Trees were manually edited with MEGA X . The DNA-A and DNA-B phylogenetic trees were rooted with the sequences of the genomic DNA of the OW monopartite begomovirus tomato yellow leaf curl virus and the DNAB component of the OW bipartite begomovirus African cassava mosaic virus , respectively.Preliminary datasets of complete sequences of 584 DNA-A and 240 DNA-B components were assembled. This included the complete nt sequences of the DNA-A and DNA-B components of: the bipartite begomoviruses from the M1-M4 samples; the TbLCuCV isolates from CU; and sequences of selected viruses retrieved from GenBank. SDT and the Recombination Detection Program version 4.0 were used to remove sequences that were identical or having nt sequence identities <70%. Final datasets of complete sequences of 488 DNA-A and 201 DNA-B components were used for recombination analyses. MSA were generated with MUSCLE within MEGA X , and the alignments were manually edited and exported as FASTA files. Detection of recombination breakpoints and identification of potential parental viruses were performed with RDP4. The recombination analysis was performed with default settings and a Bonferroni-corrected p-value cut-off of 0.05. Only recombination events detected with three or more methods coupled with significant phylogenetic support were considered bona fide events.In the phylogenetic tree generated with the complete DNA-A sequences, the TbLCuCV isolates from Hispaniola formed a strongly supported clade with the isolates from CU. Within this clade there was evidence of genetic divergence between isolates from CU and Hispaniola, grow trays consistent with geographical separation . In this tree, AbGYMV was placed on a distinct branch , which was included in a larger strongly supported clade with the TbLCuCV isolates.
This clade was part of the larger C1 clade of the AbMV lineage, which includes mostly weed-infecting begomoviruses from the Caribbean Basin , whereas the other large clade included crop- and weed-infecting begomoviruses from many countries of Latin America . The phylogenetic tree generated with the complete DNA-B sequences revealed a similar overall topology, but with some notable differences. The TbLCuCV isolates from Hispaniola and CU were placed in a strongly supported clade in the AbMV lineage . In contrast to the DNA-A tree, AbGYMV did not form a sister clade with the TbLCuCV isolates, but was placed together with the TbLCuCV isolates and other weed-infecting bipartite begomoviruses from the Caribbean Basin in the strongly supported C1 clade of the AbMV lineage . In the DNA-B tree, the C2 clade included viruses from North and Central America and the Caribbean Basin, whereas more distantly related viruses from South America were placed in a paraphyletic group . Finally, whereas the DNA-A tree clearly separates the BGYMV, Brazil, SLCuV and BoGMV lineages, these clades clustered together in a larger clade in the DNA-B tree . Taken togetherwith the SDT analysis and sequence comparisons, the results of the phylogenetic analyses are consistent with TbLCuCV and AbGYMV representing distinct but closely related species, which are most closely related to NW bipartite begomovirus species infecting weeds in the Caribbean Basin.In a preliminary experiment, N. benthamiana plants agroinoculated with the multimeric cloned DNA-A and DNA-B components of TbLCuCV-[HT:14] were stunted and newly emerged leaves showed epinasty, crumpling, deformation, mosaic and vein yellowing by 14 dpi .In the host range experiment, the infectious cloned DNA-A and DNA-B components of TbLCuCV induced stunting and golden/yellow mosaic in newly emerged leaves of all agroinoculated Malachra sp. plants by 14 dpi .
