Comparative metabolomic and transcriptomic analyses revealed the differential accumulation of secondary metabolites during the ripening process of acerola cherry (Malpighia emarginata) fruit.

BACKGROUND: Acerola cherry is a famous functional fruit containing plentiful antioxidants and other nutrients. However, studies on the variations among nutrients during the ripening process of acerola fruit are scare. RESULTS: Comparative metabolomic and transcriptomic analyses were performed and identified 31 331 unigenes and 1896 annotated metabolite features in acerola cherry fruit. K Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that several antioxidant and nutrient-related metabolic pathways, such as the flavonoids, vitamins, carotenoids, amino acids, and fatty acids metabolic pathways, were significantly changed during the ripening process. The metabolites related to the vitamin, carotenoid, and fatty acid metabolic pathways were downregulated during the ripening process. Several flavonoid biosynthesis-related genes (including dihydroflavonol 4-reductase, chalcone synthase, flavanone 3-hydroxylase, and anthocyanidin synthase), were significantly upregulated, suggesting their essential functions in the accumulation of flavonoids in mature fruit. CONCLUSION: Most of the vitamin and carotenoid metabolism-related metabolites significantly accumulated in immature fruit, suggesting that immature acerola fruit is a good material for the extraction of vitamins and carotenoids. For macronutrients, most of the amino acids accumulated in mature fruit and most of the fatty acids greatly accumulated in immature fruit. Our data revealed the differential accumulation of antioxidants and nutrients during the ripening process of acerola cherry fruit. © 2021 Society of Chemical Industry.

Transferability and characterization of microsatellite markers from Byrsonima cydoniifolia A. Juss. (MALPIGHIACEAE) in seven related taxa from Cerrado biome reveal genetic relationships.

Byrsonima Rich. is one of the largest genera of the Malpighiaceae family with 97 species occurrence in Brazil and multiple potentialities, including pharmaceutical and food industries. In this study, 17 microsatellite markers characterized in Byrsonima cydoniifolia were tested for seven related taxa, all species are native to Brazil and four are endemic. Genomic DNA was extracted from leaves tissues and 17 microsatellite markers were used to cross-amplification of microsatellite regions. Polymorphism and genetic diversity were evaluated for B. intermedia, B. verbascifolia, B. laxiflora, B. subterranea, B. umbellata, B. linearifolia. from 16 individuals and for B. viminifolia from 14 individuals. Transferred microsatellite markers panels ranged from 11 (64.8%) in B. viminifolia to 6 (35.2%) in B. umbellata. The total number of alleles per locus ranged from 5 (B. linearifolia) to 8 (B. subterranea) alleles. B. umbellata showed lower values of observed and expected heterozygosity (HO = 0.312; HE = 0.436) and B. subterranea presented the highest values (HO = 0.687; HE = 0.778). A greater number of microsatellite markers should be developed for B. umbellata. The microsatellite marker panels transferred to the species B. intermedia, B. verbascifolia, B. laxiflora, B. subterranea, B. viminifolia and B. linearifolia are very informative, with a high combined probability of exclusion of paternity (Q ≥ 0.976) and the low combined probability of identity (I ≤ 9.91 × 10-6), potentially suitable for future genetic-population studies, supporting strategies for maintaining the genetic diversity and for exploration of Byrsonima species as genetic resources.

Genetic Diversity Assessment of a Chinese Medicinal Endemic Species, Aspidopterys obcordata var. obcordata, by Combined Molecular Marker Methods (ISSR & SRAP).

Aspidopterys obcordata var. obcordata, a medicinal plant endemic to China, is a narrowly distributed species and wild resources are extremely limited. To evaluate the genetic variability and degree of genetic divergence of A. obcordata var. obcordata, and to make rational scientific decisions on its harvest and germplasm conservation, we collected 122 samples from across nearly all of its distribution area and studied genetic diversity using inter-simple sequence repeats (ISSRs), sequence-related amplified polymorphisms (SRAPs), and a method combining the two techniques. The results revealed the high genetic diversity of A. obcordata var. obcordata, mainly due to its intra-population diversity, and the top two populations with the highest levels of intra-population diversity were ML and DH, individuals of which can serve as excellent germplasm candidates during the processing of germplasm screening and conservation. In general, the combining method was prior to the ISSR analyses and SRAP analyses results, except for a slight difference in the genetic structure of individual populations. Therefore, we suggest that a combination analysis of the two marker methods is ideal for evaluating the genetic diversity and genetic relationships of A. obcordata var. obcordata.

