Vegetative compatibility groups explain variation in the virulence of Verticillium dahliae on strawberry

Verticillium dahliae infection of strawberry (Fragaria x ananassa) is a major cause of disease-induced wilting in soil-grown strawberries across the world. To understand what components of the pathogen are affecting disease expression, the presence of the known effector VdAve1 was screened in a sample of Verticillium dahliae isolates. Isolates from strawberry were found to contain VdAve1 and were divided into two major clades, based upon their vegetative compatibility groups (VCG); no UK strawberry isolates contained VdAve1. VC clade was strongly related to their virulence levels. VdAve1-containing isolates pathogenic on strawberry were found in both clades, in contrast to some recently published findings. On strawberry, VdAve1-containing isolates had significantly higher virulence during early infection, which diminished in significance as the infection progressed. Transformation of a virulent non-VdAve1 containing isolate, with VdAve1 was found neither to increase nor decrease virulence when inoculated on a susceptible strawberry cultivar. There are therefore virulence factors that are epistatic to VdAve1 and potentially multiple independent routes to high virulence on strawberry in V. dahliae lineages. Genome sequencing a subset of isolates across the two VCGs revealed that isolates were differentiated at the whole genome level and contained multiple changes in putative effector content, indicating that different clonal VCGs may have evolved different strategies for infecting strawberry, leading to different virulence levels in pathogenicity tests. It is therefore important to consider both clonal lineage and effector complement as the adaptive potential of each lineage will differ, even if they contain the same race determining effector.


V. dahliae hosts and control methods
Verticillium dahliae Kleb. is a soilborne plant pathogenic fungus which can cause wilting on over 200 plant species, including many agricultural and horticultural crops 1 . This pathogen has a rapidly expanding host range, most notably on the crops lettuce and cauliflower where it has become a major pathogen within these industries 2 ; 3,4 . V. dahliae can not act as an effective saprophyte and thus must produce resting structures to persist within the soil between infection events. The resting structures are microsclerotia, these are robust melanised hyphae which are very difficult to eradicate and can persist in the soil for up to 20 years post-formation 1 . Soil fumigation is t he only effective control measure however it relies on the use of chemicals with harmful environmental effects which have either been banned or (in the EU/UK) are due to be phased out 5 . Furthermore, crop rotation is largely ineffective as a management strategy and there are no available fungicides to combat the disease once established 6,7 . Consequently, breeding natural host disease resistance into crops remains the most effective strategy to control Verticillium wilt disease.

The Ve1 resistance gene is operational against 'race 1' isolates
Pathogens have evolved to deliver effector proteins in order to suppress host PTI (pathogen associated molecular pattern [PAMP]-triggered immunity). If successful, the pathogen will cause effector-triggered susceptibility (ETS) in the host and as such allow the pathogen to cause disease 8 . The plant cell wall is the primary barrier preventing pathogen infection. In order to overcome this barrier, pathogens secrete cell wall-degrading enzymes (CDEs) which are able to catabolise plant cell wall polysaccharides 9 . Although CWDEs are pathogenicity genes [9][10][11] , some also function as PAMPs, and trigger host PTI independent of their depolymerisation activity [9][10][11] .
Plants have an innate immune system, part of which is comprised of so called 'resistance' genes that encode receptors to recognize molecular patterns of pathogen proteins 12 and effectors 13 . Dominant plant resistance genes that bring about resistance to V. dahliae 'race 1' isolates have been identified in cotton, lettuce, tomato, and sunflower [14][15][16][17] have been deployed successful in commercial tomato (Ve1) 16 , and lettuce (Vr1) 18 cultivars. Ve1 is a cell surface like receptor operative against 'race 1' of the pathogen (defined by resistance in tomato), which carries the avirulence gene VdAve1 19 , 20 . However, Ve1 on tomato and its orthologs in other crops are not effective against 'race 2' isolates which lack VdAve1 21 .
Although attempts have been made to find tomato cultivars resistant to race 2 isolates, no high level resistance has been discovered 22 .

