Andrew A. Crowl

How to Handle Speciose Clades? Mass Taxon-Sampling as a Strategy towards Illuminating the Natural History of Campanula (Campanuloideae)

Background Speciose clades usually harbor species with a broad spectrum of adaptive strategies and complex distribution patterns, and thus constitute ideal systems to disentangle biotic and abiotic causes underlying species diversification. The delimitation of such study systems to test evolutionary hypotheses is difficult because they often rely on artificial genus concepts as starting points. One of the most prominent examples is the bellflower genus Campanula with some 420 species, but up to 600 species when including all lineages to which Campanula is paraphyletic. We generated a large alignment of petD group II intron sequences to include more than 70% of described species as a reference. By comparison with partial data sets we could then assess the impact of selective taxon sampling strategies on phylogenetic reconstruction and subsequent evolutionary conclusions. Methodology/Principal Findings Phylogenetic analyses based on maximum parsimony (PAUP, PRAP), Bayesian inference (MrBayes), and maximum likelihood (RAxML) were first carried out on the large reference data set (D680). Parameters including tree topology, branch support, and age estimates, were then compared to those obtained from smaller data sets resulting from “classification-guided” (D088) and “phylogeny-guided sampling” (D101). Analyses of D088 failed to fully recover the phylogenetic diversity in Campanula, whereas D101 inferred significantly different branch support and age estimates. Conclusions/Significance A short genomic region with high phylogenetic utility allowed us to easily generate a comprehensive phylogenetic framework for the speciose Campanula clade. Our approach recovered 17 well-supported and circumscribed sub-lineages. Knowing these will be instrumental for developing more specific evolutionary hypotheses and guide future research, we highlight the predictive value of a mass taxon-sampling strategy as a first essential step towards illuminating the detailed evolutionary history of diverse clades.

The Report of My Death was an Exaggeration: A Review for Researchers Using Microsatellites in the 21st Century

Microsatellites, or simple sequence repeats (SSRs), have long played a major role in genetic studies due to their typically high polymorphism. They have diverse applications, including genome mapping, forensics, ascertaining parentage, population and conservation genetics, identification of the parentage of polyploids, and phylogeography. We compare SSRs and newer methods, such as genotyping by sequencing (GBS) and restriction site associated DNA sequencing (RAD-Seq), and offer recommendations for researchers considering which genetic markers to use. We also review the variety of techniques currently used for identifying microsatellite loci and developing primers, with a particular focus on those that make use of next-generation sequencing (NGS). Additionally, we review software for microsatellite development and report on an experiment to assess the utility of currently available software for SSR development. Finally, we discuss the future of microsatellites and make recommendations for researchers preparing to use microsatellites. We argue that microsatellites still have an important place in the genomic age as they remain effective and cost-efficient markers.

Making next-generation sequencing work for you: approaches and practical considerations for marker development and phylogenetics

Recent reviews are setting the stage for the use of next-generation sequencing technologies in phylogenetic applications. However, the processes for developing new markers for phylogenetic analyses remain difficult to navigate for many researchers in plant systematics. We review several experimental approaches and practical considerations for developing new phylogenetic markers with next-generation sequencing technologies. We also outline a flexible framework for data acquisition that is readily adaptable to the needs of individual researchers and carefully consider cost-related issues that may be of concern to many laboratories in evolutionary biology. The next-generation and targeted sequencing approaches presented here offer considerable savings of time and money over the traditional PCR and Sanger sequencing approaches currently used in plant systematic research, particularly in cases involving large numbers of taxa and phylogenetic markers. Even with a limited research budget, next-generation sequence data can accommodate exploration of biological questions in ways that were not previously possible.

Phylogeny of Campanuloideae (Campanulaceae) with Emphasis on the Utility of Nuclear Pentatricopeptide Repeat (PPR) Genes