Identification and evaluation of reference genes for reliable normalization of real-time quantitative PCR data in acerola fruit, leaf, and flower.

Understanding into acerola (Malpighia emarginata) molecular and biochemical bases is still obscure, despite it is one of the most important natural source of vitamin C for humans. Recently, our research group published the first data on acerola transcriptome generating valuable information to identify reference genes for RT-qPCR in this species. Hence, this study aimed to identify the most stably expressed genes based on acerola transcriptome data, and further to evaluate the suitability of F-box, U3, Merad50-ATPase, TGD4, NOB1, PA-RNA, RCC1, RBL and PGAL candidates for accurate gene expression normalization in leaf, flower and fruit at 12, 16 and 20 days after anthesis using RT-qPCR analysis. Three algorithms, geNorm, NormFinder, and BestKeeper confirmed the expression stability of all nine candidate reference genes, whereas RefFinder consensually summarized a comprehensive gene ranking. Based on geNorm, the combination of the most stable reference genes RBL and U3 for leaf/flower group, TGD4, F-box and PGAL (fruit developmental stages or fruit/leaf), RCC1, PGAL and RBL (fruit/flower) and RCC1, RBL, TGD4 and PGAL (total samples) were required for accurate normalization. Moreover, the use of these reference genes to assess the expression profile of GMP1 and NAT3 genes confirmed the reliability of ranking and defined the best combination of genes recommended by geNorm and RefFinder. This work will benefit further RT-qPCR studies in these acerola organs by offering a foundation for accurate normalization of gene expression profiling.

Transcriptome analysis of acerola fruit ripening: insights into ascorbate, ethylene, respiration, and softening metabolisms.

KEY MESSAGE: The first transcriptome coupled to metabolite analyses reveals major trends during acerola fruit ripening and shed lights on ascorbate, ethylene signalling, cellular respiration, sugar accumulation, and softening key regulatory genes. Acerola is a fast growing and ripening fruit that exhibits high amounts of ascorbate. During ripening, the fruit experience high respiratory rates leading to ascorbate depletion and a quickly fragile and perishable state. Despite its growing economic importance, understanding of its developmental metabolism remains obscure due to the absence of genomic and transcriptomic data. We performed an acerola transcriptome sequencing that generated over 600 million reads, 40,830 contigs, and provided the annotation of 25,298 unique transcripts. Overall, this study revealed the main metabolic changes that occur in the acerola ripening. This transcriptional profile linked to metabolite measurements, allowed us to focus on ascorbate, ethylene, respiration, sugar, and firmness, the major metabolism indicators for acerola quality. Our results suggest a cooperative role of several genes involved in AsA biosynthesis (PMM, GMP1 and 3, GME1 and 2, GGP1 and 2), translocation (NAT3, 4, 6 and 6-like) and recycling (MDHAR2 and DHAR1) pathways for AsA accumulation in unripe fruits. Moreover, the association of metabolites with transcript profiles provided a comprehensive understanding of ethylene signalling, respiration, sugar accumulation and softening of acerola, shedding light on promising key regulatory genes. Overall, this study provides a foundation for further examination of the functional significance of these genes to improve fruit quality traits.

High levels of expression of multiple enzymes in the Smirnoff-Wheeler pathway are important for high accumulation of ascorbic acid in acerola fruits.

Acerola fruits contain abundant ascorbic acid (AsA). The gene expression levels of three upstream enzymes in the primary AsA biosynthesis pathway were correlated with AsA contents in the fruits of two acerola cultivars. Multiple overexpression of the enzymes increased AsA contents, suggesting their high expression is important for high AsA accumulation in acerola fruits and the breeding of AsA-rich plants. Abbreviations: AsA: ascorbic acid; PMI: phosphomannose isomerase; PMM: phosphomannomutase; GMP: GDP-d-mannose pyrophosphorylase; GME: GDP-d-mannose 3',5'-epimerase; GGP: GDP-l-galactose phosphorylase; GPP: l-galactose-1-phosphate phosphatase; GDH: l-galactose dehydrogenase; GLDH: l-galactono-1,4-lactone dehydrogenase.

