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Virology 330 (2004) 408–423 www.elsevier.com/locate/yviro

Comparisons of the genetic structure of populations of Turnip mosaic in West and East Eurasia

Kenta Tomimuraa, Josef Sˇpakb, Nikos Katisc, Carol E. Jennerd, John A. Walshd, Adrian J. Gibbse, Kazusato Ohshimaa,*

aLaboratory of Virology, Faculty of Agriculture, Saga University, Saga 840-8502, Japan bInstitute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, Cˇ eske´ Bude´jovice, Czech Republic cPlant Pathology Laboratory, Faculty of Agriculture, Aristotle University of Thessaloniki, 540 06 Thessaloniki, Greece dWarwick HRI, University of Warwick, Wellesbourne, Warwick CV35 9EF, UK eSchool of Botany and Zoology, Australian National University, Canberra, A. C. T. 0200, Australia

Received 16 July 2004; returned to author for revision 15 September 2004; accepted 28 September 2004

Abstract

The genetic structure of populations of Turnip mosaic virus (TuMV) in Eurasia was assessed by making host range and gene sequence comparisons of 142 isolates. Most isolates collected in West Eurasia infected whereas those from East Eurasia infected both Brassica and Raphanus plants. Analyses of recombination sites (RSs) in five regions of the genome (one third of the full sequence) showed that the protein 1 (P1 gene) had recombined more frequently than the other gene regions in both subpopulations, but that the RSs were located in different parts of the genomes of the subpopulations. Estimates of nucleotide diversity showed that the West Eurasian subpopulation was more diverse than the East Eurasian subpopulation, but the Asian-BR group of the genes from the latter subpopulation had a greater nonsynonymous/synonymous substitution ratio, especially in the P1, viral genome-linked protein (VPg) and nuclear inclusion a proteinase (NIa-Pro) genes. These subpopulations seem to have evolved independently from the ancestral European population, and their genetic structure probably reflects founder effects. D 2004 Elsevier Inc. All rights reserved.

Keywords: Genetic structure; Recombination; Selection; Population; Eurasia; TuMV;

Introduction belongs to the genus Potyvirus. This is the largest genus of the largest family of plant , the (Shukla et al., Turnip mosaic virus (TuMV) infects a wide range of plant 1994; van Regenmortel et al., 2000), which itself belongs to species, primarily from the family .Itis the picorna-like supergroup of viruses. TuMV, like other probably the most widespread and important virus infecting , is transmitted by aphids in the nonpersistent both crop and ornamental species of this family and occurs manner (Hamlyn, 1953). throughout the world (Ohshima et al., 2002; Provvidenti, Potyviruses have flexuous filamentous particles 700–750 1996). TuMV was ranked second only to Cucumber mosaic nm long, each of which contains a single copy of the virus as the most important virus infecting field-grown genome, which is a single-stranded positive sense RNA vegetables in a survey of virus disease in 28 countries and molecule of about 10,000 nucleotides (nts). The genomes regions (Tomlinson, 1987; Walsh and Jenner, 2002). TuMV have terminal untranslated regions flanking a single open reading frame. The single large polyprotein hydrolyses itself after translation into at least 10 proteins (Riechmann et al., * Corresponding author. Laboratory of Plant Virology, Faculty of Agriculture, 1-Banchi, Honjo-machi, Saga University, Saga 840-8502, 1992; Urcuqui-Inchima et al., 2001). The protein 1 (P1) Japan. Fax: +81 952 28 8709. gene, situated at 5V end of the genome, encodes a serine E-mail address: [email protected] (K. Ohshima). proteinase and is the protein with the greatest between-

0042-6822/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2004.09.040

YVIRO-02785; No. of pages: 16; 4C: 11 K. Tomimura et al. / Virology 330 (2004) 408–423 409 species variation. Even though P1 enhances amplification each area for their spatial genetic structure using different and movement of the virus, it is not strictly required for viral evolutionary assumptions. We discuss the results in terms of infectivity. The 6K protein 2 (second 6 kDa protein) is the information they provide about the changes that have involved in viral long-distance movement and symptom occurred during continent-wide evolution and adaptation of induction. The viral genome-linked protein (VPg) is a TuMV populations. multifunctional protein with several suggested roles in the viral infection cycle; it may act as a primer for RNA replicase during virus multiplication, possibly through direct inter- Results action with the viral RNA polymerase, and it has essential functions in host genotype specificity. The nuclear inclusion Biological characterization of Eurasian population a proteinase (NIa-Pro) cleaves the polyprotein. The coat protein (CP) is involved in aphid transmission, cell-to-cell The 70 West Eurasian, 72 East Eurasian, and 6 non- and systemic movement, encapsidation of the viral RNA, Eurasian TuMV isolates examined in this study are listed in and regulation of viral RNA amplification. CP sequences Table 1. Brassica rapa and Nicotiana benthamiana plants have been studied extensively to assess the difference within infected systemically with each of the West Eurasian TuMV and between potyvirus species (Choi et al., 2002; Dunoyer et isolates were homogenized and their sap mechanically al., 2004; Jenner et al., 2002, 2003; Moury et al., 2004; Spetz inoculated to young plants of B. rapa cv. Hakatasuwari, and Valkonen, 2004; Urcuqui-Inchima et al., 2001). Brassica pekinensis cv. Nozaki-1go, and Raphanus sativus Studies of the molecular evolutionary history of viruses cvs. Taibyo-sobutori and Akimasari. Sixty-six of the West help to provide understanding of such important features of Eurasian isolates infected most Brassica plants systemically their biology as changes in virulence and geographical but did not infect R. sativus. Thus, most of these isolates, ranges and their bemergenceQ as new epidemics, information despite minor differences in pathogenicity for some of the that is essential for designing strategies for controlling tested Brassica hosts (data not shown), were of the Brassica viruses. The complexity of such studies is becoming (B) infecting host type (strain). Only four of the West increasingly obvious as they involve understanding varia- Eurasian isolates infected R. sativus systemically and were tion caused by mutation, recombination, selection, and therefore of the Brassica/Raphanus (BR) host type; these adaptation (Bateson et al., 2002; Chen et al., 2002a; Garcı´a- were isolates Cal1, ITA 7, ITA 8, DEU 4, and PV0104, which Arenal et al., 2001; Monci et al., 2002; Moreno et al., 2004; had been collected in Italy and Germany from Calendula Moury et al., 2002; Ohshima et al., 2002; Rubio et al., 2001; officinalis, Raphanus raphanistrum, sp., and Simon and Bujarski, 1994; Stenger et al., 2002; Tan et al., Lactuca sativa, respectively. Isolates DEU 4 and PV0104 2004; Tomimura et al., 2003). There have been three studies are probably the same isolate. In contrast, approximately 64% of the phylodemography of TuMV (Ohshima et al., 2002; of isolates from East Eurasia were BR host type (Tan et al., Tan et al., 2004; Tomimura et al., 2003). These showed that 2004). parts of around half of the sequences were phylogenetically anomalous and had resulted from genetic recombination Molecular characterization of Eurasian population from different parental lineages, a significant feature of the genetic diversity of this virus. One of the major driving Sequences of the genes encoding the P1, C-terminus of forces of plant virus evolution is the interaction between cylindrical inclusion protein (Ct-CI, 3Vterminal 63 nts), 6K2, host and virus (Garcı´a-Arenal et al., 2001; Roossinck, VPg and NIa-Pro, and the CP of each isolate were 1997). However, despite growing interest in the molecular determined. The genomic regions encoding the P1, 6K2, epidemiology of potyviruses (Bateson et al., 2002; Bousa- VPg, and NIa-Pro of all isolates were 1086, 159, 576, and 729 lem et al., 2000; Tomimura et al., 2003), there have been nts long, respectively. The CP genes of most isolates were few attempts to determine which regions of the genome are 864 nts long, whereas those of isolates AllA, GK 1, GRC 44, subject to selective constraints (Moreno et al., 2004; Moury and GRC 45 were 861 nts long. The sequence data have been et al., 2002). We decided that to better understand the submitted to GenBank, EMBL, and DDBJ databases and evolution and host/virus interactions it was important to assigned accession codes: AB188861–AB189021. determine in more detail the molecular changes that occurred during the evolution of TuMV populations. Recombination sites Our earlier studies have shown that the East Eurasian TuMV subpopulation has probably emerged from the more We looked for evidence of recombination using the ancient West Eurasian subpopulation (Tomimura et al., concatenatedP1,Ct-CI,6K2,VPg,NIa-Pro,andCP 2003). In both regions, brassica crops are an important sequences (bconcatsQ). As the Ct-CI, 6K2, VPg, and NIa- component of local agriculture, but in West Eurasia the Pro regions are contiguous in the genome, the position of crops are mostly Brassica species whereas in East Eurasia recombination sites (RSs) within those genes, except they are both Brassica and Raphanus species. In this study, perhaps in the 5V- and 3V-terminal 100 nts, will be revealed we compared approximately 70 representative isolates from by PHYLPRO and SISCAN analyses. Similarly, RSs within 410 K. Tomimura et al. / Virology 330 (2004) 408–423