Background The Campanuloideae (Campanulaceae) are a highly diverse clade of angiosperms found mostly in the Northern Hemisphere, with the highest diversity in temperate areas of the Old World. Chloroplast markers have greatly improved our understanding of this clade but many relationships remain unclear primarily due to low levels of molecular evolution and recent and rapid divergence. Furthermore, focusing solely on maternally inherited markers such as those from the chloroplast genome may obscure processes such as hybridization. In this study we explore the phylogenetic utility of two low-copy nuclear loci from the pentatricopeptide repeat gene family (PPR). Rapidly evolving nuclear loci may provide increased phylogenetic resolution in clades containing recently diverged or closely related taxa. We present results based on both chloroplast and low-copy nuclear loci and discuss the utility of such markers to resolve evolutionary relationships and infer hybridization events within the Campanuloideae clade. Results The inclusion of low-copy nuclear genes into the analyses provides increased phylogenetic resolution in two species-rich clades containing recently diverged taxa. We also obtain support for the placement of two early diverging lineages (Jasione and Musschia-Gadellia clades) that have previously been unresolved. Furthermore, phylogenetic analyses of PPR loci revealed potential hybridization events for a number of taxa (e.g., Campanula pelviformis and Legousia species). These loci offer greater overall topological support than obtained with plastid DNA alone. Conclusion This study represents the first inclusion of low-copy nuclear genes for phylogenetic reconstruction in Campanuloideae. The two PPR loci were easy to sequence, required no cloning, and the sequence alignments were straightforward across the entire Campanuloideae clade. Although potentially complicated by incomplete lineage sorting, these markers proved useful for understanding the processes of reticulate evolution and resolving relationships at a wide range of phylogenetic levels. Our results stress the importance of including multiple, independent loci in phylogenetic analyses.

A global perspective on Campanulaceae: Biogeographic, genomic, and floral evolution

PREMISE OF THE STUDY The Campanulaceae are a diverse clade of flowering plants encompassing more than 2300 species in myriad habitats from tropical rainforests to arctic tundra. A robust, multigene phylogeny, including all major lineages, is presented to provide a broad, evolutionary perspective of this cosmopolitan clade. METHODS We used a phylogenetic framework, in combination with divergence dating, ancestral range estimation, chromosome modeling, and morphological character reconstruction analyses to infer phylogenetic placement and timing of major biogeographic, genomic, and morphological changes in the history of the group and provide insights into the diversification of this clade across six continents. KEY RESULTS Ancestral range estimation supports an out-of-Africa diversification following the Cretaceous-Tertiary extinction event. Chromosomal modeling, with corroboration from the distribution of synonymous substitutions among gene duplicates, provides evidence for as many as 20 genome-wide duplication events before large radiations. Morphological reconstructions support the hypothesis that switches in floral symmetry and anther dehiscence were important in the evolution of secondary pollen presentation mechanisms. CONCLUSIONS This study provides a broad, phylogenetic perspective on the evolution of the Campanulaceae clade. The remarkable habitat diversity and cosmopolitan distribution of this lineage appears to be the result of a complex history of genome duplications and numerous long-distance dispersal events. We failed to find evidence for an ancestral polyploidy event for this clade, and our analyses indicate an ancestral base number of nine for the group. This study will serve as a framework for future studies in diverse areas of research in Campanulaceae.

Evolution and biogeography of the endemic Roucela complex (Campanulaceae: Campanula) in the Eastern Mediterranean

Abstract At the intersection of geological activity, climatic fluctuations, and human pressure, the Mediterranean Basin – a hotspot of biodiversity – provides an ideal setting for studying endemism, evolution, and biogeography. Here, we focus on the Roucela complex (Campanula subgenus Roucela), a group of 13 bellflower species found primarily in the eastern Mediterranean Basin. Plastid and low‐copy nuclear markers were employed to reconstruct evolutionary relationships and estimate divergence times within the Roucela complex using both concatenation and species tree analyses. Niche modeling, ancestral range estimation, and diversification analyses were conducted to provide further insights into patterns of endemism and diversification through time. Diversification of the Roucela clade appears to have been primarily the result of vicariance driven by the breakup of an ancient landmass. We found geologic events such as the formation of the mid‐Aegean trench and the Messinian Salinity Crisis to be historically important in the evolutionary history of this group. Contrary to numerous past studies, the onset of the Mediterranean climate has not promoted diversification in the Roucela complex and, in fact, may be negatively affecting these species. This study highlights the diversity and complexity of historical processes driving plant evolution in the Mediterranean Basin.

A New Resource for the Development of SSR Markers: Millions of Loci from a Thousand Plant Transcriptomes

Premise of the study: The One Thousand Plant Transcriptomes Project (1KP, 1000+ assembled plant transcriptomes) provides an enormous resource for developing microsatellite loci across the plant tree of life. We developed loci from these transcriptomes and tested their utility. Methods and Results: Using software packages and custom scripts, we identified microsatellite loci in 1KP transcriptomes. We assessed the potential for cross-amplification and whether loci were biased toward exons, as compared to markers derived from genomic DNA. We characterized over 5.7 million simple sequence repeat (SSR) loci from 1334 plant transcriptomes. Eighteen percent of loci substantially overlapped with open reading frames (ORFs), and electronic PCR revealed that over half the loci would amplify successfully in conspecific taxa. Transcriptomic SSRs were approximately three times more likely to map to translated regions than genomic SSRs. Conclusions: We believe microsatellites still have a place in the genomic age—they remain effective and cost-efficient markers. The loci presented here are a valuable resource for researchers.

