A NEW PHYLOGENY FOR CHROMOPHYTE ALGAE USING 16S-LIKE RRNA SEQUENCES FROM MALLOMONAS PAPILLOSA (SYNUROPHYCEAE) AND TRIBONEMA AEQUALE (XANTHOPHYCEAE)1

The 16S-like ribosomal RNA genes from Mallomonas papillosa Harris et Bradley (Synurophyceae) and Tribonema aequale Pascher (Xanthophyceae) were sequenced and compared to those of other eukaryotes. Mallomonas is closely related to Ochromonas (Chrysophyceae) and supports the general hypothesis of a close phylogenetic relationship between the Synurophyceae and Chrysophyceae. Tribonema is specifically related to Costaria costata (C. A. Agardh) Saunders (Phaeophyceae) demonstrating an unexpected phylogenetic relationship between the Xanthophyceae and Phaeophyceae. Distance and parsimony analysis place these four chromophyte genera in a complex evolutionary assemblage that includes the Bacillariophyceae and Oomycetes but excludes the Dinophyceae. The close relationship between the chromophyte algae and the Öomycete fungi supports the hypothesis that protists with tripartite hairs form a natural assemblage.     

Schizocladia ischiensis: a new filamentous marine chromophyte belonging to a new class,Schizocladiophyceae

A new marine filamentous chromophyte Schizocladia ischiensis sp. nov. is described from Naples, Italy, and a new class, Schizocladiophyceae, is proposed to accommodate the species based on morphology, photosynthetic pigment analysis, and rbcL and 18S rRNA gene sequences. The vegetative thallus is composed of branched filaments, 3-7 microm in diameter, containing one to two light brown parietal plastids. Cell walls are composed of layered fibers containing alginates, but lacking cellulose. Plastids are of the typical chromophyte type, containing chlorophylls a and c, and abundant fucoxanthin. Zoospores are formed by direct transformation of vegetative cells or through a process including a multinucleated cell stage. Zoospores are teardrop-shaped with a longer anterior flagellum with tubular mastigonemes and a shorter smooth posterior flagellum with a basal swelling. Flagella have a single basal plate and multi-gyred transitional helix distal to the basal plate. Each zoospore has an eyespot. Phylogenetic analyses using rbcL and 18S rDNA sequences suggest the closest phylogenetic relationship with Phaeophyceae, and then with Xanthophyceae and Phaeothamniophyceae. Nevertheless, Schizocladia differs from Phaeophyceae in some essential features (i.e. cell wall lacking cellulose and plasmodesmata, presence of flagellar transitional helix). Therefore, an independent class Schizocladiophyceae is proposed to accommodate this new taxon.     

PHYLOGENETIC AFFINITIES OF THE SARCINOCHRYSIDALES AND CHRYSOMERIDALES (HETEROKONTA) BASED ON ANALYSES OF MOLECULAR AND COMBINED DATA1

Small-subunit ribosomal RNA nucleotide sequences were inferred for Giraudyopsis stellifera Dangeard (Chrysomeridales), as well as for Pulvinaria sp. and Sarcinochrysis marina Geitler (Sarcinochrysidales,). Phylogenetic analyses of the molecular data indicate that the former is weakly related to the Phaeophyceae/Xanthophyceae clade, whereas the latter two have affinities to the Pelagophyceae, and the Sarcinochrysidales sensu stricto is transferred to this class. A recent study proposed that the Pelagophyceae belongs to a larger assemblage of chromophytic species characterized by reduced flagellar apparatuses. Although the flagellar apparatus characterizing the Sarcinochrysidales is reduced relative to the Chysomeridaels and some other chromophytes, it is the most complicated to be associated with “the reduced flagellar apparatus” lineage. Cladistic analyses of a traditional data set (largely ultrastructural features of the flagellar apparatus) and a combined traditional/molecular data set were used to assess the evolutionary trends of reduction in the flagellar apparatus within the heterokont chromophytes.     