Table 1 Turnip mosaic virus isolates analyzed in this study Isolate Original host Location Year of Host typea Referencesb Sequenced regionc (city, district, country) collection or accession nos. West Eurasia A102/11 Anemone coronaria –, Liguria, Italy 1993 B (k), (o) AB093597 A64 A. coronaria –, Liguria, Italy 1991 B (o) AB093599 Al Alliaria officinalis –, Piedmont, Italy 1968 (B) (g), (k), (o) AB093598 AllA A. officinalis –, –, Denmark 1991 (B) N Mid C Cal1 Calendula officinalis –, Liguria, Italy 1979 BR (k), (o) AB093601 CZE 1 Brassica oleracea Ruzyne, –, Czech Republic b1981 B (k), (o) AB093608 CZE 2 Armoracia rusticana Ceske Budejovice, –, Czech Republic 1993 B (e) N Mid C CZE 4 B. rapa Ceske Budejovice, –, Czech Republic 1994 B (e) N Mid C CZE 5 B. rapa Ceske Budejovice, –, Czech Republic 1994 B (e) N Mid C CZE 10 A. officinalis Ceske Budejovice, –, Czech Republic 1994 B (e) N Mid C CZE 18 A. rusticana Chomutov, –, Czech Republic 1995 B (e) N Mid C CZE 19 A. officinalis Ceske Budejovice, –, Czech Republic 1995 B (e) N Mid C DEU 1 Not known –, –, Germany 1993 B (e) N Mid C DEU 4d Lactuca sativa –, –, Germany 1993 BR (e) N Mid C DEU 5 L. sativa Monchengladbach, –, Germany 1994 B (e) N Mid C DEU 7 L. sativa Frankfurt, –, Germany 1994 B (e) N Mid C DNK 2 Brassica napus –, –, Denmark b1993 B (e) N Mid C DNK 3 B. rapa –, –, Denmark b1993 B (e) N Mid C DNK 4 B. rapa –, –, Denmark b1993 B (e) N Mid C Eru1D Eruca sativa –, Piedmont, Italy 1991 B N Mid C FRA 2 B. napus –, –, France 1994 B (e) N Mid C FRD 1 B. oleracea –, –, Germany 1987 B (q) N Mid C GBR 7 Rheum rhabarbarum –, Gloucestershire, UK 1993 B (e), (k) AB076482 Mid AB076556 GBR 8 Lunaria annua –, Essex, UK 1994 B (e) N Mid C GBR 27 B. oleracea –, Dorset, UK 1999 B (d) N Mid C GBR 30 B. oleracea –, Dorset, UK 1999 B (d) N Mid C GBR 31 B. oleracea –, Dorset, UK 1999 B (d) N Mid C GBR 32 B. oleracea –, Dorset, UK 1999 B (d) N Mid C GBR 36 B. oleracea –, Dorset, UK 1999 B (d) N Mid C GBR 38 B. oleracea –, Dorset, UK 1999 B (d) N Mid C GBR 50 B. oleracea –, Yorkshire, UK 2000 B (d) N Mid C GBR 51 B. oleracea –, Yorkshire, UK 2000 B (d) N Mid C GBR 57 B. oleracea –, Conwy, UK 2000 B (d) N Mid C GBR 58 B. oleracea –, Conwy, UK 2000 B (d) N Mid C GK 1 Matthiola incana –, –, Greece b1989 B (k), (q) AB076451 Mid X96542 GRC 2 B. oleracea Thessaloniki, –, Greece 1993 B (e) N Mid C GRC 6 B. oleracea Nea Karvali, –, Greece 1993 B (e) N Mid C GRC 12 B. oleracea Nea Karvali, –, Greece 1993 B (e) N Mid C GRC 17 B. oleracea Volos, –, Greece 1993 B (e) N Mid C GRC 18 B. oleracea Xanthi, –, Greece 1994 B (e) N Mid C GRC 31 B. oleracea Nea Tenedos, –, Greece 1994 B (e) N Mid C GRC 32 B. oleracea Orestiada, –, Greece 1994 B (e) N Mid C GRC 41 Cheiranthus sp. –, –, Greece 1999 B N Mid C GRC 42 Allium sp. –, –, Greece 1999 B N Mid C GRC 43 Allium sp. –, –, Greece 1999 B N Mid C GRC 44 Allium hirsutum –, –, Greece 2000 B N Mid C GRC 45 Allium napolitanum –, –, Greece 2000 B N Mid C IS1 Allium ampeloprasum –, –, Israel 1993 B (a), (k), (o) AB093602 ITA 1 Brassica sp. –, Campania, Italy 1994 B (e), (k) AB076477 Mid AB076523 ITA 3 Brassica sp. –, Campania, Italy 1994 B (e) N Mid C ITA 4 B. rapa –, Campania, Italy 1994 B (e) N Mid C ITA 5 Brassica sp. –, Campania, Italy 1994 B (e) N Mid C ITA 6 M. incana –, Campania, Italy 1994 B (e) N Mid C ITA 7 Raphanus raphanistrum –, Campania, Italy 1994 BR (e), (k), (o) AB093600 ITA 8 Abutilon sp. –, –, Italy 1995 BR (c) N Mid C ITA 9 Cucurbita pepo –, –, Italy 1995 B N Mid C NLD 1 B. oleracea –, –, The Netherlands 1995 B (e) N Mid C NLD 2 B. oleracea –, –, The Netherlands 1995 B (e) N Mid C POL 1 B. napus Poznan, –, Poland 1993 B (e) N Mid C POL 2 Papaver somniferum Czempin, –, Poland 1993 B (e) N Mid C K. Tomimura et al. / Virology 330 (2004) 408–423 411

