Old research projects
Annonaceae
Cremastosperma (and other evolutionary digressions)
Molecular phylogenetic, biogeographic, and taxonomic studies in Neotropical
Annonaceae
(link to the complete thesis in electronic form)
Michael D. Pirie; National Herbarium of the Netherlands - Utrecht University
branch: Ph.D. thesis. Promoter: Prof. Dr. P.J.M. Maas; co-promoter: Dr. L.W.
Chatrou
Results are presented of research on a family of flowering plants, the
Annonaceae, species of which are found in tropical rainforest across the world.
The project focussed on one group of species, the genus Cremastosperma,
which is found in the South and Central American tropics. A taxonomic revision
of Cremastosperma was made. Most of the species of Cremastosperma
are found along the foothills of the Andes. Twenty-nine species were recognised,
13 of which were new to science. They are often narrow endemics, found only in
areas such as the valleys of the Magdalena River in Colombia (e.g. C.
magdalenae), and Marañon in Peru (e.g. C. bullatum). DNA sequences
were compared in order to reconstruct the phylogeny of 19 of the 29 species of
Cremastosperma. Clades were revealed which included species distributed
either west or east of the Andes mountain chain, providing further evidence to
suggest the importance of the Andean orogeny as a vicariance event in the
history of the evolution of Cremastosperma.
Many other groups of plants share this ‘Andean-centred’ distribution, and the
rising of the Andean mountain chain has thus been hypothesised to have caused
the origin of a large proportion of the high biodiversity of the New World
tropics. DNA sequences were used to reconstruct the relationships between
species of four Andean-centred genera of Annonaceae, Cremastosperma,
Klarobelia, Malmea and Mosannona, and to estimate how long ago
those species shared common ancestors. The conclusion was drawn that the Andean-centred
distribution patterns as observed are not the arbitrary result of the definition
of poly- or paraphyletic groups. The timing of species diversifications in these
groups was shown to coincide with the rise of the northern Andes, within the
last 20-30 million years.
Further results compared the timing of diversifications in Cremastosperma
and Mosannona with those in two more widely distributed and species rich
genera, Duguetia and Guatteria. The effects of taxon and character
sampling on date estimates in these genera were assessed and compared. Higher
sampling of crown group taxa of the species-rich genus Guatteria resulted
in significantly older age estimations, representing a potentially serious bias
in a widely used molecular dating method (nonparametric rate-smoothing).
Molecular dating techniques should be assessed for sensitivity to levels of
taxon sampling under differing conditions.
Finally, an ancient paralogue of the widely used chloroplast marker trnL-F was
discovered, the origin of which was inferred to have taken place in a common
ancestor of the Annonaceae. The paralogue discovered evolves at a faster rate
and appears to have complementary phylogenetic signal. It may be a useful
phylogenetic marker. Although the exon appears to be intact, some intron
sequences show signs of disruption of the secondary structure which is otherwise
conserved across land plants. Function may thus have been lost. Future research
should attempt to determine both the whereabouts and origin of both copies of
trnL-F in the Annonaceae genome.
Algae
S.G.A. Draisma and Dr W.F. Prud’homme van Reine – NHN, Leiden; Dr W.T. Stam and Prof. Dr J.L. Olsen - Dept. of Marine Biology, Centre for Ecological and Evolutionary Studies, University of Groningen.
