Download Rapid evolutionary dynamics of zucchini yellow mosaic virus

Journal of General Virology (2008), 89, 1081–1085
Short
Communication
DOI 10.1099/vir.0.83543-0
Rapid evolutionary dynamics of zucchini yellow
mosaic virus
Heather E. Simmons,1 Edward C. Holmes1,2 and Andrew G. Stephenson1
Correspondence
Edward C. Holmes
[email protected]
Received 25 October 2007
Accepted 8 January 2008
1
Center for Infectious Disease Dynamics, Department of Biology, The Pennsylvania State
University, University Park, PA 16802, USA
2
Fogarty International Center, National Institutes of Health, Bethesda, MD 20892, USA
Zucchini yellow mosaic virus (ZYMV) is an economically important virus of cucurbit crops.
However, little is known about the rate at which this virus has evolved within members of the family
Cucurbitaceae, or the timescale of its epidemiological history. Herein, we present the first analysis
of the evolutionary dynamics of ZYMV. Using a Bayesian coalescent approach we show that
the coat protein of ZYMV has evolved at a mean rate of 5.0¾10”4 nucleotide substitutions per
site, per year. Notably, this rate is equivalent to those observed in animal RNA viruses. Using the
same approach we show that the lineages of ZYMV sampled here have an ancestry that dates
back no more than 800 years, suggesting that human activities have played a central role in the
dispersal of ZYMV. Finally, an analysis of phylogeographical structure provides strong evidence
for the in situ evolution of ZYMV within individual countries.
Zucchini yellow mosaic virus (ZYMV), first isolated in
1973 and described in 1981 (Lisa et al., 1981), is the cause
of one of the most economically important diseases of the
family Cucurbitaceae, naturally infecting plants in more
than 50 countries (Desbiez & Lecoq, 1997). Symptoms
include yellowing, stunting, leaf deformations, and misshaped and discoloured fruits, which often renders the
fruits unmarketable, drastically reducing agricultural yields
(Blua & Perring, 1989; Desbiez & Lecoq, 1997; Gal-On,
2007). Although ZYMV is widespread, few viral reservoirs
have been identified, particularly in temperate regions
(Desbiez & Lecoq, 1997).
ZYMV is a single-stranded, positive-sense RNA virus of the
family Potyviridae. The primary mode of transmission is
via aphids in a non-persistent manner. Although 10 aphid
species have been reported as vectors (Katis et al., 2006;
Lisa et al., 1981), a wider range of potential aphid vectors
has been identified under experimental conditions
(Blackman & Eastop, 2000; Katis et al., 2006). While aphid
transmission is undoubtedly the main route of spread for
ZYMV, infrequent seed transmission has also been
proposed (Robinson et al., 1993; Schrijnwerkers et al.,
1991), the epidemiological importance of which is
uncertain (Johansen et al., 1994).
ZYMV has a genome of 9593 nt arranged as a single open
reading frame encoding a polyprotein precursor that is
processed into 10 putative proteins (Gal-On, 2007). Of
these, the coat protein (CP) is involved in the encapsidation
The GenBank/EMBL/DDBJ accession numbers for the sequences
reported in this paper are EU371645–EU371650.
0008-3543 G 2008 SGM
of viral RNA, vector transmission (Shukla et al., 1991;
Urcuqui-Inchima et al., 2001), the regulation of viral RNA
amplification and cell-to-cell and systemic movement
(Urcuqui-Inchima et al., 2001). Transmission occurs as a
result of the interaction between the aphid stylet, CP and the
HC-Pro protein (Pirone & Blanc, 1996), such that some
mutations in CP and HC-Pro disrupt viral transmission
(Gal-On, 2007; Pirone & Blanc, 1996; Shukla et al., 1991;
Urcuqui-Inchima et al., 2001). The CP is also extensively
used as a tool to infer the phylogenetic relationships among
viral isolates (Rybicki & Shukla, 1992; Shukla et al., 1991).
