Philippe Küpfer

Phylogeny and biogeography of Exacum (Gentianaceae): A disjunctive distribution in the Indian Ocean Basin resulted from long distance dispersal and extensive radiation

Disjunctive distributions across paleotropical regions in the Indian Ocean Basin (IOB) often invoke dispersal/vicariance debates. Exacum (Gentianaceae, tribe Exaceae) species are spread around the IOB, in Africa, Madagascar, Socotra, the Arabian peninsula, Sri Lanka, India, the Himalayas, mainland Southeast Asia including southern China and Malaysia, and northern Australia. The distribution of this genus was suggested to be a typical example of vicariance resulting from the breakup of the Gondwanan supercontinent. The molecular phylogeny of Exacum is in principle congruent with morphological conclusions and shows a pattern that resembles a vicariance scenario with rapid divergence among lineages, but our molecular dating analysis demonstrates that the radiation is too recent to be associated with the Gondwanan continental breakup. We used our dating analysis to test the results of DIVA and found that the program predicted impossible vicariance events. Ancestral area reconstruction suggests that Exacum originated in Madagascar, and divergence dating suggests its origin was not before the Eocene. The Madagascan progenitor, the most recent common ancestor of Exacum, colonized Sri Lanka and southern India via long-distance dispersals. This colonizer underwent an extensive range expansion and spread to Socotra-Arabia, northern India, and mainland Southeast Asia in the northern IOB when it was warm and humid in these regions. This widespread common ancestor retreated subsequently from most parts of these regions and survived in isolation in Socotra-Arabia, southern India-Sri Lanka, and perhaps mainland Southeast Asia, possibly as a consequence of drastic climatic changes, particularly the spreading drought during the Neogene. Secondary diversification from these surviving centers and Madagascar resulted in the extant main lineages of the genus. The vicariance-like pattern shown by the phylogeny appears to have resulted from long-distance dispersals followed by extensive range expansion and subsequent fragmentation. The extant African species E. oldenlandioides is confirmed to be recently dispersed from Madagascar.

Genetic diversity in widespread species is not congruent with species richness in alpine plant communities

The Convention on Biological Diversity (CBD) aims at the conservation of all three levels of biodiversity, that is, ecosystems, species and genes. Genetic diversity represents evolutionary potential and is important for ecosystem functioning. Unfortunately, genetic diversity in natural populations is hardly considered in conservation strategies because it is difficult to measure and has been hypothesised to co-vary with species richness. This means that species richness is taken as a surrogate of genetic diversity in conservation planning, though their relationship has not been properly evaluated. We tested whether the genetic and species levels of biodiversity co-vary, using a large-scale and multi-species approach. We chose the high-mountain flora of the Alps and the Carpathians as study systems and demonstrate that species richness and genetic diversity are not correlated. Species richness thus cannot act as a surrogate for genetic diversity. Our results have important consequences for implementing the CBD when designing conservation strategies.

Phylogeny and biogeography of Balsaminaceae inferred from ITS sequences

Sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA were acquired for 112 species of Balsaminaceae worldwide and five species of its closest relatives Marcgraviaceae and Tetrameristaceae. Phylogenetic analyses applying parsimony and distance estimates confirmed the monophyly of Balsaminaceae and suggest the monophyly of Impatiens. Within Impatiens, a few clades are recognized with strong support. Two of the most important clades are the spurless Madagascan endemic group, and the one comprising species with broadly fusiform fruits and the basic chromosome number x = 8, that shows a Southeast Asia, southern India, Africa, and Madagascar connection. Despite recognition of several strongly supported small lineages, ITS data alone could.not resolve relationships among most of the lineages with confident support values. ITS phylogenies are therefore of limited taxonomic value for Impatiens. However, ITS phylogenies do reveal that extant Impatiens species are of Southeast Asian origin, from where dispersals to boreal Eurasia and North America, to central Asia and eastern Europe via the Himalayas, and to India and Africa have occurred. The Madagascan Impatiens show an African origin. Molecular phylogenies suggest the ancestral basic chromosome number to be x = 10, and the spurred flowers and elongated linear fruits to be plesiomorphic states in Impatiens. A predominantly descending dysploid chromosome evolution, following dispersal of the clade with broadly fusiform fruits from Southeast Asia to India, Africa, and Madagascar, is also suggested.

Phylogenetics of Impatiens and Hydrocera (Balsaminaceae) Using Chloroplast atpB-rbcL Spacer Sequences

Abstract Balsaminaceae are a morphologically diverse family with ca. 1,000 representatives that are mainly distributed in the Old World tropics and subtropics. To understand the relationships of its members, we obtained chloroplast atpB-rbcL sequences from 86 species of Balsaminaceae and five outgroups. Phylogenetic reconstructions using parsimony and Bayesian approaches provide a well-resolved phylogeny in which the sister group relationship between Impatiens and Hydrocera is confirmed. The overall topology of Impatiens is strongly supported and is geographically structured. Impatiens likely originated in South China from which it colonized the adjacent regions and afterwards dispersed into North America, Africa, India, the Southeast Asian peninsula, and the Himalayan region.

