Reconstructions of phylogenetic relationships in the flowering plant family Rubiaceae have up until now relied heavily on single- or multi-gene data, primarily from the plastid compartment. With the availability of cost- and time-efficient techniques for generating complete genome sequences, the opportunity arises to resolve some of the relationships that, up until now, have proven problematic. Here, we contribute new data from complete 58 plastid genome sequences, representing 55 of the currently 65 recognized tribes of the Rubiaceae. Also contributed are new data from the nuclear rDNA cistrons for corresponding taxa. Phylogenetic analyses are conducted on two plastid data sets, one including data from the protein coding genes only, and a second where protein coding data are combined with non-coding regions, and on a nuclear rDNA data set. Our results clearly show that simply adopting a more characters approach does not resolve the relationships in the Rubiaceae. More importantly, we identify conflicting phylogenetic signals in the data. Analyses of the same plastid data, treated as nucleotides or as codon-degenerated data, resolve and support conflicting topologies in the subfamily Cinchonoideae. As these analyses use the same data, we interpret the conflict to result from erroneous assumptions in the models used to reconstruct our phylogenies. Conflicting signals are also identified in the analyses of the plastid versus the nuclear rDNA data sets. These analyses use data from different genomic compartments, with different inheritance patterns, and we interpret the conflicts as representing real conflicts, reflecting biological processes of the past.

PREMISE OF THE STUDY: Reconstruction of plant phylogeny has heavily relied on single-gene or multigene plastid data. New sequencing methods have led to an increasing number of studies based on data from the entire plastid, but the mitochondrion has rarely been used to infer plant phylogeny because of an assumed information poverty and demonstrated lateral transfer of mitochondrial gene regions between distantly related species. METHODS: We explored phylogenetic information from the plant mitochondrion using 57 representatives of the species-rich coffee family as study system and assessed consistency with previous results based (mostly) on plastid data. KEY RESULTS: We showed that the mitochondrial genome can provide structured and statistically significant information on plant phylogeny. While most of our results are consistent with those based on plastid data, some surprising and statistically significant conflicts emerge, and our study demonstrates with striking clarity that the phylogeny of Rubiaceae is far from resolved. CONCLUSIONS: It appears unlikely that conflicts between results retrieved from the different genomic compartments would be restricted to Rubiaceae. Rather, they are probably a general phenomenon and an important factor behind longstanding difficult phylogenetic questions. The biological processes responsible for the conflicting results detected here are unclear, but some conflicts are likely caused by hybridization events that occurred tens of millions of years ago. Whether such ancient events can be reconstructed based on molecular data from extant plants remains to be seen, but future studies of the nuclear genome may provide a way forward.

PREMISE OF THE STUDY: The Western Indian Ocean Region (WIOR) is a biodiversity hotspot providing an ideal setting for exploring the origins of insular biodiversity and dynamics of island colonization. We aimed to investigate the origins of the WIOR Psychotrieae alliance (Rubiaceae) with typically small, probably mainly bird-dispersed drupes, and the timing and direction or sequence of its colonization events in the region.

METHODS: We used the program BEAST to estimate divergence times and Lagrange for biogeographic reconstruction.

KEY RESULTS: The alliance has reached the WIOR at least 14 times via dispersals from Africa along with Asia and the Pacific mostly during the last 10 My, with at least one back-colonization to Africa. We inferred the earliest dispersal to Madagascar from the Pacific or Asia in the Miocene and numerous out-of-Madagascar dispersals to the nearby archipelagos but no dispersal out of those archipelagos. Gynochthodes with multiple fruits reached Madagascar twice from the Pacific possibly via ocean drifting. Psychotria with dry fruits (schizocarps) colonized Madagascar from the Pacific or Asia before reaching the Comoros from Madagascar possibly via wind dispersal.

CONCLUSIONS: This study reinforces the pivotal role of dispersal in shaping the WIOR biodiversity and as the critical initiating step in the generation of endemic biodiversity on its islands. The WIOR alliance shows strong Asian and Pacific affinities despite the proximity of the region to Africa. Madagascar has served as a stepping-stone for subsequent dispersal to the rest of the region. The Afro-Malagasy-Seychelles genus Craterispermum and the Malagasy Puffia may represent relictual lineages.

Aim The Western Indian Ocean region (WIOR) is home to a very diverse and largely unique flora that has mainly originated via long-distance dispersals. The aim of this study is to gain insight into the origins of the WIOR biodiversity and to understand the dynamics of colonization events between the islands. We investigate spatial and temporal hypotheses of the routes of dispersal, and compare the dispersal patterns of plants of the Coffeeae alliance (Rubiaceae) and their dispersers. Rubiaceae is the second most species-rich plant family in Madagascar, and includes many endemic genera. The neighbouring archipelagos of the Comoros, Mascarenes and Seychelles also harbour several endemic Rubiaceae.

Location The islands of the Western Indian Ocean.

