Authentication of Brazilian Ginseng using Bar-HRM analysis

Abstract Hebanthe eriantha (Martius) Kuntze and Pfaffia glomerata (Spreng) Pedersen are medicinal plants popularly known as "Brazilian Ginseng" due to their similarity to Panax ginseng. In Brazil, they are sold as the same herb, despite their different pharmacological and toxicological properties. The morphological identification is difficult, which facilitates their adulteration. We report the application of the Barcode DNA High-Resolution Melting (Bar-HRM) using matK gene to differentiate both species in samples sold in the Brazilian market. Using the proposed method, we could discriminate and identify both species. Bar-HRM analysis allowed discriminating and identifying both species. It allowed the identification of H. eriantha and P. glomerata in 43.6% and 56.4% of the amplified samples, respectively. Of these, only seven samples were authenticated and, in 71.4% of the cases, adulterated. We concluded that Bar-HRM has proven to be a fast alternative method to authenticate plants under the common name "Brazilian Ginseng".

Palyno-morphological investigations of halophytic taxa of Amaranthaceae through SEM from Salt range of Northern Punjab, Pakistan.

The pollen morphology of 11 salt tolerant plant species of family Amaranthaceae from the salt range of Northern Punjab, Pakistan has been studied. The palyno-morphological characters were examined using light and scanning electron microscope. The examined all salt tolerant species have a slight difference in size but have similarity in shape, pore ornamentation, and polarity. The observed morphological characters of pollen grains were pollen symmetry, size, shape, pore ornamentation, pore size, number of pores, exine thickness, polar and equatorial diameter and, P/E ratio. Apolar type of pollens has been observed in all species. Shape of pollens was spheroidal. Exine sculpturing of pollen grains was scabrate (six spp), microechinate (four spp), and microechinate-scabrate (one spp). Different pori numbers were observed in different species. The pantoporate aperturate and sunken pore ornamentation have been reported in all species. A pollen taxonomic key was developed using examined morphological characters for the accurate identification of halophytic taxa. The high fertility and low sterility of pollens confirmed that the selected halophytes are well-established in the salt region. The findings highlight the taxonomic significance of pollen morphology in correct identification and differentiation of salt tolerant plant species.

[Comparison of chemical constituents between Cyathula offinalis and adulterant and their admixture by HPLC characteristic chromatogram fingerprint combined with chemometrics].

Cyathula capitate is the main adulterant of C.offinalis. According to the literature reported, there are obvious differences in properties, taste and pharmacological activity between C. capitate and C.offinalis. Therefore, C. capitate can only be used as a local conventional medicine and can't be a substitute for C. offinalis. Since the appearance of C.capitata is very similar to the C.offinalis and the content of cyasterone also can reach the limit of the current pharmacopoeia standard, the C.capitata is mostly sold in the form of impersonation oradmixture, which seriously affected the safety of the clinical medication and the development of the genuine crude drugs. In view of this, HPLC characteristic fingerprint was used to reveal the difference of multi-ingredients of C. offinalis, C. capitata and their admixture. According to the HPLC chromatogram of C.offinalis, C. capitata. and their admixture, 65 different components were obtained to set up a peak area data matrix of 26×65, which was applied to perform the characteristic peak difference analysis, similarity analysis, hierarchical clustering analysis HCA and principal component analysis (PCA). Characteristic peak difference analysis showed that the characteristic peaks of C. capitata and their admixture are more and higher respond than those of C. offinalis. The 9 characteristic peaks were used to distinguish C. capitata, 2 of which were used to distinguish C. offinalis mixed with 5% C. capitata. UV spectra of 9 characteristic peaks are mostly similar to the end absorption spectra of saponins, indicating that C. capitata may contain a large amount of saponins. By the reference fingerprint of C.offinalis established, the similarity analysis showed that the similarity degree of C. offinalis are higher than 0.942, while the similarity degree of C. capitata, C.offinalis mixed with 5% C. capitata are less than 0.383 and 0.399. C.offinalis, C. capitata, C.offinalis mixed with 5% C. capitata could be obviously divided into 3 classes by HCA and PCA. These results showed that there are obvious difference in the chemical composition of C. offinalis, C. capitata and their admixture, which could provide evidence for their identification.

The Evolution of the FT/TFL1 Genes in Amaranthaceae and Their Expression Patterns in the Course of Vegetative Growth and Flowering in Chenopodium rubrum.