A variety of studies have explored the extent and structure
of genetic diversity in ZYMV, particularly within a
biogeographical context. Analysis of a 250 nt fragment of
160 viral isolates sampled from 23 geographical areas
revealed two major groups of ZYMV, denoted A and B,
with the former divided into three clusters (Desbiez et al.,
2002). A subsequent analysis of the CP revealed three main
groups of isolates with differing geographical distributions
(Zhao et al., 2003). Group I included the majority of
European isolates, as well as some from China and Japan,
and a single Californian isolate. Group II was exclusively
composed of viruses from Asia, while group III included
several Chinese isolates. Notably, while group I and II
isolates resulted in mosaic symptoms on leaves and fruit
distortion, group III viruses did not cause symptoms on
the fruit, but induced severe mosaic symptoms on the
leaves (Zhao et al., 2003). More phylogenetically distant
ZYMV isolates were observed in Singapore and Réunion
(and other islands in the Indian Ocean representing group
B of Desbiez et al., 2002), which probably reflects their
biographical separation (Gal-On, 2007; Zhao et al., 2003).
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H. E. Simmons, E. C. Holmes and A. G. Stephenson
More localized phylogeographical studies have revealed
that viruses can diffuse within specific localities, such as
Central Europe (Glasa & Pittnerova, 2006; Glasa et al.,
2007; Tobias & Palkovics, 2003), perhaps mediated by the
local spread of aphids. However, isolates sampled from
adjoining locations are not always related (Pfosser &
Baumann, 2002), suggesting that biogeographical structure
may, to some extent, be determined by the international
trading of infected seeds (Desbiez et al., 2002; Tobias &
Palkovics, 2003).
There has also been considerable interest in using sequence
data from plant RNA viruses to infer evolutionary dynamics.
Although a combination of intrinsically high rates of
mutation, rapid replication and large population sizes are
thought to provide RNA viruses with abundant genetic
variation, some plant RNA viruses appear more genetically
stable than their animal counterparts (Garcı́a-Arenal et al.,
2001, 2003). This could be due to a combination of
intrinsically lower rates of mutation (Malpica et al., 2002)
and a reduced fixation rate of advantageous non-synonymous mutations because of weaker immune selection (Garcı́aArenal et al., 2001). Similarly, genetic bottlenecks play a
major role in structuring genetic diversity during both
systemic infection (French & Stenger, 2003; Li & Roossinck,
2004; Sacristán et al., 2003) and horizontal transmission by
aphids (Ali et al., 2006).
Despite the agricultural importance of ZYMV, there has
been little work documenting either the rate of molecular
evolution of this virus or the age of the sampled genetic
diversity, reflected in the time to the most recent common
ancestor (TMRCA). However, this information is central to
understanding the evolutionary dynamics of plant RNA
viruses in general, and particularly whether they exhibit
reduced rates of evolutionary change, which in turn may
have major implications on their ability to emerge in new
host species.
Cucurbita pepo ssp. texana is an annual monoecious vine
that is native to northern Mexico, Texas, and the lower
Mississippi River drainage area. It is thought to be either
the wild progenitor of the cultivated squashes (C. pepo ssp.
pepo) or an early escape from cultivation (Decker &
Wilson, 1987; Decker-Walters, 1990; Decker-Walters et al.,
2002; Lira et al., 1995). ZYMV infection of plants collected
during the 2006 growing season was determined immunologically (DAS-ELISA test kit; Agdia). Leaf tissue from
infected plants was then homogenized in liquid nitrogen
and RNA extracted using a Qiagen RNeasy Plant Mini kit.
First-strand cDNA was synthesized from the extracted
RNA using Superscript III First-Strand kit (Invitrogen).
The target cDNA was then amplified directly via PCR and
sequenced. The CP-specific primers used for the cDNA,
PCR and sequencing steps were: forward, 59-AAGATTGGCACGCTA-39; reverse, 59-CGGTAAATATTAGAATTAGCTCG-39. All sequences generated here have been
submitted to GenBank and assigned accession numbers
EU371645–EU371650.
