Inter- and intra-specific variation among five Erythroxylum taxa assessed by AFLP

BACKGROUND: and Aims The four cultivated Erythroxylum taxa (E. coca var. coca, E. novogranatense var. novogranatense, E. coca var. ipadu and E. novogranatense var. truxillense) are indigenous to the Andean region of South America and have been cultivated for folk-medicine and, within the last century, for illicit cocaine production. The objective of this research was to assess the structure of genetic diversity within and among the four cultivated alkaloid-bearing taxa of Erythroxylum in the living collection at Beltsville Agricultural Research Center. METHODS: Amplified fragment length polymorphism (AFLP) fingerprinting was performed in 86 Erythroxylum accessions using a capillary genotyping system. Cluster analysis, multidimensional scaling (MDS) and analysis of molecular variance (AMOVA) were used to assess the pattern and level of genetic variation among and within the taxa. KEY RESULTS: A clear distinction was revealed between E. coca and E. novogranatense. At the intra-specific level, significant differentiation was observed between E. c. var. coca and E. c. var. ipadu, but the differentiation between E. n. var. novogranatense and E. n. var. truxillense was negligible. Erythroxylum c. var. ipadu had a significantly lower amount of diversity than the E. c. var. coca and is genetically different from the E. c. var. ipadu currently under cultivation in Colombia, South America. CONCLUSIONS: There is a heterogeneous genetic structure among the cultivated Erythroxylum taxa where E. coca and E. novogranatense are two independent species. Erythroxylum coca var. coca is most likely the ancestral taxon of E. c. var. ipadu and a founder effect may have occurred as E. c. var. ipadu moved from the eastern Andes in Peru and Bolivia into the lowland Amazonian basin. There is an indication of artificial hybridization in coca grown in Colombia.     

Flavonoids as chemotaxonomic markers for cultivated Amazonian coca

Purported ‘Amazonian coca,’ Erythroxylum coca var. ipadu Plowman (E. coca var. ipadu) was harvested from cultivated fields in Colombia, South America to determine: (a) its identity; (b) if its leaf flavonoids were complimentary to those present in leaf tissue of E. c. var. ipadu in our collection derived from Colombia; and (c) if complimentary, indicative of kinship to E. coca var. ipadu obtained from Colombia, or a related Erythroxylum taxon. Leaf extracts from Amazonian field-grown coca afforded eight O-conjugated flavonoids: two O-conjugates of taxifolin, one O-conjugate of quercetin, two O-conjugates of eriodictyol and three O-conjugates of kaempferol. Present also in leaf tissue of Amazonian field-grown coca, but lacking in leaf tissue from our collection of E. c. var. ipadu was an O-ethyl ester typically found in E. coca var. coca, kaempferols and a 7-O-rutinoside commonly encountered in the E. novogranatense taxons. Flavonoids found in our collection of E. coca var. ipadu were five O-conjugated derivatives of taxifolin and an O-conjugated quercetin. Leaf flavonoids of currently cultivated Amazonian coca are a mixture of those present in E. coca var. coca, E. coca var. ipadu and E. novogranatense var. truxillense, whereas those present in our authenticated living collection are derivatives of E. coca var. coca. Our data suggest that the Amazonian coca under cultivation in Colombia is a genetic hybrid cross between E. coca var. coca and E. novogranatense var. truxillense, occurring after 1972.     

Identification of Erythroxylum taxa by AFLP DNA analysis

Erythroxylum coca, indigenous to the Andean region of South America, is grown historically as a source of homeopathic medicine. However, in the last century, cultivation of E. coca and several closely-related species for the production of illicit cocaine has become a major global problem. Two subspecies, E. coca var. coca and E. coca var. ipadu, are almost indistinguishable phenotypically; a related cocaine-bearing species also has two subspecies (E. novogranatense var. novogranatense and E. novogranatense var. truxillense) that are phenotypically similar, but morphologically distinguishable. The purpose of this research was to discover unique AFLP DNA patterns ("genetic fingerprinting") that characterize the four taxa and then, if successful, to evaluate this approach for positive identification of the various species of coca. Of seven different AFLP primer pairs tested, a combination of five proved optimal in differentiating the four taxa as well as a non-cocaine-bearing species, E. aerolatum. This method of DNA fragment separation was selective, and faster, for coca identification, compared with analyses based on flavonoid chemotaxonomy. Using the 5-primer AFLP approach, 132 known and unknown coca leaf accessions were evaluated. Of these, 38 were collected in 1997-2001 from illicit coca fields in Colombia, and all were genetically differentiated from coca originating in Peru and Bolivia. Based on the DNA profiling, we believe that the Colombian coca now represents a hybridization of E. coca var. ipadu. Geographical profiling within Colombia also seems feasible as new coca production areas are developed or new types of coca are introduced within traditional growing areas.     

