BUILDING A SPECIES PHYLOGENY FOR THE GENUS COLEOCHAETE (COLEOCHAETALES) USING THE GENES RBCL AND ATPB

The freshwater green alga Coleochaete Breb. (Coleochaetaceae; Coleochaetales) is a key streptophyte genus and is important to the understanding of the evolutionary origin of embryophytes (land plants). To date only a few species have been available from public culture collections. To facilitate research on this genus we have isolated 17 previously uncultured species of Coloechaete from material collected in the United States, Puerto Rico, and the Dominican Republic. Sequences for the genes rbcL and atpB were determined for these new isolates of Coleochaete (and for existing cultures) and combined with sequences from representative other streptophytes. Phylogenetic analyses indicate that Coleochaete, along with Chaetosphaeridium and Chara, are closely related to embryophytes and constitute a ‘higher streptophyte’ clade. At least four well‐supported lineages exist within Coleochaete. Characteristic growth forms have been identified for these four lineages, with important characters including aspects of thallus establishment, thallus habit, zygote development and hair sheath position. These data provide an improved understanding of species diversity and character evolution in the genus Coleochaete, and facilitate examination of hypotheses concerning character evolution in the streptophytes.     

PHYLOGENY OF THE BASAL LINEAGES OF STREPTOPHYTA BASED ON RBCL AND ATPB GENE SEQUENCE DATA

The streptophytes comprise the Charophyceae sensu Mattox and Stewart (a morphologically diverse group of fresh‐water green algae) and the embryophytes (land plants). Several charophycean groups are currently recognized. These include the Charales, Coleochaetales, Chlorokybales, Klebsormidiales and Zygnemophyceae (Desmidiales and Zygnematales). Recently, SSU rRNA gene sequence data allied Mesostigma viride (Prasinophyceae) with the Streptophyta. Complete chloroplast sequence data, however, placed Mesostigma sister to all green algae, not with the streptophytes. Several morphological, ultrastructural and biochemical features unite these lineages into a monophyletic group including embryophytes, but evolutionary relationships among the basal streptophytes remain ambiguous. To date, numerous studies using SSU rRNA gene sequences have yielded differing phylogenies with varying degrees of support dependent upon taxon sampling and choice of phylogenetic method. Like SSU data, chloroplast DNA sequence data have been used to examine relationships within the Charales, Coleochaetales, Zygnemophyceae and embryophytes. Representatives of all basal streptophyte lineages have not been examined using chloroplast data in a single analysis. Phylogenetic analyses were performed using DNA sequences of rbcL (the genes encoding the large subunit of rubisco) and atpB (the beta‐subunit of ATPase) to examine relationships of basal streptophyte lineages. Preliminary analyses placed the branch leading to Mesostigma as the basal lineage in the Streptophyta with Chlorokybus, the sole representative of the Chlorokybales, branching next. Klebsormidiales and the enigmatic genus Entransia were sister taxa. Sister to these, the Charales, Coleochaetales, embryophytes and Zygnemophyceae formed a monophyletic group with Charales and Coleochaetales sister to each other and this clade sister to the embryophytes.     

THE PINGUIOPHYCEAE CLASSIS NOVA, CHROMOPHYTE ALGAE PRODUCING LARGE AMOUNTS OF OMEGA-3 FATTY ACIDS

The streptophytes comprise the Charophyceae sensu Mattox and Stewart (a morphologically diverse group of fresh-water green algae) and the embryophytes (land plants). Several charophycean groups are currently recognized. These include the Charales, Coleochaetales, Chlorokybales, Klebsormidiales and Zygnemophyceae (Desmidiales and Zygnematales). Recently, SSU rRNA gene sequence data allied Mesostigma viride (Prasinophyceae) with the Streptophyta. Complete chloroplast sequence data, however, placed Mesostigma sister to all green algae, not with the streptophytes. Several morphological, ultrastructural and biochemical features unite these lineages into a monophyletic group including embryophytes, but evolutionary relationships among the basal streptophytes remain ambiguous. To date, numerous studies using SSU rRNA gene sequences have yielded differing phylogenies with varying degrees of support dependent upon taxon sampling and choice of phylogenetic method. Like SSU data, chloroplast DNA sequence data have been used to examine relationships within the Charales, Coleochaetales, Zygnemophyceae and embryophytes. Representatives of all basal streptophyte lineages have not been examined using chloroplast data in a single analysis. Phylogenetic analyses were performed using DNA sequences of rbc L (the genes encoding the large subunit of rubisco) and atp B (the beta-subunit of ATPase) to examine relationships of basal streptophyte lineages. Preliminary analyses placed the branch leading to Mesostigma as the basal lineage in the Streptophyta with Chlorokybus , the sole representative of the Chlorokybales, branching next. Klebsormidiales and the enigmatic genus Entransia were sister taxa. Sister to these, the Charales, Coleochaetales, embryophytes and Zygnemophyceae formed a monophyletic group with Charales and Coleochaetales sister to each other and this clade sister to the embryophytes.     