The FT/TFL1 gene family controls important aspects of plant development: MFT-like genes affect germination, TFL1-like genes act as floral inhibitors, and FT-like genes are floral activators. Gene duplications produced paralogs with modified functions required by the specific lifestyles of various angiosperm species. We constructed the transcriptome of the weedy annual plant Chenopodium rubrum and used it for the comprehensive search for the FT/TFL1 genes. We analyzed their phylogenetic relationships across Amaranthaceae and all angiosperms. We discovered a very ancient phylogenetic clade of FT genes represented by the CrFTL3 gene of C. rubrum Another paralog CrFTL2 showed an unusual structural rearrangement which might have contributed to the functional shift. We examined the transcription patterns of the FT/TFL1 genes during the vegetative growth and floral transition in C. rubrum to get clues about their possible functions. All the genes except for the constitutively expressed CrFTL2 gene, and the CrFTL3 gene, which was transcribed only in seeds, exhibited organ-specific expression influenced by the specific light regime. The CrFTL1 gene was confirmed as a single floral activator from the FT/TFL1 family in C. rubrum Its floral promoting activity may be counteracted by CrTFL1 C. rubrum emerges as an easily manipulated model for the study of floral induction in weedy fast-cycling plants lacking a juvenile phase.

Evolutionary and Biogeographic Insights on the Macaronesian Beta-Patellifolia Species (Amaranthaceae) from a Time-Scaled Molecular Phylogeny.

The Western Mediterranean Region and Macaronesian Islands are one of the top biodiversity hotspots of Europe, containing a significant native genetic diversity of global value among the Crop Wild Relatives (CWR). Sugar beet is the primary crop of the genus Beta (subfamily Betoideae, Amaranthaceae) and despite the great economic importance of this genus, and of the close relative Patellifolia species, a reconstruction of their evolutionary history is still lacking. We analyzed nrDNA (ITS) and cpDNA gene (matK, trnH-psbA, trnL intron, rbcL) sequences to: (i) investigate the phylogenetic relationships within the Betoideae subfamily, and (ii) elucidate the historical biogeography of wild beet species in the Western Mediterranean Region, including the Macaronesian Islands. The results support the Betoideae as a monophyletic group (excluding the Acroglochin genus) and provide a detailed inference of relationships within this subfamily, revealing: (i) a deep genetic differentiation between Beta and Patellifolia species, which may have occurred in Late Oligocene; and (ii) the occurrence of a West-East genetic divergence within Beta, indicating that the Mediterranean species probably differentiated by the end of the Miocene. This was interpreted as a signature of species radiation induced by dramatic habitat changes during the Messinian Salinity Crisis (MSC, 5.96-5.33 Mya). Moreover, colonization events during the Pleistocene also played a role in shaping the current diversity patterns among and within the Macaronesian Islands. The origin and number of these events could not be revealed due to insufficient phylogenetic resolution, suggesting that the diversification was quite recent in these archipelagos, and unravelling potential complex biogeographic patterns with hybridization and gene flow playing an important role. Finally, three evolutionary lineages were identified corresponding to major gene pools of sugar beet wild relatives, which provide useful information for establishing in situ and ex situ conservation priorities in the hotspot area of the Macaronesian Islands.

Culturable endophytic bacteria from the salt marsh plant Halimione portulacoides: phylogenetic diversity, functional characterization, and influence of metal(loid) contamination.

Halimione portulacoides is abundant in salt marshes, accumulates mercury (Hg), and was proposed as useful for phytoremediation and pollution biomonitoring. Endophytic bacteria promote plant growth and provide compounds with industrial applications. Nevertheless, information about endophytic bacteria from H. portulacoides is scarce. Endophytic isolates (n = 665) were obtained from aboveground and belowground plant tissues, from two Hg-contaminated sites (sites E and B) and a noncontaminated site (site C), in the estuary Ria de Aveiro. Representative isolates (n = 467) were identified by 16S rRNA gene sequencing and subjected to functional assays. Isolates affiliated with Proteobacteria (64 %), Actinobacteria (23 %), Firmicutes (10 %), and Bacteroidetes (3 %). Altererythrobacter (7.4 %), Marinilactibacillus (6.4 %), Microbacterium (10.2 %), Salinicola (8.8 %), and Vibrio (7.8 %) were the most abundant genera. Notably, Salinicola (n = 58) were only isolated from site C; Hoeflea (17), Labrenzia (22), and Microbacterium (67) only from belowground tissues. This is the first report of Marinilactibacillus in the endosphere. Principal coordinate analysis showed that community composition changes with the contamination gradient and tissue. Our results suggest that the endosphere of H. portulacoides represents a diverse bacterial hotspot including putative novel species. Many isolates, particularly those affiliated to Altererythrobacter, Marinilactibacillus, Microbacterium, and Vibrio, tested positive for enzymatic activities and plant growth promoters, exposing H. portulacoides as a source of bacteria and compounds with biotechnological applications.