Table 1 (continued) Isolate Original host Location Year of Host typea Referencesb Sequenced regionc (city, district, country) collection or accession nos. West Eurasia POL 4 B. napus Grabianowo, –, Poland 1993 B (e) N Mid C PRT 1 B. oleracea Madeira, –, Portugal 1994 B (e) N Mid C PV0054 B. oleracea –, –, Germany Not known B (k), AB076457 Mid AB076531 PV0104d L. sativa Stuttgart, –, Germany 1993 BR (k), (o) AB093603 PV376-Br B. napus Braunschweig, –, Germany 1970 B (g), (o) AB093604 Rn98 Ranunculus asiaticus –, Liguria, Italy 1997 B (k) AB076444 Mid AB076520 RUS 1 A. rusticana –, –, Russia 1993 B (e), (k), (o) AB093606 RUS 2 B. napus Moscow, –, Russia Not known B (k), (o) AB093607 St48 Limonium sinuatum –, Toscana, Italy 1993 B (k), (o) AB093596 UK 1 B. napus –, Warwickshire, UK 1975 B (p) AF169561

East Eurasia 1J Raphanus sativus Saga, Saga, Japan 1977 BR (j) D83184 2J Brassica pekinensis –, Tochigi, Japan 1994 BR (k), (o) AB093622 59J R. sativus Saga, Saga, Japan 1996 BR (k), (o) AB093620 C1 Not known –, Taiwan, Not known Not known AF394601 C42J B. rapa Saga, Saga, Japan 1993 B (k), (o) AB093625 CH6 R. sativus Zengjiang, Jiangsu, China 1999 BR (n) AB179888 AB179939 AB179990 CH7 R. sativus Zengjiang, Jiangsu, China 1999 BR (n) AB179889 AB179940 AB179991 CH8 R. sativus Zengjiang, Jiangsu, China 1999 BR (n) AB179890 AB179941 AB179992 CHBJ1 R. sativus Beijing, –, China 1999 BR (n) AB179891 AB179942 AB179993 CHBJ2 R. sativus Beijing, –, China 1999 BR (n) AB179892 AB179943 AB179994 CHHH29 Brassica campestris Hengyang, Hunan, China 1999 B(R) (n) AB179893 AB179944 AB179995 CHHH30 Brassica sp. Hengyang, Hunan, China 1999 B(R) (n) AB179894 AB179945 AB179996 CHHZ3 Brassica sp. Zhuzhou, Hunan, China 1999 B (n) AB179895 AB179946 AB179997 CHK16 R. sativus Guilin, Guangxi, China 2000 BR (n) AB179896 AB179947 AB179998 CHK51 R. sativus Guilin, Guangxi, China 2001 BR (n) AB179897 AB179948 AB179999 CHK55 B. rapa Guilin, Guangxi, China 2001 B (n) AB179898 AB179949 AB180000 CHL13 R. sativus Lushun, Liaoning, China 1999 BR (n) AB179899 AB179950 AB180001 CHL14 R. sativus Lushun, Liaoning, China 1999 BR (n) AB179900 AB179951 AB180002 CHN 1 Brassica sp. –, Taiwan, China b1980 BR (k), (l), (o) AB093626 CHN 2 Brassica sp. –, Taiwan, China b1980 B (k), (l), (n) AB076475 AB179952 AB076549 CHN 3 Brassica sp. –, Taiwan, China b1980 B (k), (l), (n) AB076476 AB179953 AB076550 CHN 4 Brassica sp. –, Taiwan, China b1980 B (k), (l), (n) AB076477 AB179954 AB076551 CHN 5 Brassica sp. –, Taiwan, China b1985 B (b), (k), (n) AB076478 AB179955 AB076552 CHN 12 Not known –, –, China b1990 B (f), (h), (o), (n) AY090660 CHSC2 Brassica sp. Chengdu, Sichuan, China 1999 B(R) (n) AB179905 AB179956 AB180007 CHSE1 Brassica sp. Emei, Sichuan, China 1999 B (n) AB179906 AB179957 AB180008 CHYK19 Brassica integrifolia Kunming, Yunnan, China 2000 B (n) AB179907 AB179958 AB180009 CHYK20 B. integrifolia Kunming, Yunnan, China 2000 B (n) AB179908 AB179959 AB180010 (continued on next page) 412 K. Tomimura et al. / Virology 330 (2004) 408–423

Table 1 (continued) Isolate Original host Location Year of Host typea Referencesb Sequenced regionc (city, district, country) collection or accession nos. East Eurasia CHYK56 B. rapa Kunming, Yunnan, China 2001 B(R) (n) AB179909 AB179960 AB180011 CHZC1 Brassica juncea Hongzhou, Zhejiang, China 1998 B (n) AB179910 AB179961 AB180012 CHZC21 B. campestris Cixi, Zhejiang, China 1999 B (n) AB179911 AB179962 AB180013 CHZC22 Brassica sp. Cixi, Zhejiang, China 1999 B (n) AB179912 AB179963 AB180014 CHZH33 B. juncea Hongzhou, Zhejiang, China 2000 B (n) AB179913 AB179964 AB180015 CHZH34 B. campestris Hongzhou, Zhejiang, China 2000 B (n) AB179914 AB179965 AB180016 CHZH36 B. rapa Hongzhou, Zhejiang, China 2000 BR (n) AB179915 AB179966 AB180017 CHZJ23 R. sativus Jiande, Zhejiang, China 1999 BR (n) AB179916 AB179967 AB180018 CHZJ25 Brassica sp. Jiande, Zhejiang, China 1999 B (n) AB179917 AB179968 AB180019 CHZJ26 B. campestris Jiande, Zhejiang, China 1999 BR (n) AB179918 AB179969 AB180020 CHZJ27 B. campestris Jiande, Zhejiang, China 1999 B (n) AB179919 AB179970 AB180021 CP845J C. officinalis Kisarazu, Chiba, Japan 1997 BR (k), (o) AB093614 CQS1 B. pekinensis –, –, Korea 1998 B (n) AB179924 AB179975 AB180026 DMJ R. sativus –, Tochigi, Japan 1996 BR (k), (o) AB093623 FD21J R. sativus Shime, Fukuoka, Japan 1998 BR (k), (n) AB076416 AB179984 AB076492 FD27J R. sativus Fukuoka, Fukuoka, Japan 1998 BR (k), (o) AB093618 HBI2 B. pekinensis Hongzhou, Zhejiang, China 1998 B (n) AB179920 AB179971 AB180022 HJ1S B. juncea Hongzhou, Zhejiang, China 1998 B (k), (n) AB076479 AB179972 AB076553 HOD517J R. sativus Kimobetsu, Hokkaido, Japan 1998 BR (k), (o) AB093617 HRD R. sativus Hongzhou, Zhejiang, China 1998 BR (k), (o) AB093627 HZ5 R. sativus Xiaoshan, Zhejiang, China 1998 BR (n) AB076481 AB179973 AB076555 HZ6 Brassica sp. Xiaoshan, Zhejiang, China 1998 B (n) AB179923 AB179974 AB180025 Ka1J B. pekinensis –, Tochigi, Japan 1994 BR (k), (o) AB093624 KD32J R. sativus Nankan, Kumamoto, Japan 1998 BR (k), (o) AB093621 KYD81J R. sativus Joyo, Kyoto, Japan 1998 BR (k), (o) AB093613 MN43J Brassica sp. Nobeoka, Miyazaki, Japan 1998 BR (k), (n) AB076425 AB179989 AB076501 ND10J R. sativus Hirato, Nagasaki, Japan 1998 BR (k), (n) AB076418 AB179985 AB076494 NDJ R. sativus Takaki, Nagasaki, Japan 1997 BR (k), (o) AB093616 NPL 3 B. juncea –, Pokhara, Nepal 1993 B (e), (k), (n) AB076471 AB179978 AB076545 NPL 4 B. juncea –, Pokhara, Nepal 1993 BR (e), (k), (n) AB076472 AB179979 AB076546 NPL 5 B. juncea –, Pokhara, Nepal 1993 BR (e), (n) AB179929 AB179980 AB180031 OD11J R. sativus Hiji, Oita, Japan 1998 BR (k), (n) AB076420 AB179986 AB076496 OD14J R. sativus Amagase, Oita, Japan 1998 BR (k), (n) AB076422 AB179987 AB076498 ON16J B. rapa Yufuin, Oita, Japan 1998 BR (k), (n) AB076423 AB179988 AB076499 SD3J R. sativus Saga, Saga, Japan 1996 BR (k), (n) AB076413 AB179983 AB076489 K. Tomimura et al. / Virology 330 (2004) 408–423 413

