Sylvain G. Razafimandimbison

Recent Origin and Phylogenetic Utility of Divergent ITS Putative Pseudogenes: A Case Study from Naucleeae (Rubiaceae)

The internal transcribed spacer (ITS) of nuclear ribosomal DNA has been widely used by systematists for reconstructing phylogenies of closely related taxa. Although the occurrence of ITS putative pseudogenes is well documented for many groups of animals and plants, the potential utility of these pseudogenes in phylogenetic analyses has often been underestimated or even ignored in part because of deletions that make unambiguous alignment difficult. In addition, long branches often can lead to spurious relationships, particularly in parsimony analyses. We have discovered unusually high levels of ITS polymorphism (up to 30%, 40%, and 14%, respectively) in three tropical tree species of the coffee family (Rubiaceae), Adinauclea fagifolia, Haldina cordifolia, and Mitragyna rubrostipulata. Both secondary structure stability and patterns of nucleotide substitutions in a highly conserved region (5.8S gene) were used for distinguishing presumed functional sequences from putative pseudogenes. The combination of both criteria was the most powerful approach. The sequences from A. fagifolia appear to be a mix of functional genes and highly distinct putative pseudogenes, whereas those from H. cordifolia and M. rubrostipulata were identified as putative pseudogenes. We explored the potential utility of the identified putative pseudogenes in the phylogenetic analyses of Naucleeae sensu lato. Both Bayesian and parsimony trees identified the same monophyletic groups and indicated that the polymorphisms do not transcend species boundaries, implying that they do not predate the divergence of these three species. The resulting trees are similar to those produced by previous analyses of chloroplast genes. In contrast to results of previous studies therefore, divergent putative pseudogenes can be useful for phylogenetic analyses, especially when no sequences of their functional counterparts are available. Our studies clearly show that ITS polymorphism may not necessarily mislead phylogenetic inference. Despite using many different PCR conditions (different primers, higher denaturing temperatures, and absence or presence of DMSO and BSA-TMACl), we recovered only a few functional ITS copies from A. fagifolia and none from H. cordifolia and M. rubrostipulata, which suggests that PCR selection is occurring and/or the presumed functional alleles are located at minor loci (with few ribosomal DNA copies).

Spatial structure and the effects of host and soil environments on communities of ectomycorrhizal fungi in wooded savannas and rain forests of Continental Africa and Madagascar

Mycorrhizal fungi play a key role in mineral nutrition of terrestrial plants, but the factors affecting natural distribution, diversity and community composition of particularly tropical fungi remain poorly understood. This study addresses shifts in community structure and species frequency of ectomycorrhizal (EcM) fungi in relation to host taxa, soil depth and spatial structure in four contrasting African ecosystems. We used the rDNA and plastid trnL intron sequence analysis for identification of fungi and host plants, respectively. By partitioning out spatial autocorrelation in plant and fungal distribution, we suggest that African EcM fungal communities are little structured by soil horizon and host at the plant species and family levels. These findings contrast with patterns of vegetation in these forests and EcM fungal communities in other tropical and temperate ecosystems. The low level of host preference indirectly supports an earlier hypothesis that pioneer Phyllanthaceae may facilitate the establishment of late successional Fabaceae and potentially other EcM host trees by providing compatible fungal inoculum in deforested and naturally disturbed ecosystems of tropical Africa.

Phylogeny and classification of Naucleeae s.l. (Rubiaceae) inferred from molecular (ITS, rBCL, and tRNT-F) and morphological data

Parsimony analyses of the tribe Naucleeae sensu lato (s.l.) using the noncoding internal transcribed spacer (ITS) regions of nuclear rDNA, the protein-coding rbcL and noncoding trnT-F regions of chloroplast DNA, and morphological data were performed to construct new intratribal classification, test the monophyly of previous subtribal circumscriptions, and evaluate the generic status of Naucleeae s.l. Fifty-two ITS, 45 rbcL, and 55 trnT-F new sequences are published here. Our study supports the monophyly of the subtribes Anthocephalidae, Mitragynae, Uncariae all sensu Haviland and Naucleinae sensu Ridsdale. There was no support for Cephalanthidae sensu Haviland and Adininae sensu Ridsdale. Naucleeae can be subdivided into six highly supported and morphologically distinct subtribes, Breoniinae, Cephalanthinae, Corynantheinae, Naucleinae, and Mitragyninae, Uncarinae, plus one, Adininae, which is poorly supported. The relationships among these subtribes were largely unresolved. We maintain the following 22 genera: Adina, Adinauclea, Breonadia, Breonia, Burttdavya, Cephalanthus, Gyrostipula, Haldina, Janotia, Ludekia, Metadina, Mitragyna, Myrmeconauclea, Nauclea, Neolamarckia, Neonauclea, Ochreinauclea, Pausinystalia, Pertusadina, Sarcocephalus, Sinoadina, and Uncaria. Pseudocinchona is reestablished. Corynanthe is restricted to C. paniculata and Hallea is reincluded in Mitragyna. Our results were inconclusive for assessing the relationships among Adina, Adinauclea, Metadina, and Pertusadina due to lack of resolution.