The Transition From Algae to Embryophytes: Chloroplast Phylogenomic Evidence (II)

The transition of plant life from aquatic algae to land plants was one of the major events in the history of life. However, in hypothesizing the evolutionary path of the transition, limited shared phenotypic characters in aquatic algae and land plants (embryophytes) have been a major hinderance. Chloroplast genomes contain characters useful in tracing evolutionary histories. Embryophyte chloroplast genomes are distinguished from algal cpDNAs by the presence of over 20 group II introns and three ribosomal protein operons (rpl23, clpP and 3?rps12 operons). These phylogenomic features indicate a phylogenetic relationship of charophytes and embryophytes. In addition to these operons and introns, the evolution of rRNA and psbB operon evolution of streptophyte lineages will be incorporated with major biological phenotypic features to produce a phylogenetic tree. Basal embryophytes, the antithetic hypothesis, monophyly of embryophytes, and paraphyly of charophytes will be discussed. Strepotophytes are classified into three major groups (basal streptophytes, mid‐divergent streptophytes and late divergent charophytes‐embryophytes).     

MOLECULAR AND MORPHOLOGICAL DATA IDENTIFY A CRYPTIC SPECIES COMPLEX IN ENDOPHYTIC MEMBERS OF THE GENUS COLEOCHAETE BRÉB. (CHAROPHYTA: COLEOCHAETACEAE)1

The genus Coleochaete Bréb. is a relatively small group of freshwater microscopic green algae with about 15 recognized species. Although Coleochaete has long been considered to be a close relative of embryophytes, a comprehensive study of the genus has not been published since Pringsheim's 1860 monograph. As part of a systematic study of Coleochaete, we investigated four accessions of the genus that are morphologically similar to the endophytic species C. nitellarum Jost. Each of the four cultures was determined to be capable of endophytic growth in Nitella C. A. Agardh, a member of the closely related order Charales. Maximum likelihood and maximum parsimony analyses were performed on nucleotide data from the chloroplast genes atpB and rbcL that were sequenced from 16 members of the Coleochaetales and from other members of the Charophyceae, embryophytes, and outgroup taxa. These analyses indicate that the Coleochaetales are monophyletic and that the endophytic accessions are members of the scutata group of species. In addition, cell size and nucleotide data suggest that at least three different endophytic species may be represented. Herbivory, nutritional benefits, and substrate competition are three hypotheses that could explain the evolution and maintenance of the endophytic habit in Coleochaete. These data also imply that diversity in the genus may be markedly underestimated.     

THE OCCURRENCE AND PHYLOGENETIC SIGNIFICANCE OF PUTATIVE PLACENTAL TRANSFER CELLS IN THE GREEN ALGA COLEOCHAETE

Following fertilization, zygotes of the green alga Coleochaete orbicularis, which are retained on the haploid thallus, first enlarge, then become covered with a layer of vegetative cells. Light microscopy and high-voltage electron microscopy revealed the presence of localized wall ingrowths in vegetative cells adjacent to zygotes. These covering cells resemble the gametophytic placental transfer cells of embryophytes in their morphology, location, and time of development. If Coleochaete cells with wall protuberances function as do placental transfer cells of embryophytes, their presence is evidence that photosynthates may be transported between haploid thallus cells and zygotes. Thus, a nutritional relationship between different phases of the life cycle, similar to that which occurs in embryophytes, may also have evolved in green algae. This first report of putative placental transfer cells in a green alga supports Bower's (1908) ideas concerning the origin of land plant sporophytes and alternation of generations. The presence or absence of cells with wall ingrowths in several species of Coleochaete was correlated with estimates of zygote-plant area ratios.     