A novel regulatory element responsible for high transcriptional expression of acerola GDP-d-mannose pyrophosphorylase gene.

GDP-d-mannose pyrophosphorylase (GMP) is one of the enzymes that highly expressed in acerola plants. A promoter assay suggests the presence of a new cis-element in the -1087 to -1083 bp sequence of the MgGMP promoter. Moreover, cis-elements, present in the -1080 to -600 bp sequence of the MgGMP promoter, function as enhancers of MgGMP expression.

Genetic variability analysis of Byrsonima crassifolia germplasm collected in Pará State using ISSR markers.

Native of the Amazon, the nanche (Byrsonima crassifolia) is a fruit cultivated by family farmers and used in cooking; as such, it represents an opportunity for regional agribusiness. The Embrapa Eastern Amazon set up an active germplasm bank (BAG) consisting of 22 accessions sampled in 11 municipalities of Pará State. Due to its economic potential, there is an interest to advance the genetic breeding program of this species. The aim of this study was to characterize the BAG nanche collection using inter-simple sequence repeat (ISSR) markers. Accessions were genotyped using 23 pre-selected ISSR primers resulting in 109 amplified polymorphic and 51 monomorphic bands. With eight polymorphic bands each, the most polymorphic primers were UBC 809 and UBC 848. An unweighted pair-group method with arithmetic average cluster analysis based on Jaccard's coefficient indicated that the individuals clustered into two distinct groups. Accessions Igarapé Açu-2 and Augusto Corrêa-Pl 1 were most similar. The genetic dissimilarity values ranged from 0.10 to 0.59. We conclude that the ISSR markers were efficient in detecting polymorphisms in the nanche accessions, and that it is possible to infer the genetic variability among accessions of the collection. This demonstrate the importance of using molecular markers in poorly studied species and the advantages that this information can bring to the genetic improvement of such species.

Response of Malpighia emarginata active germplasm bank accessions to Meloidogyne enterolobii parasitism.

Malpighia emarginata is cultivated in almost all Brazil and is considered an important agricultural crop. The root-knot nematode Meloidogyne enterolobii has been described as a major threat to this crop, causing great production losses. Due to the scarcity of information about the severity of this parasite in M. emarginata plants in Brazil, this study investigated M. enterolobii resistance of ten M. emarginata genotypes from the active germplasm bank of Universidade Federal Rural de Pernambuco. The experiment was conducted adopting a completely randomized design in a factorial arrangement of 11 x 2 x 5, where M. emarginata cuttings were inoculated with 10,000 eggs in a greenhouse. After 150 days, plants were evaluated for the following parameters: gall index, egg mass index, number of eggs per root system, number of eggs per gram of root, and reproduction factor. The accessions showed different responses depending on host x pathogen interaction, from susceptibility to moderate tolerance. Accessions 027-CMF and 031-CMF were considered tolerant to the nematode and could be of great value in new breeding programs for resistance to M. enterolobii infection.

Phylogenomics and a posteriori data partitioning resolve the Cretaceous angiosperm radiation Malpighiales.

The angiosperm order Malpighiales includes ~16,000 species and constitutes up to 40% of the understory tree diversity in tropical rain forests. Despite remarkable progress in angiosperm systematics during the last 20 y, relationships within Malpighiales remain poorly resolved, possibly owing to its rapid rise during the mid-Cretaceous. Using phylogenomic approaches, including analyses of 82 plastid genes from 58 species, we identified 12 additional clades in Malpighiales and substantially increased resolution along the backbone. This greatly improved phylogeny revealed a dynamic history of shifts in net diversification rates across Malpighiales, with bursts of diversification noted in the Barbados cherries (Malpighiaceae), cocas (Erythroxylaceae), and passion flowers (Passifloraceae). We found that commonly used a priori approaches for partitioning concatenated data in maximum likelihood analyses, by gene or by codon position, performed poorly relative to the use of partitions identified a posteriori using a Bayesian mixture model. We also found better branch support in trees inferred from a taxon-rich, data-sparse matrix, which deeply sampled only the phylogenetically critical placeholders, than in trees inferred from a taxon-sparse matrix with little missing data. Although this matrix has more missing data, our a posteriori partitioning strategy reduced the possibility of producing multiple distinct but equally optimal topologies and increased phylogenetic decisiveness, compared with the strategy of partitioning by gene. These approaches are likely to help improve phylogenetic resolution in other poorly resolved major clades of angiosperms and to be more broadly useful in studies across the Tree of Life.