Table 1 (continued) Isolate Original host Location Year of Host typea Referencesb Sequenced regionc (city, district, country) collection or accession nos. East Eurasia SGD311J R. sativus Nishiazai, Shiga, Japan 1998 BR (k), (o) AB093619 ST19J B. juncea Nabeshima, Saga. Japan 1998 B (k), (n) AB076411 AB179981 AB076487 STD8J R. sativus Tosu, Saga, Japan 1997 BR (k), (n) AB076412 AB179982 AB076488 RAD1 R. sativus –, –, Korea Not known BR (n) AB179925 AB179976 AB180027 RHS1 R. sativus –, –, Korea Not known BR (n) AB179926 AB179977 AB180028 TD88J R. sativus Tokyo, Tokyo, Japan 1998 BR (k), (o) AB093615 Tu-2R1 R. sativus –, Tochigi, Japan Not known BR (m) AB105135 Tu-3 B. oleracea –, Tochigi, Japan Not known B (m) AB105134 TW Not known –, Taiwan, China Not known Not known AF394602

Other BZ1 B. oleracea –, Federal, Brazil 1996 B (k), (o) AB093611 CDN 1 B. napus –, –, Canada b1989 B (o), (q) AB039610 KEN 1 B. oleracea –, –, Kenya 1994 B (e), (k), (o) AB093605 NZ290 B. pekinensis –, Alan Stewart, New Zealand 1998 B (k), (o) AB093612 Q-Ca B. rapa –, –, Canada Not known Not known (i) D10927 USA 1 B. oleracea –, –, United States b1980 B (k), (l), (o) AB093609 a Host type B; Brassica, isolates infected B. rapa cv. Hakatasuwari systemically with mosaic symptoms on uninoculated leaves. Host type (B); isolates infected B. rapa latently and only occasionally. Host type BR; these isolates infected both B. rapa and R. sativus cvs. Akimasari and Taibyosobutori systemically, with mosaic symptoms on uninoculated leaves. Host type B(R); isolates infected B. rapa systemically with mosaic symptoms on the uninoculated leaves and infected R. sativus cvs. Akimasari and Taibyosobutori only occasionally and latently. b References: (a) Gera et al. (1997); (b) Green and Deng (1985); (c) Guglielmone et al. (2000); (d) Hughes et al. (2003); (e) Jenner and Walsh (1996); (f) Jenner et al. (2002); (g) Lisa and Lovisolo (1976); (h) Liu et al. (1990); (i) Nicolas and Laliberte´ (1992); (j) Ohshima et al. (1996); (k) Ohshima et al. (2002); (l) Provvidenti (1980); (m) Suheiro et al. (2004); (n) Tan et al. (2004); (o) Tomimura et al. (2003); (p) Tomlinson and Ward (1978); (q) Walsh (1989). c N P1; Mid C-terminus CI + 6K2 + VPg + NIa-Pro; C CP; where we sequenced in this study. d DEU 4 and PV0104 are probably the same isolate however the nucleotide sequences of genomes between the isolates were slightly different after single isolations. We therefore included both isolates for the analyses. the P1 and CP regions will be found within those regions. al., 2004). We therefore used SISCAN to check 100 nt slices However, RSs in the HC-Pro, P3, 6K1, and CI genes that lie of all Eurasian sequences for evidence of recombination between the P1 gene and the 6K2 gene will only be found as using total nt, synonymous (ds), and nonsynonymous (dn) RSs at the P1/6K2 interface of a concat when there are sites and found, for example, that isolate GBR 7 showed different parental sequences either side of that interface. significant affinities (Z values N3.0) with sequence AllA in Similarly single RSs, but not dinsertions,T within the NIb the N-terminal half of the P1 gene (nt 250–350 in total nt site region will be found at the NIa-Pro/CP interface of a concat. and nt 200–400 in silent nt site analyses), but its P1 to CP After gaps had been removed, the dphylogenetic profilesT genes (nt 500–2950 in total nt site and nt 450–3000 in silent of the concats were examined using the PHYLPRO program nt site analyses) were more similar to CHN 12 (for total nt, (Weiller, 1998). This clearly showed that some were silent, and nonsilent nt site analyses, see Figs. 1A–C, phylogenetically anomalous, in that different parts of them respectively). We also found RSs in West Eurasian isolates had different phylogenetic affinities (data not shown). The in the P1 gene, at the junction between the P1 gene and Ct-CI dsister scanningT method (Gibbs et al., 2000) was used to region and in the VPg, NIa-Pro, and CP genes. Table 2 lists determine whether these anomalies resulted from recombi- the probable recombinants among the West Eurasian isolates nation rather than convergent selection. All the East Eurasian and their RSs as indicated by SISCAN, together with the isolates that had been identified as recombinants in the same sequences closest to their parents (bparental sequencesQ). genomic regions by Tomimura et al. (2003) and Tan et al. Although both parental sequences of most West Eurasian (2004) had statistically significant dconflicting related recombinants showed Z values larger than 3 in total nt site signalsT (CRSs) in sites that differed synonymously from analyses (data not shown), some, mostly from South Europe, those in other sequences. The PHYLPRO program was used had only one definitive parental sequence (Z values larger to search for the dparental lineagesT of East Eurasian than 3) with another tentative parental sequence (Z values recombinant sequences. However, not all sequences that around 3 in silent nt site). By contrast, all the East Eurasian had different relationships in adjacent 100 nt sequences recombinants had parental sequences that gave Z values showed clear phylogenetic anomalies in PHYLPRO (Tan et greater than 3 in total and silent nt site analyses (Tan et al., 414 K. Tomimura et al. / Virology 330 (2004) 408–423

Fig. 1. Graphs showing SISCAN analyses of the concat (P1+Ct-CI+6K2+VPg+NIa-Pro+CP) sequence of isolate GBR 7 with that of CHN 12-like (black thick line) and that of AllA-like (black thin line). The sequences of CHN 12 and AllA represent the likely parental sequences of GBR 7. Note the strong support (i.e., Z value N3.0) for GBR 7 being more closely related to CHN 12 than AllA between approximately 200 and 400 nt and the reverse between 450 and 3000 nt in total nt (A), synonymous (B), and nonsynonymous (C) site analyses. For all three graphs, each window comparison involved subsequences of 100 nts with a step between window positions of 50 nts. K. Tomimura et al. / Virology 330 (2004) 408–423 415