ALGAE CONTAINING CHLOROPHYLLS a + c ARE PARAPHYLETIC: MOLECULAR EVOLUTIONARY ANALYSIS OF THE CHROMOPHYTA

Sequence comparisons of small subunit ribosomal RNA coding regions from 12 chlorophylls a + c-containing algae were used to infer phylogenetic relationships within the Chromophyta. Three chromophyte lines of descent, delineated by the Bacillariophyceae, the Phaeophyceae/Xanthophyceae, and the Chrysophyceae/Eustigmatophyceae/Synurophyceae are members of a complex evolutionary assemblage, which also includes representatives of the Oomycota (“lower” fungi). Maximum parsimony and distance matrix methods demonstrate a common evolutionary history for these lineages but their relative branching order could not be determined. Other algal species with chlorophylls a + c, including dinoflagellates and prymnesiophytes, are not members of this complex assemblage. Dinoflagellates are specifically related to apicomplexans and ciliates, and the prymnesiophyte, Emiliania huxleyi, represents an independent photosynthetic lineage that separated from other eukaryotes during the nearly simultaneous divergence of plants, animals, fungi, and a number of other protist lineages. The small subunit rRNA phylogenies of chromophytes/oomycetes were compared to those derived from comparisons of ultrastructural characters. Only tubular, tripartite mastigonemes (flagellar hairs) characterized all studied taxa of chromophytes/oomycetes as a monophyletic assemblage.     

DISTRIBUTION OF THE MITOCHONDRIAL DEVIANT GENETIC CODE AUA FOR METHIONINE IN HETEROKONT ALGAE

The DNA sequence of the cytochrome oxidase subunit I ( COX I) gene (1059 bp), was determined in a number of heterokont algae, including five species of the Phaeophyceae [ Chorda filum (Linnaeus) Stackhouse, Colpomenia bullosa (Saunders) Yamada, Ectocarpus sp., Pseudochorda nagaii (Tokida) Inagaki, Undaria pinnatifida (Harvey) Suringar], and a member of the Raphidophyceae [ Chattonella antiqua (Hada) Ono]. The distribution of a deviant mitochondrial code, the AUA codon for methionine (AUA/Met), which was previously reported in the Xanthophyceae, was inferred from these COX I sequences. Comparative analyses of these sequences revealed that all the algae described above bear the universal genetic code, including the assignment for the AUA codon. A phylogenetic tree was constructed using the obtained sequences along with already-published COX I sequences of various heterokont algae. The clusters of the Xanthophyceae and the Phaeophyceae were resolved as sister groups with high bootstrap support, excluding a bacillariophycean species, a raphidophycean species, and three species of the Eustigmatophyceae. Taking the distribution of the deviant code and the COX I phylogenetic tree together, the genetic code change most probably occurred in an ancestor of the Xanthophyceae after it had branched off from the Phaeophyceae.     

CHARACTERIZATION AND PHYLOGENETIC POSITION OF THE ENIGMATIC GOLDEN ALGA PHAEOTHAMNION CONFERVICOLA: ULTRASTRUCTURE, PIGMENT COMPOSITION AND PARTIAL SSU rDNA SEQUENCE

The morphology, ultrastructure, photosynthetic pigments, and nuclear-encoded small subunit ribosomal DNA (SSU rDNA) were examined for Phaeothamnion confervicola Lagerheim strain SAG119.79. The morphology of the vegetative filaments, as viewed under light microscopy, was indistinguishable from the isotype. Light microscopy, including epifluorescence microscopy, also revealed the presence of one to three chloroplasts in both vegetative cells and zoospores. Vegetative filaments occasionally transformed to a palmelloid stage in old cultures. An eyespot was not visible in zoospores when examined with light microscopy, but small droplets, similar to eyespot droplets, were apparent beneath the shorter flagellum when cells were viewed with electron microscopy. Zoospores had two flagella that were laterally inserted in the cell approximately one-third of the cell length from the apex. The longer flagellum was directed anteriorly and the shorter flagellum was directed posteriorly. Electron microscopy revealed the presence of tubular tripartite flagellar hairs on the longer flagellum, but no lateral filaments were found on the tripartite hairs. The general organization of the flagellar root system was similar to that of zoospores belonging to the Xanthophyceae and Phaeophyceae. However, the transitional region of the flagella contained a transitional helix with four to six gyres. Microtubular root R₁ consisted of six microtubules at its proximal end and one microtubule at its distal end. Roots R₂ and R₄ consisted of one microtubule each and root R₃ consisted of two microtubules. No rhizoplast was found. Thin-layer chromatography revealed the presence of fucoxanthin, diadinoxanthin, neoxanthin, and heteroxanthin as well as chlorophylls a, c₁ and c₂. High-performance liquid chromatography revealed the presence of fucoxanthin, diadinoxanthin, diatoxanthin, heteroxanthin, and β,β-carotene as well as chlorophylls a and c. The complete sequence of the SSU rDNA could not be obtained, but a partial sequence (1201 bases) was determined. Parsimony and neighbor-joining distance analyses of SSU rDNA from Phaeothamnion and 36 other chromophyte algae (with two Öomycete fungi as the outgroup) indicated that Phaeothamnion was a weakly supported (bootstrap = <50%, 52%) sister taxon to the Xanthophyceae representatives and that this combined clade was in turn a weakly supported (bootstrap = <50%, 67%) sister to the Phaeophyceae. Based upon ultrastructural observations, pigment analysis, and SSU rDNA phylogenetic analysis, Phaeothamnion is not a member of the Chrysophyceae and should be classified as incertae sedis with affinities to the Xanthophyceae and Phaeophyceae.     