Race structure in current crops
It was thought that genome rearrangement was responsible for the widespread success of V. dahliae as a pathogen across multiple host species 23 ; 24 , however, a similar pattern of rearrangement has been observed across the 10 species within the Verticillium genus, including those which are plant pathogens with a limited host range 25 . It is now proposed that gene loss is more important in defining differences in speciation and phenotypic diversity within the Verticillium spp. 25 .
V. dahliae has linage specific genome regions enriched in effectors which have arisen by segmental duplication to allow for functional divergence [24][25][26] . By contrast, the effector VdAve1 was most likely acquired by horizontal gene transfer from the host plant on a single occasion and this effector has undergone multiple independent gene loss events within different 'race 2' isolates of V. dahliae 20 ; 27 . Indeed, VdAve1 is flanked by several 'young' transposable elements in a lineage specific region of V. dahliae , which, it is believed provide a mechanism of gene loss 20 ; 27,28 . Furthermore, it was suggested that 'race 2' isolates should be divided into discrete groups based on the sequences in the genomic region around VdAve1 gene 24 with these regions representing each VdAve1 loss event. Greater complication of the race structure has arisen, whereby 'race 2' isolates infecting tomato in Japan can be split into two further races based on their differential pathogenicity on resistant rootstocks 29 , however further work is required to determine the causative effector(s).
A study investigating race distribution in Californian tomato exclusively isolated 'race 2' strains from tomato, despite isolating 'race 1' strains from other crop hosts. It was proposed that this is due to the predominant use of Ve1 containing tomato cultivars 30 . Likewise, V. dahliae strains from cotton, mint, olive, and potato were exclusively found to be 'race 2' isolates whereas all isolates from artichoke were 'race 1' and isolates from lettuce, pepper and Californian strawberry contained both races 31 . It is clear that there is a global overlapping distribution on 'race 1' and 'race 2' isolates however, the host determines the proportion of the races isolated 31 .
A complex race structure in strawberry has been defined through a series of pathology experiments by Govorova and Govorov (1992), who identified six newly described races of Russian V. dahliae isolates based on resistance associated with five cultivars 32 . This work did not seek to quantify race structure in terms of VdAve1 presence nor the effector complexes however there is support for a complex series of plant -pathogen interactions. Strawberry wilt resistance was identified at the QTL level, in wilt segregating progenies planted within a mixed inoculum field, resistance was not monogenic, but quantitative, thus providing further evidence for complex plant-pathogen interaction 33 .
V. dahliae is an asexual organism comprised of several discrete clonal lineages . Vegetative compatibility groups (VCGs) determine whether V. dahliae strains can form hyphal anastomosis bridges, leading to genetic exchange through nuclear fusion and subsequent haploidisation. VCGs limit the transmission of genetic material between unrelated strains and thus promote the maintenance of distinct clonal lineages 34

(4) Objective of this study
The objective of this study was to characterise whether UK strawberry cultivars were infected by both 'race 1' and 'race 2' isolates of V. dahliae and if so whether VdAve1 played any role in virulence or avirulence.

PCR detection of vegetative compatibility group
The vegetative compatibility group of the isolates used within the pathogenicity assay was determined through PCR amplification of a lineage specific region using the primers DB19 and DB22 35,37 . The PCR was conducted using Q5 High-Fidelity DNA polymerase with conditions of 98 o C 30 s; 35 cycles of 98 o C 10 s, 55 o C 30 s, 72 o C 30 s and 72 o C at 2 min.
Amplicon production was confirmed through gel electrophoresis on a 1 % agarose gel before PCR clean up using the Machery-Nagel Nucleospin® Gel and PCR clean up kit and sequenced in two directions using Sanger sequencing.

Phylogeny of VC groups
Multiple sequence alignment of the region amplified by the DB19/22 primer set was performed using MUSCLE 38 . A neighbour-joining phylogeny was constructed using the HKY85 substitution model, with support from 1000 Bootstrap replicates in the Geneious v10 software package ( www.geneious.com ) 39 . The resulting dendrogram was visualised using FigTree ( http://tree.bio.ed.ac.uk/software/figtree/ ).

Pathogenicity assay on strawberry
The wilt susceptible strawberry cv. Hapil was used to determine the pathogenicity of isolates. Conidial inoculum of thirteen isolates were prepared through transfer of five, 1 cm plugs of mycelia growing on PDA into 50 ml of PDB media incubated in the dark at 25°C and 150 rpm for one week. After filtration through 13 μm particle retention filter paper, the conidial density was adjusted to 1x10 6 spores.ml -1 using sterile distilled water. Three-week old strawberry runners were up-rooted from pots, excess soil was removed with water, and trimmed 2 cm from the root tips. Roots were dipped in spore suspensions for 5 min and re-planted in the pot. Plants were kept in a growth room with 16:8 hr light -dark cycles and at a constant temperature of 22 °C . Disease severity was surveyed following five categories: 0healthy shoots, all leaves green; 1 -single leaf yellow-brown; 2 -<25% leaves yellow-brown; 3-26-50% leaves yellow-brown; 4-51-75% leaves yellow-brown; 5-76-100% leaves yellow-brown, the plants dead 40 .