Determination of cocaine in some south american species of Erythroxylum using mass fragmentography

Thirteen South American species of Erythroxylum have been analyzed for their cocaine content. Cocaine was found only in E. coca Lam., E. novogranatense (Morris) Hieron. and E. novogranatense var. truxillense (Rusby) Machado. The amount of cocaine was determined by mass fragmentography using deuterium labelled cocaine as internal standard     

Cocaine and Cinnamoylcocaine Content of Erythroxylum Species

The dried leaves of 31 species and two varieties of the genusErythroxylum were quantitatively analysed for cocaine and bothcis- and trans-cinnamoylocaine, using a stable-isotope dilutionmethod and gas chromatography-mass spectrometry selected-ionmonitoring. Both cocaine and cinnamoylcocaine were detectedin all four varieties of cultivated coca (E. coca var. coca,E. coca var. ipadu, E. novogranatense var. novogranatense andE. novogranatense var.truxillense). Cinnamoylcocaines (cis andtrans) were found in much higher concentrations in both varietiesof E. novogranatense than in either variety of E. coca. Amazoniancoca (E. coca var. ipadu) contained consistently lower cocainelevels than the montane variety (E. coca var. coca). Twenty-ninewild species of Erythroxylum, selected to represent morphological,ecological and taxonomic diversity, were analysed for the samealkaloids; cocaine was detected in 13 neotropical species, representingfive sections of the genus. No cocaine was detected in the OldWorld species. Two wild species from Venezuela, E. recurrensand E. steyermarkii, contained cocaine levels comparable tothose found in the commercially cultivated species. Erythroxylaceae, Erythroxylum, coca, cocaine, cinnamoylcocaine, alkaloids, chemotaxonomy     

A phylogenetic analysis based on nucleotide sequence of a marker linked to the brittle rachis locus indicates a diphyletic origin of barley

BACKGROUND AND AIMS: Barley (Hordeum vulgare ssp. vulgare) cultivation started between 9500 and 8400 years ago, and was a major part of ancient agriculture in the Near East. The brittle rachis is a critical trait in the domestication process. METHODS: A DNA sequence closely linked to the brittle rachis complex was amplified and resequenced in a collection of cultivated barleys, wild barleys (H. vulgare ssp. spontaneum) and weedy brittle rachis varieties (H. vulgare ssp. vulgare var. agriocrithon). The sequence was used to construct a phylogenetic tree. KEY RESULTS: The phylogeny separated the W- (btr1-carrying) from the E- (btr2-carrying) cultivars. The wild barleys had a high sequence diversity and were distributed throughout the W- and E-clades. Some of the Tibetan var. agriocrithon lines were closely related to the E-type and others to the W-type cultivated barleys, but an Israeli var. agriocrithon line has a complex origin. CONCLUSIONS: The results are consistent with a diphyletic origin of barley. The W- and E-type cultivars are assumed to have evolved from previously diverged wild barley via independent mutations at Btr1 and Btr2.     

Flavonoids as chemotaxonomic markers for Erythroxylum australe

Methanolic leaf extracts of Erythroxylum australe F. Muell. produced eight O-conjugated flavonoids. Six of the flavonoid aglycones were dihydroisoflavones (all dihydro-orobol derivatives), one a flavanone, eriodictyol, and one a flavonol, quercetin. The major glycosides of the flavonoids included mono-glucosyl-rhamnosyls and dirhamnosyl-glucosides with either 3, 5, 7 or 3', 4' linkage or a combination thereof The array of flavonoids present in E. australe suggests kinship to E. ulei and linkage to the four cultivated alkaloid-bearing Erythroxylum, especially the ancestral E. coca var. coca. Because of the uniqueness of the flavonoids present in leaf tissue of E. australe they are unambiguously useful as chemotaxonomic markers for the taxon.     