The structure and phylogenetic significance of the flagellar transition region in the chlorophyll c-containing algae.

An electron-dense helix is the most conspicuous structure in the flagellar transition region of members of the algal class Chrysophyceae. This “transitional helix” (TH) lies immediately distal to a partition across the flagellar axoneme which occurs exactly at the level at which the flagellum enters the cell body. The helix surrounds the central axonemal pair and lies at a distance of 10 nm from the 9 peripheral doublets. From the new data presented and a survey of published observations on the structure of the transition region of all the chlorophyll c-containing classes of algae, it is shown that a TH characteristic of the Chrysophyceae, Xanthophyceae and Eustigmatophyceae. The number of TH gyres varies from 3 to 6 in the Xanthophyceae and from 1 to 8 in the Chrysophyceae. In any one species, however, the TH is the same size in both the long flagellum which bears tubular mastigonemes and in the short smooth flagellum, though in some chrysophytes where the short flagellum is vestigial the number is fewer than in the normal flagellum. A TH appears to be absent from the Rhaphidophyceae and zoids of the Bacillariophyceae and Phaeophyceae though the structure of the transition region in these groups otherwise resembles that of the Chrysophyceae, Xanthophyceae and Eustigmatophyceae.The value of transition region variation in determining evolutionary relationships among the chlorophyll c-containing algal classes is assessed against a background of current ideas on their taxonomy and phylogeny. The relevant structural and biochemical features are tabled, and a phylogenetic scheme is presented which appears most logically to interpret these data. It is suggested that the line leading to the Eustigmatophyceae probably diverged from that leading to the strictly heterokont classes Xanthophyceae, Chrysophyceae, Phaeophyceae and Bacillariophyceae before evolution of a girdle lamella in the chloroplast and a photoreceptor apparatus involving a swelling at the proximal end of the short flagellum and an intraplastidial eyespot. The possession of a TH by both the Chrysophyceae and Xanthophyceae adds further support to the concept of their close relationship based on a range of other features. The exceptional absence of a TH from the chrysophycean genera Pedinella and Pseudopedinella reinforces the idea that these taxa are remote from the main chrysophycean line. Absence of a TH from the Phaeophyceae and Bacillariophyceae which otherwise share many important features with the Chrysophyceae and Xanthophyceae is probably a result of loss owing to the functional and morphological specialization of the zoids of these two groups. Transition region structure does not clarify the possible relationships of the Rhaphidophyceae, Prymnesiophyceae, Cryptophyceae or Dinophyceae.The proposed phylogeny supports the idea of a mutually related “heterokont” protist assemblage comprising the Chrysophyceae, Xanthophyceae, Phaeophyceae, Bacillariophyceae and possibly Rhaphidophyceae and the Oomycetes (water moulds) though in the latter the TH is replaced by a dense cylinder with a corrugated wall which may or may not be homologous with it. Structures resembling a TH have been described in a wide variety of other flagellated cells including the prasinophyte Pyraminonas orientalis, one species of the colourless flagellate genus Bicosoeca and the proteromonads Karotomorpha and Proteromonas. Only in the latter genera does homology with a TH seem likely on present evidence, suggesting that flagellates of this type may have evolved from chrysomonad-like ancestors.     

Inventaire des algues et phanérogames marines benthiques de la Tunisie

Abstract

Check list of the benthic marine flora of Tunisia. — 393 species, varieties, formae and stadii of algae and seagrasses are listed: 6 Bangiophyceae, 239 Florideophyceae, 1 Xanthophyceae, 82 Phaeophyceae, 20 Chlorophyceae, 45 Bryopsidophyceae and 5 seagrasses.     