Table 2 List of probable recombinants and their recombination sites within the concat region as revealed by SISCAN analysis Region RS positiona Isolate dParentalT-like SiScan (Z value)b Type of recombinant-likec (Gene) isolate All sites Synonymous sites Nonsynonymous sites P1 Nt 100–150 CZE 10 GRC 45 Â HZ5 4.91 3.23 3.18 UD Â Asian-BR Nt 100–150 CZE 19 GRC 45 Â HZ5 4.91 3.23 3.18 UD Â Asian-BR Nt 200–250 AllA TW Â GBR 7 5.48 3.39 4.22 world-B Â basal-B Nt 200–250 DEU 7 TW Â GBR 7 5.73 3.92 4.22 world-B Â basal-B Nt 250–300 GRC 45d A64 Â ITA 1 3.97 2.75 2.71 basal-B Â basal-B Nt 300–350 A102/11d CZE 19 Â GRC 45 4.57 2.75 4.57 UD Â basal-B Nt 300–350 ITA 1d RUS 1 Â GRC 45 6.72 2.94 3.71 UD Â basal-B Nt 300–350 ITA 9d CZE 19 Â GRC 45 4.25 2.67 2.5N UD Â basal-B Nt 400–450 GBR 7 AllA Â CHN 12 6.28 4.20 3.50 basal-B Â world-B Nt 400–450 CZE 10d HZ5 Â GRC 45 4.91 2.92 2.94 UD Â basal-B Nt 400–450 CZE 19d HZ5 Â GRC 45 4.91 2.92 2.94 UD Â basal-B Nt 450–500 GBR 57d TW Â GRC 44 2.87 2.60 2.5N world-B Â basal-B Nt 450–500 GBR 58 CZE 4 Â GRC 44 3.59 3.96 2.5N world-B Â basal-B Nt 450–500 St48 OD11J Â GRC 44 3.46 3.68 2.5N world-B Â basal-B P1-Ct-CI Nt 950–1000 DNK 2 FRA 2 Â CZE 1 4.77 5.81 2.83 world-B Â world-B CZE 2 FD27J Â ND10J 5.5 3.32 3.44 world-B Â world-B GBR 27 FD27J Â ND10J 6.21 4.14 4.51 world-B Â world-B GBR 30 FD27J Â ND10J 5.2 3.39 3.44 world-B Â world-B GBR 32 FD27J Â ND10J 5.2 3.39 3.44 world-B Â world-B GBR 51 FD27J Â ND10J 5.43 3.30 3.85 world-B Â world-B GRC 2 FD27J Â ND10J 4.66 3.45 3.44 world-B Â world-B GRC 17 FD27J Â ND10J 5.31 3.50 4.63 world-B Â world-B KEN 1 FD27J Â ND10J 5.49 4.03 3.69 world-B Â world-B NLD 2 FD27J Â ND10J 5.93 4.07 4.51 world-B Â world-B UK 1 FD27J Â ND10J 5.5 3.32 3.44 world-B Â world-B VPg Nt 1250–1300 CZE 18 DNK 2 Â DNK 3 5.46 3.82 2.5N world-B Â world-B Nt 1250–1300 RUS 2 DNK 2 Â DNK 3 4.6 3.82 2.5N world-B Â world-B NIa-Pro Nt 1850–1900 IS1 UK 1 Â GRC 43 3.76 3.56 2.5N world-B Â basal-B Nt 2000–2050 GBR 36 NLD 1 Â DEU 5 4.64 4.22 2.5N world-B Â world-B Nt 2000–2050 GBR 38d NLD 1 Â CZE 4 3.69 2.82 2.5N CP Nt 2550–2560 CZE 1 DEU 5 Â DNK 2 5.53 4.23 2.5N world-B Â world-B CZE 4 CZE 18 Â CHSE1 3.41 3.01 2.5N world-B Â world-B CZE 18 DNK 3 Â DNK 2 5.88 4.51 2.5N world-B Â world-B RUS 2 DNK 3 Â DNK 2 6.04 3.95 2.5N world-B Â world-B Nt 2800–2850 ITA 1d GRC 43 Â USA 1 4.06 2.97 2.5N a P1 nt 1-972, Ct-CI nt 973-1035, 6K2 nt 1036-1194, VPg nt 1195-1770, NIa-Pro nt 1771-2499 and CP nt 2500-3315. b One of parents of all recombinants showed Z values greater than 3 in total nucleotide and nonsynonymous site analyses, therefore lower Z values of one of parents are shown. c Type of recombinants was estimated from the phylogenies constructed from various parts of genomic sequences. Parental lineages are listed but those of some recombinants were difficult to define, thus shown as UD (undefined). d The dtentativeT recombinants namely those with only one dparentT giving a Z values lower than 3 in total nucleotide or nonsynonymous site analysis are marked, but ddefinitiveT recombinants, namely, those that have both dparentsT giving Z values greater than 3 in total nucleotide and nonsynonymous site analyses are not additionally labeled.

2004). Thus, the RSs were more difficult to identify in West Eurasian isolates were found to be recombinants compared Eurasian isolates than those from East Eurasia. with the West Eurasian isolates (80% compared with 42%), Our analyses using SISCAN showed that as larger and similarly more of the BR host type isolates were numbers of sequences are obtained and compared, sequences recombinants than the B host type isolates (74% compared closer to the parents of tentative recombinants may have been with 54%). However, neither the BR nor B host type included in our study and so tentative recombinants may phenotype was uniquely linked with the presence or absence dbecomeT definitive recombinants as was found for the of recombination in a particular region of the genome, Japanese isolate Tu-3 (Suehiro et al., 2004; Tan et al., although RSs were noticeably more common in the VPg gene 2004). However, no other new recombinants were found of BR host type isolates (62%) than in the same region of B among East Eurasian isolates and thus a total of 56 out of 72 host type isolates (2%). Because RSs in the different (80%) of the East Eurasian isolates were found to be subpopulations may show the relationship between them, recombinants. Table 3 summarizes the numbers of isolates we looked in more detail at the sequences around the RSs in which have RSs in particular genes and are probably the East and West Eurasian isolates (Fig. 2) and confirmed recombinants from parental sequence within two Eurasian that the locations of the RSs in West and East Eurasian subpopulations. It can be seen that twice as many of the East isolates were not identical. 416 K. Tomimura et al. / Virology 330 (2004) 408–423

Table 3 Numbers of isolates which have recombination sites in protein gene/region and probable recombinants within Eurasian subpopulation/host type Numbers of isolates which have recombination sites in particular gene/regions Total numbers Total numbers b c Subpopulation/ Within P1 Between Within Within VPg Within Between Within of recombinants of isolates host typea P1 and 6K2 6K2 NIa-Pro NIa-Pro and CP CP West Eurasian subpopulation B host type 12 (18.5%) 10 (15.4%) 0 2 (3.1%) 3 (4.6%) 0 5 (7.7%) 30 (46.2%) 65 BR host type 0 0 0 0 0 0 0 0 5 Total 12 (17.1%) 10 (14.2%) 0 2 (2.9%) 3 (4.3%) 0 5 (7.1%) 30 (42.3%) 70

East Eurasian subpopulation B host type 0 19 (76.0%) 0 0 0 0 0 19 (76.0%) 25 BR host type 21 (46.7%) 36 (80.0%) 2 (4.4%) 31 (68.9%) 1 (2.2%) 0 1 (2.2%) 37 (82.2%) 45 Total 21 (30.0%) 55 (78.6%) 2 (2.9%) 31 (44.3%) 1 (1.4%) 0 1 (1.4%) 56 (80.0%) 70

Eurasian subpopulations B host type 12 (13.3%) 29 (32.2%) 0 2 (2.2%) 3 (3.3%) 0 5 (5.6%) 49 (54.4%) 90 BR host type 21 (42.0%) 36 (72.0%) 2 (4.0%) 31 (62.0%) 1 (2.0%) 0 1 (2.0%) 37 (74.0%) 50 Total 33 (23.6%) 65 (46.4%) 2 (1.4%) 33 (23.6%) 4 (2.9%) 0 6 (4.3%) 86 (61.4%) 140 a BR or B host type includes BR and B(R) host types or B and (B) host types, respectively, listed in Table 1. b Note that some isolates have multiple recombination sites. c Isolate of unknown host type is excluded.