Statistical analysis
Wilt severity scores ( Y i ) [= 0, 1, 2, 3, 4 and 5] were analysed using a cumulative logit model in which Y i is assumed to follow a multinomial distribution. A cumulative logit model is a regression model for a cumulative logit 41 : where x k represents the k th explanatory variate, the k effect of x k , and α j the intercept for each cumulative logit. The larger the value of , the higher the probability of Y i falling in a category at the upper end of the category scale. It is the extension of the common logistic model with only two possible outcomes (i.e. diseased or healthy). estimates the change in the cumulative odds ratio (on log e scale) for one unit increase in the explanatory variate x . In the present study, there were three categorical explanatory variates: VCG, host and race group.

Generation of VdAve1 knock-in mutants and induction of VdAve1 expression
The known avirulence gene gene VdAve1 and its native promoter (called PAve1) were cloned from the 'race 1' isolate 12067 using the primer pair VdAve1C-F/R and NPAve1-F/R, respectively (Supplementary Table 1). The VdAve1 random insertion vector was constructed using USER-Brick vector system, 42 . Core USER Bricks, as well as the constitutive fungal promoter PgpdA and the selective marker Hygromycin, were amplified from the plasmid pRF-HU2-F/R and pRF-HUE-F/R using the primers specified in 42 ). Two different insertion vectors were assembled; one containing the Ave1 native promoter, and another using the constitutive promoter PgpdA. Correct vector assembly was then verified by PCR. The valid vectors were transformed into Agrobacterium tumefaciens isolate GV3101. After A.
tumefaciens -mediated transformation (ATMT) of V. dahliae , the hygromycin B-resistant transformants were purified through the single-spore purification process as described previously 43 . Amplicon insertion in the VdAve1 knock-in isolates was confirmed through PCR with the primer pairs Hyg-F/R and VdAve1-F/R (Supplementary Table 1) and qRT-PCR detection with the housekeeping genes of elongation factor 1-α ( EF-F/R) and ß-tubulin (btubulin-F/R), as well as the specific primers for VdAve1 (qAve1-F/R, Supplementary Table   1). Moreover, in order to induce VdAve1 expression, cultures of five VdAve1 knock-in mutants of each vector were incubated on PDA and Capek-Dox agar media for 5 days at 25°C before qRT-PCR quantification. Illumina data adaptor sequences and low-quality data were removed using fastqc-mcf (available from https://expressionanalysis.github.io/ea-utils/) and the output was assembled with SPAdes 44 . RepeatMasker and TransposonPSI were used to identify repetitive sequence regions 45 46 .

Genome assembly and annotation
Long reads generated from Pacbio sequencing of isolate 12008 were assembled using Canu and polished using Illumina MiSeq reads in Pilon to correct erroneous SNPs and InDels 47,48 . The assembly was edited in accordance with recommendations from the NCBI contamination screen (run as part of submission to Genbank in November 2016 for isolate 12008 and July 2017 for illumina-only isolates) with contigs split, trimmed or excluded as required. Quast 49 was used to summarise assembly statistics and BUSCO 50 was used to assess completeness of gene space within the assembly. Gene prediction was performed using Braker1 and CodingQuarry 51,52 and functional annotations predicted for these proteins using InterproScan 53 . Sequence data and annotated genomes were deposited at DDBJ/EMBL/GenBank within Bioprojects PRJNA344737 (isolate 12008) and PRJNA352681 (isolates 12151, 12153, 12158 and 12161).
For newly sequenced genomes and for the existing JR2, VdLs17 54 and Va.Ms.102 28 , putative virulence factors and effector candidates were identified within predicted gene models. Secreted proteins were identified using SignalP 4.1 55 , removing those that were predicted to contain a transmembrane domain (TMHMM) or a GPI anchor (GPIsom). Small secreted cysteine-rich proteins (SSCP) were identified from secretomes, detecting proteins shorter than 300 amino acid and having a cysteine content of greater than 3 %. Secreted cell wall degrading enzymes were identified within from those proteins predicted as carbohydrate active enzymes (CAZYmes) from the CAZY database 56 . Further effector candidates were identified in the predicted secretome using EffectorP 57 . LysM proteins were identified using CAZY hmm model CBM50.hmm. NPP1 proteins were identified from Interproscan function annotations ( IPR008701 ) .

