Genome skimming (shallow whole-genome sequencing) offers time- and cost-efficient production of large amounts of DNA data that can be used to address unsolved evolutionary questions. Here we address phylogenetic relationships and topological incongruence in the tribe Anthospermeae (Rubiaceae), using phylogenomic data from the mitochondrion, the nuclear ribosomal cistron, and the plastome. All three genomic compartments resolve relationships in the Anthospermeae; the tribe is monophyletic and consists of three major subclades. Carpacoce Sond. is sister to the remaining clade, which comprises an African subclade and a Pacific subclade. Most results, from all three genomic compartments, are statistically well supported; however, not fully consistent. Intergenomic topological incongruence is most notable in the Pacific subclade but present also in the African subclade. Hybridization and introgression followed by organelle capture may explain these conflicts but other processes, such as incomplete lineage sorting (ILS), can yield similar patterns and cannot be ruled out based on the results. Whereas the null hypothesis of congruence among all sequenced loci in the individual genomes could not be rejected for nuclear and mitochondrial data, it was rejected for plastid data. Phylogenetic analyses of three subsets of plastid loci identified using the hierarchical likelihood ratio test demonstrated statistically supported intragenomic topological incongruence. Given that plastid genes are thought to be fully linked, this result is surprising and may suggest modeling or sampling error. However, biological processes such as biparental inheritance and inter-plastome recombination have been reported and may be responsible for the observed intragenomic incongruence. Mitochondrial insertions into the plastome are rarely documented in angiosperms. Our results indicate that a mitochondrial insertion event in the plastid trnSGGA - rps4 IGS region occurred in the common ancestor of the Pacific clade of Anthospermeae. Exclusion/inclusion of this locus in phylogenetic analyses had a strong impact on topological results in the Pacific clade. Genomic data have provided new opportunities to address phylogenetic incongruence and its bases. Our work shows notable intergenomic incongruence in a tribe of the coffee family, and the null hypothesis of congruence among all sequenced loci in the individual genomes was surprisingly rejected for plastid data. We further detected a (rarely reported) insertion of mitochondrial data into the plastome that had a decisive impact on tree topology.image

In this study of evolutionary relationships in the subfamily Rubioideae (Rubiaceae), we take advantage of the off-target proportion of reads generated via previous target capture sequencing projects based on nuclear genomic data to build a plastome phylogeny and investigate cytonuclear discordance. The assembly of off-target reads resulted in a comprehensive plastome dataset and robust inference of phylogenetic relationships, where most intratribal and intertribal relationships are resolved with strong support. While the phylogenetic results were mostly in agreement with previous studies based on plastome data, novel relationships in the plastid perspective were also detected. For example, our analyses of plastome data provide strong support for the SCOUT clade and its sister relationship to the remaining members of the subfamily, which differs from previous results based on plastid data but agrees with recent results based on nuclear genomic data. However, several instances of highly supported cytonuclear discordance were identified across the Rubioideae phylogeny. Coalescent simulation analysis indicates that while ILS could, by itself, explain the majority of the discordant relationships, plastome introgression may be the better explanation in some cases. Our study further indicates that plastomes across the Rubioideae are, with few exceptions, highly conserved and mainly conform to the structure, gene content, and gene order present in the majority of the flowering plants.

Premise: The isoetalean lineage has a rich fossil record that extends to the Devonian, but the age of the living clade is unclear. Recent results indicate that it is young, from the Cenozoic, whereas earlier work based on less data from a denser taxon sampling yielded Mesozoic median ages.

Methods: We analyzed node ages in Isoetes using two genomic data sets (plastome and nuclear ribosomal cistron), three clock models implemented in MrBayes (ILN, WN, and TK02 models), and a conservative approach to calibration.

Results: While topological results were consistently resolved in Isoetes estimated crown group ages range from the latest Paleozoic (mid-Permian) to the Mesozoic depending on data type and clock model. The oldest estimates were retrieved using the autocorrelated TK02 clock model. An (early) Cenozoic age was only obtained under one specific condition (plastome data analyzed with the uncorrelated ILN clock model). That same plastome data set also yielded the oldest (mid-Permian) age estimate when analyzed with the autocorrelated TK02 clock model. Adding the highly divergent, recently established sister species Isoetes wormaldii to the data set approximately doubled the average median node depth to the Isoetes crown group.