Molecular phylogenetics of the subtribeGentianinae (Gentianaceae) inferred from the sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA

The internal transcribed spacer (ITS) regions of 18S–25S nuclear ribosomal DNA from representatives of 23 species of the subtribeGentianinae and one outgroup species (Centaurium capitatum) were analyzed by polymerase chain reaction amplification and direct DNA sequencing. Within the taxa analyzed, the length of the ITS1 region varied from 221 to 233 bp, ITS2 from 226 to 234 bp. Of the aligned sequences of 497 positions, 151 sites involved gaps or nucleotide ambiguity, 133 were invariable and 213 showed divergence. In pairwise comparisons among the taxa of the subtribeGentianinae and the outgroup, sequence divergence ranged from 1.3% to 34.1% in ITS1, from 0 to 28.1% in ITS2 and from 0.6% to 27.5% in combined ITS1 and ITS2. Phylogenetic trees generated from ITS sequences were highly resolutive and principally concordant with morphological classifications for the major phylogenetic divisions in the subtribe. An ancient divergence leading to two evolutionary lines was suggested in the subtribe by both DNA sequence and morphological data. One line encompasses the generaGentiana, Crawfurdia andTripterospermum, morphologically characterized by their glands on the base of ovary and their plicate corolla, while the other line involves all other members of the subcribe surveyed, characterized by their epipetalous glands and simple corolla without plicae.Megacodon, with glands on the base of ovary but without plicae on its corolla, was revealed to be more related to the latter group than to the former.Comastoma, Gentianella andGentianopsis were shown to be well-defined monophyletic genera.Pterygocalyx showed much closer affinity toGentianopsis than to any other genus. Some conflictions were detected in the genusSwertia.

Monophyly and relationships of the tribe Exaceae (Gentianaceae) inferred from nuclear ribosomal and chloroplast DNA sequences.

Both chloroplast trnL (UAA) intron and nuclear ribosomal ITS sequences highly confirmed the monophyly of the tribes of the Gentianaceae defined by the recent classification, and revealed the tribe Exaceae as a basal clade just next to the basal-most lineage, the tribe Saccifolieae. Within the tribe Exaceae, Sebaea (except Sebaea madagascariensis) appeared as the most basal clade as the sister group to the rest of the tribe. The Madagascan endemic genera Gentianothamnus and Tachiadenus were very closely related to each other, together standing as sister to a clade comprising Sebaea madagascariensis, Ornichia, and Exacum. The saprophytic genus Cotylanthera nested deeply inside Exacum. Sebaea madagascariensis was shown closer to the Madagascan endemic genus Ornichia than to any other sampled Sebaea species. Exacum appeared as the most derived taxon within this tribe. The topology of the phylogenetic trees conform with the Gondwana vicariance hypothesis regarding the biogeography of Exaceae. However, no evidence for matching the older relationships within the family to the tectonic history could be corroborated with various divergence time analyses. Divergence dating estimated a post-Gondwana diverging of the Gentianaceae about 50 million years ago (MYA), and the tribe Exaceae as about 40 MYA. The Mozambique Channel land-bridge could have played an important role in the biogeographic history of the tribe Exaceae.

Genetic structure and evolution of Alpine polyploid complexes: Ranunculus kuepferi (Ranunculaceae) as a case study

The alpine white‐flowered buttercup, Ranunculus kuepferi Greuter & Burdet, is a polyploid complex with diploids endemic to the southwestern Alps and polyploids – which have been previously described as apomictic – widespread throughout European mountains. Due to the polymorphic status of both its ploidy level and its reproductive mode, R. kuepferi represents a key species for understanding the evolution of polyploid lineages in alpine habitats. To disentangle the phylogeography of this polyploid taxon, we used cpDNA sequences and AFLP (amplified fragment length polymorphism) markers in 33 populations of R. kuepferi representative of its ploidy level and distribution area. Polyploid individuals were shown to be the result of at least two polyploidization events that may have taken place in the southwestern Alps. From this region, one single main migration of tetraploids colonized the entire Alpine range, the Apennines and Corsica. Genetic recombination among tetraploids was also observed, revealing the facultative nature of the apomictic reproductive mode in R. kuepferi polyploids. Our study shows the contrasting role played by diploid lineages mostly restricted to persistent refugia and by tetraploids, whose dispersal abilities have permitted their range extension all over the previously glaciated Alpine area and throughout neighbouring mountain massifs.