Phylogenetic relationships pair of sequence variants) for each genomic region (P1, Ct-CI, 6K2, VPg, NIa-Pro, and CP genes). These analyses We initially calculated trees from the concats of the 148 showed appreciable differences between the genomic isolates, including all the recombinants identified previously regions. The P1 gene was the most variable when all (Ohshima et al., 2002; Tan et al., 2004; Tomimura et al., isolates are compared, but not, as would be expected, when 2003) and in this study. However, there was inconsistencies different groups of the isolates are considered separately. in, and poor bootstrap support for, some lineages in the The West Eurasian subpopulation was much more diverse resulting trees as previously found for P1 and CP sequences than the East Eurasian subpopulation in all the genes (Ohshima et al., 2002). We therefore calculated trees from examined (Table 4A). On the other hand, estimation of the the concats of 112 isolates, only excluding recombinants diversity between phylogenetic groups showed that the derived from parents from the different major group lineage basal-B group was the most diverse in all genes, whereas the (i.e., interlineage recombinants). We investigated the rela- Asian-BR group was the least diverse. tionships of the isolates by maximum likelihood (ML), To estimate the degree of selective constraints on each maximum parsimony (MP), and neighbor joining (NJ), but genomic region, dn and ds (i.e., nonsynonymous and only the ML tree is shown (Fig. 3). All the trees partitioned synonymous) substitutions were analyzed separately (Table most of the sequences into the same four consistent groups 4B). The number of dn was always smaller than ds and as reported earlier (Ohshima et al., 2002; Tan et al., 2004; varied considerably between genomic regions (data not Tomimura et al., 2003): basal-B, basal-BR, Asian-BR, and shown). This suggests that there is selection against (i.e., world-B. West Eurasian isolates fell into basal-B, basal-BR, negative selection) most amino acid changes although the and world-B groups, whereas East Eurasian isolates fell into degree of selection varies for different genomic regions. All basal-BR, Asian-BR, and Asian lineage of world-B. The genomic regions we examined had a dn/ds ratio in the range isolate DNK 4 collected in Denmark fell into the East reported for most other DNA and plant RNA viruses Eurasia lineage of world-B group. The basal-B group split (Garcı´a-Arenal et al., 2001; Rubio et al., 2001). The P1 into two subgroups, which we call basal-B1 and basal-B2 gene had the largest dn/ds ratio (0.0729). We estimated the subgroups, and these were all from Mediterranean countries dn/ds ratios of the different genes and of different such as Italy and Greece. The basal-B1 group was sister phylogenetic groups within the West and East Eurasian group to all others in the ML, MP, and NJ phylogenies. Star subpopulations. The ratios were almost the same in the two phylogenies were seen in the basal-BR and world-B groups, subpopulations. On the other hand, estimates of dn/ds ratios but not in basal-B group. of the genes in different phylogenetic groups showed that the P1, VPg, and NIa-Pro genes of Asian-BR group had Variability and selection larger dn/ds ratios than those of other groups. For instance, dn/ds ratios of the P1 and NIa-Pro genes of Asian-BR group The major variant sequences were used to estimate the nt were two to three times and three to seven times larger than diversity (average number of nt substitutions per site in each those of basal-B, basal-BR, and world-B, respectively. The K. Tomimura et al. / Virology 330 (2004) 408–423 417

Fig. 2. Recombination maps of TuMV genomes; the estimated nt positions of the RSs are shown relative to the 5Vend of the P1 gene. The positions of nt correspond to those of 1J sequence (Ohshima et al., 1996). The positions were approximately estimated from the data shown in Table 2, together with that published by Ohshima et al. (2002), Tomimura et al. (2003), and Tan et al. (2004). The wide box in grey shows the concat regions sequenced in this study, the narrow open box shows the regions described previously. The recombination sites in the narrow boxes need further analysis using representative isolates.

dn/ds ratios of 6K2 and CP genes were almost identical VPg genes were found in isolates of the East Eurasian between groups. subpopulation. These results may indicate that some RSs in the P1 and VPg genes are adaptively advantageous in both or one of subpopulation(s), or at least do not incur a Discussion fitness penalty, so that such recombinants persist as a successful dfounders.T An earlier study of the East Eurasian This study reports evidence that recombination has subpopulation showed that RSs were more likely to be the occurred during the evolution of TuMV in not only East surviving progeny from single recombination events rather but also West Eurasian subpopulations. Comparisons of than the progeny of multiple events occurring at recombi- recombinational, mutational, and phylogenetical analyses nation hotspots (Tan et al., 2004). This study for RSs in between the two subpopulations showed that the subpopu- the continent-wide subpopulations also supports this lations could be considered to be different populations and conclusion. were likely to have evolved independently from an ancestral The small number of ddefinitiveT recombinants found in population. South and East Europe perhaps results from mutation Recombination is an important source of genetic variation having erased evidence of recombination (Rubio et al., for TuMV (Ohshima et al., 2002; Tan et al., 2004; Tomimura 2001; Tan et al., 2004). Another possibility is that too few et al., 2003) and perhaps for other potyvirus species (Moreno samples from this area were collected and examined. As two et al., 2004; Moury et al., 2002; Revers et al., 1996). subpopulations within a single potyvirus species (TuMV) Recombination between potyviral sequences also occurs in had a different pattern of distribution of RSs, it is likely that experiments where different viral constructs containing they will be distributed differently in the genomes of other homologous regions of the viral genome have been shown potyvirus species, although we cannot exclude the possi- to recombine (Varrelmann et al., 2000). The distribution bility that, when enough sequences are compared, patterns and total number of RSs in the genomes of potyvirus shared at the family, genus, and species levels will emerge. species are largely unknown because most of the studies The number of RSs in a genome may be related to the length involved relatively small numbers of isolates, or isolates of its evolutionary history and extent of functional selection. that came from a limited collecting area (Tan et al., 2004). The large number of recombinants found in this study In the case of TuMV, RSs were clearly distributed among TuMV isolates has important practical implications if throughout the genome, and some RSs were common to their presence is correlated with the ability of the population a large number of recombinant(s). When crossover sites to adapt. For instance, caution must be taken to avoid the identified by SISCAN were compared between the two introduction of exotic TuMV sequences into a region for subpopulations, the locations of all RSs of the West transgenic plant resistance or cross protection, as they might Eurasian subpopulation were different from those of the recombine and produce TuMV isolates with new biological East Eurasian subpopulations. There were at least 12 RSs properties. in the West Eurasian subpopulation and at least 20 RSs in The dn versus ds ratios we have found (Table 4B) provide the East Eurasian subpopulation. RSs were identified evidence of strong selection against amino acid change as a within the P1 gene in both subpopulations, although we driving force for TuMV evolution. This implies that changes do not know whether this indicates that the P1 gene is in most of the third codon positions of the P1, 6K2, VPg, recombinationally more frequent than other parts of the NIa-Pro, and CP genes have a different influence on the genome or whether a larger proportion of recombinants of evolution of TuMV than changes in most of their first and this region survive. On the other hand, the most RSs in second codon positions, which have a stronger tendency to 418 K. Tomimura et al. / Virology 330 (2004) 408–423

