The application of high-throughput sequencing for taxonomy: The case of Plantago subg. Plantago (Plantaginaceae).

Plantago is a cosmopolitan genus including over 250 species, concentrated in temperate and high-elevation tropical regions. The taxonomy of Plantago is very difficult, mainly because of its reduced morphology, which features relatively few characters for species classification. Consequently, the infrageneric classification of the genus remains controversial and inadequate. In this study we applied high-throughput plastid genome skimming to provide powerful phylogenetic resolution to clarify the relationships within subg. Plantago, which is the largest, most broadly distributed and poorest understood subgenus of Plantago. Ninety-four samples covering ~56% of all species and representing all sections of subg. Plantago as well as an outgroup were successfully sequenced. The resulting phylogenetic topology was used, complemented by field and herbarium studies, to revise the sectional classification of subg. Plantago and present a complete listing of the accepted species in the subgenus. Our phylogenetic results were also tested for their usefulness in clarifying the taxonomic placement of some taxonomically complicated species in the subgenus. We conclude that a combination of morphological studies and state-of-the art high-throughput DNA data provide a useful toolbox for resolving outstanding taxonomic puzzles exemplified by the genus Plantago.

Local adaptation at range edges: comparing elevation and latitudinal gradients.

Local adaptation at range edges influences species' distributions and how they respond to environmental change. However, the factors that affect adaptation, including gene flow and local selection pressures, are likely to vary across different types of range edge. We performed a reciprocal transplant experiment to investigate local adaptation in populations of Plantago lanceolata and P. major from central locations in their European range and from their latitudinal and elevation range edges (in northern Scandinavia and Swiss Alps, respectively). We also characterized patterns of genetic diversity and differentiation in populations using molecular markers. Range-centre plants of P. major were adapted to conditions at the range centre, but performed similarly to range-edge plants when grown at the range edges. There was no evidence for local adaptation when comparing central and edge populations of P. lanceolata. However, plants of both species from high elevation were locally adapted when compared with plants from high latitude, although the reverse was not true. This asymmetry was associated with greater genetic diversity and less genetic differentiation over the elevation gradient than over the latitudinal gradient. Our results suggest that adaptation in some range-edge populations could increase their performance following climate change. However, responses are likely to differ along elevation and latitudinal gradients, with adaptation more likely at high-elevation. Furthermore, based upon these results, we suggest that gene flow is unlikely to constrain adaptation in range-edge populations of these species.

Variations of the composition of the leaf cuticular wax among Chinese populations of Plantago major.

Plantago major L. grows in a very wide range of regions in China and exhibits great variations among populations. The analysis of the cuticular-wax composition provides a potential approach to classify populations of P. major confronting different environmental conditions. Twelve populations of P. major and five populations of P. depressa Willd., distributed over regions with average annual temperatures ranging from -2.0 to 18.4°, were sampled, the variation of the composition of their cuticular waxes was analyzed, and their values of average chain length (ACL) and carbon preference index (CPI) were calculated. Great intra- and interspecies variations were observed for the total wax contents. The average annual temperature of the habitats was significantly correlated with the relative contents of the dominant n-alkanes with an odd number of C-atoms, but not with the wax contents. With an increasing average annual temperature, the relative contents of n-alkanes C29 and C31 decreased, whereas those of C33 and C35 as well as the values of ACLtotal and ACL27-33 increased. Cluster analysis based on the pattern of the n-alkane distribution allowed to clearly separate the populations of P. major according to the average annual temperature of their habitats, but not to separate the populations of the two species. Hence, the pattern of the n-alkane distribution might be a good taxonomic marker for P. major at the intraspecies level, but not at the interspecies level. Nevertheless, a small difference between the populations of the two species was observed concerning the values of ACLtotal and CPItotal , implying the potential use of these indices for the classification of the populations of the two species at the interspecies level.

