Michael Gruenstaeudl

Phylogeny of Barnadesioideae (Asteraceae) inferred from DNA sequence data and morphology

Subfamily Barnadesioideae (Asteraceae) consists of nine genera and 91 species endemic to South America. They include annual and perennial herbs, arching shrubs and trees up to 30m tall. Presumed sister to all other Asteraceae, its intergeneric relationships are key to understanding the early evolution of the family. Results of the only molecular study on the subfamily conflict with relationships inferred from morphology. We investigate inter- and intrageneric relationships in Barnadesioideae with novel DNA sequence data and morphological characters using parsimony, likelihood and Bayesian inference. All results verify Barnadesioideae as monophyletic and sister to the rest of the family. A basal split within the subfamily is recognized, with Chuquiraga, Doniophyton and Duseniella in one clade, and Arnaldoa, Barnadesia, Dasyphyllum, Fulcaldea, Huarpea and possibly Schlechtendalia in another. The largest genus, Dasyphyllum, is revealed as biphyletic with the two clades separating along subgeneric and geographic lines. Schlechtendalia, suggested as the earliest diverging lineage of the subfamily by morphological studies and parsimony analyses, is found in a more derived position under model-based inference methods. Competing phylogenetic hypotheses, both previous and present, are evaluated using likelihood-based tests. Evolutionary trends within Barnadesioideae are inferred: hummingbird pollination has developed convergently at least three times. An early vicariance in the subfamilys distribution is revealed. X=9 is supported as the ancestral base chromosome number for both Barnadesioideae and the family as a whole.

Phylogenetic analyses of Tolpis Adans. (Asteraceae) reveal patterns of adaptive radiation, multiple colonization and interspecific hybridization

The plant genus Tolpis (Asteraceae) has been the subject of several investigations on the evolution of oceanic island plants. Its insular species were utilized in studies of artificial hybrid fertility, testing the validity of Baker’s law, the application of DNA barcodes, and the phylogenetic utility of inter‐simple sequence repeat markers. Despite this considerable interest in Tolpis, little is known about its phylogenetic history. Past investigations were unable to resolve most of the interspecific relationships, especially within the Canary Islands, where the genus is particularly diverse. Incomplete taxon sampling, the use of ambiguous outgroups and the limited utility of slowly evolving chloroplast DNA markers precluded detailed reconstructions. The present investigation presents a comprehensive molecular phylogeny of Tolpis. By utilizing highly variable nuclear DNA markers and a comprehensive taxon set, we have resolved the majority of interspecific relationships in the genus. Evaluations of competing tree topologies and ancestral area reconstructions complemented the analyses. Our results highlight the presence of three dominant mechanisms of island plant evolution—island colonization, adaptive radiation and interspecific hybridization—in Tolpis: (i) the extant distribution of the genus is the result of two independent colonization pathways, (ii) Tolpis has colonized at least one archipelago multiple times, (iii) the present insular diversity is the product of adaptive radiation, (iv) potential hybridization was detected between species now inhabiting different islands and archipelagoes, indicating sympatric historical distributions, and (v) several undescribed species await taxonomic recognition.

Posterior predictive checks of coalescent models: P2C2M, an R package

Bayesian inference operates under the assumption that the empirical data are a good statistical fit to the analytical model, but this assumption can be challenging to evaluate. Here, we introduce a novel r package that utilizes posterior predictive simulation to evaluate the fit of the multispecies coalescent model used to estimate species trees. We conduct a simulation study to evaluate the consistency of different summary statistics in comparing posterior and posterior predictive distributions, the use of simulation replication in reducing error rates and the utility of parallel process invocation towards improving computation times. We also test P2C2M on two empirical data sets in which hybridization and gene flow are suspected of contributing to shared polymorphism, which is in violation with the coalescent model: Tamias chipmunks and Myotis bats. Our results indicate that (i) probability‐based summary statistics display the lowest error rates, (ii) the implementation of simulation replication decreases the rate of type II errors, and (iii) our r package displays improved statistical power compared to previous implementations of this approach. When probabilistic summary statistics are used, P2C2M corroborates the assumption that genealogies collected from Tamias and Myotis are not a good fit to the multispecies coalescent model. Taken as a whole, our findings argue that an assessment of the fit of the multispecies coalescent model should accompany any phylogenetic analysis that estimates a species tree.