Phylogeny, biogeography and ecological diversification of Sarcocornia (Salicornioideae, Amaranthaceae).

BACKGROUND AND AIMS: Sarcocornia comprises about 28 species of perennial succulent halophytes distributed worldwide, mainly in saline environments of warm-temperate and subtropical regions. The genus is characterized by strongly reduced leaves and flowers, which cause taxonomic difficulties; however, species in the genus show high diversity in growth form, with a mat-forming habit found in coastal salt marshes of all continents. Sarcocornia forms a monophyletic lineage with Salicornia whose species are all annual, yet the relationship between the two genera is poorly understood. This study is aimed at clarifying the phylogenetic relationship between Sarcocornia and Salicornia, interpreting biogeographical and ecological patterns in Sarcocornia, and gaining insights into putative parallel evolution of habit as an adaptation to environmental factors. METHODS: A comprehensively sampled and dated phylogeny of Sarcocornia is presented based on nuclear ribosomal DNA (external transcribed spacer) and chloroplast DNA (atpB-rbcL, rpl32-trnL) sequences; representative samples of Salicornia were also included in the analyses. To infer biogeographical patterns, an ancestral area reconstruction was conducted. KEY RESULTS: The Sarcocornia/Salicornia lineage arose during the Mid-Miocene from Eurasian ancestors and diversified into four subclades: the Salicornia clade, the American Sarcocornia clade, the Eurasian Sarcocornia clade and the South African/Australian Sarcocornia clade. Sarcocornia is supported as paraphyletic, with Salicornia nested within Sarcocornia being sister to the American/Eurasian Sarcocornia clade. The American and the South African/Australian Sarcocornia clade as well as the Salicornia clade were reconstructed to be of Eurasian origin. The prostrate, mat-forming habit arose multiple times in Sarcocornia. CONCLUSIONS: Sarcocornia diversified in salt-laden environments worldwide, repeatedly evolving superficially similar prostrate, mat-forming habits that seem advantageous in stressed environments with prolonged flooding, high tidal movement and frost. Some of these prostrate-habit types might be considered as ecotypes (e.g. S. pacifica or S. pillansii) while others represent good ecospecies (e.g. S. perennis, S. decumbens, S. capensis), hence representing different stages of speciation.

Produção de mudas de penicilina (Alternanthera brasiliana (L. ) Kuntze) via estaquia / Seedling production of penicillin (Alternanthera brasiliana (L. ) Kuntze) by cuttings

A penicilina (Alternanthera brasiliana (L.) Kuntze), pertence à família Amaranthaceae e tem sido reconhecida por suas propriedades anti-inflamatória, analgésica, e antiviral. O trabalho teve como objetivo avaliar a concentração de ácido indolbutírico (AIB) mais adequada para a indução do enraizamento de estacas da penicilina. Foram selecionados ramos herbáceos de plantas matrizes, coletadas em novembro de 2010, para confecção de estacas com 6 cm de comprimento e 2 folhas apicais. As estacas foram desinfestadas através de solução de hipoclorito de sódio (0,5%) por 15 minutos. Em seguida, as estacas da penicilina tiveram suas bases imersas por 10 segundos em soluções de AIB nas concentrações de 0, 250, 500, 750 ou 1000 mg L-1, e foram plantadas em tubetes, sob irrigação em casa de vegetação. O delineamento experimental foi inteiramente casualizado, com 80 estacas por tratamento. Após 30 dias não foram observados resultados com diferenças significativas entre os tratamentos utilizados quanto ao enraizamento, crescimento das raízes, mortalidade, massa fresca e seca. No entanto, houve aumento do número de raízes das estacas tratadas nas concentrações mais altas de AIB (750 e 1000 mg L-1) quando comparadas ao controle e aos demais tratamentos. As porcentagens de enraizamento foram acima de 94% para essa espécie, podendo concluir que a propagação via estaquia é viável sem o uso de reguladores para induzir a formação de raízes dessa espécie, a qual pode ser considerada de fácil enraizamento.