Embracing discordance: Phylogenomic analyses provide evidence for allopolyploidy leading to cryptic diversity in a Mediterranean Campanula (Campanulaceae) clade

The Mediterranean Basin harbors a remarkable amount of biodiversity, a high proportion of which is endemic to this region. Here, we present an in‐depth study of an angiosperm species complex, in which cryptic taxonomic diversity has been hypothesized. Specifically, we focus on four currently recognized species in the Roucela complex, a well‐supported clade in the Campanulaceae/Campanuloideae: Campanula creutzburgii, C. drabifolia, C. erinus, and C. simulans. This study takes a phylogenomic approach, utilizing near‐complete plastomes and 130 nuclear loci, to uncover cryptic diversity and test hypotheses regarding hybridization and polyploidy within this clade. Genome size estimates recovered tetraploid and octoploid lineages within the currently recognized, widespread species C. erinus, showing an east‐west geographic pattern. Though genomic data clearly differentiate these two cytotypes, we failed to discern morphological differences. The formation of a cryptic octoploid lineage, distributed across the eastern Mediterranean, is hypothesized to be the result of an allopolyploid event in which one parental morphology is retained. The tetraploid C. erinus and C. creutzburgii (also a tetraploid) are implicated as parental lineages. Our results highlight the utility of target‐enrichment approaches for obtaining genomic datasets for thorough assessments of species diversity and the importance of carefully considering gene‐tree discordance within such datasets.

Origins of East Asian Campanuloideae (Campanulaceae) diversity

The Campanulaceae comprises approximately 2300 species that are distributed among five major lineages: Campanuloideae, Cyphioideae, Cyphocarpoideae, Lobelioideae, and Nemacladoideae. Of these, the Campanuloideae, a primarily Old World clade, has the largest diversity in East Asia. In this study, we reconstruct the phylogeny of East Asian Campanuloideae based on one nuclear gene (i.e., PPR70) and five plastid markers (i.e., atpB, matK, petD, rbcL, and trnL-trnF). We then use this phylogenetic framework to reconstruct the biogeographical history of the genus. Our molecular dataset includes 376 of the 1045 currently recognized species in the Campanuloideae. Of the 376 sampled species, 116 are from East Asia, representing ca. 60% of the East Asian Campanuloideae. Our PPR dataset included sequences for 156 accessions, representing 54 species, while our plastid dataset included sequences for 305 accessions, representing 354 species. Phylogenetic analyses recovered three large clades containing East Asian taxa: Campanulinae, Platycodinae, and Wahlenberginae. The historical assembly of Campanuloideae diversity in East Asia appears to have resulted from numerous, independent movements from Africa, Europe/W. Asia, and North America. Africa was inferred as the ancestral range for the Campanuloideae. Movement of the largest East Asian clade (Platycodinae) occurred at approximately 53.1 Ma (46.6-58.73 95% HPD) from Africa, with much of the current diversity found in East Asia having resulted from in situ diversification. Thirteen additional movements into East Asia, primarily from Europe/Western Asia, occurred subsequently. One dispersal event from western North America was also inferred. In contrast, only six movements out of East Asia were found. Our results suggest that East Asia has acted primarily as a sink for Campanuloideae diversity, with Europe, Western Asia, and Africa representing major source areas.

Range change evolution of peatmosses (Sphagnum) within and between climate zones

Peat mosses (Sphagnum) hold exceptional importance in the control of global carbon fluxes and climate because of the vast stores of carbon bound up in partially decomposed biomass (peat). This study tests the hypothesis that the early diversification of Sphagnum was in the Northern Hemisphere, with subsequent range expansions to tropical latitudes and the Southern Hemisphere. A phylogenetic analysis of 192 accessions representing the moss class Sphagnopsida based on four plastid loci was conducted in conjunction with biogeographic analyses using BioGeoBEARS to investigate the tempo and mode of geographic range evolution. Analyses support the hypothesis that the major intrageneric clades of peat-forming species accounting for >90% of peat moss diversity originated and diversified at northern latitudes. The genus underwent multiple range expansions into tropical and Southern Hemisphere regions. Range evolution in peat mosses was most common within latitudinal zones, attesting to the relative difficulty of successfully invading new climate zones. Allopolyploidy in Sphagnum (inferred from microsatellite heterozygosity) does not appear to be biased with regard to geographic region nor intrageneric clade. The inference that Sphagnum diversified in cool-or cold-climate regions and repeatedly expanded its range into tropical regions makes the genus an excellent model for studying morphological, physiological, and genomic traits associated with adaptation to warming climates.