Morphological convergence characterizes the evolution of Xanthophyceae (Heterokontophyta): evidence from nuclear SSU rDNA and plastidial rbcL genes

Xanthophyceae are a group of heterokontophyte algae. Few molecular studies have investigated the evolutionary history and phylogenetic relationships of this class. We sequenced the nuclear-encoded SSU rDNA and chloroplast-encoded rbcL genes of several xanthophycean species from different orders, families, and genera. Neither SSU rDNA nor rbcL genes show intraspecific sequence variation and are good diagnostic markers for characterization of problematic species. New sequences, combined with those previously available, were used to create different multiple alignments. Analyses included sequences from 26 species of Xanthophyceae plus three Phaeothamniophyceae and two Phaeophyceae taxa used as outgroups. Phylogenetic analyses were performed according to Bayesian inference, maximum likelihood, and maximum parsimony methods. We explored effects produced on the phylogenetic outcomes by both taxon sampling as well as selected genes. Congruent results were obtained from analyses performed on single gene multiple alignments as well as on a data set including both SSU rDNA and rbcL sequences. Trees obtained in this study show that several currently recognized xanthophycean taxa do not form monophyletic groups. The order Mischococcales is paraphyletic, while Tribonematales and Botrydiales are polyphyletic even if evidence for the second order is not conclusive. Botrydiales and Vaucheriales, both including siphonous taxa, do not form a clade. The families Botrydiopsidaceae, Botryochloridaceae, and Pleurochloridaceae as well as the genera Botrydiopsis and Chlorellidium are polyphyletic. The Centritractaceae and the genus Bumilleriopsis also appear to be polyphyletic but their monophyly cannot be completely rejected with current evidence. Our results support morphological convergence at any taxonomic rank in the evolution of the Xanthophyceae. Finally, our phylogenetic analyses exclude an origin of the Xanthophyceae from a Vaucheria-like ancestor and favor a single early origin of the coccoid cell form.     

Analysis of the Cluster of Ribosomal Protein Genes in the Plastid Genome of a Unicellular Red Alga Cyanidioschyzon merolae: Translocation of the str Cluster as an Early Event in the Rhodophyte-Chromophyte Lineage of Plastid Evolution

The nucleotide sequence of a cluster of ribosomal protein genes in the plastid genome of a unicellular red alga, Cyanidioschyzon merolae, which has been supposed to be the most primitive alga, was determined. The phylogenetic tree inferred from the amino acid sequence of ribosomal proteins of two rhodophytes, a chromophyte, a glaucophyte, two chlorophytes (land plants), a cyanobacterium, and three eubacteria suggested a close relationship between the cyanobacterium Synechocystis PCC6803 and the plastids of various species in the kingdom Plantae, which is consistent with the hypothesis of the endosymbiotic origin of plastids. In this tree, the two species of rhodophytes were grouped with the chromophyte, and the glaucophyte was grouped with the chlorophytes. Analysis of the organization of the genes encoding the ribosomal proteins suggested that the translocation of the str cluster occurred early in the lineage of rhodophytes and chromophytes after these groups had been separated from chlorophytes and glaucophytes. Received: 2 June 1997 / Accepted: 15 July 1997     

Morphology,fine structure,life cycle and phylogenetic analysis of Phyllosiphon arisari,a siphonous parasitic green alga

Phyllosiphon arisari Kühn (Phyllosiphonaceae, Chlorophyta) commonly occurs in Arisarum leaves in coastal Mediterranean areas of the Iberian Peninsula and Balearic islands. The genus Phyllosiphon was first considered to be a member of the Xanthophyceae but was later transferred to the chlorophytes. However, there are few data about its morphology, ultrastructure, ecology or phylogenetic affinities. In this paper we describe the morphology of Phyllosiphon, as studied in field material and in culture; the fine structure, analysed by transmission electron microscopy; and phylogenetic relationships, inferred from DNA sequences. The siphonous filaments were seen to divide and penetrate leaf tissues. The cytoplasm divided into spherical or subspherical sporocysts producing autospores inside. Cytoplasmic remains could be observed between autospores or on their cell walls. Phylogenetic analysis of 18S rDNA and 16S rDNA sequences showed that the closest relatives of Phyllosiphon are subaerial strains of Heterochlorella, Heveochlorella and Kalinella, demonstrating that Phyllosiphon should be transferred to Trebouxiophyceae. An evolution from unicells to a siphonous thallus, and from aerophytic to endophytic and parasitic habits, is proposed for Trebouxiophyceae.     

In situ microscopy reveals reversible cell wall swelling in kelp sieve tubes: one mechanism for turgor generation and flow control?