OCCURRENCE AND PHYLOGENETIC SIGNIFICANCE OF “SPECIAL WALLS” AT MEIOSPOROGENESIS IN COLEOCHAETE

Within germinating zygotes of Coleochaete pulvinata, meiospores are individually surrounded by chamber walls which are ultrastructurally and chemically different from vegetative cell walls of the same species. Meiospore chamber walls exhibit the staining reactions typical of callose. They thus resemble the “special walls” present during sporogenesis in embryophytes. Their presence suggests that the charophycean green algal ancestors of land plants may have possessed spore development preadaptations influential in the evolution of walled spores, an important plant adaptation to terrestrial life.     

PHYLOGENY OF THE GENUS COLEOCHAETE (COLEOCHAETALES,CHAROPHYTA) AND RELATED TAXA INFERRED BY ANALYSIS OF THE CHLOROPLAST GENE rbcL1

The genus Coleochaete Bréb. is considered to be a key taxon in the evolution of green algae and embryophytes (land plants), but only a few of the approximately 15 species have been studied with molecular phylogenetic methods. We report here the sequences of the gene rbcL from six new cultures of Coleochaete and two of Chaetosphaeridium Klebahn. These sequences were combined with 32 additional sequences, and phylogenetic analyses were performed with maximum likelihood, distance optimality, and parsimony methods. Important subgroups within Coleochaete include two primary lineages, one marked by fully corticated zygotes and the other by naked or weakly corticated zygotes. In the first lineage there is a subclade with tightly joined filaments and distinctive (“T‐shaped”) cell division, an assemblage of strains that resembles the endophytic species Coleochaete nitellarum Jost, and a clade with loosely joined filaments and “Y‐shaped” cell divisions. Consistent with recent multigene phylogenies, these analyses support the monophyly of the Coleochaetales, place the Charales as the sister taxon to land plants, and indicate that Chaetosphaeridium is far more closely related to Coleochaete than to Mesostigma Lauterborn.     

Aeroterrestrial Coleochaete (Streptophyta, Coleochaetales) models early plant adaptation to land

? Premise of the study: The streptophyte water-to-land transition was a pivotal, but poorly understood event in Earth history. While some early-diverging modern streptophyte algae are aeroterrestrial (living in subaerial habitats), aeroterrestrial survival had not been tested for Coleochaete, widely regarded as obligately aquatic and one of the extant green algal genera most closely related to embryophytes. This relationship motivated a comparison of aeroterrestrial Coleochaete to lower Paleozoic microfossils whose relationships have been uncertain. ? Methods: We tested the ability of two species of the experimentally tractable, complex streptophyte algal genus Coleochaete Bréb. to (1) grow and reproduce when cultivated under conditions that mimic humid subaerial habitats, (2) survive desiccation for some period of time, and (3) produce degradation-resistant remains comparable to enigmatic Cambrian microfossils. ? Key results: When grown on mineral agar media or on quartz sand, both species displayed bodies structurally distinct from those expressed in aquatic habitats. Aeroterrestrial Coleochaete occurred as hairless, multistratose, hemispherical bodies having unistratose lobes or irregular clusters of cells with thick, layered, and chemically resistant walls that resemble certain enigmatic lower Paleozoic microfossils. Whether grown under humid conditions or air-dried for a week, then exposed to liquid water, aeroterrestrial Coleochaete produced typical asexual zoospores and germlings. Cells that had been air-dried for periods up to several months maintained their integrity and green pigmentation. ? Conclusions: Features of modern aeroterrestrial Coleochaete suggest that ancient complex streptophyte algae could grow and reproduce in moist subaerial habitats, persist through periods of desiccation, and leave behind distinctive microfossil remains.     

Streptophyte algae and the origin of embryophytes

Background

Land plants (embryophytes) evolved from streptophyte green algae, a small group of freshwater algae ranging from scaly, unicellular flagellates (Mesostigma) to complex, filamentous thalli with branching, cell differentiation and apical growth (Charales). Streptophyte algae and embryophytes form the division Streptophyta, whereas the remaining green algae are classified as Chlorophyta. The Charales (stoneworts) are often considered to be sister to land plants, suggesting progressive evolution towards cellular complexity within streptophyte green algae. Many cellular (e.g. phragmoplast, plasmodesmata, hexameric cellulose synthase, structure of flagellated cells, oogamous sexual reproduction with zygote retention) and physiological characters (e.g. type of photorespiration, phytochrome system) originated within streptophyte algae.