These symptoms were similar to those observed in Malachra sp. plants in the field in Hispaniola , thereby fulfilling Koch’s postulates for the golden/yellow mosaic disease of Malachra sp. TbLCuCV also induced stunting and epinasty and crumpling of newly emerged leaves of agroinoculated N. tabacum and N. glutinosa plants, and stunting and epinasty, deformation, chlorosis and mosaic of newly emerged leaves of agroinoculated common bean plants by 14 dpi . D. stramonium plants agroinoculated with TbLCuCV developed chlorotic spots in newly emerged leaves, whereas symptomless DNA-A and DNA-B infections were detected in some agroinoculated tomato plants by 14 dpi . TbLCuCV did not infect Cayenne long pepper , pumpkin and C. amaranticolor plants. N. benthamiana plants agroinoculated with the multimeric cloned DNA-A and DNA-B components of AbGYMV were stunted and developed mild symptoms of epinasty and crumpling in newly emerged leaves and no obvious mosaic or vein yellowing by 14 dpi . These symptoms became progressively milder by 21 dpi . In the host range experiment all Abutilon sp. plants agroinoculated with the infectious DNA-A and DNA-B components of AbGYMV were stunted and developed epinasty and striking golden/yellow mosaic of newly emerged leaves by 14 dpi . Moreover, these symptoms were similar to those observed in Abutilon sp. plants in the DO , thereby fulfilling Koch’s postulates for the golden/yellow mosaic disease of Abutilon sp. in the DO. In contrast, agroinoculated Malachra sp. plants developed no symptoms and only a small number of plants had DNA-A only infections . AbGYMV induced mild upward leaf curling symptoms in N. glutinosa, and very mild symptoms of leaf epinasty in common bean by 14 dpi . Symptomless DNA-A and DNA-B infections were detected in agroinoculated N. tabacum and D. stramonium plants, whereas symptomless DNA-A only infections were detected in some tomato by 14 dpi . AbGYMV did not infect Cayenne long pepper, pumpkin, C. amaranticolor and A. indicum plants.
In all these experiments, the presence of the inoculated DNA-A and DNA-B components was confirmed in newly emerged leaves of representative symptomatic and in all non-symptomatic plants by PCR tests with component-specific primers . Plants agroinoculated with the empty vector or bombarded with gold particles alone did not develop symptoms and were negative for the TbLCuCV/AbGYMV DNA-A and DNA-B components.To further investigate the relationship between TbLCuCV and AbGYMV, pseudorecombination experiments were conducted in N. benthamiana and Malachra sp. plants . In N. benthamiana, pseudorecombinants formed with the TbLCuCV DNA-A and AbGYMV DNA-B or AbGYMV DNA-A and TbLCuCV DNA-B were highly infectious and induced severe symptoms by 14 dpi. The TA + AbB PR induced epinasty, crumpling, deformation, mosaic and vein yellowing symptoms, which were more similar to those induced by the TbLCuCV parent . In contrast, the AbA + TB PR induced mostly epinasty and crumpling symptoms, which were more similar to those induced by the AbGYMV parent . Thus, the symptoms induced by these PRs were associated with the source of the DNA-A component. Furthermore, the symptoms induced by both PRs were more severe than those induced by the AbGYMV parent . Taken together, these results suggest an important role for the DNA-A component in symptom development in this host. In equivalent experiments conducted in Malachra sp., both PRs were infectious, but at lower rates than in N. benthamiana. Furthermore, the PRs differed markedly in infectivity, with the TA + AbB PR having an infection rate of 80%, whereas that of the AbA + TB was only 22%. The symptoms induced by these PRs were different compared with those induced by the parental viruses. Thus, both PRs induced more severe symptoms than those induced by the AbGYMV parent . Furthermore, the TA + AbB PR induced epinasty, crumpling and deformation, but little yellow mosaic ; whereas the AbA + TB PR induced epinasty, crumpling, deformation as well as yellow mosaic by 14 dpi . These results suggest an important role for the DNA-A component in infectivity and a role for the DNA-B component in symptom development in Malachra sp. In PCR tests with component-specific primers, the inoculated DNA components were detected in newly emerged leaves of all symptomatic plants. Together, these results established that the components of these viruses are interchangeable, consistent with the conservation of critical CR sequences and their close phylogenetic relationship . Moreover, grow systems for weed infectivity and symptoms were host-dependent, involved both components and revealed evidence of differential adaptation of these viruses.Since the advent of agriculture humans have encountered plants that have frustrated their goal to manage their environment. Today, we call the plant pests that interfere with agriculture ‘weeds’. In the last few centuries, humans have taken an increasing interest in preserving and otherwise maintaining the biodiversity of more ‘natural’ [i.e., ‘less managed’ ] communities. Here, too, plant pests frustrate human intentions. In such situations, these plants are called ‘invasives’. Weeds and invasives are problematic plants at ends of a continuum of how intensively humans manage an ecosystem, with manicured lawns and cultivated croplands at one end, through forest plantations and rangelands, to natural, deliberately lightly managed, areas at the other end. Thus, the distinction between weeds and invasives, though often clear, is occasionally fuzzy or arbitrary.