Kelps, brown algae (Phaeophyceae) of the order Laminariales, possess sieve tubes for the symplasmic long‐distance transport of photoassimilates that are evolutionarily unrelated but structurally similar to the tubes in the phloem of vascular plants. We visualized sieve tube structure and wound responses in fully functional, intact Bull Kelp (Nereocystis luetkeana [K. Mertens] Postels & Ruprecht 1840). In injured tubes, apparent slime plugs formed but were unlikely to cause sieve tube occlusion as they assembled at the downstream side of sieve plates. Cell walls expanded massively in the radial direction, reducing the volume of the wounded sieve elements by up to 90%. Ultrastructural examination showed that a layer of the immediate cell wall characterized by circumferential cellulose fibrils was responsible for swelling and suggested that alginates, abundant gelatinous polymers of the cell wall matrix, were involved. Wall swelling was rapid, reversible and depended on intracellular pressure, as demonstrated by pressure‐injection of silicon oil. Our results revive the concept of turgor generation and buffering by swelling cell walls, which had fallen into oblivion over the last century. Because sieve tube transport is pressure‐driven and controlled physically by tube diameter, a regulatory role of wall swelling in photoassimilate distribution is implied in kelps.     

PHOSPHOTUNGSTIC ACID-CHROMIC ACID: A SELECTIVE STAIN FOR ALGAL PLASMA MEMBRANES1,2

A phosphotungstic acid-chromic acid mixture selectively stains the plasma membrane of whole cells of selected members of the Chlorophyceae, Charophyceae, Euglenophyceae, Xanthophyceae, Bacillariophyceae, Chrysophyceae, and Rhodophyceae, and the plasma membrane in cell-free fractions of Mougeotia (Chlorophyceae). The procedure is not effective on the plasma membrane of the cyanophycean Scytonema or the cyanophycean endosymbiont of Glaucocystis. Staining of the cell walls of Chlamydomonas, Bangia, and Scytonema and the pellicle and sliding junction of Euglena and Astasia suggest that PTA-CrO₃ reactivity may be associated with glycoproteins in the cell walls and plasma membranes.     

The marine algae of Orkney

Six species are described and compared with related algae. All were isolated in culture from terrestrial habitats on Signy Island, South Orkney Islands, Antarctica. They are: Botrydiopsis constricta sp. nov. (Mischococcales, Xanthophyceae), Heterothrix antarctica sp. nov. (Tribonematales, Xanthophyceae), Sphaerocystis oleifera sp. nov. (Chlorococcales, Chlorophyceae), Sphaerocystis signiensis sp. nov., Sphaerocystis bilobata sp. nov. and Fottea pyrenoidosa sp. nov. (Ulothricales, Chlorophyceae).     

Aurearenophyceae classis nova, a new class of Heterokontophyta based on a new marine unicellular alga Aurearena cruciata gen. et sp. nov. inhabiting sandy beaches

A new heterokontophyte alga, Aurearena cruciata gen. et sp. nov., was isolated from sandy beaches in Japan. Isolates were characterized by light and electron microscopy, spectroscopy of pigment composition, and molecular phylogenetic analyses using 18S rDNA and rbcL. The alga usually possessed a cell wall but also retained two heterokont flagella beneath the cell wall. Each walled cell first produced only a single flagellate cell that subsequently divided into two flagellate cells. Electron-opaque vesicles, possibly associated with cell wall formation, were observed beneath the cell membrane. The chloroplast consisted of two compartments, each enclosed by a chloroplast envelope and the inner membrane of the chloroplast endoplasmic reticulum; these two compartments were surrounded by a common outer membrane of chloroplast endoplasmic reticulum. Molecular phylogenetic trees suggested that this alga was a new and independent member of the clade that included the Phaeophyceae and Xanthophyceae (PX clade). A new class, Aurearenophyceae classis nova was proposed for A. cruciata.     

