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Impact assessments of virus resistance transgene introgression into wild, free-living populations are important for determining whether these transgenes present a risk to agriculture or the environment. Transgenic virus-resistant Cucurbita pepo ssp. ovifera var. ovifera L. (squash) cultivars have been commercialized, and may be cultivated in close proximity to cross-compatible wild, free-living relatives (C. pepo subsp. pepo vars. ozarkana and texana). Therefore, the potential impact of these virus resistance transgenes was studied by surveying the incidence and fluctuations of virus infection (as assayed by ELISA), virus symptoms (which may not be seen in an infected plant) and population size in forty-three free-living C. pepo populations in Illinois, Missouri, Arkansas, Mississippi, Louisiana, and Texas. Ten of these populations were studied over three consecutive seasons. Depending on the year, 61% to 78% percent of the populations had at least one individual infected by at CMV, ZYMV or WMV2, but the median incidence of infection within populations was 13%. The observed infection level in free-living populations was consistent with levels defined as "low" in field plot experiments conducted by others, leading to the conclusion that transgenic virus resistance should not provide a significant fitness advantage to the free-living populations examined. Viral symptoms were detected in only 2% of plants observed, indicating that severity of viral infection was low. CMV, ZYMV, and WMV2 were not the only viruses infecting these populations, further reducing the likelihood that resistance to these viruses would release populations from constraints imposed by virus diseases.

Nucleotide substitutions (i.e., point mutations) are the primary driving force in generating DNA variation upon which selection can act. Substitutions called transitions, which entail exchanges between purines (A = adenine, G = guanine) or pyrimidines (C = cytosine, T = thymine), typically outnumber transversions (e.g., exchanges between a purine and a pyrimidine) in a DNA strand. With an increasing number of plant studies revealing a transversion rather than transition bias, we chose to perform a detailed substitution analysis for the plant family Cucurbitaceae using data from several short plastid DNA sequences. We generated a phylogenetic tree for 19 taxa of the tribe Benincaseae and related genera and then scored conservative substitution changes (e.g., those not exhibiting homoplasy or reversals) from the unambiguous branches of the tree. Neither the transition nor (A+T)/(G+C) biases found in previous studies were supported by our overall data. More importantly, we found a novel and symmetrical substitution bias in which Gs had been preferentially replaced by A, As by C, Cs by T, and Ts by G, resulting in the G-->A-->C-->T-->G substitution series. Understanding this pattern will lead to new hypotheses concerning plastid evolution, which in turn will affect the choices of substitution models and other tree-building algorithms for phylogenetic analyses based on nucleotide data.