Fig. 3. A maximum likelihood (ML) tree calculated from the concat sequences (P1+Ct-CI+6K2+VPg+NIa-Pro+CP) of 112 isolates of TuMV that did not include the interlineage recombinants identified in this study and by Tan et al. (2004). The isolates collected in West Eurasian (acronyms in black), East Eurasian (acronyms in red), and other continents (acronyms in blue) are separately listed. Numbers at each node indicate the percentage of supporting puzzling steps (or bootstrap samples) (only values N50 are shown) in ML, MP, and NJ, respectively. Horizontal branch length is drawn to scale with the bar indicating 0.1 nt replacements per site. The homologous sequences of two isolates (mild and j1) of JYMV and an isolate of ScaMV were used as the outgroup. An asterisk indicates an intralineage recombinant. K. Tomimura et al. / Virology 330 (2004) 408–423 419

Table 4 Nucleotide diversity (A) and nonsynonymous and synonymous substitution ratios (B) of TuMV gene/regions and group/subpopulations Region/gene Subpopulation/group All isolates(112) West Eurasian(57) East Eurasian(49) Basal-B(12) Basal-BR(8) Asian-BR(10) World-B(82) A concat 0.1251 0.1409 0.0945 0.1451 0.0967 0.0194 0.0744 [nt 1–2979, 2979b] P1 [nt 1–627, 627b] 0.1772 0.1960 0.1418 0.2248 0.1616 0.0210 0.1053 6K2 [nt 691–849, 159b] 0.1698 0.2000 0.1009 0.1772 0.1056 0.0253 0.1166 VPg [nt 850–1425, 576b] 0.1487 0.1599 0.1220 0.1555 0.1086 0.0197 0.0885 NIa-Pro [nt 1426–2154, 729b] 0.1220 0.1425 0.0857 0.1380 0.0867 0.0218 0.0709 CP [nt 2155–2979, 825b] 0.0673 0.0779 0.0496 0.0843 0.0505 0.0151 0.0366