Methods Phylogenetic relationships and divergence times were reconstructed from plastid DNA data of an ingroup sample of 340 species, using Bayesian inference. Ancestral areas and range evolution history were inferred by a maximum likelihood method that takes topological uncertainty into account.

Results At least 15 arrivals to Madagascar were inferred, the majority of which have taken place within the last 10 Myr. Most dispersal events were supported as being from mainland Africa, but Catunaregam may have dispersed from Asia. Although most Coffeeae alliance lineages are zoochorous, the general pattern of dispersals from Africa is incongruent with the biogeographic origins of the extant Malagasy volant frugivores. Several out-of-Madagascar dispersals were inferred to the neighbouring islands, as well as back-colonizations of Africa.

Main conclusions The African flora has been of foremost importance as source of dispersal to the islands of the Western Indian Ocean. Following the colonization of Madagascar, rapid radiations appear to have taken place in some clades, and Madagascar has also been an important source area for subsequent dispersal to the Comoros, Mascarenes and Seychelles.

Background: The Philippine archipelago is globally one of the most important model island systems for studying evolutionary processes. However, most plant species on this archipelago have not yet been studied in sufficient detail. The main aim of this study is to unravel the evolutionary history and biogeographic relationships of the Philippine members of the pantropical genus Ixora. Results: The complex plastid and nuclear divergence patterns in Philippine Ixora, documented using tree and network approaches, reveal a highly dynamic evolution in Ixora, involving several phases of radiation and recolonization. Philippine Ixora comprises at least five lineages, of which one is most closely related to species from Wallacea, and the remaining four to species from Asia. Conclusions: Our study highlights the importance of Philippine species for understanding phytogeographic patterns in the Indomalayan-Australasian eco-region. The overall genetic differentiation, as well as the incongruence between genealogies based on the biparentally inherited nucleome and the maternally inherited plastome in Ixora, reflect the complex tectonic history of the Philippine archipelago. The Ixora lineage related to Wallacean species supports the delimitation of different ecozones along Huxley's line, because it is absent from Palawan. The remaining four lineages are all allied with Asian taxa, reflecting several waves of colonization. Close relationships between some widespread Philippine species and locally adapted narrow endemics suggest that the widespread, genetically diverse species act as pools for the formation of new species in a process of ongoing speciation. Our results suggest that the species concepts of some of the more widespread taxa need to be revised.

Divergence time analyses in the coffee family (Rubiaceae) have all relied on the same Gentianales crown group age estimate, reported by an earlier analysis of the asterids, for defining the upper age bound of the root node in their analyses. However, not only did the asterid analysis suffer from several analytical shortcomings, but the estimate itself has been used in highly inconsistent ways in these Rubiaceae analyses. Based on the original data, we here reanalyze the divergence times of the asterids using relaxed-clock models and 14 fossil-based minimum age constraints. We also expand the data set to include an additional 67 taxa from Rubiaceae sampled across all three subfamilies recognized in the family. Three analyses are conducted: a separate analysis of the asterids, which completely mirrors the original asterid analysis in terms of taxon sample and data; a separate analysis of the Gentianales, where the result from the first analysis is used for defining a secondary root calibration point; and a combined analysis where all taxa are analyzed simultaneously. Results are presented in the form of a time-calibrated phylogeny, and age estimates for asterid groups, Gentianales, and major groups of Rubiaceae are compared and discussed in relation to previously published estimates. Our updated age estimates for major groups of Rubiaceae provide a significant step forward towards the long term goal of establishing a robust temporal framework for the divergence of this biologically diverse and fascinating group of plants.

This is the first phylogenetic study focused on the Pavetteae, one of the most species-rich and morphologically diverse tribes within the coffee family (Rubiaceae). Fifteen of the 17 currently recognized genera, represented by 85 taxa, were sequenced for rps16, trnT-F and ITS and analysed using Bayesian inference and maximum likelihood methods. The monophyly of the Pavetteae is confirmed. Four major lineages are identified, but their phylogenetic relationships are not fully resolved. The continental African genera Rutidea, Nichallea and Tennantia, the Madagascan genera Homollea and Robbrechtia, and the paleotropical genus Pavetta are monophyletic. Other genera are paraphyletic in their current circumscriptions and the following changes are made: Homolliella is placed in synonymy with Paracephaelis, and Coleactina and Dictyandra with Leptactina, resulting in four new combinations. The large paleotropical genus Tarenna is shown not to be monophyletic. In the future, the name Tarenna should not be used for continental African species. Most of these could be transferred to the hitherto monospecific genus Cladoceras, but other species might constitute altogether new genera. The relationship between the monophyletic Asian-Pacific and Madagascan Tarenna species remains unclear. The phylogeny of the Madagascan genera of the Pavetteae is largely unresolved and the largest Madagascar-centred genus Coptosperma was not recovered as monophyletic. The low resolution for the Madagascan taxa can be considered as an indication of rapid radiation. Further molecular and morphological studies are necessary to clarify the phylogeny of the Pavetteae, especially regarding the African Tarenna species and the Madagascan genera of the tribe.