Generation of a genome-wide phylogenetic tree
In order to study the genomic region surrounding the VdAve1 locus, Illumina MiSeq reads of the five UK 'race 2' isolates were mapped to the reference genome of JR2 using Bowtie2 and the alignment visually inspected using IGV 58 . The phylogenetic tree of these five 'race 2' isolates along with the isolates of VdLs17 and JR2 was generated using RealPhy 59 using V.
dahliae isolate JR2 as a reference genome.

Orthogroup analysis
Gene expansion was investigated through clustering proteins into ortholog groups and then identifying the number of genes from each organism within that orthogroup. The proteomes of the five sequenced isolates and reference isolates JR2, VdLs17 and VaM102 were clustered using OrthoMCL 60 using a cut-off length of 50 amino acids and an inflation value of 5.

UK strawberry isolates do not contain VdAve1
In order to investigate whether there is any evidence for the presence of VdAve1 in V.
dahliae in the UK, we screened a total of 38 isolates isolated from UK strawberry, hop, acer, raspberry, potato, phlox, cotinus, tomato and chrysanthemum by PCR using a pair of primers targeting a 351 bp region within the VdAve1 gene (see Table 1 and Supplementary  1A).

VdAve1 is not associated with a single VC group
Further characterisation of a subset of 30 isolates from the the initial screen for VdAve1 revealed variation in vegetative compatibility groups of the isolates in this study.
Contrary to recent, but not all reports, VdAve1 was found in two major VC clades-VC subclade II-1 and VC subclade II-2 ( Fig. 1) and not just a single VCG. The two VC clades identified in this study contain the previously reported VC groups on UK strawberry: VGC4B (nested within Subclade II-1) and VGC2B 824 (Subclade II-2) 35,36 . The presence of VdAve1 in a VC clade other than VCG2A (Subclade II-1) is in contrast to the recent finding of Jimenez-Dıaz but consistent with earlier studies by de Jonge 26,27 .

UK strawberry isolates display a range of virulence on a common susceptible cultivar
In order to understand variation in virulence of VdAve1 -containing isolates on strawberry, isolate pathogenicity was determined using a the susceptible cultivar of Fragaria x ananassa. Seven 'race one' VdAve1-containing isolates (two from UK hop, two from UK acer, three from Californian strawberry) and six 'race two' isolates (one from Californian strawberry and six from UK strawberry) were tested on the V. dahliae susceptible strawberry cv. Hapil. A wide range of symptoms were observed (Fig. 2a) ranging from full plant collapse to mild stunting. No clear association was observed between the presence of VdAve1 and the virulence of the isolate by the end of the experiment, at six weeks post-inoculation (Fig.   2b).

VdAve1 isolates, when infected on strawberry have higher virulence early in the infection process and VC group is significantly associated with overall virulence level
A cumulative logit model was used to assess the severity of disease over time. From the results presented in Table 2, a clear pattern is apparent. VdAve1 containing isolates across the VC groups display higher virulence early in the infection process, with the most pronounced effects at three weeks post inoculation. This variation is less visible as infection progresses and is undetectable at six weeks post-inoculation. Variation in pathogenicity associated with VC subclade grows in importance over time, with isolates in subclade II-2 displaying far higher pathogenicity, than isolates in subclade II-1. Isolate 12008 was found to be the most pathogenic isolate, with the highest level of disease recorded six weeks post inoculation (Fig. 2b). This isolate lacks VdAve1 but is within subclade II-2.