Banana (Musa spp) from peel to pulp: ethnopharmacology, source of bioactive compounds and its relevance for human health.

ETHNOPHARMACOLOGICAL RELEVANCE: Banana is a fruit with nutritional properties and also with acclaimed therapeutic uses, cultivated widely throughout the tropics as source of food and income for people. Banana peel is known by its local and traditional use to promote wound healing mainly from burns and to help overcome or prevent a substantial number of illnesses, as depression. AIM OF THE STUDY: This review critically assessed the phytochemical properties and biological activities of Musa spp fruit pulp and peel. MATERIALS AND METHODS: A survey on the literature on banana (Musa spp, Musaceae) covering its botanical classification and nomenclature, as well as the local and traditional use of its pulp and peel was performed. Besides, the current state of art on banana fruit pulp and peel as interesting complex matrices sources of high-value compounds from secondary metabolism was also approached. RESULTS: Dessert bananas and plantains are systematic classified into four sections, Eumusa, Rhodochlamys, Australimusa, and Callimusa, according to the number of chromosomes. The fruits differ only in their ploidy arrangement and a single scientific name can be given to all the edible bananas, i.e., Musa spp. The chemical composition of banana's peel and pulp comprise mostly carotenoids, phenolic compounds, and biogenic amines. The biological potential of those biomasses is directly related to their chemical composition, particularly as pro-vitamin A supplementation, as potential antioxidants attributed to their phenolic constituents, as well as in the treatment of Parkinson's disease considering their contents in l-dopa and dopamine. CONCLUSION: Banana's pulp and peel can be used as natural sources of antioxidants and pro-vitamin A due to their contents in carotenoids, phenolics, and amine compounds, for instance. For the development of a phytomedicine or even an allopathic medicine, e.g., banana fruit pulp and peel could be of interest as raw materials riches in beneficial bioactive compounds.

[Identification of plantaginis semen based on ITS2 and psbA-trnH sequences].

In order to evaluate the efficiency of ITS2 and psbA-trnH sequences used as DNA barcodes to distinguish Plantaginis Semen from its adulterants, we collected 71 samples of Plantaginis Semen and its adulterants. The ITS2 and psbA-trnH sequences were aligned through Clustal W, and the genetic distances were calculated by kimura 2-parameter (K2P) model and the Neighbor-Joining (NJ) phylogenetic trees were constructed using MEGA 5.1. The results indicated that the ITS2 sequence lengths of Plantago asiatica and P. depressa were 199 bp and 200 bp, respectively; the maximum intra-specific K2P distance were lower than the minimum inter-specific K2P distance; the NJ tree based on ITS2 sequence indicated that Plantaginis Semen and its adulterants could be distinguished clearly. The sequence lengths of psbA-trnH of both P. asiatica and P. depressa were 340 bp; the maximum intra-specific K2P distances were lower than the minimum inter-specific K2P distance; the NJ tree based on psbA-trnH sequence showed that Plantaginis Semen can be distinguished clearly from its adulterants except for P. major. Therefore, ITS2 sequences can be used as an ideal DNA barcode to distinguish Plantaginis Semen from its adulterants.

Role of plant ß-glucosidases in the dual defense system of iridoid glycosides and their hydrolyzing enzymes in Plantago lanceolata and Plantago major.