Biosystematic studies of the Elymus hispidus (Poaceae: Triticeae) group in Iran

Biosystematic studies on the Elymus hispidus group included investigations on morphological characters and variability in cultivation, crossing experiments, hybrid fertility and meiotic configuration in hybrids. Elymus gentry! is a distinct species with a different altitudinal distribution from that of E. hispidus. Morphological characters which have been used to split E. hispidus into distinct species or subspecies are not constant in cultivation. The majority of the cultivated plants were morphologically similar to the maternal individuals from which seed was collected in the field. However, in some accessions the offspring represented different morphological variants when cultivated. Hybrids between different variants were fertile and showed regular meiosis. Different variants within the E. hispidus group tend to grow in different habitats and as characters to some extent show constancy in cultivation, three varieties, var. hispidus , var. podperae and var. villosus are recognized for E. hispidus in Iran.     

Coca chewing for exercise: hormonal and metabolic responses of nonhabitual chewers

Favier, Roland, Esperanza Caceres, Laurent Guillon, BrigitteSempore, Michel Sauvain, Harry Koubi, and Hilde Spielvogel. Cocachewing for exercise: hormonal and metabolic responses of nonhabitualchewers. J. Appl. Physiol. 81(5):1901-1907, 1996.To determine the effects of acute coca use onthe hormonal and metabolic responses to exercise, 12 healthynonhabitual coca users were submitted twice to steady-state exercise(~75% maximal O₂ uptake). Onone occasion, they were asked to chew 15 g of coca leaves 1 h beforeexercise, whereas on the other occasion, exercise was performed after 1 h of chewing a sugar-free chewing gum. Plasma epinephrine,norepinephrine, insulin, glucagon, and metabolites (glucose, lactate,glycerol, and free fatty acids) were determined at rest before andafter coca chewing and during the 5th, 15th, 30th, and 60th min ofexercise. Simultaneously to these determinations, cardiorespiratoryvariables (heart rate, mean arterial blood pressure, oxygen uptake, andrespiratory gas exchange ratio) were also measured. At rest, cocachewing had no effect on plasma hormonal and metabolic levels exceptfor a significantly reduced insulin concentration. During exercise, theoxygen uptake, heart rate, and respiratory gas exchange ratio weresignificantly increased in the coca-chewing trial compared with thecontrol (gum-chewing) test. The exercise-induced drop in plasma glucoseand insulin was prevented by prior coca chewing. These results contrastwith previous data obtained in chronic coca users who display duringprolonged submaximal exercise an exaggerated plasma sympatheticresponse, an enhanced availability and utilization of fat (R. Favier,E. Caceres, H. Koubi, B. Sempore, M. Sauvain, and H. Spielvogel.J. Appl. Physiol. 80: 650-655, 1996). We conclude that, whereas coca chewing might affect glucose homeostasis during exercise, none of the physiological data provided bythis study would suggest that acute coca chewing in nonhabitual userscould enhance tolerance to exercise.

     

AFLP Phylogeny of 36 Erythroxylum Species

Four taxa of the plant genus Erythroxylum; Erythroxylum coca var. coca (Ecc), Erythroxylum coca var. ipadu (Eci), Erythroxylum novogranatense var. novogranatense (Enn) and Erythroxylum novogranatense var. truxillense (Ent) are cultivated primarily for the illicit extraction and processing of cocaine. Despite their economic and medical importance, the evolutionary history of these species remains unknown in a modern phylogenetic framework. The aims of this study were to: (a) investigate the relationship among the cultivated and a select number of non-cultivated taxa, and (b) test Plowman??s (Journal of Psychodelic Drugs 11:103?C117, 1979b) linear progression hypothesis of the cultivated Erythroxylum taxa versus Johnson??s et al. (Annals of Botany 95:601?C608, 2005) hypothesis that Ec and En are sister species. AFLP phylogeny was used to compare the relationships among 36 Erythroxylum species (133 accessions) spanning the geographic distribution of the genus. A Maximum Parsimony tree revealed both geographic and taxonomic partitioning into clades representing species from Africa, Asia-Pacific and the New World (Tropical Americas). Ec and En formed distinct clades, indicating they are sister species and a cluster of non-cultivated species were the most closely related to the cultivated species. Multivariate ordination analysis was used to evaluate the relationship between cultivated and non-cultivated Erythroxylum taxa from the Tropical Americas. Our results support the hypothesis that the cultivated species are more closely related to each other than to any other species of Erythroxylum, but refute the hypothesis that Ent (and Enn) descended from Ecc. Instead our data suggest an independent, non-linear evolutionary relationship between Ec and En. Finally, the AFLP analyses identified significantly different genetic groups within Erythroxylum suggesting that the current intrageneric classification of this genus be revised.     