Genetic variability in accessions of the acerola germplasm bank of Universidade Federal Rural de Pernambuco, Brazil.

Brazil is the world's largest producer of acerola, Malpighia emarginata (Malpighiaceae); the Northeast is responsible for 60% of the national production. The culture of acerola in Brazil has great genetic variability; plantings have high phenotypic diversity and are not very productive, often originating from propagation by seed. We evaluated the genetic diversity of 42 accessions from the Acerola Active Germplasm Bank of Universidade Federal de Pernambuco. Using 15 RAPD primers, 182 markers were obtained, of which 166 were polymorphic and 16 were monomorphic. We found high genetic variability among the accessions (HE = 0.29), with no redundancy. Considering the accessions from the states of Pernambuco, Bahia and Pará as distinct groups, there was greater diversity in accessions from Bahia than from the other two states.

Development of microsatellite markers in Byrsonima crassifolia (Malpighiaceae).

PREMISE OF THE STUDY: We developed and characterized microsatellite markers for Byrsonima crassifolia (Malpighiaceae), a widely distributed neotropical fruit tree. METHODS AND RESULTS: Eight polymorphic and two monomorphic microsatellite loci were identified and screened in 60 samples from four geographically disparate populations (Bolivia, Brazil, Mexico, and Panama). Each locus exhibited between two and 11 alleles. Expected heterozygosity ranged from 0 to 0.839. All loci amplify in the congeners B. variabilis and B. basiloba, four amplify in B. bucidaefolia, and seven amplify in B. variabilis, although levels of polymorphism have not been assessed. CONCLUSIONS: These loci will provide novel tools for comparing genetic diversity present in cultivated and noncultivated populations of B. crassifolia throughout its range, and may prove valuable in related species.

Pterandra pyroidea: a case of pollination shift within neotropical Malpighiaceae.

BACKGROUND AND AIMS: Most Neotropical species of Malpighiaceae produce floral fatty oils in calyx glands to attract pollinating oil-collecting bees, which depend on this resource for reproduction. This specialized type of pollination system tends to be lost in members of the family that occur outside the geographic distribution (e.g. Africa) of Neotropical oil-collecting bees. This study focused on the pollination ecology, chemical ecology and reproductive biology of an oil flower species, Pterandra pyroidea (Malpighiaceae) from the Brazilian Cerrado. Populations of this species consist of plants with oil-secreting (glandular) flowers, plants with non-oil-secreting flowers (eglandular) or a mix of both plant types. This study specifically aims to clarify the role of eglandular morphs in this species. METHODS: Data on pollinators were recorded by in situ observations. Breeding system experiments were conducted by isolating inflorescences and by enzymatic reactions. Floral resources, pollen and floral oils offered by this species were analysed by staining and a combination of various spectroscopic methods. KEY RESULTS: Eglandular flowers of P. pyroidea do not act as mimics of their oil-producing conspecifics to attract pollinators. Instead, both oil-producing and oil-free flowers depend on pollen-collecting bees for reproduction, and their main pollinators are bumble-bees. Floral oils produced by glandular flowers are less complex than those described in closely related genera. CONCLUSIONS: Eglandular flowers represent a shift in the pollination system in which oil is being lost and pollen is becoming the main reward of P. pyroidea flowers. Pollination shifts of this kind have hitherto not been demonstrated empirically within Neotropical Malpighiaceae and this species exhibits an unusual transition from a specialized towards a generalized pollination system in an area considered the hotspot of oil-collecting bee diversity in the Neotropics. Transitions of this type provide an opportunity to study ongoing evolutionary mechanisms that promote the persistence of species previously involved in specialized mutualistic relationships.

Botanical aspects of Heteropterys umbellata (Malpighiaceae): a cytological and palynological approach.