The typical defense compounds of Plantaginaceae are the iridoid glycosides, which retard growth and/or enhance mortality of non-adapted herbivores. In plants, glycosidic defense compounds and hydrolytic enzymes often form a dual defense system, in which the glycosides are activated by the enzymes to exert biological effects. Yet, little is known about the activating enzymes in iridoid glycoside-containing plants. To examine the role of plant-derived ß-glucosidases in the dual defense system of two common plantain species, Plantago lanceolata and Plantago major, we determined the concentration of iridoid glycosides as well as the ß-glucosidase activity in leaves of different age. To investigate the presence of other leaf metabolites potentially involved in plant defense, we used a metabolic fingerprinting approach with ultra-high performance liquid chromatography coupled with time-of-flight-mass spectrometry. According to the optimal defense hypothesis, more valuable parts such as young leaves should be better protected than less valuable parts. Therefore, we expected that both, the concentrations of defense compounds as well as the ß-glucosidase activity, should be highest in younger leaves and decrease with increasing leaf age. Both species possessed ß-glucosidase activity, which hydrolyzed aucubin, one of the two most abundant iridoid glycosides in both plant species, with high activity. In line with the optimal defense hypothesis, the ß-glucosidase activity in both Plantago species as well as the concentration of defense-related metabolites such as iridoid glycosides correlated negatively to leaf age. When leaf extracts were incubated with bovine serum albumin and aucubin, SDS-PAGE revealed a protein-denaturing effect of the leaf extracts of both plantain species, suggesting that iridoid glycosides and plant ß-glucosidase interact in a dual defense system.

Identification and quantification of phytochemical composition and anti-inflammatory, cellular antioxidant, and radical scavenging activities of 12 Plantago species.

Twenty-eight seed samples of 12 Plantago species were investigated for their chemical compositions and anti-inflammatory, cellular antioxidant, and radical scavenging properties. A new UPLC-UV procedure was developed and applied to quantify acteoside and geniposidic acid, the characteristic constituents of the genus Plantago. The amounts of acteoside and geniposidic acid ranged from 0.07 to 15.96 mg/g and from 0.05 to 10.04 mg/g in the tested samples, respectively. Furthermore, 26 compounds were tentatively identified by UPLC/Q-TOF-MS analysis. The Plantago samples significantly differed in their phytochemical compositions. The extracts of Plantago seeds also showed inhibitory effects on LPS-induced IL-1ß, IL-6, and COX-2 mRNA expression in RAW 264.7 mouse macrophage cells. Additionally, significant variations were observed among different samples on cellular antioxidant activities in HepG2 cells, as well as DPPH and hydroxyl radical scavenging capacities. The results from this study may be used to promote the use of the genus Plantago in improving human health.

Variable changes in genome size associated with different polyploid events in Plantago (Plantaginaceae).

The genus Plantago (Plantaginaceae) in New Zealand comprises species that can be difficult to delimit and contains a wide range of ploidy levels from diploid to 16-ploid. To investigate whether there have been changes in genome size associated with polyploid events and whether pollen and guard cell size can be used as an initial guide to identify polyploids, we have used flow cytometry with propidium iodide as the stain to measure the genome size (DNA C-value) in a range of Plantago species with different ploidy levels. Light microscopy was used to measure pollen diameter and guard cell length in these plants to see whether these could be used for ploidy level identification. Large differences in C-value were observed between species but the extent of changes associated with different polyploid events was highly variable. Pollen diameter was correlated with C-value but not necessarily with ploidy level. Guard cell lengths were different between diploids and polyploids but not between the different polyploid species. The significance of the changes in genome size and its relationship to pollen and stomatal guard cell size is discussed.

Estimation of heritability, evolvability and genetic correlations of two pollen and pistil traits involved in a sexual conflict over timing of stigma receptivity in Collinsia heterophylla (Plantaginaceae).

