codons. The prior selection analysis was searched for possible recombination events, because it may contribute to a false inference of positive selection (Scheffler et al., 2006). With no evidence of recombination, the nucleotide segment was not subdivided for further selection analysis (Scheffler et al., 2006). For the CP of PYVV, three different algorithms, designed to detect selection in a particular codon, coincided in suggesting that codon 205 is undergoing diversifying selection (Fig. 2). Positive selection in codon 147 is supported only by the REL algorithm. The amino acid at position 205 in the majority of the isolates corresponded to a phenylalanine, except for isolates JF718317 (ST) from Cundinamarca, JF718292 (SP) from Nariño and AJ557129 (ST) from Peru, which codified for a serine. The dN/dS ratio of CP sequences analyzed in the MEGA program, where dN is the ratio of non-synonymous substi- tutions and dS is the ratio of synonymous substitutions, is 0.19. This value is in the range for viruses that infect plants TABLE 3. Deduced PYVV haplotypes. Haplotype Isolates H1 JF718307, JF718306 H2 CCYV H3 JF718309 H4 JF718300 H5 JF718317 H6 JF718299 H7 HQ620547, HQ620546, GQ397985, GQ397982, AJ557129 H8 JF718298, GQ397981 H9 JF718294 H10 HQ620551, GQ397983, GQ397979 H11 GQ344830 H12 GQ397984 H13 JF718292 H14 JF718303 H15 JF718305 H16 JF718310, JF718287, JF718286, JF718285, JF718284 H17 JF718304, GQ397978 H18 JF718301 H19 JF718302 H20 AJ560291, AJ586116, AJ586115 H21 JF718312 H22 HQ620553, HQ620552, JF718296, GQ397977 H23 JF718297 H24 JF718316 H25 HQ620550, HQ620548, GQ397986, AJ586117, HQ620545, GQ397976, GQ397975 H26 JF718290 H27 JF718314 H28 JF718289, JF718311, AJ586113, HQ620549, GQ397972, JF718288, AJ586114, GQ397973, JF718313, JF718315 H29 HQ620554 H30 JF718295, JF718293, JF718291 H31 JF718308 H32 JF718318 H33 GQ397987, GQ397980, GQ397974 167Chaves-Bedoya, Guzmán-Barney, and Ortíz-Rojas: Genetic structure and evidence of putative Darwinian diversifying selection in the Potato yellow vein virus (PYVV) (Garcia-Arenal et al., 2001). dN/dS = 0.19 indicates a nega- tive selection pressure for the amino acid change in this genomic region. However, the results of selection pressure which fall in the average dN/dS range of a region of interest may have a poor statistical power to detect positive selec- tion because only a few sites may be undergoing selection (Kosakovsky Pond and Frost, 2005). In a site to site search for selection, algorithms FEL (P-value 0.030), REL (Bayes Factor 279 379) and MEME (P-value 0.044) indicated that there is enough statistical evidence to suggest that codon 169 of the alignment is undergoing positive selection (Fig. 2). Unlike FEL and MEME, REL analysis further suggested that codon 111 may also be undergoing positive selection (Tab. 4). Isolates that have amino acid changes at two positions un- dergoing putative diversifying selection included JF718292 (SP), JF718317 (ST) and AJ557129 (ST), the latter reported in Peru. For Colombian PYVV isolates, at position 147 (111), there was a change of isoleucine to valine due to the transition of GTT → ATT. However, in this position, 24 other isolates presented a valine. For position 205 (169), only isolates JF718292, JF718 317 and AJ557129 had the amino acid change of phenylalanine to a serine, due to the transition of TTC → TCC. Phenylalanine has been reported as an important amino acid in the differentiation of viral strains in Citrus tristeza virus (CTV) at position 124 of the CP. This aminoacid causes the epitope responsible for the reaction with an- tibody MCA 13, which differentiates between severe and soft strains of CTV with 95% confidence (Pappu et al., 1993; Permar et al., 1990). Our in silico results could be coincidental, and the amino acid variation of serine or phenylalanine could indicate a change in epitope recogni- tion for PYVV. However, this is a hypothesis that should be demonstrated because in this virus, there are no reports on strains expressing different symptoms. Amino acids