The genus Heteropterys is one of the major genera in Malpighiaceae. However, few cytological and palynological studies were reported. The present work described for the first time the chromosome number, heterochromatin pattern, meiotic behavior, pollen viability and palynological aspects of Heteropterys ubellata, a very spread species. One large Brazilian population was evaluated using conventional techniques for meiotic studies and acetolyse to access the pollen morphology. The species showed 2n = 20 chromosomes, normal meiotic development and viable pollens. Great blocks of heterochromatin were observed around the centromers. DAPI staining was positive for centroeric heterochroatin, hile CMA3 ark as observed just at terinal regions of one pair of hoologues chromosomes. This result and the presence of one chromosome pair attached to the nucleoli during the pachytene and diakinesis suggested the presence of only one pair of NORs. Palynological analysis revealed that pollen grains are apolar, 6 porate and with colpoids associated to all pores. The pollen content was positive for the starch test, and the exine was rugulate with little psilate regions.

Increase in ascorbate content of transgenic tobacco plants overexpressing the acerola (Malpighia glabra) phosphomannomutase gene.

Phosphomannomutase (PMM; EC 5.4.2.8) catalyzes the interconversion of mannose-6-phosphate to mannose-1-phosphate in the Smirnoff-Wheeler pathway for the biosynthesis of l-ascorbic acid (AsA). We have cloned the PMM cDNA from acerola (Malpighia glabra), a plant containing an enormous amount of AsA. The AsA contents correlate with the PMM gene expression of the ripening fruits and leaves. The PMM activities in the leaves of acerola, tomato and Arabidopsis correlate with their respective AsA contents. Transgenic tobacco plants overexpressing the acerola PMM gene showed about a 2-fold increase in AsA contents compared with the wild type, with a corresponding correlation with the PMM transcript levels and activities.

Intra-plant variation in cyanogenesis and the continuum of foliar plant defense traits in the rainforest tree Ryparosa kurrangii (Achariaceae).

At the intra-plant level, temporal and spatial variations in plant defense traits can be influenced by resource requirements, defensive priorities and storage opportunities. Across a leaf age gradient, cyanogenic glycoside concentrations in the rainforest understory tree Ryparosa kurrangii B.L. Webber were higher in young expanding leaves than in mature leaves (2.58 and 1.38 mg g(-1), respectively). Moreover, cyanogens, as an effective chemical defense against generalist herbivores, contributed to a defense continuum protecting foliar tissue during leaf development. Chemical (cyanogens and phenolic compounds) and phenological (delayed greening) defense traits protected young leaves, whereas mature leaves were largely protected by physical defense mechanisms (lamina toughness; explained primarily by leaf mass per area). Cyanogen concentration was considerably higher in floral tissue than in foliar tissue and decreased in floral tissue during development. Across contrasting tropical seasons, foliar cyanogenic concentration varied significantly, being highest in the late wet season and lowest during the pre-wet season, the latter coinciding with fruiting and leaf flushing. Cyanogens in R. kurrangii appear to be differentially allocated in a way that maximizes plant fitness but may also act as a store of reduced nitrogen that is remobilized during flowering and leaf flushing.

Chloroplast genomes of Byrsonima species (Malpighiaceae): comparative analysis and screening of high divergence sequences.

Byrsonima is the third largest genus (about 200 species) in the Malpighiaceae family, and one of the most common in Brazilian savannas. However, there is no molecular phylogeny available for the genus and taxonomic uncertainties at the generic and family level still remain. Herein, we sequenced the complete chloroplast genome of B. coccolobifolia and B. crassifolia, the first ones described for Malpighiaceae, and performed comparative analyses with sequences previously published for other families in the order Malpighiales. The chloroplast genomes assembled had a similar structure, gene content and organization, even when compared with species from other families. Chloroplast genomes ranged between 160,212 bp in B. crassifolia and 160,329 bp in B. coccolobifolia, both containing 115 genes (four ribosomal RNA genes, 28 tRNA genes and 83 protein-coding genes). We also identified sequences with high divergence that might be informative for phylogenetic inferences in the Malpighiales order, Malpighiaceae family and within the genus Byrsonima. The phylogenetic reconstruction of Malpighiales with these regions highlighted their utility for phylogenetic studies. The comparative analyses among species in Malpighiales provided insights into the chloroplast genome evolution in this order, including the presence/absence of three genes (infA, rpl32 and rps16) and two pseudogenes (ycf1 and rps19).

Cloning and expression of GDP-D-mannose pyrophosphorylase gene and ascorbic acid content of acerola (Malpighia glabra L.) fruit at ripening stages.