Recent Progress

Phylogenetic studies have demonstrated that Mesostigma (flagellate) and Chlorokybus (sarcinoid) form the earliest divergence within streptophytes, as sister to all other Streptophyta including embryophytes. The question whether Charales, Coleochaetales or Zygnematales are the sister to embryophytes is still (or, again) hotly debated. Projects to study genome evolution within streptophytes including protein families and polyadenylation signals have been initiated. In agreement with morphological and physiological features, many molecular traits believed to be specific for embryophytes have been shown to predate the Chlorophyta/Streptophyta split, or to have originated within streptophyte algae. Molecular phylogenies and the fossil record allow a detailed reconstruction of the early evolutionary events that led to the origin of true land plants, and shaped the current diversity and ecology of streptophyte green algae and their embryophyte descendants.

Conclusions

The Streptophyta/Chlorophyta divergence correlates with a remarkably conservative preference for freshwater/marine habitats, and the early freshwater adaptation of streptophyte algae was a major advantage for the earliest land plants, even before the origin of the embryo and the sporophyte generation. The complete genomes of a few key streptophyte algae taxa will be required for a better understanding of the colonization of terrestrial habitats by streptophytes.Key words: Chlorophyta, Streptophyta, Embryophyta, Charales, Coleochaetales, Zygnematales, viridiplant phylogeny, land plants, genome evolution, freshwater adaptation, sporophyte origin, diversification, extinction     

The charophycean green algae provide insights into the early origins of plant cell walls

Numerous evolutionary innovations were required to enable freshwater green algae to colonize terrestrial habitats and thereby initiate the evolution of land plants (embryophytes). These adaptations probably included changes in cell-wall composition and architecture that were to become essential for embryophyte development and radiation. However, it is not known to what extent the polymers that are characteristic of embryophyte cell walls, including pectins, hemicelluloses, glycoproteins and lignin, evolved in response to the demands of the terrestrial environment or whether they pre-existed in their algal ancestors. Here we show that members of the advanced charophycean green algae (CGA), including the Charales, Coleochaetales and Zygnematales, but not basal CGA (Klebsormidiales and Chlorokybales), have cell walls that are comparable in several respects to the primary walls of embryophytes. Moreover, we provide both chemical and immunocytochemical evidence that selected Coleochaete species have cell walls that contain small amounts of lignin or lignin-like polymers derived from radical coupling of hydroxycinnamyl alcohols. Thus, the ability to synthesize many of the components that characterize extant embryophyte walls evolved during divergence within CGA. Our study provides new insight into the evolutionary window during which the structurally complex walls of embryophytes originated, and the significance of the advanced CGA during these events.     

Flourishing in water: the early evolution and diversification of plant receptor-like kinases

Receptor-like kinases (RLKs) play significant roles in mediating innate immunity and development of plants. The evolution of plant RLKs has been characterized by extensive variation in copy numbers and domain configurations. However, much remains unknown about the origin, evolution, and early diversification of plant RLKs. Here, we perform phylogenomic analyses of RLKs across plants (Archaeplastida), including embryophytes, charophytes, chlorophytes, prasinodermophytes, glaucophytes, and rhodophytes. We identify the presence of RLKs in all the streptophytes (land plants and charophytes), nine out of 18 chlorophytes, one prasinodermophyte, and one glaucophyte, but not in rhodophytes. Interestingly, the copy number of RLKs increased drastically in streptophytes after the split of the clade of Mesostigmatophyceae and Chlorokybophyceae and other streptophytes. Moreover, phylogenetic analyses suggest RLKs from charophytes form diverse distinct clusters, and are dispersed along the diversity of land plant RLKs, indicating that RLKs have extensively diversified in charophytes and charophyte RLKs seeded the major diversity of land plant RLKs. We identify at least 81 and 76 different kinase-associated domains for charophyte and land plant RLKs, 23 of which are shared, suggesting that RLKs might have evolved in a modular fashion through frequent domain gains or losses. We also detect signatures of positive selection for many charophyte RLK groups, indicating potential functions in host–microbe interaction. Taken together, our findings provide significant insights into the early evolution and diversification of plant RLKs and the ancient evolution of plant–microbe symbiosis.     