The heterokont class Phaeophyceae, better known as the brown algae, have continued to fascinate systematists throughout the last century for the simple reason that they present an extensive range of morphological form—from microscopic filaments to the giant, structurally complex thalli of kelps. The comparative morphological approach has, therefore, been supplied with a rich source of characters including thallus construction, modes of growth, type of sexual reproduction and characteristics of the chloroplast. Silva and De Reviers (2000) reviewed the classical taxonomic literature and catalogued some 39 ordinal names, including 17 descriptive names and 22 names based solely on specific genera. Numerous classifications have been proposed (e.g. Bold & Wynne 1985, Van den Hoek et al. 1995, De Reviers & Rousseau 1999, Graham & Wilcox 2000). At present, 13-17 orders of Phaeophyceae are recognised: Ascoseirales, Chordariales, Cutleriales, Desmarestiales, Dictyosiphonales, Dictyotales, Durvillaeales, Ectocarpales, Fucales, Laminariales, Ralfsiales (nomen nudum), Scytosiphonales, Scytothamnales, Sphacelariales, Sporochnales, Syringodermatales and Tilopteridales. De Reviers and Rousseau (1999) downgraded the Durvillaeales (and the Notheiales) to family status within the Fucales. They merged the Chordariales, Dictyosiphonales (including the Punctariales), Ectocarpales and Scytosiphonales into the Ectocarpales sensu lato (s.l.) although they were not the first authors to propose this.
Evolutionary relationships within the Phaeophyceae have long been a source of discussion and speculation. Various phylogenetic hypotheses have been put forth, based on a limited number of characters and in the absence of any formal analyses. For example, Wynne and Loiseaux (1976) envisioned two deep sister-lineages, one including the Fucales and Durvillaeales and the other including all of the remaining orders with the Ectocarpales s.s. most primitive. Clayton (1984) suggested that the Dictyotales represented an independent evolutionary lineage, not closely related to any of the other orders. This was based on the presence of unique uniflagellate spermatozoids and meiosporangia that differed from the more typical unilocular sporangia found in brown algae. She also hypothesized that the Fucales were derived from the Chordariales or Dictyosiphonales. Based on their simple filamentous construction, Van den Hoek et al. (1995) considered the Ectocarpales s.s. to be the most primitive brown algae; likewise they considered the Fucales and Durvillaeales as the most advanced based on the lack of a free-living gametophytic phase.
With the advent of DNA sequence data and formal analysis methods in the early 1990s, the picture of the evolutionary history of the brown algae has been steadily emerging. The first studies were coarse and involved extremely limited taxon sampling. Gradually, however, representatives from all of the principal orders have been surveyed. The nuclear rDNA small subunit gene (18S) has provided the bulk of the data. Unfortunately, the resolving power of the 18S has proven disappointing owing to the apparent relative recency of the various phaeophycean lineages. Partial sequences of the rDNA large subunit gene (26S) have also been used with similar results (Rousseau & De Reviers 1999, Rousseau et al. 2000). The rDNA phylogeny of De Reviers and Rousseau (1999) provides the best taxon sampling so far, but the tree remains poorly resolved, there are no support values and no non-phaeophycean outgroup. The authors suggested that rapid divergence has resulted in a "brown algal crown radiation". The chloroplast encoded, large subunit RUBISCO gene (rbcL) has recently been added to the arsenal (e.g. Siemer et al. 1998) of useful sequences for brown algal phylogeny. To date, the available rbcL data has been restricted to a few groups so that full-scale comparisons across the phaeophytes have necessarily remained limited. The observation that rbcL has a slightly faster mutation rate than ribosomal genes within the phaeophytes may yet allow us to recover some of the branching order within the "brown algal crown radiation".