Some plants can become weeds and/or invasives with the appropriate ecological opportunity and without any genetic change. But an increasing body of research has revealed that some plants have evolved to become pests. Following the publication of the book, The Genetics of Colonizing Species , evolutionary biologists began to focus on how weeds might evolve . The idea of evolution as a potential route to invasiveness has become rapidly accepted in the last two decades, not only for plants, but also for animals and microbes . With the goal of understanding whether and how weediness and invasiveness evolve, empirical studies are accumulating that compare problematic lineages with their putative ancestral populations, in plants as well as other organisms . Some of these studies compare genetic marker variation, often identifying changes in diversity and population genetic structure. Other descriptive studies compare phenotypic or ecological differences of the invasive or weed and those of putative source populations . The latter can suggest evolutionary changes, but ‘common garden’ experiments in both the invaded and the native range are often necessary to demonstrate genetically-based phenotypic or ecological differences between problematic organisms and their presumed progenitors . A classical case is that of a variety of barnyard grass [Echinochloa crus-galli var. oryzicola P. Beauv.], a noxious weed that has evolved to mimic domesticated rice . Barrett grew seedlings of E. crus-galli var. oryzicola, its progenitor, E. crus-galli var. crus-galli, and O. sativa in a common garden experiment measuring numerous morphological characters. Multivariate analysis of 15 quantitative characters revealed that, in their vegetative phase, rice and its weedy mimic are not significantly different morphologically from each other, despite being in different genera. However, both differed significantly from E. crus-galli var. crus-galli . Morphological crop mimicry is an adaptation that is the result of continued selection by visually based human weeding. Indeed, barnyard grass individuals in Japanese rice fields that most closely resemble cultivated rice plants morphologically are less likely to be removed from rice fields by hand-weeding . Apparently, thousands of years of hand-weeding rice selected for a crop mimic that is almost vegetatively indistinguishable from rice. Similar studies have been conducted for invasives. In a common garden experiment conducted in California, Dlugosch and Parker compared invasive California populations of the shrub Canary Islands St. John’s wort with the native populations of that species, including the genetically-determined precise source population . They found that California populations had evolved an increased growth rate relative to the source population. They also found a diversification of flowering phenology of the California plants that correlated with their latitudinal origins. Such apparently adaptive evolutionary changes are not uncommon, although some authors caution that alternative explanations can account equally well for the appearance of adaptation . Only a handful of experimental studies report no evidence for adaptive evolution in invasives relative to their putative source populations . The example of Dlugosch and Parker is exceptional for invasives in that the progenitor population was precisely identified, allowing for the appropriate experimental comparison of progenitor and derived genotypes. But most often detailed information about source populations is, at best, lacking or at worst, complicated by an unknowable number of multiple introductions to multiple locations over decades with little knowledge about the time and place of initial invasion.A subset of weeds and invasives has evolved from domesticated ancestors, presenting certain advantages for study. We note that weeds and invasives can evolve from domesticate plants by two different pathways . Some, like California’s weedy rye are directly descended from a crop , though not all endoferal plant pests necessarily arise via evolutionary change. Other problematic plants, such as Europe’s weed beet , are descended from hybrids between a crop and another, usually wild, taxon .