BACKGROUND AND AIMS: Heritable genetic variation is crucial for selection to operate, yet there is a paucity of studies quantifying such variation in interactive male/female sexual traits, especially those of plants. Previous work on the annual plant Collinsia heterophylla, a mixed-mating species, suggests that delayed stigma receptivity is involved in a sexual conflict: pollen from certain donors fertilize ovules earlier than others at the expense of reduced maternal seed set and lower levels of pollen competition. METHODS: Parent-offspring regressions and sib analyses were performed to test for heritable genetic variation and co-variation in male and female interactive traits related to the sexual conflict. KEY RESULTS: SOME heritable variation and evolvability were found for the female trait (delayed stigma receptivity in presence of pollen), but no evidence was found for genetic variation in the male trait (ability to fertilize ovules early). The results further indicated a marginally significant correlation between a male's ability to fertilize early and early stigma receptivity in offspring. However, despite potential indirect selection of these traits, antagonistic co-evolution may not occur given the lack of heritability of the male trait. CONCLUSIONS: To our knowledge, this is the first study of a plant or any hermaphrodite that examines patterns of genetic correlation between two interactive sexual traits, and also the first to assess heritabilities of plant traits putatively involved in a sexual conflict. It is concluded that the ability to delay fertilization in presence of pollen can respond to selection, while the pollen trait has lower evolutionary potential.

Phylogenetic perspectives on diversification, biogeography, and floral evolution of Collinsia and Tonella (Plantaginaceae).

PREMISE OF THE STUDY: Collinsia was the subject of classic biosystematic studies by Garber and colleagues and is increasingly investigated to address major evolutionary questions. Lack of phylogenetic data from more than one gene region and one taxonomic exemplar has left relationships, diversity, and phytogeography of Collinsia in question and has limited understanding of its diversification. METHODS: Phylogenetic analyses representing 179 populations of Collinsia and closely related Tonella were conducted based on DNA sequences of nuclear ribosomal transcribed spacers, the single-copy nuclear gene CYCLOIDEA-1, and part of the chloroplast matK/trnK intron region to reexamine systematic hypotheses and extend understanding of the importance of floral characters, chromosome evolution, interfertility, crossability, hybridization, edaphic factors, and ecogeographic barriers to diversification in the group. KEY RESULTS: Informal "sections" of Collinsia are artificial, although pedicel length and other traditional deep-level taxonomic characters are more conservative evolutionarily than flower size. Evolutionary loss of crossability and interfertility in Collinsia appears to be largely a byproduct of divergence. Although most taxa appear to have arisen by divergent evolution, multiple lines of evidence indicate a homoploid hybrid constitution of C. tinctoria, possibly explaining an occurrence of convergent chromosome evolution. Phylogeographic and cryptic diversity is extensive. CONCLUSIONS: Diversity in Collinsia is greater than previously documented. Recently divergent lineages are often associated with distinct habitat (including soil) and geographic factors, different flower sizes, and contrasting chromosomal arrangements. Evidence for a hybrid constitution of diploid C. tinctoria is consistent with lack of strong intersterility barriers between closely related taxa.

Potencial alelopático de extratos aquosos de Melissa officinalis L. e Mentha x villosa L. na germinação e vigor de sementes de Plantago major L. / Allelopathic effect of extracts of Melissa officinallis L. and Mentha x villosa L. on seed germination and vigor of Plantago major L.

O objetivo do trabalho foi avaliar o efeito dos extratos aquosos de hortelã e melissa na germinação e vigor de sementes de tanchagem. O experimento foi conduzido no Departamento de Fitotecnia da Universidade Federal de Viçosa, no mês de abril de 2011. O delineamento utilizado foi o inteiramente casualizado com cinco tratamentos (testemunha, 25 por cento, 50 por cento, 75 por cento e 100 por cento do extrato aquoso de hortelã e melissa) e quatro repetições. Os extratos foram preparados utilizando a parte aérea das plantas, na proporção de 100g de planta para 1 L de água destilada. Foram utilizadas 50 sementes de tanchagem em cada caixa gerbox, previamente umedecido com os extratos aquosos (correspondentes aos tratamentos), estas foram levadas para germinar em câmara de germinação, em temperatura de 20°C, fotoperíodo de 16 horas luz e 8 horas escuro, por 14 dias. Durante esse tempo foram feitas às contagens diárias das sementes germinadas para calcular o índice de velocidade de germinação e ao final dos 14 dias foi calculada a porcentagem de germinação. Sementes de tanchagem não submetidas aos extratos aquosos de melissa e hortelã apresentaram-se mais vigorosas quando comparado com as concentrações, evidenciando o potencial alelopático de melissa e hortelã sobre a sua qualidade fisiológica.