In order to better assess the current state of phaeophycean phylogeny, we compiled all currently available rbcL, 18S and 26S rDNA sequences from the EMBL/GenBank database; and added new rbcL sequences of our own. We then developed three new alignments that were designed to maximize taxon sampling while minimizing information loss due to partial sequences. Phylogenetic analyses were performed on separate and combined data sets (with and without taxa from the sister-classes Tribophyceae and Phaeothamniophyceae as outgroups) using a variety of assumption sets, tree-drawing algorithms (parsimony, neighbor joining and likelihood) and resampling methods (bootstrap, decay, jackknife). Partition homogeneity testing (PHT) by codon position within rbcL showed that all positions could be used despite mild third position saturation. PHT by gene and domain within rDNA showed that the 26S D1 and D2 regions do not enhance phylogenetic signal even when combined with the 18S. The rbcL and rDNA (excluding the 26S D1 and D2) could be combined under PHT. The topology of the combined tree was the same as that of the rbcL tree alone but bootstrap support was consistently higher in the combined analysis, applied to more branches, and enabled the establishment of sister-group relationships among six orders. Although the taxon sampling for the combination tree was lower (N=22) than for individual gene analyses (N=58 for rbcL and N=59 for rDNA), results show that the Laminariales (previously reported) and Sphacelariales (new) are both paraphyletic. Choristocarpus tenellus is the most basal phaeophyte and the Dictyotales the most basal order. In contrast, the Laminariales s.s. and Ectocarpales s.l. are the most derived. For phylogenetic studies in the Phaeophyceae, rbcL has more resolving power than rDNA though the reason for this is unclear based on the fact that both genes are highly conserved.
Phylogeny of the Sphacelariales
S.G.A. Draisma and Dr W.F. Prud’homme van Reine – NHN, Leiden; Dr W.T. Stam and Prof. Dr J.L. Olsen - Dept. of Marine Biology, Centre for Ecological and Evolutionary Studies, University of Groningen.
The cosmopolitan Sphacelariales (Phaeophyceae) was erected by Migula (1909). The order is characterised by blackening of the cell walls when treated with bleaching liquid and growth by conspicuous apical cells. Transverse divisions of the subapical cells (or primary segments) result in secondary segments and are followed by longitudinal and secondary transverse divisions. In this matter a parenchymatous construction is achieved. Other general features include an isomorphic diplohaplontic life history and asexual reproduction in some genera from distinctively shaped vegetative propagules. Two species have been recorded from freshwater, the others are marine. Oltmanns (1922) divided the order into three families based on modes of growth and distinctive branching patterns. These include the Cladostephaceae (1 genus, 1 species), the Sphacelariaceae (2 genera, ca. 45 species) and the Stypocaulaceae (5 genera, 14 species). The family Choristocarpaceae Kjellman 1891 (4 genera, 5 species) was also placed in the Sphacelariales by Fritsch (1945) based on the presence of growth by prominent apical cells. However, the members of this family do not show the characteristic transverse division of subapical cells, nor do their cell walls blacken in bleach. Therefore, the former three families are often referred to as the Sphacelariales sensu stricto (s.s.), whereas the term Sphacelariales sensu lato (s.l.) also includes the latter family.
A molecular phylogenetic study has now shown that the Sphacelariales is paraphyletic. Using both sequences of the chloroplast-encoded large subunit of the RUBISCO gene (rbcL) and partial sequences of the small and large subunit of the nuclear-encoded ribosmal cistron, we were able to identify three clades. These included the Sphacelariales s.s., a clade containing the genera Onslowia and Verosphacella and a clade containing Choristocarpus by itself. The latter three genera were members of the Chorsitocarpaceae. A third choristocarpacean genus, Discosporangium, was not included in the study. However, it is clear that the Choristocarpaceae do not belong to the Sphacelariales. Draisma and Prud’homme van Reine (submitted) separated Onslowia and Verosphacella from the Choristocarpaceae and placed them in the newly created family Onslowiaceae. Both the Choristocarpaceae and the Onslowiaceae were considered incertae sedis for the time being.
Within the Sphacelariales s.s. phylogenetic relationships were furhter examined using rbcL sequences and sequences of its adjacent RUBISCO spacer. All but one were the northern hemisphere specimens. It turned out that the monotypic Cladostephaceae and the Stypocaulaceae were nested within the Sphacelariaceae. Stypocaulaceae remained, however, monophyletic. Nomenclatural changes have not yet been made. Radical options are to call everything Sphacelaria (although Cladostephus is the oldest name) or to raise several Sphacelaria species and subgenera to the genus or family level. However, we suggest to await sequences of southern hemisphere genera and subgenera before nomenclatural changes are implemented.