Conclusions: There is no consistent support for a Cenozoic origin of the living clade Isoetes. We obtained seemingly well-founded, yet strongly deviating results depending on data type and clock model. The single most important future improvement is probably to add calibration points, which requires an improved understanding of the isoetalean fossil record or alternative bases for calibration.

Background: Merlin's grass (Isoetes, Isoetaceae, Lycopsida), is the extant remnant of the isoetalean wood-producing lycopsids that originated during the Paleozoic, possibly in aquatic or boggy habitats. Modern day species are aquatic, semi-aquatic or terrestrial and occur almost worldwide. They display little morphological variation; the lobed corm has helically arranged leaves with internal air channels and basal sporangia. Genetic variation has also proven limited, which has hampered phylogenetic inference. We investigate evolutionary relationships in Isoetes, using molecular data and an extended sample of species compared to previous work, adding species that have never before been included in a phylogenetic study.

Results: Our results reveal an unexpected discovery of an Amborella syndrome in Isoetaceae: a single poorly known species is sister to the remaining family. The species, Isoetes wormaldii, is a rare endemic to the Eastern Cape of South Africa. Its leaves are flattened with a rounded point, which sharply contrasts with the awl-shaped leaves of most other species of Isoetes. The remaining species of Isoetes are resolved in five major clades, also indicated in previous work. While the phylogeny shows geographic structure, the patterns are complex. For example, tropical-southern African species occur in at least five clades, and Indian, Australian and Mediterranean species in at least three clades each.

Conclusion: The evolutionary and biogeographical history of Isoetes is not easily explained, and may conceivably include ample extinction and a mixture of ancient and more recent processes. Previously shown difficulties with node age estimation increase the problem. The here demonstrated sister-relationship between the phylogenetically, morphologically and genetically distinct Isoetes wormaldii and the remaining family appears to bridge the morphological gap between Isoetes and its extinct relatives, although further studies are needed. Moreover, it shortens the branch length to its living sister genus Selaginella, and may enhance node age estimation in future studies. Isoetes wormaldii is critically endangered, known only from one (to a few) minor populations. Immediate actions need to be taken if we want to prevent this unique species from going extinct.

• Background and aims The tribe Danaideae (Rubiaceae) is almost exclusively endemic to the Western Indian Ocean Region (WIOR), and encompasses the genera Danais, Payera and Schismatoclada that occur in humid, sub-humid and mountain and mountain bio climate zones. Much of the species diversity is endemic to restricted, remote and/or mountainous areas of Madagascar and recent field work on the island indicates substantial unknown diversity of the Danaideae. Furthermore, the monophyly of the Malagasy genera Payera and Schismatoclada has been questioned in previous work, species delimitations and phylogenetic relationships within the genera are poorly understood, and the distribution and evolution of gross morphological features have not been assessed.
• Methods We conducted morphological investigations, and produced robust phylogenies of Danaideae based on nuclear and plastid sequence data from 193 terminals. Ample plant material has been newly collected in the WIOR for the purpose of the present study, including potentially new species unknown to science. We performed Bayesian non-clock and relaxed-clock analyses employing three alternative clock models of a dataset with a dense sample of taxa from the entire geographical ranges of Danaideae. Based on the results, we discuss species diversity and distribution, relationships, and morphology in Danaideae.
• Key results Our results demonstrate the monophyly of Danaideae, its three genera and 42 species. Nine species are resolved as non-monophyletic. Many geographically distinct but morphologically heterogeneous lineages were identified, and morphological features traditionally considered diagnostic of subgroups of the genera, used for example in species identification keys, are not clade-specific.
• Conclusions Our results demonstrate that Madagascar contains ample previously undocumented morphological and species diversity of Danaideae. Our novel approach to molecular phylogenetic analyses as a precursor to taxonomic revisions provides numerous benefits for the latter. There are tentative indications of parallel northward diversification in Payera and Schismatoclada on Madagascar, and of geographical phylogenetic clustering despite the anemochorous condition of Danaideae.