Polyphyly of Mussaenda inferred from ITS and trnT-F data and its implication for generic limits in Mussaendeae (Rubiaceae).

Although recognition of Mussaenda as a separate genus has been widely accepted, its generic circumscriptions have always been controversial. In this first molecular phylogenetic study focused specifically on Mussaenda sensu lato (s.l.) and its allied genera, parsimony analyses were based on both ITS and trnT-F sequence data to (1) test the monophyly of Mussaenda s.l. as presently circumscribed, (2) assess the phylogenetic relationships within the tribe Mussaendeae as currently delimited, (3) evaluate the phylogenetic value of the morphological characters traditionally and/or currently used to circumscribe Mussaendeae, (4) and make inferences on the biogeographical origin of Mussaenda. Of the 63 trnT-F and 38 ITS sequences included in our studies, 52 and 36 sequences, respectively, are newly published here. Our results highly support the polyphyly of Mussaenda s.l. as currently delimited but further support the monophyly of Mussaendeae sensu Bremer and Thulin. The Malagasy Mussaenda are more closely related to Landiopsis than they are to the African and Asian Mussaenda. Pseudomussaenda and the Afro-Asian Mussaenda clade are resolved as sister groups. Aphaenandra is nested within the Afro-Asian Mussaenda clade. As a result, we merge Aphaenandra in Mussaenda, which is now restricted to include only the African and Asian Mussaenda representatives. We describe a new genus Bremeria to accommodate all Indian Ocean (Madagascar and the Mascarenes) Mussaenda species and make 19 new combinations. The newly delimited Mussaenda is diagnosed by reduplicate-valvate aestivation and glabrous styles, whereas Bremeria can be distinguished from the remaining Mussaendeae genera by having both reduplicate- and induplicate-valvate aestivation and densely pubescent styles. Our studies strongly suggest an African origin of the newly delimited Mussaenda. Finally, descriptions of the newly circumscribed Mussaenda and Bremeria are provided.

Evolution and trends in the Psychotrieae alliance (Rubiaceae)--a rarely reported evolutionary change of many-seeded carpels from one-seeded carpels.

Bayesian and parsimony analyses of five plastid gene and nrITS regions from 58 Rubioideae (Rubiaceae) taxa further support the sister-group relationship between the African monotypic genus Schizocolea and the Psychotrieae alliance sensu Bremer & Manen. Our analyses show that the Psychotrieae alliance can be subdivided into in four well-supported clades: Schizocolea, (Schradereae(Gaertnereae(Mitchelleae-Morindeae s.s.))), Palicoureeae-Psychotrieae s.s., and Craterispermeae-Prismatomerideae. The relationships between the latter three clades remain unsettled. Our study further reveals much higher numbers of molecular autapomorphies of the tribes compared with those of molecular synapomorphies of two sister tribes or groups of tribes. Within the newly delimited Psychotrieae alliance a one-seeded carpel was inferred as ancestral and many- and two-seeded carpels evolved once each. We describe Mitchelleae to accommodate Damnacanthus and Mitchella and restrict Morindeae to include only Appunia, Coelospermum, Gynochthodes, Morinda, Pogonolobus, and Syphonandrium. Mitchelleae is characterized e.g., by placentae inserted near the top of the septum and a single campylotropous ovule per carpel, while Morindeae s.s. has massive and T-shaped placentae inserted in the middle of the septum and two anatropous ovules per carpel.