B concat 0.0601 0.0571 0.0620 0.0591 0.0636 0.1097 0.0736 [nt 1–2979, 2979b] P1 [nt 1–627, 627b] 0.0729 0.0859 0.0518 0.1236 0.0994 0.2973 0.1143 6K2 [nt 691–849, 159b] 0.0295 0.0297 0.0256 0.0232 0.0446 0.0542 0.0405 VPg [nt 850–1425, 576b] 0.0430 0.0376 0.0489 0.0299 0.0380 0.1658 0.0502 NIa-Pro [nt 1426–2154, 729b] 0.0097 0.0094 0.0108 0.0050 0.0088 0.0360 0.0143 CP [nt 2155–2979, 825b] 0.0526 0.0506 0.0515 0.0458 0.0408 0.0567 0.0581 Nucleotide (nt) diversity was estimated according to Kimura’s two-parameter method (A). Nt diversity is the average number of nt substitutions per site in each pair of sequence variants. Nonsynonymous (dn) and synonymous (ds) substitution (dn/ds) ratios in pairwise comparisons were estimated from TuMV gene/ region sequences using codeml of PAML package version 3.14 (Yang, 1997) (B). The isolates used in these analyses were listed in Fig. 3. The concats were aligned with the sequences of JYMV and ScaMV as the outgroup used to construct the phylogeny. The isolates that were identified to have recombination sites of interlineage parents (parents from different lineage) in the protein genes were removed, and the nt diversities and the dn/ds ratios were estimated. Numbers of isolate sequences in each subpopulation/group used for the estimation are shown in parentheses. be unchanged while the evolution of the third codon within the East Eurasian subpopulation may indicate that position is much more relaxed. The degree of selection in this subpopulation is relatively younger than the West genes can be estimated by comparing the rate between nt Eurasian subpopulation. For a more detailed comparison of diversity at dn versus ds positions and a strong conservative TuMV populations, we estimated the nt diversity of West selection operates on most animal and plant viruses and East Eurasian isolates for concats and for each genomic (Domingo et al., 2001; Garcı´a-Arenal et al., 2001). Different region(s) (Table 4A), and this indicated that the two TuMV evolutionary dynamics for different genomic regions have subpopulations are from different populations. The presence been described for plant viruses, a phenomenon that has of most ddefinitiveT recombinants and a unique phylogenetic been analyzed in detail for Citrus tristeza virus (D’Urso et group with larger dn/ds ratio in the East Eurasian sub- al., 2003; Lo´pez et al., 1998), Tobacco mild green mosaic population, the presence of some dtentativeT recombinants in virus (Fraile et al., 1996), Rice yellow mottle virus (Fargette West Eurasian subpopulation, and more diversity in West et al., 2004), and in 6K2 and the N-terminus of CP of some Eurasian subpopulation add credibility to our conclusion potyviruses (Moury et al., 2002). In the case of TuMV, the that TuMV in East Eurasia is a more recently emerged P1 gene had the largest dn/ds ratio whereas the NIa-Pro the population than that in West Eurasia. smallest, indicating that different protein genes are under Phylogenetic analysis of the 112 isolate sequences different evolutionary constraints. This has also been showed that most fell into four well-defined groups, reported in Watermelon mosaic virus where the P1 gene although East Eurasian isolates seemed to form a single had the largest dn/ds ratio compared to those of the CI and lineage in the world-B group. Star phylogenies, as found in CP genes (Moreno et al., 2004). We also analyzed dn/ds epidemic populations of Simian and Human immunodefi- ratios in different phylogenetic groups. The greatest dn/ds ciency viruses, and Cucumber mosaic virus, may indicate a ratios were found in isolates of the Asian-BR group, recent emergence with minimal selection (Myers et al., suggesting that different lineages are under different evolu- 1993; Roossinck et al., 1999). Such star phylogenies were tionary constraints and that the overall substitution rate has seen in world-B and basal-BR but not in basal-B groups, changed on the infection of a new species such as R. sativus. supporting the hypothesis that the basal-B group is the It is possible that the evolution of TuMV in nature is greatly oldest (Ohshima et al., 2002). Sa´nchez et al. (2003) affected by stochastic processes, including population analyzed approximately 40 CP sequences using RFLP and bottlenecks that may occur among hosts during trans- noted that basal-B group could represent several lineages. In mission, and within hosts. Recently, random effects due to the present study, the basal-B group split into two genetic drift of Tobacco mosaic virus within an infected subgroups, basal-B1 and basal-B2. The most variable group, tobacco plant were reported (Sacrista´n et al., 2003). The the basal-B1, was sister group to other (sub)groups and had existence of an Asian-BR group with large dn/ds ratios bootstrap support in all ML, MP, and NJ trees that supported 420 K. Tomimura et al. / Virology 330 (2004) 408–423 its separation. The isolates in the basal-B1 group originated benthamiana plants. Plants infected systemically with each from nonbrassica plants. This may indicate that the detailed of the TuMV isolates were homogenized in 0.01 M comparison of the B host types of the isolates in basal-B and potassium phosphate buffer (pH 7.0) and mechanically world-B groups using wide range of brassica host species is inoculated to young plants of B. rapa cv. Hakatasuwari, B. necessary. The simplest interpretation of the phylogenetic pekinensis cv. Nozaki-1go, Brassica napus cv. Norin-32go, analyses is that the original TuMV population is Southeast and R. sativus cvs. Taibyo-sobutori and Akimasari. Inocu- European as the basal-B1 group, although we need to lated plants were kept for at least four weeks in a glasshouse analyze phylogenetic relationships including Minor at 25 8C. and mid-Eurasian isolates. The basal-B group isolates seemed to give rise to the two BR lineages, that in the Extraction of viral RNA basal-BR group (found in West and East Eurasia) and the Asian-BR group (confined to East Eurasia). On the other The viral RNAs were extracted from purified virions hand, some East Eurasian isolates in world-B group were (Choi et al., 1977) or TuMV-infected B. rapa and N. BR or B(R) host type, which occasionally latently infect R. benthamiana leaves using RNeasy Plant Mini Kit (Qiagen). sativus, and the B(R) host type may be a progenitor of the The RNAs were reverse transcribed and amplified using BR host type. Our recombinational and phylogenetic study high-fidelity Platinum Pfx DNA polymerase (Invitrogen). therefore may show that an ancestral isolate(s) in basal-B The cDNAs were separated by electrophoresis in agarose obtained virulence toward R. sativus by the mutations and gels and purified using the QIAquick Gel Extraction Kit by the recombination during the migration to East Eurasia. (Qiagen). The purified cDNAs were cloned into pBluescript Recently, Suehiro et al. (2004) produced infectious clones of II SK+. Plasmids were maintained in Escherichia coli XL1- the isolates Tu-3 and Tu-2R1, which are B and BR host Blue (Stratagene). types, respectively, but both from the world-B group (Fig. 3), and suggested that the P3 protein is an important factor Sequencing in determining the host range. Our phylogenetic studies show that although that major groupings correlate with the Sequences from each isolate were determined using two geographical origin of the isolate and its host, nonetheless independent RT-PCR products and cloned plasmids. Each some isolates do not fit this pattern, and these indicate that cloned plasmid and RT-PCR product was sequenced by TuMV is able to cross species boundaries fairly freely. primer walking in both directions using the BigDye In conclusion, from a biological and molecular point of Terminator v3.0 Cycle Sequencing Ready Reaction Kit view, the West and East Eurasian isolates analyzed here (Applied Biosystems) and an Applied Biosystems Genetic could be considered to be two different subpopulations Analyzer DNA Model 310; ambiguous nts in any sequence having evolved separately, each with little diversity and with were checked in sequences obtained from at least four other close evolutionary relationships, suggesting that founder independent plasmids. Sequence data were assembled using effects might shape their genetic structure. Our analysis of BioEdit version 5.0.9 (Hall, 1999). representative TuMV isolates from West and East Eurasia provides useful information about the evolution and genetic Recombinant analyses conservation of potyviruses and plant RNA viruses (Garcı´a- Arenal et al., 2001), and to our knowledge, this is perhaps The sequences of the 148 isolates were used for evolu- the first report of such a detailed analysis in plant viruses, tionary analyses. First, we joined the nt sequences of the P1, using a large number of isolates collected from the Eurasian part Ct-CI, 6K2, VPg, NIa-Pro, and CP genes and called this continent. the dconcat,T namely a concatenated sequence analogous to a contiguous sequence or dcontig.T Among the 29 phylogenies obtained from the aligned gene sequences or genomic slices, Materials and methods only those from the region around nt 5501–6500 in degapped sequences, which we call region 12 (R12; see Tomimura et Virus isolates and host tests al., 2003), and which encodes from the Ct-CI to the middle of NIa-Pro nt sequences, were almost identical to those Details of the TuMV isolates, their country of origin, constructed from entire genomic sequences in all three ML, original host plant, year of isolation, and host type (strain) MP, and NJ trees. The concats were used for evolutionary are shown in Table 1, together with details of the isolates for analyses. The corresponding regions of two sequences of which the complete genomic sequences have already been Japanese yam mosaic virus (JYMV) (Fuji and Nakamae, reported (Suehiro et al., 2004; Tomimura et al., 2003; 1999, 2000), one of Scallion mosaic virus (ScaMV) (Chen GenBank accession nos. AF394601 and AF394602). All the et al., 2002b), and one of Narcissus yellow stripe virus isolates were inoculated to Chenopodium quinoa and (NYSV) (Chen et al., 2003) were used to align the TuMV serially cloned through single lesions at least three times. concats as BLAST searches had shown them to be the They were propagated in B. rapa cv. Hakatasuwari or N. sequences in the international sequence databases most K. Tomimura et al. / Virology 330 (2004) 408–423 421 closely and consistently related to those of TuMV; TuMV al., 1985). For MP analyses, the heuristic search option P1 genes were more closely related to those of JYMV than and 1000 bootstrap resamplings were used. For NJ ScaMV, whereas for some other TuMV regions/genes such analyses, distance matrices were calculated by DNADIST as Ct-CI+6K2+VPg+NIa-Pro it was the converse, except with the Kimura two-parameter option (Kimura, 1980), that the TuMV CP gene is most closely related to that of and trees constructed from these matrices by the NJ NYSV. We thus aligned all 148 P1 sequences with those of method (Saitou and Nei, 1987). A bootstrap value for each two JYMV isolates as the outgroup, the Ct-CI+6K2+VPg+ internal node of the NJ trees was calculated using 1000 NIa-Pro sequences with those of JYMV and ScaMV, and the random resamplings with SEQBOOT (Felsenstein, 1985). CP sequences with that of NYSV using CLUSTAL X The calculated trees were displayed by TREEVIEW (Page, (Jeanmougin et al., 1998). However, this procedure resulted 1996). One ScaMV and two JYMV sequences were used in some gaps, which were not in multiples of three nts. as the outgroup to construct a phylogenetic tree of the Therefore, the amino acid sequences corresponding to concat regions because the CP sequence of only one individual regions with the appropriate outgroups shown NYSV isolate was available (Chen et al., 2003). The above were aligned using CLUSTAL X with TRANS- JYMV and ScaMV sequences corresponding to individual ALIGN (kindly supplied by Georg Weiller) to maintain the gene regions within the concats were aligned as the degapped alignment of the encoded amino acids and then encoded amino acids using CLUSTAL X with TRANS- reassembled to form sequences 3315 nt long. The aligned ALIGN to maintain the degapped alignment of the nts and sequences were checked for incongruent relationships, then reassembled to form sequences 2979 nt long. which might have resulted from recombination, using SISCAN Version 2 (Gibbs et al., 2000) and PHYLPRO (Weiller, 1998). These analyses also assessed which non- Acknowledgments recombinant sequences have regions that are closest to regions of the recombinant sequences and hence indicate the We thank Ryo Hirota, Chikage Morodomi, and Erina likely lineages that provided those regions to the recombi- Tsuji (Saga University, Japan) for their careful technical nants. For simplicity, we call these the dparentalT isolates of assistance. We thank Drs. K. Bech (Denmark), I. Cooper recombinants, although they are merely those that include (United Kingdom), M. J. Gibbs (Australia), P. Hunter (United the most closely related regions. Parental lineages of the Kingdom), C. Kerlan (France), R. Loewe (Germany), B. recombinants were estimated from the phylogenies con- McKeown (United Kingdom), K. Ostrowka (Poland), P. structed from successive sliced concats; constructed depend- Pradhanang (Nepal), A. Ragozzino (Italy), T. van der Horst ent on the sequences of each 5Vand 3Vside of RS boundaries (The Netherlands), J. Verhoeven (The Netherlands), and H. J. of the recombinants. The transversion and transition rates of Vetten (Germany) for supplying TuMV isolates. This work all pairs of sequences were calculated using the DAMBE was supported by Grant-in-Aid for Scientific Research no. program (Xia and Xie, 2001). 15580036 from the Japan Society for the Promotion of Science. Variation analyses

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