A "Total Evidence" Analysis of the Phylogenetic Relationships among the Photosynthetic Stramenopiles

Phylogenetic relationships among the nine major autotrophic stramenopile taxa were inferred in a combined analysis of the rbcL, SSU rDNA, partial LSU rRNA, carotenoid, and ultrastructural data sets. The structure of the shortest combined tree is: (Outgroup, ((((Bacillariophyceae, (Pelagophyceae, Dictyochophyceae)),((Phaeophyceae, Xanthophyceae), Raphidophyceae)), Eustigmatophyceae),(Chrysophyceae, Synurophyceae))). The Synurophyceae/Chrysophyceae is the best supported group followed by the Phaeophyceae/Xanthophyceae and the Pelagophyceae/Dictyochophyceae clades. The monophyletic groups composed of Bacillariophyceae/Pelagophyceae/Dictyochophyceae and Phaeophyceae/Xanthophyceae/Raphidophyceae received the lowest Bremer support values. The optimal combined tree suggests that the diatom frustule is derived from the siliceous "skeleton" in Dictyochophyceae, that the reduced flagellar apparatus arose once in the Bacillariophyceae/Dictyochophyceae/Pelagophyceae clade, and that the specific photoreceptor-eyespot apparatus in Chrysophyceae and the Phaeophyceae/Xantophyceae clade originated independently within the autotrophic stramenopiles. Despite conflicts in tree structure between the most-parsimonious combined phylogeny and the optimal tree(s) of each data partition, it cannot be concluded that extensive incongruence exists between the data sets.     

Current state of seaweed resources in Spain

Commercial seaweeds in Spain are harvested on the north and northwest coasts. They are mainly agarophytes and carrageenophytes (Gelidium spp. and some Irish moss-like species, respectively), although some Phaeophyceae species (Fucus spp. and Laminaria spp.) are also exploited for alginates. No industrial seaweed cultivation is carried out in Spain at present. Spain's total commercial seaweed harvest can be estimated at 6,528 ± 2,076 t dry wt year^–1. Gelidium spp. are by far the most harvested, attaining 5,135 ± 1,761 t dry wt y^–1. To date, Spain's commercial seaweed harvest is low compared with that of other countries, but data on field standing crops and productivities of commercial taxa suggest that harvesting could be increased greatly.     

Sequence analysis of duplicated actin genes in Lagenidium giganteum and Pythium irregulare (Oomycota)

Southern analysis of genomic DNA identified multiple-copy actin gene families in Lagenidium giganteum and Pythium irregulare (Oomycota). Polymerase chain reaction (PCR) protocols were used to amplify members of these actin gene families. Sequence analysis of genomic coding regions demonstrated five unique actin sequences in L. giganteum (Lg-Ac 1, 2, 3, 4, 5) and four unique actin sequences in P. irregulare (Pi-Acl, 2, 3, 4); none were interrupted by introns. Maximum parsimony analysis of the coding regions demonstrated a close phylogenetic relationship between oomycetes and the chromophyte alga Costaria costata. Three types of actin coding regions were identified in the chromophyte/oomycete lineage. The type 1 actin is the single-copy coding region found in C. costata. The type 2 and type 3 actins are found in the oomycetes and are the result of a gene duplication which occurred soon after the divergence of the oomycetes from the chromophyte algae. The type 2 coding regions are the single-copy sequence of Phytophthora megasperma, the Phytophthora infestans actB gene, Lg-Ac5 and Pi-Ac2. The type 3 coding regions are the single-copy sequence of Achlya bisexualis, the P. infestans actA gene, Lg-Ac1, 2, 3, 4 and Pi-Acl, 3, 4. Correspondence to: D. Bhattacharya     

Micromorphological studies of the leaf epidermis of Iranian Cotoneaster Medik

In this study, micromorphological features of the leaf epidermal cells of 17 species of Iranian Cotoneaster were determined. The studied species were compared with each other and with phylogenetic relationships established by previous studies. Three populations of C. integerrimus and C. multiflorus were studied as representatives of C. subgen. Cotoneaster and Chaenopetalum, respectively. To evaluate quantitative and qualitative characters, multiple correspondence analysis (MCA) and factor analysis of mixed data (FAMD) were performed. Finally, relationships among the taxa were illustrated by hierarchical clustering analysis. Three stomatal types were identified, i.e. anomocytic, stephanocytic and actino‐stephanocytic. The wax and cuticle density was higher in species from semi‐moist habitats than in those from dry and semi‐dry habitats. The patterns formed by the anticlinal walls of the epidermal cells were sinuous, repand and straight‐curved. Interestingly, there was a significant relationship between the length of leaves, the density of the indumentum and the pattern formed by the anticlinal cell walls. The leaves of species with large leaves also have lower hair density and more curved anticlinal cell walls. It appears probable that climatic conditions affected the evolution of leaf micro‐morphological characters during the early diversification of the genus, but that these characters have subsequently been permanently fixed by phylogenetic constraints and thus appear diagnostic for extant species.