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A total of six ZYMV CP, newly acquired here, were
combined with 49 collected from GenBank (accession
numbers available from the authors on request), producing
a total dataset of 55 CP sequences, 815 nt in length. To
determine the evolutionary relationships among all 55
sequences we employed the maximum-likelihood (ML)
method available within the PAUP* package (Swofford,
2003). The best-fit model of nucleotide substitution was
determined by MODELTEST (Posada & Crandall, 1998) as
TIM+I+C4 and this was used as the basis for tree
bisection-reconnection
branch-swapping
(parameter
values available from the authors on request). A bootstrap
resampling approach (1000 replications), employing the
ML substitution model, was used to assess the support for
individual nodes. To determine the strength of phylogenetic clustering by country of virus isolation we employed a
parsimony character mapping approach (Carrington et al.,
2005). Each ZYMV sequence was therefore assigned a
character state reflecting its country (or continent) of
origin. Given the ML phylogeny for these sequences, the
minimum number of state changes needed to produce the
observed distribution of country character states was
estimated using parsimony (excluding ambiguous
changes). To determine the expected number of changes
under the null hypothesis of complete mixing among
countries, the states of all isolates were randomized 1000
times. The difference between the mean number of
observed and expected state changes indicates the level of
geographical isolation, with statistical significance assessed
by comparing the total number of observed state changes
to the number expected under random mixing. All analyses
were performed using PAUP* (Swofford, 2003).
The rate of nucleotide substitution per site, as well as the
TMRCA of the ZYMV CP sequences were estimated using
the Bayesian Markov chain Monte Carlo approach
implemented in the BEAST package (Drummond &
Rambaut, 2007). This approach analyses the distribution
of tip times on millions of plausible sampled phylogenies,
so that estimates are set within a rigorous statistical
framework. As this analysis requires time-structured data,
where the date of sampling of each isolate is known, it was
restricted to a subset of 35 CP sequences for which the year
of sampling was available, representing a 22 year period
from 1984 to 2006. In the case of eight Chinese viruses,
sampling dates were known only to the nearest two
possible years. To account for this uncertainty, analyses
were repeated using the different sampling times available.
We also compared the demographical models of a constant
population size and exponential population growth,
employing both strict and relaxed (uncorrelated lognormal) molecular clocks. Bayes factors were used to
determine the best supported model. Because the
TIM+I+C4 substitution model is unavailable in the
BEAST package, the closely related GTR+I+C4 model was
used in its place. The extent of statistical uncertainty in
parameter estimates is reflected in the 95 % highest
probability density values. Finally, site-specific selection
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Journal of General Virology 89
Evolution of zucchini yellow mosaic virus
pressures in the 55 CP dataset were estimated as the ratio of
non-synonymous (dN) to synonymous substitutions (dS)
per site (ratio dN/dS) using both the single likelihood
ancestor counting (SLAC) and fixed effects likelihood
(FEL) methods, available at the Datamonkey facility
(Kosakovsky Pond & Frost, 2005).
In accord with other studies of the phylogeography of
ZYMV, distinct clusters of viral isolates are apparent in the
ML tree of 55 CP sequences (Fig. 1). These clusters
represent: (i) a large group of isolates sampled from a
variety of locations in Asia (China, Japan, Korea and
Taiwan), Europe and the Middle-East (Austria, Germany,
Israel, Italy, Hungary and Slovenia), and USA, and
previously denoted as groups I and II; (ii) China
(previously denoted group III); and (iii) Singapore and
the Réunion Island (previously unclassified). We found no
compelling evidence for the existence of group II isolates
(from Asia), as these fell within the phylogenetic diversity
of group I viruses, and suggest that those isolates from
Singapore and the Réunion Island are so phylogenetically
distinct that they be assigned to their own group.
A number of inferences can be made from this spatial
pattern. First, the greatest level of genetic diversity,
including the deepest phylogenetic split, is seen in Asia
(particularly China), including the presence of one clade of
viruses that has only been observed (to date) in China.