Subfamily Rubioideae is the largest of the main lineages in the coffee family (Rubiaceae), with over 8,000 species and 29 tribes. Phylogenetic relationships among tribes and other major clades within this group of plants are still only partly resolved despite considerable efforts. While previous studies have mainly utilized data from the organellar genomes and nuclear ribosomal DNA, we here use a large number of low-copy nuclear genes obtained via a target capture approach to infer phylogenetic relationships within Rubioideae. We included 101 Rubioideae species representing all but two (the monogeneric tribes Foonchewieae and Aitchinsonieae) of the currently recognized tribes, and all but one non-monogeneric tribe were represented by more than one genus. Using data from the 353 genes targeted with the universal Angiosperms353 probe set we investigated the impact of data type, analytical approach, and potential paralogs on phylogenetic reconstruction. We inferred a robust phylogenetic hypothesis of Rubioideae with the vast majority (or all) nodes being highly supported across all analyses and datasets and few incongruences between the inferred topologies. The results were similar to those of previous studies but novel relationships were also identified. We found that supercontigs [coding sequence (CDS) + non-coding sequence] clearly outperformed CDS data in levels of support and gene tree congruence. The full datasets (353 genes) outperformed the datasets with potentially paralogous genes removed (186 genes) in levels of support but increased gene tree incongruence slightly. The pattern of gene tree conflict at short internal branches were often consistent with high levels of incomplete lineage sorting (ILS) due to rapid speciation in the group. While concatenation- and coalescence-based trees mainly agreed, the observed phylogenetic discordance between the two approaches may be best explained by their differences in accounting for ILS. The use of target capture data greatly improved our confidence and understanding of the Rubioideae phylogeny, highlighted by the increased support for previously uncertain relationships and the increased possibility to explore sources of underlying phylogenetic discordance.

Knowledge of relationships among plants has improved dramatically but for many groups, it still rests solely on information from the plastid genome. Consequently, only parts of the organisms' evolutionary history are revealed. For the ancient gymnosperms of Ephedra (Gnetales), previous conclusions were based on both plastid and nuclear ribosomal DNA data, but results were typically poorly resolved and supported, presumably because of information poverty in the utilized gene regions. With the aim of resolving phylogenetic questions using more data, we sequenced the plastid genome and the nuclear ribosomal cistron for 50 specimens of Ephedra, largely covering the diversity of the genus. Phylogeny and node ages were estimated using maximum likelihood and Bayesian methods. However, instead of clarifying a few remaining uncertainties, we were left with new questions and incongruent results. Age estimates of the crown group of Ephedra vary considerably depending on utilized software and specified tree prior. Furthermore, previous estimates of the phylogeny of Ephedra have largely reflected information from nrITS, despite utilization of plastid gene regions as well. With analyses based on the entire plastid genome, completely new results emerge. Earlier conclusions of deep divergences in the genus are not supported, and there are ample examples of phylogenetic incongruence. Our study overturns conclusions in previous work and highlights that we still know fairly little about evolution in the ancient Ephedra lineage. How many species of Ephedra are there, and how are they related? How old is the crown group? Many species appear affected by a history of hybridization/introgression and/or polyploidy, but other processes may result in similar patterns and reasons for the detected incongruences must be further analyzed, preferably using population-level sampling and low-copy nuclear data.

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 research. With wind-pollinated flowers and partly temperate distribution, the tribe Anthospermeae stands out in the otherwise mostly animal-pollinated and tropical coffee family (Rubiaceae). Nevertheless, few attempts to resolve the phylogeny of the group have been made, and inter- and infrageneric relationships have been only partly addressed. Here we investigate evolutionary relationships and generic and subtribal delimitations of Anthospermeae. We assess the influence of alternative evolutionary rate models on topology and node support. Methodology. Using sequence data from the nuclear (nrITS and nrETS) and plastid (atpB-rbcL, ndhF, rbcL, rps16, and trnT-trnF) genomes collected for a broad sample of taxa, we conducted Bayesian analyses using nonclock, strict clock, and relaxed clock models. The resulting topologies and support values were compared, and the relative fit of evolutionary models to our data was evaluated. Marginal likelihood estimates were used to discriminate between the competing rate models. Pivotal results. The monophyly of Anthospermeae was confirmed with Carpacoce resolved as sister to the remaining species. We found several cases of supported topological conflict between results based on nuclear and plastid data, but the deepest splits of the tribe were congruent among all analyses and incompatible with traditional subtribal delimitations of Anthospermeae. Monophyly of the genera Anthospermum, Nenax, and Coprosma was not supported. While the relaxed clock model was consistently favored over the nonclock and strict clock models for all data sets, the use of the different models had little impact on phylogenetic results. Conclusions. We propose a revised subtribal classification of Anthospermeae, including a new subtribe, the monogeneric Carpacocinae. Introgression/hybridization and incomplete lineage sorting are the most likely causes for the plastid-nuclear incongruences detected for Anthospermeae, but their relative contribution could not be concluded.

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.