Penicillin (Alternanthera brasiliana (L.) Kuntze) belongs to the Amaranthaceae and has been recognized for its anti-inflammatory, analgesic and antiviral properties. This study aimed to evaluate the most appropriate concentration of indole butyric acid (IBA) to induce rooting of penicillin cuttings. We selected herbaceous branches, collected in November of 2010, to prepare 6 cm long cuttings with two apical leaves and disinfected them in solution of sodium hypochlorite (0.5%) for 15 minutes. Then, the penicillin cuttings had their bases immersed for 10 seconds in IBA at the concentrations of 0, 250, 500, 750 or 1000 mg L-1, planted in small plastic tubes, irrigated and maintained in a greenhouse. The experimental design was completely randomized, with 80 cuttings per treatment. After 30 days, we observed no significant different results among treatments on rooting, root growth, mortality, fresh and dry mass. There was an increase in the number of roots in the cuttings immersed in higher concentrations (750 and 1000 mg L-1) of IBA compared to the control and other treatments. The rooting percentages were above 94% for this species, and we could conclude that the propagation by cuttings is feasible without the use of regulators to induce root formation in this species, which can be considered as easy to root.

Fruit and seed anatomy of Chenopodium and related genera (Chenopodioideae, Chenopodiaceae/Amaranthaceae): implications for evolution and taxonomy.

A comparative carpological study of 96 species of all clades formerly considered as the tribe Chenopodieae has been conducted for the first time. The results show important differences in the anatomical structure of the pericarp and seed coat between representatives of terminal clades including Chenopodium s.str.+Chenopodiastrum and the recently recognized genera Blitum, Oxybasis and Dysphania. Within Chenopodium the most significant changes in fruit and seed structure are found in members of C. sect. Skottsbergia. The genera Rhagodia and Einadia differ insignificantly from Chenopodium. The evolution of heterospermy in Chenopodium is discussed. Almost all representatives of the tribe Dysphanieae are clearly separated from other Chenopodioideae on the basis of a diverse set of characteristics, including the small dimensions of the fruits (especially in Australian taxa), their subglobose shape (excl. Teloxys and Suckleya), and peculiarities of the pericarp indumentum. The set of fruit and seed characters evolved within the subfamily Chenopodioideae is described. A recent phylogenetic hypothesis is employed to examine the evolution of three (out of a total of 21) characters, namely seed color, testa-cell protoplast characteristics and embryo orientation.

A broader model for C4 photosynthesis evolution in plants inferred from the goosefoot family (Chenopodiaceae s.s.).

C(4) photosynthesis is a fascinating example of parallel evolution of a complex trait involving multiple genetic, biochemical and anatomical changes. It is seen as an adaptation to deleteriously high levels of photorespiration. The current scenario for C(4) evolution inferred from grasses is that it originated subsequent to the Oligocene decline in CO(2) levels, is promoted in open habitats, acts as a pre-adaptation to drought resistance, and, once gained, is not subsequently lost. We test the generality of these hypotheses using a dated phylogeny of Amaranthaceae s.l. (including Chenopodiaceae), which includes the largest number of C(4) lineages in eudicots. The oldest chenopod C(4) lineage dates back to the Eocene/Oligocene boundary, representing one of the first origins of C(4) in plants, but still corresponding with the Oligocene decline of atmospheric CO(2). In contrast to grasses, the rate of transitions from C(3) to C(4) is highest in ancestrally drought resistant (salt-tolerant and succulent) lineages, implying that adaptation to dry or saline habitats promoted the evolution of C(4); and possible reversions from C(4) to C(3) are apparent. We conclude that the paradigm established in grasses must be regarded as just one aspect of a more complex system of C(4) evolution in plants in general.

Leaf surfaces of Gomphrena spp. (Amaranthaceae) from Cerrado biome.

