Our analyses of new samples with new tools provide a revised view of paleopolyploidy in the Compositae. Together with results from a high density Lactuca linkage map, our results suggest that the Compositae and Calyceraceae have a common paleotetraploid ancestor and that most Compositae are descendants of a paleohexaploid. Although paleohexaploids have been previously identified, this is the first example where the paleotetraploid and paleohexaploid lineages have survived over tens of millions of years. The complex polyploidy in the ancestry of the Compositae and Calyceraceae represents a unique opportunity to study the long-term evolutionary fates and consequences of different ploidal levels.

Diseases of soybean caused by Cercospora spp. are endemic throughout the world’s soybean production regions. Species diversity in the genus Cercospora has been underestimated due to overdependence on morphological characteristics, symptoms, and host associations. Currently, only two species (Cercospora kikuchii and C. sojina) are recognized to infect soybean; C. kikuchii causes Cercospora leaf blight (CLB) and purple seed stain (PSS), whereas C. sojina causes frogeye leaf spot. To assess cryptic speciation among pathogens causing CLB and PSS, phylogenetic and phylogeographic analyses were performed with isolates from the top three soybean producing countries (USA, Brazil, and Argentina; collectively accounting for ~80% of global production). Eight nuclear genes and one mitochondrial gene were partially sequenced and analyzed. Additionally, amino acid substitutions conferring fungicide resistance were surveyed, and the production of cercosporin (a polyketide toxin produced by many Cercospora spp.) was assessed. From these analyses, the long-held assumption of C. kikuchii as the single causal agent of CLB and PSS was rejected experimentally. Four cercosporin-producing lineages were uncovered with origins (about 1 Mya) predicted to predate agriculture. Some of the Cercospora spp. newly associated with CLB and PSS appear to represent undescribed species; others were not previously reported to infect soybeans. Lineage 1, which contained the ex-type strain of C. kikuchii, was monophyletic and occurred in Argentina and Brazil. In contrast, lineages 2 and 3 were polyphyletic and contained wide-host range species complexes. Lineage 4 was monophyletic, thrived in Argentina and the USA, and included the generalist Cercospora cf. flagellaris. Interlineage recombination was detected, along with a high frequency of mutations linked to fungicide resistance in lineages 2 and 3. These findings point to cryptic Cercospora species as underappreciated global considerations for soybean production and phytosanitary vigilance, and urge a reassessment of host-specificity as a diagnostic tool for Cercospora.

Promoters of most seed proteins in legumes contain one or more 5'-CATGCAT-3' elements. To test if these elements have a function in the expression of these genes, the 2.3 kilobase pairs Gy2 glycinin promoter was ligated to a j6-glucuronidase reporter sequence and transformed into tobacco. Elimination of a 5'-CATGCAT-3' element 101 base pairs upstream from the transcription start site in the construction caused about a 10-fold reduction in the amount of j6-glucuronidase activity compared with when the element was present in the gene. Elimination of 1.9 kilobase pairs from the 5'-end of the promoter caused a twoto threefold reduction in activity. The results show that the 5'-CATGCAT-3' element plays a role in regulating the amount of expression from the gene, but that there are also other factors farther upstream from the gene that affect the level of expression.A DNA element with the sequence 5'-CATGCAT-3' has been implicated in the regulation of seed protein genes in legumes (6). It was first identified as part of a sequence that was absent from the 5'-flanking region of a lectin null-allele from Phaseolus vulgaris (8). Subsequently, sequences in which 7 of the 8 base pairs in the element were conserved were identified in the first 400 base pairs of promoters in 20 of 21 legume seed protein genes but were absent in the same region of nonseed protein genes. Because of the wide distribution of the 5'-CATGCAT-3' element among seed protein genes, a more general role for the elements in gene expression was proposed (6).The participation of 5'-CATGCAT-3' elements in gene expression is not based on a demonstration of function. Indeed, a number of unsuccessful efforts to demonstrate the binding of nuclear proteins to 5'-CATGCAT-3' elements have been reported (13,15 without the element. Evidence is presented that modification of the element results in a large decrease in ,r-glucuronidase activity in mature seeds from transformed tobacco plants. MATERIALS AND METHODS DNA ClonesThe promoter from the Gy2 glycinin gene from soybean was used for these studies. The gene containing it was derived from the genomic clone XDA28-30 (14). Gy2 was subcloned into pUC8 and sequenced as described by Thanh et al. (18). Tobacco TransformationPlasmid constructions were purified and used to transform the LB4404 strain ofAgrobacterium tumefaciens as described by An et al. (1). Minipreparations of Agrobacterium DNA were isolated from the transformants and checked for rearrangements by restriction mapping. The DNA sequence across the borders where ligation occurred was determined to assure that small deletions or insertions had not occurred. Tobacco of the variety SRI (gift from R. Schilperoort) was transformed by the leafdisc transformation method ofHorsch et al. (9). Shoots and roots were selected on medium containing 200 and 100 /ig/mL kanamycin, respectively. A single leaf from each primary transformant of tobacco was collected and the DNA from it purified. Polymerase chain reactions programmed with this DNA were carried out to ens...

Phakopsora pachyrhizi has dispersed globally and brought severe economic losses to soybean growers. The fungus has been established in Brazil since 2002 and is found nationwide. To gather information on the temporal and spatial patterns of genetic variation in P. pachyrhizi, we sequenced the nuclear internal transcribed spacer regions (ITS1 and ITS2). Total genomic DNA was extracted using either lyophilized urediniospores or lesions removed from infected leaves sampled from 26 soybean fields in Brazil and one field in South Africa. Cloning prior to sequencing was necessary because direct sequencing of PCR amplicons gave partially unreadable electrophoretograms with peak displacements suggestive of multiple sequences with length polymorphism. Sequences were determined from four clones per field. ITS sequences from African or Asian isolates available from the GenBank were included in the analyses. Independent sequence alignments of the ITS1 and ITS2 datasets identified 27 and 19 ribotypes, respectively. Molecular phylogeographic analyses revealed that ribotypes of widespread distribution in Brazil displayed characteristics of ancestrality and were shared with Africa and Asia, while ribotypes of rare occurrence in Brazil were indigenous. The results suggest P. pachyrhizi found in Brazil as originating from multiple, independent long-distance dispersal events.

Molecular phylogeography can lead to a better understanding of the interaction between past climate events, large-scale vegetation shifts, and the evolutionary history of Neotropical seasonal forests. The endangered timber tree species Cedrela fissilis is associated with seasonal forests and occurs throughout South America. We sampled C. fissilis from 56 sites across the species' range in Brazil and Bolivia and obtained sequence data for nuclear and chloroplast DNA. Most specimens (149 out of 169) exhibited intraindividual polymorphism for the nuclear internal transcribed spacer (ITS). Cloning and an array of complementary sequence analyses indicated that the multiple copies of ITS were functional paralogs--concerted evolution in C. fissilis appeared to be incomplete. Independent Bayesian analyses using either ITS or cpDNA data revealed two separate phylogenetic lineages within C. fissilis that corresponded to populations located in separate geographic regions. The divergence occurred in the Early Pliocene and Late Miocene. We argue that climate-mediated events triggered dispersal events and split ancestral populations into at least two large refugial areas of seasonal forest that were located to the east and west of the present day Cerrado. Upon recent climate amelioration, formerly isolated lineages reconnected and intraspecific hybridization gave rise to intraindividual polymorphism and incomplete concerted evolution in C. fissilis.