Chloroplast gene arrangement variation within a closely related group of green algae (Trebouxiophyceae, Chlorophyta)

The 22 published chloroplast genomes of green algae, representing sparse taxonomic sampling of diverse lineages that span over one billion years of evolution, each possess a unique gene arrangement. In contrast, many of the >190 published embryophyte (land plant) chloroplast genomes have relatively conserved architectures. To determine the phylogenetic depth at which chloroplast gene rearrangements occur in green algae, a 1.5-4 kb segment of the chloroplast genome was compared across nine species in three closely related genera of Trebouxiophyceae (Chlorophyta). In total, four distinct gene arrangements were obtained for the three genera Elliptochloris, Hemichloris, and Coccomyxa. In Elliptochloris, three distinct chloroplast gene arrangements were detected, one of which is shared with members of its sister genus Hemichloris. Both species of Coccomyxa examined share the fourth arrangement of this genome region, one characterized by very long spacers. Next, the order of genes found in this segment of the chloroplast genome was compared across green algae and land plants. As taxonomic ranks are not equivalent among different groups of organisms, the maximum molecular divergence among taxa sharing a common gene arrangement in this genome segment was compared. Well-supported clades possessing a single gene order had similar phylogenetic depth in green algae and embryophytes. When the dominant gene order of this chloroplast segment in embryophytes was assumed to be ancestral for land plants, the maximum molecular divergence was found to be over two times greater in embryophytes than in trebouxiophyte green algae. This study greatly expands information about chloroplast genome variation in green algae, is the first to demonstrate such variation among congeneric green algae, and further illustrates the fluidity of green algal chloroplast genome architecture in comparison to that of many embryophytes.     

轮藻和陆地植物系统发育及其进化

Charophytic algae and land plants together make up a monophyletic group, streptophytes, which represents one of the main lineages of multicellular eukaryotes and has contributed greatly to the change of the environment on earth in the Phanerozoic Eon. Significant progress has been made to understand phylogenetic relationships among members of this group by phylogenetic studies of morphological and molecular data over the last twenty-five years. Mesostigma viride is now regarded as among the earliest diverging unicellular organisms in streptophytes. Characeae are the sister group to land plants. Liverworts represent the first diverging lineage of land plants. Hornworts and lycophytes are extant representatives of bryophytes and vascular plants, respectively, when early land plants changed from gametophyte to sporophyte as the dominant generation in the life cycle. Equisetum, Psilotaceae, and ferns constitute the monophyletic group of monilophytes, which are sister to seed plants. Gnetales are related to conifers, not to angiosperms as previously thought. Amborella, Nymphaeales, Hydatellaceae, Illiciales, Trimeniaceae, and Austrobaileya represent the earliest diverging lineages of extant angiosperms. These phylogenetic results, together with recent progress on elucidating genetic and developmental aspects of the plant life cycle, multicellularity, and gravitropism, will facilitate evolutionary developmental studies of these key traits, which will help us to gain mechanistic understanding on how plants adapted to environmental challenges when they colonized the land during one of the major transitions in evolution of life.     

EST analysis of the scaly green flagellate Mesostigma viride (Streptophyta): Implications for the evolution of green plants (Viridiplantae)

Background  

The Viridiplantae (land plants and green algae) consist of two monophyletic lineages, the Chlorophyta and the Streptophyta. The Streptophyta include all embryophytes and a small but diverse group of freshwater algae traditionally known as the Charophyceae (e.g. Charales, Coleochaete and the Zygnematales). The only flagellate currently included in the Streptophyta is Mesostigma viride Lauterborn. To gain insight into the genome evolution in streptophytes, we have sequenced 10,395 ESTs from Mesostigma representing 3,300 independent contigs and compared the ESTs of Mesostigma with available plant genomes (Arabidopsis, Oryza, Chlamydomonas), with ESTs from the bryophyte Physcomitrella, the genome of the rhodophyte Cyanidioschyzon, the ESTs from the rhodophyte Porphyra, and the genome of the diatom Thalassiosira.     

Starting from scratch: Evolution of the lichen thallus in the basidiolichen Dictyonema (Agaricales: Hygrophoraceae)