The aim of this work was to evaluate the effect of aqueous extracts of Melissa officinallis and Mentha x villosa for the germination and vigor of Plantago major seeds. The experiment was conducted at the Plant Science Department, Federal University of Viçosa, in April 2011. The design was a completely randomized design with five treatments (control, 25 percent, 50 percent, 75 percent and 100 percent of the aqueous extract of Mentha x villosa and Melissa Officinallis) and four replications. The extracts were prepared utulizando the shoots of plants at the rate of 100g of plant to 1 L of distilled water. We used 50 Plantago major seeds in each box incubator and these were subsequently placed in a germination chamber. 14 days during the counting of germinated seeds per day to calculate the index of germination speed and the end of 14 days was the germination percentage. Plantago major seeds not subjected to aqueous extracts of lemon balm and mint showed higher values in the variables studied when compared to other concentrations, showing the allelopathic potencial of Melissa officinallis and Mentha x villosa on the development of plantain, determined by germinating.

Phenylethanoid and iridoid glycosides in the New Zealand snow hebes (Veronica, Plantaginaceae).

Snow hebes are the alpine cushion-forming plants of New Zealand Veronica, formerly classified as Chionohebe. The chemical compositions of Veronica pulvinaris and Veronica thomsonii were studied and 33 water-soluble compounds were isolated. The structures of 14 previously unknown esters of phenylethanoid glycosides were elucidated by spectroscopic analyses. Further, eight known phenylethanoids, nine iridoids, 6'-feruloyl-sucrose and mannitol are also reported. It was found that the iridoid profile of the snow hebes was different from the other species of Veronica in New Zealand but similar to the alpine Northern Hemisphere representatives of the genus.

Evidence for a vicariant origin of Macaronesian-Eritreo/Arabian disjunctions in Campylanthus Roth (Plantaginaceae).

The numerous disjunct plant distributions between Macaronesia and eastern Africa-Arabia suggest that these could be the relicts of a once continuous vegetation belt along the southern Tethys, which has been fragmented by Upper Miocene-Pliocene aridification. We tested this vicariance hypothesis with a phylogenetic analysis of Campylanthus (Plantaginaceae), based on nuclear and plastid DNA sequence data. Our results indicate a basal split within Campylanthus giving rise to Macaronesian and Eritreo-Arabian lineages in the Pliocene/Upper Miocene. This is consistent with the vicariance hypothesis, thus obviating the need to postulate trans-Saharan long-distance dispersal. The biogeography of Campylanthus may parallel patterns in other plant groups and the implications for our understanding of the biogeography of northern and eastern Africa, and Arabia are discussed.

[Studies on the Herba Plantaginis of different kinds and habitats by FTIR reflection spectroscopy-chemometrics analysis].

In the present paper, FTIR of different species obtained from different regions of sibling plantaginis were determined by Fourier transform infrared reflection spectroscopy (FTIRS), and thirty-five comparatively typical absorption peaks were selected and used to study genetic relationship, combined with chemometric methods. The phylogenetic cluster analysis revealed that three species could be divided into two groups based on the distance of 0.036, among which Plantago depressa Willd. was clustered with Plantago asiatica L. based on distance of 0.033, while clustered with Plantago virginica L. based on distance of 0.042, and the result was consistent with that of traditional taxology. The principal component analysis result revealed that the distances of Plantago asiatica L. in the similar environment are similar in three-dimensional FTIR chart, however, it is dispersive when obtained from different regions. This method is scientific, simple and direct, and has important theoretical and practical application value.

Can differences in autonomous selfing ability explain differences in range size among sister-taxa pairs of Collinsia (Plantaginaceae)? An extension of Baker's Law.