identi- fied as undergoing positive selection in PYVV could be in a region of interaction with proteins of the vector and positive selection reflects gain or loss of affinity for the interaction with the vector, since no correlation between the encoded amino acid and the host was found. Conclusions The low heterogeneity found in PYVV is possibly due to its rapid expansion as a result of whitefly population growth and expansion, or similar selection pressures in the differ- ent species of Solanum. The proposed functions of the positively selected ami- noacids in the CP of PYVV are speculative. We are ex- trapolating the function of another two suggested amino acids undergoing positive selection in an unrelated gene of virus species belonging to a different genus in the fam- ily Closteroviridae. Not much can be concluded from the small number of amino acid variants without a biological relationship. Although this does suggest selection pressure, one cannot guess what the selection would be without an obvious biological trait. For now, this change only suggests some limited variation among PYVV isolates. Further FIGURE 2. Section of PYVV’s CP analyzed. The alignment of the amino acid sequence from position 36 (1) to position 231 (195) is indicated. The sequence of 9 amino acids at the beginning and end of the target region is conserved in all sequences. Arrows indicate the approximate position of codons 147 (111) and 205 (169) with reference to the ab- solute position and (analyzed region) may be undergoing positive selec- tion according to algorithm 1 (*) and algorithms 3 (**). TABLE 4. Amino acid positions undergoing positive selection in the CP of PYVV detected with at least one algorithm. FEL REL MEME PYVV CP segment Codon dN-dS P -value dN-dS Posterior probability Bayes factor P-value aa36 (1) – aa231 (195) 147 (111) - - 3.85363 0.982815 127.193 - 205 (169) 79.5713 0.0309 4.06015 0.9921 279.379 0.04498 Anayzed region (Position in aminoacids) * Absolute position PYVV CP PROTEIN 168 Agron. Colomb. 31(2) 2013 studies must be carried out to determinate the biological significance of this variation. Literature cited Alicai, T., N.S. Fenby, R.W. Gibson, E. Adipala, H.J. Vetten, G.D. Foster, and S.E. Seal. 1999. Occurrence of two serotypes of sweet potato chlorotic stunt virus in East Africa and their associated dierences in coat protein and HSP70 homologue gene sequences. Plant Pathol. 48, 718-726. Cavatorta, J.R., A.E. Savage, I. Yeam, S.M. Gray, and M.M. Jahn. 2008. Positive Darwinian selection at single amino acid sites conferring plant virus resistance. J. Mol. Evol. 67, 551-559. Delport, W., A.F. Poon, S.D. Frost, and S.L. Kosakovsky Pond. 2010. Datamonkey 2010: a suite of phylogenetic analysis tools for evolutionary biology. Bioinformatics 26, 2455-2457. Delport, W., K. Scheffler, and C. Seoighe. 2009. Models of coding sequence evolution. Brief. Bioinform. 10, 97-109. Domingo, E. and J.J. Holland. 1997. RNA virus mutations and fitness for survival. Annu. Rev. Microbiol. 51, 151-178. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783-791. Garcia-Arenal, F., A. Fraile, and J.M. Malpica. 2001. Variability and genetic structure of plant virus populations. Annu. Rev. Phytopathol. 39, 157-186. Garcia-Arenal, F., A. Fraile, and J.M. Malpica. 2003. Variation and evolution of plant virus populations. Int. Microbiol. 6, 225-232. Ge, L., J. Zhang, X. Zhou, and H. Li. 2007. Genetic structure and population variability of tomato yellow leaf curl China virus. J. Virol. 81, 5902-5907. Gojobori, T., E.N. Moriyama, and M. Kimura. 1990. Molecular clock of viral evolution, and the neutral theory. Proc. Natl. Acad. Sci. USA 87, 10015-10018. Goldbach, R., J. Wellink, J.Verver, A. Van Kammen, D. Kasteel, and J. Van Lent. 1994. Adaptation of positive-strand RNA viruses to plants. Arch. Virol. Suppl. 9, 87-97. Guzmán, M., E. Ruiz, N. Arciniegas, and R.H. Coutts. 2006. Oc- currence and variability of Potato yellow vein virus in three departments of Colombia. J.Phytopatol. 154, 748-750. Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111-120. Kosakovsky Pond, S.L. and S.D. Frost. 2005. Not so different after all: a comparison of methods for detecting amino acid sites under selection. Mol. Biol. Evol. 22, 1208-1222. Kosakovsky Pond, S.L., D. Posada, M.B. Gravenor, C.H. Woelk, and S.D. Frost. 2006. Automated phylogenetic detection of recombination using a genetic algorithm. Mol. Biol. Evol. 23, 1891-1901. Livieratos, I.C., E. Eliasco, G. Muller, R.C. Olsthoorn, L.F. Salazar, C.W. Pleij, and R.H. Coutts. 2004. Analysis of the RNA of Potato yellow vein virus: evidence for a tripartite genome and conserved 3’-terminal structures among members of the genus Crinivirus. J. Gen. Virol. 85, 2065-2075. Marco, C.F. and M.A. Aranda. 2005. Genetic diversity of a natural population of Cucurbit yellow stunting disorder virus. J. Gen. Virol. 86, 815-822. Martin, S., M.L. Garcia, A. Troisi, L. Rubio, G. Legarreta, O. Grau, D. Alioto, P. Moreno, and J. Guerri. 2006. Genetic variation of populations of Citrus psorosis virus. J. Gen. Virol. 87, 3097-3102. Offei, S.K., N. Arciniegas, G. Muller, M. Guzman, L.F. Salazar, and R.H. Coutts. 2004. Molecular variation of Potato yellow vein virus isolates. Arch. Virol. 149, 821-827. Pappu, H., S. Pappu, C. Niblett, R. Lee, and E. Civerolo. 1993. Com- parative sequence analysis of the coat proteins of biologically distinct citrus tristeza closterovirus isolates. Virus Genes 7, 255-264. Permar, T.A., S.M. Gransey, D.J. Gumpf, and R.F. Lee. 1990. A monoclonal antibody wich discriminates strains of Citrus tristeza virus. Phytopathology 80, 224-228. Pond, S.L., and S.D. Frost. 2005. Datamonkey: rapid detection of selective pressure on individual sites of codon alignments. Bioinformatics 21, 2531-2533. Price, E.W. and I. Carbone. 2005. SNAP: workbench management tool for evolutionary population genetic analysis. Bioinformat- ics 21, 402-404. Rangel, E.A., A. Alfaro-Fernandez, M.I. Font-San-Ambrosio, M. Luis-Arteaga, and L. Rubio. 2011. Genetic variability and evo- lutionary analyses of the coat protein gene of Tomato mosaic virus. Virus Genes 43, 435-438. Rodriguez, P., G. Chaves-Bedoya, L. Franco-Lara, and M. Guzmán. 2010. Low molecular variability of Potato yellow vein virus (PYVV) isolates of Solanum phureja and Solanum tuberosum from Colombia. Phytopathology 100, s176. Rubio, L., Y. Abou-Jawdah, H.X. Lin, and B.W. Falk. 2001a. Geo- graphically distant isolates of the crinivirus Cucurbit yellow stunting disorder virus show very low genetic diversity in the coat protein gene. J. Gen. Virol. 82, 929-933. Rubio, L., M.A. Ayllon, P. Kong, A. Fernández, M. Polek, J. Guerri, P. Moreno, and B.W. Falk. 2001b. Genetic variation of Citrus tristeza virus isolates from California and Spain: evidence for mixed infections and recombination. J. Virol. 75, 8054-8062. Rubio, L., J. Soong, J. Kao, and B.W. Falk. 1999. Geographic distribu- tion and molecular variation of isolates of three whitefly-borne closteroviruses of cucurbits: lettuce infectious yellows virus, cucurbit yellow stunting disorder virus, and beet pseudo- yellows virus. Phytopathology 89, 707-711. Saitou, N. and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406-425. Salazar, L.F., G. Miller, M. Querci, J.L. Zapata, and R.A. Owens. 2000. Potato yellow vein virus: its host range, distribution in South America and identification as a crinivirus transmitted by Trialeurodes vaporariorum. Ann. Appl. Biol. 137, 7-19. Scheffler, K., D.P. Martin, and C. Seoighe. 2006. Robust inference of positive selection from recombining coding sequences. Bioinformatics 22, 2493-2499. Tamura, K.,D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar. 2011. MEGA5: molecular evolutionary genetics analy- sis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28(10), 2731-9. Vives, M.C., L. Rubio, L. Galipienso, L. Navarro, P. Moreno, and J. Guerri. 2002. Low genetic variation between isolates of Citrus leaf blotch virus from different host species and of different geographical origins. J. Gen. Virol. 83, 2587-2591.