Phylogenetic studies indicate that the basidiolichen genus Dictyonema s.lat., often thought to represent only a single genus with few species, includes several well-supported genus-level clades, all of which form associations with a unique lineage of obligately lichenized cyanobacteria (Rhizonema). In an attempt to elucidate the evolution and genus- and species-level diversification in Dictyonema s.lat., we generated 68 new sequences of the nuclear large subunit rDNA (nuLSU), the internal transcribed spacer (ITS), and the RNA polymerase II subunit (RPB2), for 29 species-level lineages representing all major clades of Dictyonema s.lat. and most of the species currently known. The multilocus phylogeny obtained via maximum likelihood and Bayesian approaches indicates the presence of five genus-level groups: a basal clade, Cyphellostereum, that is sister to the rest of the species, a paraphyletic grade representing Dictyonema s.str., and three clades representing the genera Acantholichen, Cora, and Corella. To determine the evolutionary transformations of the lichenized thallus in the group, ancestral character state reconstruction was done using six characters (lichenisation, thallus type, cortex type, hyphal sheath and haustorial type, photobiont morphology, and basidiocarp type). Our analysis indicates a progressive development of the lichenized thallus from loosely organized filamentous crusts with separate, cyphelloid basidiocarps in Cyphellostereum, to filamentous crusts with derived hyphal sheath and cyphelloid–stereoid basidiocarps partially incorporated into the lichen thallus in Dictyonema, to squamulose–foliose thalli with corticioid basidiocarps entirely supported by the lichen thallus in Cora. These results indicate a remarkable evolutionary integration of lichenized and reproductive tissues in Dictyonema s.lat., supporting the hypothesis that, at least in this case, lichenized thalli may have evolved from reproductive structures in their nonlichenized ancestors.     

Amino Acid Compositional Shifts During Streptophyte Transitions to Terrestrial Habitats

Across the streptophyte lineage, which includes charophycean algae and embryophytic plants, there have been at least four independent transitions to the terrestrial habitat. One of these involved the evolution of embryophytes (bryophytes and tracheophytes) from a charophycean ancestor, while others involved the earliest branching lineages, containing the monotypic genera Mesostigma and Chlorokybus, and within the Klebsormidiales and Zygnematales lineages. To overcome heat, water stress, and increased exposure to ultraviolet radiation, which must have accompanied these transitions, adaptive mechanisms would have been required. During periods of dehydration and/or desiccation, proteomes struggle to maintain adequate cytoplasmic solute concentrations. The increased usage of charged amino acids (DEHKR) may be one way of maintaining protein hydration, while increased use of aromatic residues (FHWY) protects proteins and nucleic acids by absorbing damaging UV, with both groups of residues thought to be important for the stabilization of protein structures. To test these hypotheses we examined amino acid sequences of orthologous proteins representing both mitochondrion- and plastid-encoded proteomes across streptophytic lineages. We compared relative differences within categories of amino acid residues and found consistent patterns of amino acid compositional fluxuation in extra-membranous regions that correspond with episodes of terrestrialization: positive change in usage frequency for residues with charged side-chains, and aromatic residues of the light-capturing chloroplast proteomes. We also found a general decrease in the usage frequency of hydrophobic, aliphatic, and small residues. These results suggest that amino acid compositional shifts in extra-membrane regions of plastid and mitochondrial proteins may represent biochemical adaptations that allowed green plants to colonize the land.     

Evolutionary history and speciation modes in the cyprinid genus Barbus.

Phylogenetic relationships and evolutionary patterns in the genus Barbus were examined through the analysis of the complete sequences of three mitochondrial genes: ATPases 8 and 6, which overlap slightly, and cytochrome b. This complex genus includes diploid as well as tetraploid and hexaploid species that are distributed throughout the Palaearctic, Ethiopian and Asiatic biogeographical regions. Given that genome duplication is an important evolutionary mechanism in eukaryotes, in the present report we attempt to describe its role in the evolution of the genus Barbus, as well as drawing systematic and phylogenetic conclusions. The phylogenetic results indicated the splitting of the current Barbus genus into five main mitochondrial lineages corresponding to (i) the genus Barbus sensu stricto (tetraploid, which is subdivided into the subgenera Barbus and Luciobarbus), (ii) the hexaploid species, (iii) the Ethiopian tetraploid species, (iv) the African diploid species, and (v) the Asian diploid species. The branching of 'foreign' genera as sister groups of some of these monophyletic assemblages (such as Aulopyge is to Barbus sensu stricto or Varicorhinus is to the hexaploid barbels) demonstrates the polyphyly of the group. Moreover, the relationships between the proposed lineages also show that genome duplication may be considered as a homoplasic character since it must have occurred over at least three independent periods and/or in three independent areas. In relation to the possible saltational evolutionary model for the polyploid species examined here, it was found that, although feasible at the nuclear level, the mitochondrial markers looked at do not appear to have undergone this type of evolution. Rather, they seem to have experienced more or less constant change through time.