Species with greater selfing ability are predicted to be better adapted for colonizing new habitats (Baker's Law). Here, we tested an expansion of this hypothesis: that species proficient at autonomous selfing have larger range sizes than their less proficient sister taxa. We also tested competing hypotheses regarding seed production and niche breadth on range size. Floral traits affecting the proficiency of autonomous selfing were measured and seed production was calculated for six sister-taxa pairs in the clade Collinsia. We tested for the hypothesized effects of these variables on elevational distribution and range size. We found that species most proficient at selfing had significantly larger range sizes than their sister-taxa that were less proficient at selfing. Species proficient at autonomous selfing occupied a higher mean elevation than their sister taxa, but they did not differ in their total elevational range. Species with greater seed production did not have larger range sizes. Our results extend Baker's Law, suggesting that species proficient at autonomous selfing are better adapted to establish new populations and thus can more readily expand their range. Autonomous selfing ability may play a vital role in explaining variance in range size among other species.

Species delimitation and phylogeny of a New Zealand plant species radiation.

BACKGROUND: Delimiting species boundaries and reconstructing the evolutionary relationships of late Tertiary and Quaternary species radiations is difficult. One recent approach emphasizes the use of genome-wide molecular markers, such as amplified fragment length polymorphisms (AFLPs) and single nucleotide polymorphisms (SNPs), to identify distinct metapopulation lineages as taxonomic species. Here we investigate the properties of AFLP data, and the usefulness of tree-based and non-tree-based clustering methods to delimit species and reconstruct evolutionary relationships among high-elevation Ourisia species (Plantaginaceae) in the New Zealand archipelago. RESULTS: New Zealand Ourisia are shown to comprise a geologically recent species radiation based on molecular dating analyses of ITS sequences (0.4-1.3 MY). Supernetwork analyses indicate that separate tree-based clustering analyses of four independent AFLP primer combinations and 193 individuals of Ourisia produced similar trees. When combined and analysed using tree building methods, 15 distinct metapopulations could be identified. These clusters corresponded very closely to species and subspecies identified on the basis of diagnostic morphological characters. In contrast, Structure and PCO-MC analyses of the same data identified a maximum of 12 and 8 metapopulations, respectively. All approaches resolved a large-leaved group and a small-leaved group, as well as a lineage of three alpine species within the small-leaved group. We were unable to further resolve relationships within these groups as corrected and uncorrected distances derived from AFLP profiles had limited tree-like properties. CONCLUSION: Ourisia radiated into a range of alpine and subalpine habitats in New Zealand during the Pleistocene, resulting in 13 morphologically and ecologically distinct species, including one reinstated from subspecies rank. Analyses of AFLP identified distinct metapopulations consistent with morphological characters allowing species boundaries to be delimited in Ourisia. Importantly, Structure analyses suggest some degree of admixture with most species, which may also explain why the AFLP data do not exhibit sufficient tree-like properties necessary for reconstructing some species relationships. We discuss this feature and highlight the importance of improving models for phylogenetic analyses of species radiations using AFLP and SNP data.

Amplified fragment length polymorphisms and sequence data in the phylogenetic analysis of polyploids: multiple origins of Veronica cymbalaria (Plantaginaceae).

The origin of polyploid Veronica cymbalaria (Plantaginaceae) was investigated using DNA sequence data and amplified fragment length polymorphism (AFLP) fingerprints to reveal the parentage of this taxon. The use of AFLP fingerprints in phylogenetic analysis is problematic and various methods have therefore been compared. DNA sequence data (for the internal transcribed spacer (ITS) region and the plastid trnL-F region (trnL intron, 3'exon, and trnL-F spacer)) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the ITS region suggested a reliable hypothesis for the evolution of the V. cymbalaria complex. This hypothesis allowed evaluation of the effect of different distance measures (Jaccard and Nei-Li) in phenetic, character-state weighted parsimony, and Bayesian analyses of AFLP markers. The study establishes that tetraploid V. cymbalaria originated at least twice in the eastern Mediterranean, with one parent differing in the two separate origins. Hexaploid V. cymbalaria originated even more often. The results illustrate that even subtle differences in the analyses of AFLP markers can lead to drastically different conclusions. The study reveals multiple origins of a Mediterranean polyploid species. Furthermore, it demonstrates that the analysis of a complex marker system such as AFLP fingerprints using only one type of analysis can easily be misleading.