Plastid genome structure and phylogenomics of Nymphaeales: conserved gene order and new insights into relationships

The plastid genomes of early-diverging angiosperms were among the first land plant plastomes investigated. Despite their importance to understanding angiosperm evolution, no investigation has so far compared gene content or gene synteny of these plastid genomes with a focus on the Nymphaeales. Here, we report an evaluation and comparison of gene content, gene synteny and inverted repeat length for a set of 15 plastid genomes of early-diverging angiosperms. Seven plastid genomes of the Nymphaeales were newly sequenced for this investigation. We compare gene order and inverted repeat (IR) length across all genomes, review the gene annotations of previously published genomes, generate a multi-gene alignment of 77 plastid-encoded genes and reconstruct the phylogenetic relationships of the taxa under study. Our results show that gene content and synteny are highly conserved across early-diverging angiosperms: All species analyzed display complete gene synteny when accounting for expansions and contractions of the IRs. This conservation was initially obscured by ambiguous and potentially incorrect gene annotations in previously published genomes. We also report the presence of intact open reading frames across all taxa analyzed. The multi-gene phylogeny displays maximum support for the families Cabombaceae and Hydatellaceae, but no support for a clade of all Nymphaeaceae. It further indicates that the genus Victoria is embedded within Nymphaea. Plastid genomes of Trithuria were found to deviate by numerous substitutions and length changes in the IRs. Phylogenetic analyses further indicate that a previously published plastome named Nymphaea mexicana falls into a clade of N. odorata and should be re-evaluated.

Community trees: Identifying codiversification in the Páramo dipteran community.

Groups of codistributed species that responded in a concerted manner to environmental events are expected to share patterns of evolutionary diversification. However, the identification of such groups has largely been based on qualitative, post hoc analyses. We develop here two methods (posterior predictive simulation [PPS], Kuhner–Felsenstein [K–F] analysis of variance [ANOVA]) for the analysis of codistributed species that, given a group of species with a shared pattern of diversification, allow empiricists to identify those taxa that do not codiversify (i.e., “outlier” species). The identification of outlier species makes it possible to jointly estimate the evolutionary history of co‐diversifying taxa. To evaluate the approaches presented here, we collected data from Páramo dipterans, identified outlier species, and estimated a “community tree” from species that are identified as having codiversified. Our results demonstrate that dipteran communities from different Páramo habitats in the same mountain range are more closely related than communities in other ranges. We also conduct simulation testing to evaluate this approach. Results suggest that our approach provides a useful addition to comparative phylogeographic methods, while identifying aspects of the analysis that require careful interpretation. In particular, both the PPS and K–F ANOVA perform acceptably when there are one or two outlier species, but less so as the number of outliers increases. This is likely a function of the corresponding degradation of the signal of community divergence; without a strong signal from a codiversifying community, there is no dominant pattern from which to detect an outlier species. For this reason, both the magnitude of K–F distance distribution and outside knowledge about the phylogeographic history of each putative member of the community should be considered when interpreting the results.

WARACS: Wrappers to Automate the Reconstruction of Ancestral Character States

Premise of the study: Reconstructions of ancestral character states are among the most widely used analyses for evaluating the morphological, cytological, or ecological evolution of an organismic lineage. The software application Mesquite remains the most popular application for such reconstructions among plant scientists, even though its support for automating complex analyses is limited. A software tool is needed that automates the reconstruction and visualization of ancestral character states with Mesquite and similar applications. Methods and Results: A set of command line–based Python scripts was developed that (a) communicates standardized input to and output from the software applications Mesquite, BayesTraits, and TreeGraph2; (b) automates the process of ancestral character state reconstruction; and (c) facilitates the visualization of reconstruction results. Conclusions: WARACS provides a simple tool that streamlines the reconstruction and visualization of ancestral character states over a wide array of parameters, including tree distribution, character state, and optimality criterion.

Phylogeography and population genetics of the riparian relict tree Pterocarya fraxinifolia (Juglandaceae) in the South Caucasus

We aimed to (i) assess the extant genetic diversity of the riparian relict tree Pterocarya fraxinifolia across its current distribution range in the South Caucasus, including the past refugial areas Colchis and Hyrcan, and (ii) test if a separation of these areas is reflected in its phylogeographic history. Genetic diversity of natural populations was examined using nuclear microsatellite and plastid DNA markers. Spatial genetic structure was evaluated using Bayesian clustering methods and the reconstruction of plastid DNA networks. Divergence times of Colchic and Hyrcanian populations were estimated via divergence dating using a relaxed molecular clock. Allelic richness, private allelic richness, and expected heterozygosity were significantly higher in Hyrcan than in Colchis and the Greater Caucasus, and significant genetic differentiation was revealed between the two groups. Whereas only two plastid haplotypes were detected for the Colchic and Caucasian populations, the Hyrcanian populations displayed 11 different haplotypes. Significant isolation by distance was detected in Hyrcan. The most recent common ancestor of all P. fraxinifolia haplotypes was dated to a time well before a suggested glaciation period in the Caucasus during the late Pliocene (5.98 Ma [11.3–2.48 Ma HPD]). The widespread Colchic haplotype that also occurs along the southern slope of the Greater Caucasus and reaches south-eastern Azerbaijan has appeared more recently (0.24 Ma [1.41–0 Ma HPD]). This diversification pattern of Colchic haplotypes from ancient Hyrcanian haplotypes suggests a colonization of the region from south-east to north-west that predates the last glacial maximum (LGM). Natural populations of P. fraxinifolia show low-to-intermediate levels of genetic diversity and a significant decrease of diversity from Hyrcan to Colchis. However, the genetic differentiation between Colchic-Caucasian and Hyrcanian populations for nuclear markers suggests that independent gene pools existed in both areas at least since the LGM. Particular attention to conservation seems justified for the more diverse Hyrcanian populations.