The presence of VdAve1 alone is neither correlated with gain or loss of virulence on strawberry in artificial transformation of a highly virulent isolate
In order to examine whether the virulence of 12008 could be increased by the action of VdAve1 we cloned the VdAve1 gene from a 'race 1' isolate 12067 from tomato and then transformed the VdAve1 expression vector into isolate 12008, a highly virulent isolate from UK strawberry using Agrobacterium tumefaciens mediated transformation. Vectors were constructed with either the inclusion of the native promoter region or use of a constitutive fungal promoter. In both cases five independent transformants (with the construct integrated at a random location in the genome) were selected for analysis. Strawberry plants inoculated with VdAve1 knock-in conidia cultured in PDA media showed comparable wilt disease severity to the wild type (WT) isolate 12008 irrespective of whether the native promoter or constitutive promoter was used. Furthermore, even though qRT-PCR results confirmed that VdAve1 knock-in mutants of PA4 with the native promoter and PG1 with a constitutive promoter had a similar VdAve1 expression level with that of the 'race 1' isolate 12067, no significant difference was found between disease scores of the two mutants compared with the wild type isolate 12008 (Fig. 3a). qRT-PCR results with cultures of VdAve1 knock-in mutants incubated on PDA or Czapek-Dox plates showed that VdAve1 expression levels were induced ( to differing degrees) on both PDA media and Czapek-Dox media (Fig. 3b). In conclusion the presence of VdAve1 did not increase the virulence of V. dahliae on strawberry, when transformed into a virulent isolate.  (Table 3).

Genome sequencing and assembly of selected isolates
Putative secreted effector proteins were identified using EffectorP and through searches for SSCPs. Results of effector prediction were broadly similar between the five sequenced isolates, with 187-196 EffectorP candidates and 125-140 SSCPs (Table 3) (Table 3). Putative NPP1 (necrosis-inducing phytophthora protein) effectors were also identified, with isolate 12161 carrying an additional NPP1 protein that was not present in the other four isolates (Table 3).

VdAve1 loss differ between VC groups
Even though the five isolates of 12008, 12251, 12253, 12158 and 12161 from UK strawberry were 'race 2' isolates, their virulence on strawberry was divergent as 12008 and 12251 can cause severe wilt disease while the isolate 12161 had very low virulence (Fig. 3A).
After sequencing, short reads of each genome were mapped to the reference genome JR2 using bowtie, which is a V. dahliae 'race 1' isolate. The read coverage drop flanking VdAve1 gene occurred earlier in the isolates of 12158 and 12161 than in isolates of 12008, 12251 and around 672 kb of the JR2 contig while no read coverage was observed over this region in the other three isolates (Fig. 4, panel A), indicating that these five 'race 2' isolates could be divided into two distinct groups.
Phylogenetic analysis of RealPhy using whole-genome sequencing reads of these 5 'race 2' isolates and the reference genome JR2 showed that V. dahliae isolates from UK strawberry should be divided into two clusters, consistent with the coverage mapping result.
In the phylogenetic tree, the isolates of 12158 and 12161 were clustered together in a separate clade from the other isolates (Fig. 4, panel B). Interestingly, the isolates of 12008, 12251, 12253, JR2 and VdLs17 were clustered together (Fig. 4, panel B), which is concordant with the VC group analysis (Fig. 1).

Effector expansion and losses in VC groups
To further identify those genes determining differences in virulence, orthology analysis was performed on the proteomes of the five UK isolates, V. dahliae reference isolates JR2 and VdLs17 and the V. alfalfae isolate VaMs102. In total, 80518 proteins clustered into 9697 orthogroups and a further 3110 proteins were considered orphan proteins, not clustering using OrthoMCL 60 . Results showed that the majority of proteins were conserved between V.
dahliae and V. alfalfae , with 7285 orthogroups common to all isolates representing 81 % of the proteins (8542) from 12008. A further 501 orthogroups were conserved between all V.
dahliae isolates, representing a further 5 % of proteins from 12008. A total of 361 orthogroups contained proteins predicted as effectors. Expansion of effector genes was investigated between VC subclades II-1 and II-2; 18 orthogroups were identified that showed patterns of gain or loss congruent with VC subclade (Table 4). Putative effector genes containing a glycoside hydrolase domain in orthogroup19 and a galactose oxidase domain in orthogroup73 were expanded in the higher virulence group isolates 12008, 12251 and 12253 ( Table 2,   Table 4), which may be responsible for aggressive performance. Nevertheless, effector genes containing the FAS1 domain in orthogroup700, necrosis inducing proteins in orthogroup736 and Zn (2)-C6 fungal-type DNA-binding domain in orthogroup824 were more abundant in the lower virulence isolates of 12158 and 12161 (Table 4, Supplementary Table 2).