Tribal delimitation of Naucleeae (Cinchonoideae, Rubiaceae): inference from molecular and morphological data

The circumscription of the tribe Naucleeae has been under debate for a long time. This is the first study to test if the previous circumscriptions are supported as monophyletic. Parsimony analyses of the rbcL sequence data of Rubiaceae, followed by separate and combined analyses of the ITS, rbcL, and morphological data were performed in attempt to propose a new tribal delimitation for Naucleeae. Results did not support any of the previous delimitations of Naucleeae. All favoured a broader circumscription, which includes not only all members of Naucleeae sensu Ridsdale, but also Cephalanthus, Hallea, Mitragyna, and Uncaria, formerly placed in Naucleeae, Corynanthe and Pausinystalia. Naucleeae sensu Ridsdale were shown to be monophyletic without support in the morphology- and combined ITS-rbcL-morphology-based trees. The tribe Coptosapelteae sensu Andersson & Persson appeared polyphyletic. A new tribe Hymenodictyoneae is created to accommo- date Hymenodictyon and Paracorynanthe. Descriptions and diagnostic features of the newly circum- scribed Naucleeae and Hymenodictyoneae are provided.

Deep divergences in the coffee family and the systematic position of Acranthera

Despite extensive efforts, there are unresolved questions on evolutionary relationships in the angiosperm family Rubiaceae. Here, information from six loci and 149 Rubiaceae taxa provide new insights. Acranthera and Coptosapelta are strongly supported as sisters. Pollen grains of Acranthera possess several features common in Rubiaceae, but amongst potential similarities with the unusual grains of Coptosapelta are the nature of the apertures and the structure of the sexine. Luculia, Acranthera and Coptosapelta are excluded from the three subfamilies Ixoroideae, Cinchonoideae and Rubioideae. Sipaneeae and Condamineeae form a clade, sister to remaining Ixoroideae. Rondeletieae and Guettardeae are sisters to remaining Cinchonoideae. Colletoecema is sister to remaining Rubioideae, followed by the Urophylleae–Ophiorrhizeae clade. Nuclear ITS provided structured information at all phylogenetic levels, but the main gain from adding nrITS was the increased resolution. Average support values also increased but were generally high also without nrITS and the increase was not statistically significant.

Molecular phylogenetics and generic assessment in the tribe Morindeae (Rubiaceae-Rubioideae): how to circumscribe Morinda L. to be monophyletic?

Most of the species of the family Rubiaceae with flowers arranged in head inflorescences are currently classified in three distantly related tribes, Naucleeae (subfamily Cinchonoideae) and Morindeae and Schradereae (subfamily Rubioideae). Within Morindeae the type genus Morinda is traditionally and currently circumscribed based on its head inflorescences and syncarpous fruits (syncarps). These characters are also present in some members of its allied genera, raising doubts about the monophyly of Morinda. We perform Bayesian phylogenetic analyses using combined nrETS/nrITS/trnT-F data for 67 Morindeae taxa and five outgroups from the closely related tribes Mitchelleae and Gaertnereae to rigorously test the monophyly of Morinda as currently delimited and assess the phylogenetic value of head inflorescences and syncarps in Morinda and Morindeae and to evaluate generic relationships and limits in Morindeae. Our analyses demonstrate that head inflorescences and syncarps in Morinda and Morindeae are evolutionarily labile. Morinda is highly paraphyletic, unless the genera Coelospermum, Gynochthodes, Pogonolobus, and Sarcopygme are also included. Morindeae comprises four well-supported and morphologically distinct major lineages: Appunia clade, Morinda clade (including Sarcopygme and the lectotype M. royoc), Coelospermum clade (containing Pogonolobus and Morinda reticulata), and Gynochthodes-Morinda clade. Four possible alternatives for revising generic boundaries are presented to establish monophyletic units. We favor the recognition of the four major lineages of Morindeae as separate genera, because this classification reflects the occurrence of a considerable morphological diversity in the tribe and the phylogenetic and taxonomic distinctness of its newly delimited genera.