The leaf structure and micromorphology characterize plant species and reflex its interactions with the environment. Leaf epidermis sculptures aid high transpiration plants on light reflection. The form and distribution of epicuticular wax crystalloids are important to characterize the surface. Aiming to know the micromorphology and the ultrastructure of G. arborescens, G. pohlii and G. virgata, leaves of these Cerrado native species were collected in Brasília, Distrito Federal, Brazil, at the Olympic Center of the Universidade de Brasília and at Reserva Ecológica do Roncador. Leaves of G. globosa, an Indian native species, were also studied for comparison. Leaves were fractionated, fixed and treated for observation under optical and scanning electron microscope. A description of the leaf epidermis is provided, along with some quantitative data to help the species taxonomy and support future studies on their physiology: all species are amphistomatic and have Stomatal Index between 7.27 and 18.99. The Gomphrena spp. studied have epicuticular wax platelets and wax sculptures over their larger trichome, which are relevant for their taxonomy. Over the Cerrado species cuticle, epicuticular wax is damaged by fungi hyphae development. The presence of epicuticular wax on Gomphrena spp. leaves corroborates the phylogenetical alliance between Amaranthaceae and Chenopodiaceae.

Leaf surfaces of Gomphrena spp. (Amaranthaceae) from Cerrado biome

The leaf structure and micromorphology characterize plant species and reflex its interactions with the environment. Leaf epidermis sculptures aid high transpiration plants on light reflection. The form and distribution of epicuticular wax crystalloids are important to characterize the surface. Aiming to know the micromorphology and the ultrastructure of G. arborescens, G. pohlii and G. virgata, leaves of these Cerrado native species were collected in Brasília, Distrito Federal, Brazil, at the Olympic Center of the Universidade de Brasília and at Reserva Ecológica do Roncador. Leaves of G. globosa, an Indian native species, were also studied for comparison. Leaves were fractionated, fixed and treated for observation under optical and scanning electron microscope. A description of the leaf epidermis is provided, alo ng with some quantitative data to help the species taxonomy and support future studies on their physiology: all species are amphistomatic and have Stomatal Index between 7.27 and 18.99. The Gomphrena spp. studied have epicuticular wax platelets and wax sculptures over their larger trichome, which are relevant for their taxonomy. Over the Cerrado species cuticle, epicuticular wax is damaged by fungi hyphae development. The presence of epicuticular wax on Gomphrena spp. leaves corroborates the phylogenetical alliance between Amaranthaceae and Chenopodiaceae.

Leaf surfaces of Gomphrena spp. (Amaranthaceae) from Cerrado biome

The leaf structure and micromorphology characterize plant species and reflex its interactions with the environment. Leaf epidermis sculptures aid high transpiration plants on light reflection. The form and distribution of epicuticular wax crystalloids are important to characterize the surface. Aiming to know the micromorphology and the ultrastructure of G. arborescens, G. pohlii and G. virgata, leaves of these Cerrado native species were collected in Brasília, Distrito Federal, Brazil, at the Olympic Center of the Universidade de Brasília and at Reserva Ecológica do Roncador. Leaves of G. globosa, an Indian native species, were also studied for comparison. Leaves were fractionated, fixed and treated for observation under optical and scanning electron microscope. A description of the leaf epidermis is provided, alo ng with some quantitative data to help the species taxonomy and support future studies on their physiology: all species are amphistomatic and have Stomatal Index between 7.27 and 18.99. The Gomphrena spp. studied have epicuticular wax platelets and wax sculptures over their larger trichome, which are relevant for their taxonomy. Over the Cerrado species cuticle, epicuticular wax is damaged by fungi hyphae development. The presence of epicuticular wax on Gomphrena spp. leaves corroborates the phylogenetical alliance between Amaranthaceae and Chenopodiaceae.(AU)

Vertical distribution of a soil microbial community as affected by plant ecophysiological adaptation in a desert system.

The spatial and temporal patterns of resource distribution in a desert system have been shown to influence a number of soil biota components and processes. The pattern of possible different resources supplied by two typical halophyte shrubs with different ecophysiological adaptations, Atriplex halimus and Hammada scoparia, was found to be the trigger in the present study on the microbial community temporal and spatial (vertical) scale. The uniqueness of A. halimus, a saltbush plant, is in the creation of 'islands of salinity' in its rhizosphere system and the relatively high percentage of organic matter beneath its canopy, whereas the H. scoparia shrub has a leafless stem with a relatively lower amount of organic matter accumulation beneath its canopy. Soil samples from 0 to 50 cm depths were collected at 10-cm intervals during each of the four seasons. The vertical distribution of the microbial community was measured in the vicinity of the above plant shrubs and in the open spaces between them. The results obtained from the field study demonstrated that plant ecophysiological adaptation played an important role in the temporal and spatial distribution of abiotic conditions and microbial community levels such as microbial biomass, CO(2) evolution, and colony-forming units (CFUs) of both bacteria and fungi. However, we did not find consistency in the positive effect of plant cover on the microbial community. The shrub effect was observed to have a major outcome on microbial variables not only on a temporal but also on a spatial (vertical) basis. This contributed to the understanding of the controversy regarding the difference between the shrubs.

