Lars W. Chatrou

Angiosperm phylogeny based on matK sequence information

Plastid matK gene sequences for 374 genera representing all angiosperm orders and 12 genera of gymnosperms were analyzed using parsimony (MP) and Bayesian inference (BI) approaches. Traditionally, slowly evolving genomic regions have been preferred for deep-level phylogenetic inference in angiosperms. The matK gene evolves approximately three times faster than the widely used plastid genes rbcL and atpB. The MP and BI trees are highly congruent. The robustness of the strict consensus tree supercedes all individual gene analyses and is comparable only to multigene-based phylogenies. Of the 385 nodes resolved, 79% are supported by high jackknife values, averaging 88%. Amborella is sister to the remaining angiosperms, followed by a grade of Nymphaeaceae and Austrobaileyales. Bayesian inference resolves Amborella + Nymphaeaceae as sister to the rest, but with weak (0.42) posterior probability. The MP analysis shows a trichotomy sister to the Austrobaileyales representing eumagnoliids, monocots + Chloranthales, and Ceratophyllum + eudicots. The matK gene produces the highest internal support yet for basal eudicots and, within core eudicots, resolves a crown group comprising Berberidopsidaceae/Aextoxicaceae, Santalales, and Caryophyllales + asterids. Moreover, matK sequences provide good resolution within many angiosperm orders. Combined analyses of matK and other rapidly evolving DNA regions with available multigene data sets have strong potential to enhance resolution and internal support in deep level angiosperm phylogenetics and provide additional insights into angiosperm evolution.

Phylogenetic Analyses of Basal Angiosperms Based on Nine Plastid, Mitochondrial, and Nuclear Genes

DNA sequences of nine genes (plastid: atpB, matK, and rbcL; mitochondrial: atp1, matR, mtSSU, and mtLSU; nuclear: 18S and 26S rDNAs) from 100 species of basal angiosperms and gymnosperms were analyzed using parsimony, Bayesian, and maximum likelihood methods. All of these analyses support the following consensus of relationships among basal angiosperms. First, Amborella, Nymphaeaceae, and Austrobaileyales are strongly supported as a basal grade in the angiosperm phylogeny, with either Amborella or Amborella and Nymphaeales as sister to all other angiosperms. An examination of nucleotide substitution patterns of all nine genes ruled out any possibility of analytical artifacts because of RNA editing and GC‐content bias in placing these taxa at the base of the angiosperm phylogeny. Second, Magnoliales are sister to Laurales and Piperales are sister to Canellales. These four orders together constitute the magnoliid clade. Finally, the relationships among Ceratophyllum, Chloranthaceae, monocots, magnoliids, and eudicots are resolved in different ways in various analyses, mostly with low support. Our study indicates caution in total evidence approaches in that some of the genes employed (e.g., mtSSU, mtLSU, and nuclear 26S rDNA) added signal that conflicted with the other genes in resolving certain parts of the phylogenetic tree.

Molecular phylogenetics reveal multiple tertiary vicariance origins of the African rain forest trees

BackgroundTropical rain forests are the most diverse terrestrial ecosystems on the planet. How this diversity evolved remains largely unexplained. In Africa, rain forests are situated in two geographically isolated regions: the West-Central Guineo-Congolian region and the coastal and montane regions of East Africa. These regions have strong floristic affinities with each other, suggesting a former connection via an Eocene pan-African rain forest. High levels of endemism observed in both regions have been hypothesized to be the result of either 1) a single break-up followed by a long isolation or 2) multiple fragmentation and reconnection since the Oligocene. To test these hypotheses the evolutionary history of endemic taxa within a rain forest restricted African lineage of the plant family Annonaceae was studied. Molecular phylogenies and divergence dates were estimated using a Bayesian relaxed uncorrelated molecular clock assumption accounting for both calibration and phylogenetic uncertainties.ResultsOur results provide strong evidence that East African endemic lineages of Annonaceae have multiple origins dated to significantly different times spanning the Oligocene and Miocene epochs. Moreover, these successive origins (c. 33, 16 and 8 million years – Myr) coincide with known periods of aridification and geological activity in Africa that would have recurrently isolated the Guineo-Congolian rain forest from the East African one. All East African taxa were found to have diversified prior to Pleistocene times.ConclusionMolecular phylogenetic dating analyses of this large pan-African clade of Annonaceae unravels an interesting pattern of diversification for rain forest restricted trees co-occurring in West/Central and East African rain forests. Our results suggest that repeated reconnections between the West/Central and East African rain forest blocks allowed for biotic exchange while the break-ups induced speciation via vicariance, enhancing the levels of endemicity. These results provide an explanation for present day distribution patterns and origins of endemicity for African rain forest trees. Moreover, given the pre-Pleistocene origins of all the studied endemic East African genera and species, these results also offer important insights for setting conservation priorities in these highly diversified but threatened ecosystems.