Agriculture and food from the Roman to the Islamic Period in the North-East of the Iberian peninsula: archaeobotanical studies in the city of Lleida (Catalonia, Spain)

The urban archaeological excavations carried out in the city of Lleida (Catalonia, Spain) have opened the way for an interdisciplinary project on the ancient landscape, agriculture and food. Here we present the results of the archaeobotanical study of seeds and fruits from this project, centred on the Roman and Islamic periods of the city, between the 2nd century B.C. and the 11th century A.D. During the whole of this period the cultivation and consumption of cereals was found. The most important cereals were hulled barley and naked wheat. This was already known from the later prehistoric era in the area and is similar to findings at other sites from the same period in the western Mediterranean. These cereals are accompanied by some leguminous crops and the presence of grapes and figs is very significant. The expansion of vine cultivation, together with a certain amount of tree growing was one of the basic contributions of the Roman world to proto-historical (Bronze- and Iron Age) agriculture in western Catalonia, as in other parts of Europe. In the Islamic period, there seems to have been an increase in the number of fruit tree species; however for taphonomical reasons this has to be confirmed by future investigations. Flax, already known in prehistoric times, and Gold-of-pleasure must be added to the finds. Also since the Roman period some other taxa, such as celery or fennel, might have been grown. During the time period considered in this paper, there was a wide range of plants grown and consumed. This clearly contrasts with everything known about earlier periods in western Catalonia, during which the only cultivated plants were cereals and flax.     

782. SALVIA INVOLUCRATA

Salvia involucrata Cav., the Rose‐leaf sage, is a species endemic to east Mexico which has been in cultivation since at least the late 18th century. It is little‐known as a native species but is widely cultivated and various cultivars and cultivated hybrids are also known. Salvia puberula Fern. is treated as belonging to S. involucrata but has been subject to different interpretations over the years.     

Tequila and other <Emphasis Type="Italic">Agave</Emphasis> spirits from west-central Mexico: current germplasm diversity,conservation and origin

Current germplasm diversity used in the production of Agave spirits in west-central México is in danger of erosion due to an expansion in the cultivation of the clone A. tequilana Weber var. azul, used for the elaboration of the famous drink “Tequila”. In order to define critical areas of in situ conservation and to determine the role of local native and mestizo cultures in the generation and maintenance of diversity, an ethnobotanical exploration was conducted in the center and south of the state of Jalisco. Results situate the nucleus of greatest diversity at present in the south of Jalisco and indicate that this is a result of a continuous process of selection initiated by the indigenous population for the production of food and fermented drinks, which continued into the final years of the 16th century but with a new objective: distillation using the Filipino technology introduced to west-central Mexico through Colima. More than 20 variants were found to be cultivated by the traditional farmers, the majority relating to the A. angustifolia Haw. complex. We discuss the possibilities of in situ germplasm conservation and its legal protection.     

Plant remains from the early medieval lakeshore settlement Fonyód-Bélatelep (Lake Balaton,Hungary) with especial emphasis on the history of fruit cultivation in Pannonia

During the excavation of an early medieval lakeside settlement at Fonyód-Bélatelep, Balaton (7th-9th century A.D.), remains of cultivated and wild plants were found in the culture layer. The main cereal crops of the Avar culture were Hordeum, Triticum and Secale. Panicum also played an important role. In addition to the cereals, 13 species of fruit trees were present. It appears that fruit cultivation survived from Roman times into the early medieval period.     

A taxonomic revision of Crambe, sect. Leptocrambe (Brassicaceae)

As part of a revision of the genus Crambe based on the morphological study of herbaria and cultivated material, the systematics of sect. Leptocrombe DC. is presented here. Section Leptocrambe is considered to comprise five species: C. kilimandscharica O. E. Schulz, C. sinuatodentata Hochst. ex Petri, C. hispanica L., C. filiformis Jacq. and C. kralikii Coss. C. hispanica includes three subspecies, subsp. hispanica , subsp. glabrata (DC.) Cout. and subsp. abyssnica (Hochst. ex R. E. Fr.) stat. nov. which includes var. dyssinica and var. meyeri (O. E. Schulz) comb. nov. C. kralikii includes two subspecies, subsp. kralikii and subsp. garamas (Maire) Podlech.     

745. TROPAEOLUM MINUS

Tropaeolum minus L. was the first nasturtium to appear in European gardens. It was already widespread in the 16th century, and hence it must have been among the first American plants to be cultivated widely in European gardens. It is a smaller species than T. majus L., the well‐known garden nasturtium, and has lost popularity since the latter was introduced. Its cultivation and uses are discussed.     