[Intra-individual heterostyly in Atriplex halimus L. (Amaranthaceae)]. / Hétérostylie intra-individuelle chez Atriplex halimus L. (Amaranthaceae).

Classically, the Atriplex halimus L. female flower is perianthless, has two bracteoles, one ovary and one style. Considering bracteoles, one can distinguish, among the representatives of two Tunisian populations, three phenotypes of female flowers, each of them being distributed in three types according to the style length. Male flowers produce three types of pollen. This is the only known example of intra-individual heterostyly in unisexual flowers. These results question the classical concept of individual. The authors discuss a possible process implying indirectly hybridisation and introgression between A. halimus and A. nummularia.

Discriminating between Achyranthis Bidentatae Radix and Cyathulae Radix in Chinese medicine preparations by nested PCR and DNA sequencing methods.

The widely used Achyranthis Bidentatae Radix (Niu Xi) in Chinese medicine preparation is easily misused and confused with Cyathulae Radix (Chuan Niu Xi). Taiwan authority has ruled that Achyranthis Bidentatae Radix and Cyathulae Radix should be individually entered in the given preparations. In this study, with the internal transcribed spacer (ITS) as a DNA marker, nested PCR and DNA sequencing were used to distinguish the two herbs in preparations. Authentic Achyranthis Bidentatae Radix and Cyathulae Radix were collected from the original country, mainland China, and twenty two samples of Chinese medicine preparations labeled with Niu Xi as the ingredient were purchased locally. The total DNA of all samples were extracted by organic solution and purified using a kit. For a standard, ITS regions of authentic Achyranthis Bidentatae Radix and Cyathulae Radix were amplified with PCR and analyzed by an auto-sequencer. The ITS regions of preparation samples of DNA were amplified by nested PCR and the data of sequences received from an auto-sequencer. We compared the ITS sequences of preparation samples with the sequences of a standard and Genbank database to check which Achyranthis Bidentatae Radix or Cyathulae Radix is in the preparation samples. The results showed that only one sample contained Achyranthis Bidentatae Radix and the others contained Cyathulae Radix. The Cyathulae Radix components in four preparation samples do not fit into Taiwan authority rules. And our method can be applied for the differentiation of Achyranthis Bidentatae Radix and Cyathulae Radix in Chinese medicine preparations.

[Advances in study on chemical constituents from plants of genus Pfaffia and their bioactivities].

The progress in the studies on chemical constituents and pharmacological activity of the genus Pfaffia is summarized in recent 20 years. These plants contain various chemical constituents and have broad bioactivities such as sthenic, anti-tumor, analgesic and anti-inflammatory and should be further investigated.

Comparative analysis of phylogenetic relationships of grain amaranths and their wild relatives (Amaranthus; Amaranthaceae) using internal transcribed spacer, amplified fragment length polymorphism, and double-primer fluorescent intersimple sequence repeat markers.

The most economically important group of species in the genus Amaranthus is the A. hybridus species complex, including three cultivated grain amaranths, A. cruentus, A. caudatus, and A. hypochondriacus, and their putative wild progenitors, A. hybridus, A. quitensis, and A. powellii. Taxonomic confusion exists among these closely related taxa. Internal transcribed spacer (ITS) of nuclear ribosomal DNA, amplified fragment length polymorphism (AFLP), and double-primer fluorescent intersimple sequence repeat (ISSR) were employed to reexamine the taxonomic status and phylogenetic relationships of grain amaranths and their wild relatives. Low ITS divergence in these taxa resulted in poorly resolved phylogeny. However, extensive polymorphisms exist at AFLP and ISSR loci both within and among species. In phylogenetic trees based on either AFLP or ISSR or the combined data sets, nearly all intraspecific accessions can be placed in their corresponding species clades, indicating that these taxa are well-separated species. The AFLP trees share many features in common with the ISSR trees, both showing a close relationship between A. caudatus and A. quitensis, placing A. hybridus in the same clade as all grain amaranths, and indicating that A. powellii is the most divergent taxon in the A. hybridus species complex. This study has demonstrated that both AFLP and double-primer fluorescent ISSR have a great potential for generating a large number of informative characters for phylogenetic analysis of closely related species, especially when ITS diversity is insufficient.