Argostemmateae (Rubiaceae, subfam. Rubioideae) are a mostly tropical Asian group of about 200 species currently classified in four morphologically distinct genera (Argostemma, Mouretia, Mycetia, Neohymenopogon). The monophyly of the tribe and Mycetia is strongly supported by molecular data, however, the tropical Asian genus Myrioneuron, traditionally associated with Mycetia based on its berry fruits, has not previously been investigated. The Bornean and Sulawesian genus Cyanoneuron, described based on the species of Myrioneuron with drupaceous fruits, had not been sequenced. Therefore, the phylogenetic positions of Cyanoneuron and Myrioneuron within Rubiaceae and their generic status have yet to be assessed with molecular data. These genera have tentatively been placed in tribe Spermacoceae (Rubioideae). We reconstructed a robust phylogeny of Rubioideae with sequence data from five plastid regions of 176 accessions and using the Bayesian Markov chain Monte Carlo and parsimony methods. Once the positions of Cyanoneuron and Myrioneuron were revealed, a robust phylogeny of the Spermacoceae alliance was reconstructed with the combined plastid and nuclear data (nrETS, nrITS) from 61 accessions to reassess its tribal limits. Mycetia and Myrioneuron are non-monophyletic and intermixed, and formed a well-supported clade diagnosed by berry fruits. We formally transfer Myrioneuron to Mycetia (older name), and present nine new combinations in the latter genus. Cyanoneuron was resolved with high support as monophyletic, and appears to be closely related to the Chinese monogeneric tribes Foonchewieae and Dunnieae. A new tribe Cyanoneuroneae is described to accommodate Cyanoneuron. This tribe is morphologically distinct from related tribes by its stipules apically divided into multiple linear segments, condensed-cymose inflorescences and drupe-like fruits with numerous small seeds. A new key to the genera of Argostemmateae is provided.

Hedyotis and related genera (here called the Hedyotis-Oldenlandia complex) are highly debated groups in the Rubiaceae family with no consensus to date on their generic delimitations. The present study focuses on Asian-Pacific taxa from these groups and aims at resolving taxonomic inconsistencies by describing monophyletic genera within the complex. The generic circumscriptions presented in our study are based on the phylogenetic trees of nuclear (ITS, ETS) and plastid (petD, rps16) sequence data inferred using Bayesian and maximum likelihood methods. Morphological key features of the group such as habit, fruit type, seed form, and pollen type are studied and compared with the phylogeny to characterize the clades. Based on these results, the Asian-Pacific members are placed in 14 monophyletic groups across the Hedyotis-Oldenlandia complex. Of these, we accept and circumscribe 13 monophyletic genera: Debia, Dentella, Dimetia, Edrastima, Exallage, Hedyotis, Involucrella, Kadua, Kohautia, Leptopetalum, Neanotis, Oldenlandia, and Scleromitrion. Two of these, Debia and Involucrella, are here described as new genera.

In this study we explore what historical biogeographic events are responsible for the wide and disjunct distribution of extant species in Lasiantheae, a pantropical group of trees and shrubs in the coffee family. Three of the genera in the group, Lasianthus, Saldinia, and Trichostachys, are found to be monophyletic, while there are indications that the fourth, Ronabea, is paraphyletic. We also address how the uncertainty in topology and divergence times affects the level of confidence in the biogeographic reconstruction. A data set consisting of chloroplast and nuclear ribosomal DNA data was analyzed using a Bayesian relaxed molecular clock approach to estimate phylogenetic relationships and divergence times, and the dispersal-extinction-cladogenesis (DEC) method to reconstruct geographic range evolution. Our results show that the Lasiantheae stem lineage originated in the neotropics, and the group expanded its range to the palaeotropics during the Eocene, either by continental migration through the boreotropics or by transatlantic long-distance dispersal. Two cases of Oligocene/Miocene over water-dispersal were also inferred, once from the paleotropics to the neotropics within Lasianthus, and once to Madagascar, concurrent with the origin of Saldinia. A lot of the diversification within Lasianthus took place during the Miocene and may have been influenced by climatic factors such as a period of markedly warm and moist climate in Asia and the aridification of the interior of the African continent. When biogeographic reconstructions were averaged over a random sample of 1000 dated phylogenies, the confidence in the biogeographic reconstruction decreased for most nodes, compared to when a single topology was used. A good understanding of phylogenetic relationships is necessary to understand the biogeographic history of a group, bit since the phylogeny is rarely completely known it is important to include phylogenetic uncertainty in biogeographic analysis. For nodes where the resolution is uncertain, the use of a single best topology as a basis for biogeographic analysis will result in inflated confidence in a biogeographic reconstruction which may be just one of several possible reconstructions.