High paraphyly of Swertia L. (Gentianaceae) in the Gentianella -lineage as revealed by nuclear and chloroplast DNA sequence variation

Abstract. The genus Swertia L., as currently defined, is polymorphic and mainly distributed in temperate regions of the northern hemisphere. Phylogenetic relationships between Swertia and the other genera of the Swertiinae sensu Struwe et al. (unpubl. data) are discussed based on cladistic analyses of DNA sequence data. The sequences used for this purpose include the trnL (UAA) intron, the intergenic spacers (IGS) between trnL (UAA) and trnF (GAA) exons, and between trnS (UGA) and ycf 9 exons of cpDNA, as well as the ITS region of nrDNA. Although moderately resolved, the phylogenies resulting from the separate analyses of nuclear and chloroplast data are congruent, and the incongruence length difference test (Farris et al. 1995) detected no character incongruence. The phylogeny suggested by the analysis of combined data sets defines Swertia as strongly paraphyletic in relation to the other genera. This taxon may have acted as a stem group, giving rise to diverse lineages, some of which are morphologically distinct and have been recognised at the generic level. Latouchea and Obolaria are closely related and occupy the basalmost position in the molecular tree. Swertia species are distributed in 9 different clades, three of which share a basal polytomy with Bartonia, Frasera, Gentianopsis, Halenia, Megacodon, Pterygocalyx and Veratrilla. Two lineages have an intermediate position. The remaining 4 clades occupy a more derived position. Two of the latter clades show a close relation with species of Gentianella s. str., and one is included in a large clade comprising Comastoma, Jaeschkea and Lomatogonium. Selected character states and their proposed polarity, such as number and structure of nectaries, stylar and seedcoat characteristics, pollen morphology, fusion of floral parts and chromosome number are discussed in the context of molecular data. Rugose, spinose, or winged seeds are found mainly in basal lineages, while smooth ones are typical for derived species. Chromosome numbers follow a similar pattern with x=13 restricted to basal lineages, while in more derived clades, x is always smaller than 13. With respect to the molecular phylogeny, taxonomic circumscriptions in the Swertiinae sensu Struwe et al. (unpubl. data) does not seem to reflect phyletic relationships.

Chromosome and breeding system evolution of the genus Mercurialis (Euphorbiaceae): implications of ITS molecular phylogeny

Abstract. The internal transcribed spacers (ITS1 and ITS2) of nuclear ribosomal DNA were amplified and sequenced from 19 samples representing all species of the genus Mercurialis and two outgroup species, Ricinuscommunis and Acalyphahispida. The length of ITS1 in the ingroups ranged from 223 to 246 bp and ITS2 from 210 to 218 bp. Sequence divergence between pairs of species ranged from 1.15% to 25.88% among the ingroup species in the combined data of ITS1 and ITS2. Heuristic phylogenetic analyses using Fitch parsimony on the combined data of ITS1 and ITS2 with gaps treated as missing generated 45 equally parsimonious trees. The strict consensus tree was principally concordant with morphological classification. Within the genus, the ITS sequences recognised two main infrageneric clades: the M. perennis complex including three Eurasian stoloniferous species (M.␣leiocarpa, M. ovata and M. perennis) and the western Mediterranean group including eight both annual and perennial species. Of the western Mediterranean clade, the annual and perennial species grouped respectively into two different groups, and the annual life form is revealed as a synapomorphic character derived from perennial, whereas in the Eurasian clade ITS phylogeny suggested M. leiocarpa as basal clade sister to M.␣perennis and M. ovata. ITS phylogeny failed to resolve the relationships among the different cytotypes of M.ovata and M. perennis. ITS phylogeny also suggested rapid karyotypic evolution for the genus. The karyotypic divergence among the perennial species of western Mediterranean region did not corroborate the nucleotide sequence divergence among the species. Optimisation of chromosome numbers onto the ITS phylogeny suggested x=8 to be the ancestral basic chromosome number of the genus. ITS phylogeny confirmed that the androdioecy of M. ambigua is derived from dioecy. The nucleotide heterozygosity and additivity in ITS sequences clearly confirm the interspecific hybridisation in the genus Mercurialis.

Recognition of Martellidendron, a new genus of Pandanaceae, and its biogeographic implications

Pandanaceae are an ancient family of dioecious monocots dating from the early to mid-Cretaceous, and comprising three extant genera Sararanga, Freycinetia and Pandanus. We present a cladistic analysis of Pandanaceae based on DNA sequences of four cpDNA fragments (the trnL intron and three intergenic spacers, trnL-F, trnS-ycJ9 and atpB-rbcL) in order to elucidate intergeneric relationships within the family. The results show that Pandanus, as currently circumscribed, is biphyletic, with members of the Indian Ocean subg. Martellidendron sister to the Indo-Malesian genus Freycinetia in a clade that also includes the Malesian genus Sararanga, and that the remaining members of Pandanus form a separate well supported clade. Martellidendron is thus recognized as a distinct genus, thereby circumscribing a monophyletic Pandanus. Martellidendron comprises seven species (for which new combinations are proposed). The high level of morphological differentiation between the genera of Pandanaceae suggests rapid early radiation as seen in other monocot groups. The molecular data also indicate that the distinct lineages of Pandanaceae in the Indian Ocean basin are likely the result of both vicariance (Martellidendron) and more recent step-wise long-distance dispersal from Asia across the Indian Ocean (e.g., monocarpellate spiniform species in lowland eastern Madagascar).