Evaluation of morphological and molecular variation in Plantago asiatica var. densiuscula, with special reference to the systematic treatment of Plantago asiatica var. yakusimensis.

Morphological and molecular variations in Plantago asiatica L. var. densiuscula Pilg. were analyzed to evaluate the genetic basis for recognizing the dwarf variety P. asiatica var. yakusimensis (Masam.) Ohwi. Considerable variation in the leaf size of P. asiatica var. densiuscula was observed, and no morphological discontinuities were found between the dwarf types of P. asiatica var. densiuscula and P. asiatica var. yakusimensis. Morphological analysis of plants grown under standardized conditions revealed that both environmental plasticity and genetic differentiation contributed to the dwarfisms. Molecular phylogenetic analysis of rDNA internal transcribed spacer (ITS) regions and the SUC1 locus encoding a sucrose transporter revealed that P. asiatica var. yakusimensis was genetically unique although the differentiation level was low. From the above results, we concluded that P. asiatica var. yakusimensis should be reduced to a form of P. asiatica var. densiuscula. Furthermore, the geographic distribution of the SUC1 genotype suggested multiple origins of dwarves, and possible hypotheses for the origins of dwarves are discussed.

Chemotaxonomy of Veroniceae and its allies in the Plantaginaceae.

In a chemosystematic investigation of tribe Veroniceae (Plantaginaceae), representatives of Camptoloma, Sibthorpia, Veronica subg. Pentasepalae and subg. Hebe, Veronicastrum, Wulfenia, and the related Ellisiophyllum and Globularia were examined for non-flavonoid glycosides. From the 14 species studied, 28 different iridoid glucosides and 10 caffeoyl phenylethanoid glucosides (CPGs), as well as salidroside and arbutin were isolated and characterized by NMR; of these, five compounds were previously unknown. It was found that the representatives of Veroniceae, as well as Globularia, were characterized by mannitol, aucubin, catalpol and catalpol esters. Each of the three studied species of Veronica subg. Hebe contained at least one of the 6-O-catalpol esters typical for Veronica s. str. (verminoside), supporting the inclusion of Hebe in Veronica. However, their main constituents were esters of 6-O-rhamnopyranosylcatalpol; a CPG, hebeoside (2'-beta-xylopyranosyl-verbascoside) was isolated from V. (Hebe) salicifolia. The two species of Veronicastrum also contained 6-O-rhamnopyranosylcatalpol esters, including the previously unknown 2'',3''- and 3'',4''-dicinnamoyl derivatives and, in contrast to the earlier reports, they lacked 6-O-catalpol esters. The main iridoid constituents in the three investigated species of Wulfenia were 10-O-aucubin and 10-O-catalpol esters (isoscrophularioside or globularin) while baldaccioside (10-O-cinnamoyl asystasioside E) was isolated from W. baldaccii. Globularia vulgaris contained 10-O-catalpol esters (e.g., globularin) and, in addition, asperuloside together with its benzoyl analogue named besperuloside. The representatives of Sibthorpia and Ellisiophyllum were almost completely devoid of iridoids; this, however, together with the CPGs present implied a close relationship between the two genera. Camptoloma lyperiiflorum lacked hexitols but contained esters of 6-O-rhamnopyranosylcatalpol different from those found in Veroniceae but known from Buddleja, Scrophularia and Verbascum (Scrophulariaceae s. str.).