Bioinformatic Workflows for Generating Complete Plastid Genome Sequences—An Example from Cabomba (Cabombaceae) in the Context of the Phylogenomic Analysis of the Water-Lily Clade

The sequencing and comparison of plastid genomes are becoming a standard method in plant genomics, and many researchers are using this approach to infer plant phylogenetic relationships. Due to the widespread availability of next-generation sequencing, plastid genome sequences are being generated at breakneck pace. This trend towards massive sequencing of plastid genomes highlights the need for standardized bioinformatic workflows. In particular, documentation and dissemination of the details of genome assembly, annotation, alignment and phylogenetic tree inference are needed, as these processes are highly sensitive to the choice of software and the precise settings used. Here, we present the procedure and results of sequencing, assembling, annotating and quality-checking of three complete plastid genomes of the aquatic plant genus Cabomba as well as subsequent gene alignment and phylogenetic tree inference. We accompany our findings by a detailed description of the bioinformatic workflow employed. Importantly, we share a total of eleven software scripts for each of these bioinformatic processes, enabling other researchers to evaluate and replicate our analyses step by step. The results of our analyses illustrate that the plastid genomes of Cabomba are highly conserved in both structure and gene content.

Towards resolving the evolutionary history of Caucasian pears (Pyrus, Rosaceae) - Phylogenetic relationships, divergence times and leaf trait evolution

With approximately 25 endemic species, the genus Pyrus (pears) is highly diverse in the Caucasus ecoregion. The majority of Caucasian pears inhabit xerophytic open woodlands or similar habitats, to which they display morphological adaptations, such as narrow leaves. The other species, both Caucasian and non‐Caucasian taxa, mainly inhabit mesophytic forests and display broad leaves. Using a representative taxon sampling of Pyrus from the Caucasus, Europe and Asia, we reconstruct phylogenetic relationships in the genus based on multiple plastid regions. We also estimate the divergence times of major clades in Pyrus, reconstruct the evolution of leaf shapes, and discuss the emergence of xeromorphic leaf traits. Our results confirm the monophyly of Pyrus and the existence of two major clades: (a) an E Asian clade with a crown group age of 15.7 (24.02–8.37 95% HPD) My, and (b) a W Eurasian clade that comprises species from Europe, SW Asia and the Caucasus and that displays a slightly younger crown group of 12.38 (19.02–6.41 95% HPD) My. The existing infrageneric classification of Pyrus was found partially incongruent with the inferred phylogenetic trees. Several currently accepted species were not recovered as monophyletic, indicating that current species limits require re‐evaluation. Ancestral character state reconstructions revealed several independent transitions from broad‐ to narrow‐shaped leaves in Pyrus, probably via intermediate‐shaped leaves.

Evolutionary diversification of the African achyranthoid clade (Amaranthaceae) in the context of sterile flower evolution and epizoochory

Background and Aims Many African genera of the Amaranthaceae exhibit unique inflorescences that include sterile flowers modified to hooks or spines. Considering that the abundance of large terrestrial herbivores increased on the African continent with the expansion of grassland and savannah ecosystems, modified sterile flowers could have been an innovation that boosted the diversification of an African achyranthoid clade of Amaranthaceae, with large animals serving dispersal. Methods We generated an extensively sampled phylogeny comprising 26 of the 31 achyranthoid genera as well as representatives of all other lineages of Amaranthaceae. Phylogenetic tree inference employed four genomic regions, using parsimony, likelihood and Bayesian inference methods. We estimated divergence times, evaluated trait-dependant changes and species diversification rates using state-dependent speciation and extinction models, and reconstructed ancestral character states for modified sterile flowers. Key Results The achyranthoids were found to be a major clade of the Amaranthaceae, comprising mostly African members. Phylogenetic relationships within this clade were well resolved and supported two main subclades. Several genera were found to be polyphyletic. Our results indicate that the achyranthoids started to diversify ~28 million years ago, and that modified sterile flowers evolved multiple times. An asymmetry in transition rates towards the gain of sterile flowers was observed, whereas no trait-dependent increase in species diversification rates was detected. Bayesian rate heterogeneity analyses indicated that the achyranthoids diversified without significant rate shifts. Conclusions The accumulation of modified sterile flowers within achyranthoids appears to result from the higher transition rates in favour of modified sterile flowers. Multiple gains suggest an adaptive value for this trait. However, epizoochory does not appear to fuel species diversification, possibly due to extensive gene flow through regularly migrating mammals, which limits the possibility of speciation by isolation.