Discussion
Our results clearly demonstrate that defining the clonal structure of Verticillium is key to understanding variability in pathogenicity and patterns of effector gain and loss. We conclude that VC subclade II-2 is associated with high virulence on the host plant strawberry, while VC subclade II-1, on the whole, contains isolates of lower virulence. To our knowledge this is the first time that variation in virulence on strawberry has been linked to VC group. However, comparable studies on the hosts artichoke and watermelon have also described an association of VC subclade II-2 with high virulence [61][62][63] indicating that this may be a common phenomena. High virulence was associated with VCG 1A on olive and cotton hosts, w hich may be explained by the presence of the high defoliating pathotype (D) within this lineage 64 ; 65 . In contrast, no significant association was found between any VCG and virulence of V.
dahliae on inoculated woody ornamentals, including Acer species 66 . We found no significant association between host of origin and variation in virulence on strawberry in our set of isolates (Table 2), which may suggest that the variation in virulence observed on some hosts (e.g. strawberry) that is associated with VC group may not be replicated in other hosts. This requires further study over a much larger number of isolates before a robust conclusion can be drawn. This requires further testing to determine the causative elements responsible for the differences in virulence seen between the two VCG subclades. It also remains to be seen whether there is any difference in the resistance of cultivars to the two different clonal lineages of V. dahliae . Previous QTL mapping experiments in strawberry have been conducted under field conditions in highly infested plots which were artificially inoculated with many different culture of V. dahliae 33 . Further work using inoculations with single isolates is needed to understand whether resistance responses vary between isolates that differ in VCG.
A recent study showed that 'race 2' isolates are obtained with high frequency in many crops with the exception of several crops including strawberry in California, where 'race 1' isolates are found at a higher frequency 31 . This suggests that an ortholog of Ve1 is either absent or non-functional in the octoploid strawberry. However, when screening 27 V. dahliae isolates from UK strawberry, all of them were found to be non-VdAve1 -containing 'race 2' isolates. Inoculation of common UK-grown wilt-susceptible cultivars of strawberry plants with 'race 1' isolates from other origins can cause wilt disease, indicating the lack of 'race 1' isolates on strawberry in the UK is not due to differences in host response between Californian and UK strawberry material. This indicates that Ve1-mediated resistance is not operating in the octoploid strawberry 20,70,71 .
It is possible that geographical isolation plays a role in the frequency of VdAve1 in the population. For example, all the artichoke isolates tested from California were 'race 1', though race 2 artichoke isolates from Tunisia have been reported, indicating that although the global migration of genotypes in V. dahliae has likely occurred and continues to play an important role in the diversification of fungal populations, within-field variation may be low and may be dominated by widespread clonal types. Thus, race structure in specific crops and commodities may exist, depending on the geography. Geography alone though does not fully explain the abundance of non-VdAve1 containing isolates sampled from UK strawberry fields, as it is clear that VdAve1 -containing isolates are present in the UK, as was revealed through extended sampling ( Supplementary Fig. 1). It is of course possible that VdAve1-containing isolates have remained associated with their respective hosts and have not spread to infect strawberry growing regions of the UK. More detailed and systematic sampling would allow the spatial and host-associated distributions of VdAve1-containing isolates to be determined.
The results of pathogenicity assays using 'race 1' isolates from Californian strawberries revealed that VdAve1 does enhance early development of pathogenic symptoms, the significance of which is attenuated as the disease progresses ( Isolate 12008 is the most virulent isolate, based upon the pathogenicity test that we carried out (Fig. 2) and therefore we conclude that other factors present in the 12008 genome provide an alternative route to high virulence, such that the effect of VdAve1 is masked.
Our work highlights that it is important that the nomenclature of V. dahliae is altered to move the classification of 'race' based on resistance responses in a single plant species, to one that is guided both by clonal lineage and effector complement as the adaptive potential of each lineage will differ, even if they contain the same effector.        T  a  b  l  e  3   G  e  n  o  m  i  c  s  t  a  t  i  s  t  i  c  s  o  f  V  e  r  t  i  c  i  l  l  i  u  m  s  t  r  a  i  n  s  .   S  t  r  a  i  n  1  2  0  0  8  1  2  2  5  1  1  2  2  5  3  1  2  1  5  8  1  2  1  6  1  J  R  2  *  L  s  1 V  C  s  u  b  c  l  a  d  e  I  I  -2  V  C  s  u  b  c  l  a  d  e  I  I  -1   A  n  n  o  t  a  t  i  o  n  1  2  0  0  8  1  2  2  5  1  1  2  2  5  3  L  s  1