Evolutionary trends, major lineages, and new generic limits in the dioecious group of the tribe Vanguerieae (Rubiaceae): Insights into the evolution of functional dioecy

Abstract New generic circumscriptions of the Paleotropical tribe Vanguerieae in the subfamily Ixoroideae s.l. (Rubiaceae) have recently been established as a result of a series of phylogenetic studies conducted by Lantz and Bremer. The genus Canthium Lam. was shown in their study to be highly polyphyletic, and a largely dioecious group was for the first time identified within Vanguerieae. The dioecious group sensu Lantz and Bremer comprises about 140 species classified in eight genera: Canthium (Canthium subg. Bullockia Bridson), Dinocanthium Bremek., Leroya Cavaco, Neoleroya Cavaco, Peponidium (Baill.) Arènes, Pseudopeponidium Homolles ex Arènes, Pyrostria Comm. ex Juss., and Scyphochlamys Balf. f. We sequenced 79 Vanguerieae taxa and performed phylogenetic analyses based on sequence data from two nuclear (ETS and ITS) markers to: (1) pinpoint the phylogenetic positions of the Comorean and Indian Ocean Canthium and the Southeast Asian Canthium confertum Korth. group in Vanguerieae; (2) evaluate the phylogenetic utility of three taxonomic characters (bract type, locule number, and fruit shape) previously and currently used for delimiting genera in the dioecious group; and (3) assess the evolution of functional dioecy in Vanguerieae. The results support a further disintegration of Canthium s.l., as the Comorean and Malagasy Canthium species are shown for the first time to be closely related to Peponidium. Similarly, C. confertum appears to have a close affinity with Cyclophyllum Hook. f. The combined analyses show that the dioecious group can be subdivided into four morphologically distinct clades formally recognized as genera: Bullockia (Bridson) Razafim., Lantz & B. Bremer, here elevated from Canthium subgen. Bullockia Bridson, as well as Cyclophyllum, Peponidium (including all Comorean, Malagasy, and Seychellean Canthium species), and Pyrostria (including Dinocanthium, Leroya, Neoleroya, Pseudopeponidium, and Scyphochlamys). All described species of Canthium subgen. Bullockia are transferred to Bullockia: B. dyscriton (Bullock) Razafim., Lantz & B. Bremer, B. fadenii (Bridson) Razafim., Lantz & B. Bremer, B. impressinervia (Bridson) Razafim., Lantz & B. Bremer, B. mombazensis (Baill.) Razafim., Lantz & B. Bremer, B. pseudosetiflora (Bridson) Razafim., Lantz & B. Bremer, and B. setiflora (Hiern) Razafim., Lantz & B. Bremer. Furthermore, the results seem to point to a single origin of functional dioecy followed by subsequent reversals back to the hermaphroditic conditions at least within the Cyclophyllum–Canthium confertum clade and Pyrostria s.l. The Malagasy Bullockia species seem to have an African ancestor, whereas the Comorean Peponidium and the African Pyrostria appear to have originated from Malagasy progenitors.

Phylogeny, evolutionary trends and classification of the Spathelia-Ptaeroxylon clade : morphological and molecular insights

BACKGROUND AND AIMS The Spathelia-Ptaeroxylon clade is a group of morphologically diverse plants that have been classified together as a result of molecular phylogenetic studies. The clade is currently included in Rutaceae and recognized at a subfamilial level (Spathelioideae) despite the fact that most of its genera have traditionally been associated with other families and that there are no obvious morphological synapomorphies for the clade. The aim of the present study is to construct phylogenetic trees for the Spathelia-Ptaeroxylon clade and to investigate anatomical characters in order to decide whether it should be kept in Rutaceae or recognized at the familial level. Anatomical characters were plotted on a cladogram to help explain character evolution within the group. Moreover, phylogenetic relationships and generic limits within the clade are also addressed. METHODS A species-level phylogenetic analysis of the Spathelia-Ptaeroxylon clade based on five plastid DNA regions (rbcL, atpB, trnL-trnF, rps16 and psbA-trnH) was conducted using Bayesian, maximum parsimony and maximum likelihood methods. Leaf and seed anatomical characters of all genera were (re)investigated by light and scanning electron microscopy. KEY RESULTS With the exception of Spathelia, all genera of the Spathelila-Ptaeroxylon clade are monophyletic. The typical leaf and seed anatomical characters of Rutaceae were found. Further, the presence of oil cells in the leaves provides a possible synapomorphy for the clade. CONCLUSIONS The Spathelia-Ptaeroxylon clade is well placed in Rutaceae and it is reasonable to unite the genera into one subfamily (Spathelioideae). We propose a new tribal classification of Spathelioideae. A narrow circumscription of Spathelia is established to make the genus monophyletic, and Sohnreyia is resurrected to accommodate the South American species of Spathelia. The most recent common ancestor of Spathelioideae probably had leaves with secretory cavities and oil cells, haplostemonous flowers with appendaged staminal filaments, and a tracheidal tegmen.