Identifying clades in Asian Annonaceae: monophyletic genera in the polyphyletic Miliuseae

The tribe Miliuseae (Annonaceae) comprises six genera distributed in Asia: Alphonsea, Mezzettia, Miliusa, Orophea, Platymitra, and Phoenicanthus. A phylogenetic study to investigate the putative monophyly of the tribe and the intergeneric relationships is presented here. Nucleotide sequences of the plastid gene rbcL, trnL intron, and trnL-trnF intergenic spacer were analyzed from 114 Annonaceae taxa, including 24 Miliuseae species and two outgroups using maximum parsimony and Bayesian inference. The two data sets (rbcL and the trnL-trnF regions) were analyzed separately and in combination. Miliuseae were found to be polyphyletic due to the position of Mezzettia and are part of a large, predominantly Asian and Central-American clade (miliusoid clade). Although intergeneric relationships were poorly resolved, all genera, except Polyalthia, were monophyletic, supporting previous generic delimitation based on morphology. A group of three Polyalthia species seems the most likely sister group of Miliusa. Several infrageneric groups of Miliusa, Orophea, and Polyalthia are supported by both molecular and morphological data. No morphological synapomorphies have yet been found for the miliusoid clade. Molecular clades within the miliusoid clade, however, can be characterized by size and the shape of the outer petals, number of ovules per carpel, and the size of the fruits.

Diversification of myco-heterotrophic angiosperms: Evidence from Burmanniaceae

BackgroundMyco-heterotrophy evolved independently several times during angiosperm evolution. Although many species of myco-heterotrophic plants are highly endemic and long-distance dispersal seems unlikely, some genera are widely dispersed and have pantropical distributions, often with large disjunctions. Traditionally this has been interpreted as evidence for an old age of these taxa. However, due to their scarcity and highly reduced plastid genomes our understanding about the evolutionary histories of the angiosperm myco-heterotrophic groups is poor.ResultsWe provide a hypothesis for the diversification of the myco-heterotrophic family Burmanniaceae. Phylogenetic inference, combined with biogeographical analyses, molecular divergence time estimates, and diversification analyses suggest that Burmanniaceae originated in West Gondwana and started to diversify during the Late Cretaceous. Diversification and migration of the species-rich pantropical genera Burmannia and Gymnosiphon display congruent patterns. Diversification began during the Eocene, when global temperatures peaked and tropical forests occurred at low latitudes. Simultaneous migration from the New to the Old World in Burmannia and Gymnosiphon occurred via boreotropical migration routes. Subsequent Oligocene cooling and breakup of boreotropical flora ended New-Old World migration and caused a gradual decrease in diversification rate in Burmanniaceae.ConclusionOur results indicate that extant diversity and pantropical distribution of myco-heterotrophic Burmanniaceae is the result of diversification and boreotropical migration during the Eocene when tropical rain forest expanded dramatically.

Evolution of syncarpy and other morphological characters in African Annonaceae : A posterior mapping approach

The congenital fusion of carpels, or syncarpy, is considered a key innovation as it is found in more than 80% of angiosperms. Within the magnoliids however, syncarpy has rarely evolved. Two alternative evolutionary origins of syncarpy were suggested in order to explain the evolution of this feature: multiplication of a single carpel vs. fusion of a moderate number of carpels. The magnoliid family Annonaceae provides an ideal situation to test these hypotheses as two African genera, Isolona and Monodora, are syncarpous in an otherwise apocarpous family with multicarpellate and unicarpellate genera. In addition to syncarpy, the evolution of six other morphological characters was studied. Well-supported phylogenetic relationships of African Annonaceae and in particular those of Isolona and Monodora were reconstructed. Six plastid regions were sequenced and analyzed using maximum parsimony and Bayesian inference methods. The Bayesian posterior mapping approach to study character evolution was used as it accounts for both mapping and phylogenetic uncertainty, and also allows multiple state changes along the branches. Our phylogenetic analyses recovered a fully resolved clade comprising twelve genera endemic to Africa, including Isolona and Monodora, which was nested within the so-called long-branch clade. This is the largest and most species-rich clade of African genera identified to date within Annonaceae. The two syncarpous genera were inferred with maximum support to be sister to a clade characterized by genera with multicarpellate apocarpous gynoecia, supporting the hypothesis that syncarpy arose by fusion of a moderate number of carpels. This hypothesis was also favoured when studying the floral anatomy of both genera. Annonaceae provide the only case of a clear evolution of syncarpy within an otherwise apocarpous magnoliid family. The results presented here offer a better understanding of the evolution of syncarpy in Annonaceae and within angiosperms in general.