Acerola (Malpighia glabra L.) is one of the richest natural sources of L-ascorbic acid (AsA; vitamin C). GDP-D-mannose pyrophosphorylase (GMP; EC 2.7.7.13) was found to play a major role in the proposed AsA biosynthetic pathway in plants, considering that Arabidopsis vtc1-1 mutant with point mutation in this gene has a highly reduced AsA content. GMP cDNA was isolated from acerola fruits, designated MgGMP, using rapid amplification of cDNA ends (RACE), and its expression was monitored during fruit ripening. The full-length cDNA was found to have an ORF of 1083bp encoding a polypeptide of 361 amino acids. In silico analysis of the predicted amino acid sequence showed a pI of 6.45 and molecular mass of 39.7kD. MgGMP showed over 80% amino acid sequence identity with other plant GMP homologues. The phylogenetic tree shows the close relation of MgGMP to the GMP of other plants as against those from parasite, yeasts and mammals. Southern analysis indicated that M. glabra contains not less than two copies of GMP genes. Northern blot analysis showed the transcript abundance of MgGMP in all the organs of acerola examined, with the fruit having the highest expression. The relative transcript abundance of MgGMP mRNA levels in the fruits changes as the ripening process progresses, with the unripe green fruits having the highest relative mRNA level, and the lowest was found in the fruits at advanced ripening stage. A strong correlation was also observed between the relative MgGMP mRNA levels and the AsA contents of acerola during fruit ripening.

Structures, biogenetic relationships, and cytotoxicity of pimarane-derived diterpenes from Petalostigma pubescens.

Extraction of Petalostigma pubescens heartwood followed by chromatographic purifications and crystallizations afforded five tricyclic diterpenes: 5,9-syn-rosanes petalostigmones A and B (1 and 2), the erythroxylane petalostigmone C (3), the norditerpene lactone pubescenone (4), and the known ent-cleistanthane diterpene (-)-sonderianol (5). The structures and relative stereochemistry were elucidated by means of spectroscopic methods, chemical correlations, and, in the cases of 1 and 4, by X-ray crystallographic analyses. The new isolates 1-4 are assumed to belong to the same absolute configurational family (9alphaCH3) of ent-pimarane-derived diterpenes as the known co-occurring (-)-5 (10alphaCH3). Biogenetic schemes originating from a common ent-copalyl diphosphate intermediate are presented to rationalize the structures of these natural products. A novel ring contraction-ring expansion mechanism is suggested to account for the 7-membered B ring of pubescenone. Compounds 1-5 were evaluated for their cytotoxicity; sonderianol (5) showed the highest activity against mouse leukemia cell lines L1210, P388 and mouse liver cancer cells HEPA1c1c7.

Phylogeny of Acridocarpus-Brachylophon (Malpighiaceae): implications for tertiary tropical floras and Afroasian biogeography.

A major tenet of African Tertiary biogeography posits that lowland rainforest dominated much of Africa in the late Cretaceous and was replaced by xeric vegetation as a response to continental uplift and consequent widespread aridification beginning in the late Paleogene. The aridification of Africa is thought to have been a major factor in the extinction of many African humid-tropical lineages, and in the present-day disparity of species diversity between Africa and other tropical regions. This primarily geologically based model can be tested with independent phylogenetic evidence from widespread African plant groups containing both humid- and xeric-adapted species. We estimated the phylogeny and lineage divergence times within one such angiosperm group, the acridocarpoid clade (Malpighiaceae), with combined ITS, ndhF, and trnL-F data from 15 species that encompass the range of morphological and geographic variation within the group. Dispersal-vicariance analysis and divergence-time estimates suggest that the basal acridocarpoid divergence occurred between African and Southeast Asian lineages approximately 50 million years ago (mya), perhaps after a southward ancestral retreat from high-latitude tropical forests in response to intermittent Eocene cooling. Dispersion of Aeridocarpus from Africa to Madagascar is inferred between approximately 50 and 35 mya, when lowland humid tropical forest was nearly continuous between these landmasses. A single dispersal event within Acridocarpus is inferred from western Africa to eastern Africa between approximately 23 and 17 mya, coincident with the widespread replacement of humid forests by savannas in eastern Africa. Although the spread of xeric environments resulted in the extinction of many African plant groups, our data suggest that for others it provided an opportunity for further diversification.