Archaeological evidence of coca (Erythroxylum coca,erythroxylaceae) in the upper mantaro valley,Peru

Prehistoric remains of coca (Erythroxylum spp.) are rarely uncovered by archaeologists or positively identified by botanists because of their fragile nature and the lack of rigorous archaeological collection techniques. This absence of plant evidence has made evolutionary studies of diffusion and use of coca difficult. From special depositional conditions in the Mantaro area of central Peru, one coca leaf and two endocarps have been uncovered and identified as Erythroxylum coca var. coca. These three specimens came from elite-status contexts dating to the Late Intermediate and the Late Horizon-Early Colonial Periods. These remains provide the first highland evidence for access to coca-producing, ceja de montaña farms, which lie more than 50 km away on the eastern slope of the Andes.     

Temporal changes in genetic diversity of common bean (Phaseolus vulgaris L.) accessions cultivated between 1800 and 2000

Fourteen microsatellite markers were used to describe genetic diversity in a sample of 128 common bean (Phaseolus vulgaris L.) accessions cultivated within the territory of Slovenia and its nearby regions between 1800 and 2000. The accessions were grouped into three periods, Period I comprising accessions from the beginning of the 19th century, while the other two periods included accessions from the middle (Period II) and the end of the 20th century (Period III). Seven control accessions of known Mesoamerican and Andean origin were also included in the study. A total of 130 alleles were generated. Allelic richness, in terms of number of alleles per locus, was 6.07 for Period I, 6.71 for Period II and 6.07 for Period III. In the UPGMA dendrogram, all studied accessions were intermixed in three main clusters, indicating that the diversity in the time periods overlapped. Two clusters consisted of accessions of Andean and Mesoamerican origin, while the third represents additional variation, which existed in this area already 200 years ago. The analysis of molecular variance showed that a great part of genetic diversity has been preserved till today, confirming the results of cluster analysis. The calculation of number of alleles per locus revealed no significant quantitative change in genetic diversity over the last 200 years of common bean cultivation. However, the calculation of genetic distances indicated slight qualitative shifts in genetic diversity of common bean germplasm over time, while the calculations of allelic frequency variation and polymorphic information content revealed recent decline of some alleles' frequencies. These findings should stress the need for establishing an appropriate strategy of genetic resources management.     

Temporal changes in genetic diversity of common bean (Phaseolus vulgaris L.) accessions cultivated between 1800 and 2000

Fourteen microsatellite markers were used to describe genetic diversity in a sample of 128 common bean (Phaseolus vulgaris L.) accessions cultivated within the territory of Slovenia and its nearby regions between 1800 and 2000. The accessions were grouped into three periods: period I comprising accessions from the beginning of the 19th century, while the other two periods included accessions from the middle (period II) and the end of the 20th century (period III). Seven control accessions of known Mesoamerican and Andean origin were also included in the study. A total of 130 alleles were generated. Allelic richness, in terms of number of alleles per locus, was 6.07 for period I, 6.71 for period II, and 6.07 for period III. In the UPGMA dendrogram, all studied accessions were intermixed in three main clusters, indicating that the diversity in the time periods overlapped. Two clusters consisted of accessions of Andean and Mesoamerican origin, while the third represents additional variation, which existed in this area already 200 years ago. The analysis of molecular variance showed that a great part of genetic diversity has been preserved till today, confirming the results of cluster analysis. The calculation of number of alleles per locus revealed no significant quantitative change in genetic diversity over the last 200 years of common bean cultivation. However, the calculation of genetic distances indicated slight qualitative shifts in genetic diversity of common bean germplasm over time, while the calculations of allelic frequency variation and polymorphic information content revealed recent decline of some alleles’ frequencies. These findings should stress the need for establishing an appropriate strategy of genetic resources management. The text was submitted by the authors in English.     

Henry Hurd Rusby: The father of economic botany at the New York Botanical Garden

Henry Hurd Rusby (1855–1940) is one of the outstanding personalities in the history of the New York Botanical Garden (NYBG). He played a significant role, not only in the founding of NYBG in 1891, but also in establishing a strong precedent of research and exploration in the field of economic botany at the new institution. As a result of Rusby’s influence and activity, the study of useful plants formed an important part of NYBG’s original mandate, an institutional commitment that was rejuvenated nearly a century later, with the formation of the Institute of Economic Botany. An indefatigable researcher both in the field and in the herbarium, Rusby left behind a voluminous corpus of published work in systematic and economic botany that is a legacy for modern botanists and pharmacologists.