Ancient paralogy in the cpDNA trnL-F region in Annonaceae: implications for plant molecular systematics

The plastid trnL-F region has proved useful in molecular phylogenetic studies addressing diverse evolutionary questions from biogeographic history to character evolution in a broad range of plant groups. An important assumption for phylogenetic reconstruction is that data used in combined analyses contain the same phylogenetic signal. The trnL-F region is often used in combined analyses of multiple chloroplast markers. These markers are assumed to contain congruent phylogenetic signal due to lack of recombination. Here we show that trnL-F sequences display a phylogenetic signal conflicting with that of other chloroplast markers in Annonaceae, and we demonstrate that this conflict results from ancient paralogy. TrnL-F copy 2 diverged from trnL-F copy 1 (as used in family-wide phylogenetic analyses) in a direct ancestor of the Annonaceae. Although this divergence dates back 88 million years or more, the exons of both copies appear to be intact. In this case, assuming that (putative) chloroplast markers contain the same phylogenetic signal results in an incorrect topology and an incorrect estimate of ages. Our study demonstrates that researchers should be cautious when interpreting gene phylogenies, irrespective of the genome from which they are presumed to have been sampled.

Assessment of age and greenness of herbarium specimens as predictors for successful extraction and amplification of DNA

Age and the greenness of leaves have been frequently used as indicators for selecting herbarium specimens for molecular studies. Although plant DNA extraction and amplification have been common lab procedures for the past 20 years, no studies specifically investigated the success of these indicators. Here the predictive value of age and the greenness for extraction and amplification success is assessed, using a large number of herbarium specimens from different plant groups. The investigation of these indicators is important because herbarium material is a precious commodity, and is often the only remaining floral record of now extinct ecosystems. In cases where little leaf material is available, most researchers still attempt to extract DNA. This study shows that age and greenness of leaves are unreliable indicators of extraction and amplification success, although together they can have limited usefulness. Furthermore, we found that the amount of extracted DNA from herbarium specimens decreases with c. 1% per year in age of the specimens. Therefore, researchers sometimes should refrain from using old rare specimens because chances of success are unpredictable and precious herbarium material might be wasted. The uncritical use of indicators such as age or leaf colour is therefore not recommendable. Furthermore, botanists should annotate how specimens were collected and dried because this information is essential for successful DNA extraction. Hopefully, similar studies will be reported in order to identify the best approaches to extract DNA from herbarium specimens.

Little ecological divergence associated with speciation in two African rain forest tree genera

BackgroundThe tropical rain forests (TRF) of Africa are the second largest block of this biome after the Amazon and exhibit high levels of plant endemism and diversity. Two main hypotheses have been advanced to explain speciation processes that have led to this high level of biodiversity: allopatric speciation linked to geographic isolation and ecological speciation linked to ecological gradients. Both these hypotheses rely on ecology: in the former conservation of ecological niches through time is implied, while in the latter adaptation via selection to alternative ecological niches would be a prerequisite. Here, we investigate the role of ecology in explaining present day species diversity in African TRF using a species level phylogeny and ecological niche modeling of two predominantly restricted TRF tree genera, Isolona and Monodora (Annonaceae). Both these genera, with 20 and 14 species, respectively, are widely distributed in African TRFs, with a few species occurring in slightly less humid regions such as in East Africa.ResultsA total of 11 sister species pairs were identified most of them occurring in allopatry or with little geographical overlap. Our results provide a mixed answer on the role of ecology in speciation. Although no sister species have identical niches, just under half of the tests suggest that sister species do have more similar niches than expected by chance. PCA analyses also support little ecological differences between sister species. Most speciation events within both genera predate the Pleistocene, occurring during the Late Miocene and Pliocene periods.ConclusionsEcology is almost always involved in speciation, however, it would seem to have had a little role in species generation within Isolona and Monodora at the scale analyzed here. This is consistent with the geographical speciation model for TRF diversification. These results contrast to other studies for non-TRF plant species where ecological speciation was found to be an important factor of diversification. The Pliocene period appears to be a vital time in the generation of African TRF diversity, whereas Pleistocene climatic fluctuations have had a smaller role on speciation than previously thought.Ecological niche modeling, species level phylogeny, ecological speciation, African tropics, Isolona, Monodora, Annonaceae

Classification of a large and widespread genus of Neotropical trees, Guatteria (Annonaceae) and its three satellite genera Guatteriella, Guatteriopsis and Heteropetalum

A phylogenetic study of the genus Polycarpon was made using DNA sequence data from the chloroplast rpsl6 intron and nuclear RPB2 regions. Polycarpon as currently recognised is shown to be polyphyletic. The South American species P. coquimbense and P. suffruticosum are more closely related to Haya obovata (Socotra) and Polycarpaea spicata (paleotropical) than to the remaining species of Polycarpon, and the widespread tropical species P. prostratum is nested with Macaronesian species of Polycarpaea. All three should therefore be excluded from Polycarpon. The remaining species, the P. tetraphyllum group, has its main diversity in the Mediterranean region. It is a polyploid complex with morphologically very similar taxa mostly separated only by partially overlapping characters. The members of this group form a strongly supported, but largely unresolved clade. It is proposed that they are all included in a widely circumscribed P. tetraphyllum. The dehiscence of the capsules in P. tetraphyllum, by valves that elastically roll inwards and finally throw out the seeds, is unique in Caryophyllaceae and provides a strong synapomorphy for Polycarpon in this new restricted sense.