Although this is compatible with the lineages of ZYMV
sampled here having an origin in Asia, this will need to be
confirmed with a larger sample of isolates. Second, other
than a virus sampled in Florida in 1984, all other USA
isolates, sampled between 1992 and 2006 and including
those newly obtained from Pennsylvania, have a single
common ancestor (Fig. 1). Although the sample size is
small, this suggests that there has been some in situ
evolution of ZYMV in the USA since this time, without the
importation of new viral material. Our parsimony analysis
of geographical structure also revealed a strongly significant clustering by country of origin compared with that
expected by chance alone (P,0.001). A similarly strong
clustering was observed by continent (Americas, Asia,
Europe and the Middle-East, Indian Ocean; P,0.001).
Hence, although ZYMV is able to cross geographical
boundaries as indicated by the many countries represented
within groups I/II, such gene flow is not sufficiently
frequent to eradicate geographical structure. More generally, this strong spatial clustering suggests that there is
little vertical transmission of ZYMV through cultivated
curcubits, because commercial seeds of cultivated species
are likely to be frequently transported across national
borders.
The best supported evolutionary model for the CP of
ZYMV under our Bayesian coalescent analysis was that of
exponential population growth under a relaxed molecular
clock (Table 1). Under this model the mean rate of
evolutionary change for ZYMV was 5.061024 nucleotide
substitutions per site, per year. Similar rates were obtained
under different demographic and molecular clock models,
incorporating the different possible sampling times for
those viruses for which the exact year of sampling was
unknown, and using a range of prior values for the
Fig. 1. ML tree of 55 ZYMV CP sequences.
For viruses where the year of sampling is
available, these dates are given in parentheses.
Those viruses sampled as part of this study are
shaded grey. The group nomenclature
depicted represents that previously proposed
for ZYMV (Zhao et al., 2003). The tree is drawn
to a scale of 0.05 nt substitutions per site and
bootstrap values (.90 %) are shown next to
relevant nodes. The tree is mid-point rooted for
clarity only.
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H. E. Simmons, E. C. Holmes and A. G. Stephenson
Table 1. Bayesian estimates of population dynamic and evolutionary parameters of the CP gene of
ZYMV
HPD, Highest probability density (95 %).
Parameter estimate
Date range (years)
Sample size
Substitution rate
TMRCA
Mean (HPD)
22 (1984–2006)
35 sequences
5.061024 subs per site per year (1.8–8.861024 subs per site per year)
408 years (119–771 years)
substitution rate, indicating that they are robust (results
available from the authors on request). This high
evolutionary rate falls within the normal range observed
in RNA viruses, most of which represent animal RNA
viruses (Jenkins et al., 2002; Hanada et al., 2004). As such,
we find no evidence that ZYMV evolves any more slowly
than animal RNA viruses that are subject to the same,
error-prone replication.
the cultivated cucurbits (as observed in a contemporary
setting; Perring et al., 1992), and (iii) the cultivation, in close
proximity, of cucurbit crops with diverse origins, which
allowed the virus to jump to new genera of the family
Cucurbitaceae. Overall, our study highlights the utility of
gene sequence data to reveal key aspects of the epidemiological history of plant RNA viruses.
Although repeated population bottlenecks undoubtedly
influence the genetic structure of viral populations in the
short term (Li & Roossinck, 2004), they will have no effect
on long term evolutionary rates if most substitutions are
selectively neutral. Similarly, although a weaker immune
response against plant RNA viruses will reduce the rate at
which some non-synonymous mutations accumulate
(Garcı́a-Arenal et al., 2001), the fact that these normally
constitute a minor fraction of the total number of
nucleotide substitutions means that they are unlikely to
have a major impact on long-term evolutionary rates. In
support of this we found no evidence for positive selection
acting on the CP of ZYMV using either the SLAC or FEL
methods; the predominant evolutionary pressure was that
of negative (purifying) selection, with a mean dN/dS of
0.108 and 106 of 271 codons negatively selected under the
SLAC method. This agrees with previous studies of the CPs
of plant RNA viruses, which indicate that they are subject
to relatively strong purifying selection (Chare & Holmes,
2004). Further, the lack of positive selection suggests that
experimental passage has not had a major impact on our
analyses. Although the rapid evolutionary rates observed
here for ZYMV will need to be verified for a wider range of
plant RNA viruses, the implication from this work is that
mutational and replicatory dynamics are similar across a
broad range of RNA viruses.
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