Recent Evolutionary Radiation Research Articles

Parallel evolution, atavism, and extensive introgression explain the radiation of Epimedium sect. Diphyllon (Berberidaceae) in southern East Asia.

East Asia is the richest region of plant biodiversity in the northern temperate zone, and its radiation provides key insights for understanding rapid speciation, including evolutionary patterns and processes. However, it is challenging to investigate the recent evolutionary radiation among plants because of the lack of genetic divergence, phenotypic convergence, and interspecific gene flow. Epimedium sect. Diphyllon is a rarely studied plant lineage endemic to East Asia, especially highly diversified in its southern part. In this study, we report a robust phylogenomic analysis based on genotyping-by-sequencing data of this lineage. The results revealed a clear biogeographic pattern for Epimedium sect. Diphyllon with recognition into two major clades corresponding to the Sino-Himalayan and Sino-Japanese subkingdoms of East Asian Flora and rapid diversification of the extant species dated to the Pleistocene. Evolutionary radiation of Epimedium sect. Diphyllon is characterized by recent and predominant parallel evolution and atavism between the two subkingdom regions, with extensive reticulating hybridization within each region during the course of diversification in southern East Asia. A parallel-atavism-introgression hypothesis is referred to in explaining the radiation of plant diversity in southern East Asia, which represents a potential model for the rapid diversification of plants under global climate cooling in the late Tertiary. Our study advances our understanding of the evolutionary processes of plant radiation in East Asia as well as in other biodiversity hotspot regions.

Banksia (Proteaceae) contains less phylogenetic diversity than expected in Southwestern Australia

AbstractBanksia L.f., an iconic genus of the Proteaceae, is endemic to Australia, with its highest species richness and phylogenetic diversity (PD) in southwestern Western Australia. Analysis of the phylogenetic component of richness and endemism uncovered important patterns of Banksia evolutionary history that are not seen at the species level. We found that Banksia is significantly phylogenetically clustered in this region, likely due to recent evolutionary radiations. We also found significant concentrations of phylogenetic endemism in this region, both neoendemism (short, range‐restricted evolutionary branches) and paleoendemism (long, range‐restricted evolutionary branches). There is a striking northwest to southeast divide in phyloturnover in southwestern Western Australia. The majority of the variation in turnover patterns can be explained by environmental factors, with climate representing the largest covariate. This study adds to the growing body of evidence that indicates the importance of integrating phylogenetic and biodiversity data to inform conservation planning.

Strong conservatism of floral morphology during the rapid diversification of the genus Helianthemum.

Divergence of floral morphology and breeding systems are often expected to be linked to angiosperm diversification and environmental niche divergence. However, available evidence for such relationships is not generalizable due to different taxonomic, geographical and time scales. The Palearctic genus Helianthemum shows the highest diversity of the family Cistaceae in terms of breeding systems, floral traits, and environmental conditions as a result of three recent evolutionary radiations since the Late Miocene. Here, we investigated the tempo and mode of evolution of floral morphology in the genus and its link with species diversification and environmental niche divergence. We quantified 18 floral traits from 83 taxa and applied phylogenetic comparative methods using a robust phylogenetic framework based on genotyping-by-sequencing data. We found three different floral morphologies, putatively related to three different breeding systems: type I, characterized by small flowers without herkogamy and low pollen to ovule ratio; type II, represented by large flowers with approach herkogamy and intermediate pollen to ovule ratio; and type III, featured by small flowers with reverse herkogamy and the highest pollen to ovule ratio. Each morphology has been highly conserved across each radiation and has evolved independently of species diversification and ecological niche divergence. The combined results of trait, niche, and species diversification ultimately recovered a pattern of potentially non-adaptive radiations in Helianthemum and highlight the idea that evolutionary radiations can be decoupled from floral morphology evolution even in lineages that diversified in heterogeneous environments as the Mediterranean Basin.

Genome-Wide SNPs Clarify a Complex Radiation and Support Recognition of an Additional Cat Species.

Phylogenetic reconstruction and species delimitation are often challenging in the case of recent evolutionary radiations, especially when postspeciation gene flow is present. Leopardus is a Neotropical cat genus that has a long history of recalcitrant taxonomic problems, along with both ancient and current episodes of interspecies admixture. Here, we employ genome-wide SNP data from all presently recognized Leopardus species, including several individuals from the tigrina complex (representing Leopardus guttulus and two distinct populations of Leopardus tigrinus), to investigate the evolutionary history of this genus. Our results reveal that the tigrina complex is paraphyletic, containing at least three distinct species. While one can be assigned to L. guttulus, the other two remain uncertain regarding their taxonomic assignment. Our findings indicate that the “tigrina” morphology may be plesiomorphic within this group, which has led to a longstanding taxonomic trend of lumping these poorly known felids into a single species.

Molecular and Chemical Markers to Illustrate the Complex Diversity of the Genus Lupinus (Fabaceae)

The potential of secondary metabolites as systematic markers to get new insights in an intricate phylogeny of a recent evolutionary radiation is explored. A chemosystematic study of the genus Lupinus (Fabaceae) was performed, using quinolizidine (QA) and piperidine alkaloids (ammodendrine) as diagnostic characters. Seven major QA and the piperidine alkaloid ammodendrine were found to be the most frequent compounds. Two groups were supported according to their geographic origin: an Old World/Atlantic American group and a West New World group and this pattern is concordant with molecular data (here, based on an original barcode approach using the nuclear marker ITS). However, QA profiles are less informative at the species level. Despite a lack of resolution within the two groups, the alkaloid profiles agree with well supported clades based on DNA molecular characters. The combined use of chemical and barcode genetic markers represents a viable alternative for separating recent evolutionary lineages to a first approximation without having to resort to an expensive and sophisticated molecular arsenal such as next generation sequencing.

Filling Linnean shortfalls increases endemicity patterns: conservation and biogeographical implications for the extreme case ofLiolaemus(Liolaemidae, Squamata) species

AbstractLinnean shortfalls, or the discrepancy between formally described species and the number of existing species, imply noteworthy biodiversity conservation constraints. The case of Argentinian reptiles is unique in this context. The genus Liolaemus represents ~60% of the Argentine reptile fauna and has a current species description rate of 3.25 spp./year, leading to a significant increase in the number of known species. Filling Linnean shortfalls within this genus has revealed an extraordinary recent evolutionary radiation that strongly modifies the spatial accumulation patterns of richness and endemism, mainly in the Patagonian steppe and Monte ecoregions of Argentina. Thus, while 40 years ago none of the studied species was considered endemic at the ecoregional level, today the genus represents an essential element to define the southernmost terrestrial ecoregions of the world. Furthermore, the remarkable decrease in the average extent of occurrence (EOO) of Liolaemus species implies an increased extinction risk in light of threats caused by humans. Based on the thresholds for the IUCN Red List B1 criteria, and 11 well-studied clades of Liolaemus, we show that, while in 1980 87% of the described species would not qualify as threatened based on EOO, today 80% of the species have an EOO smaller than 20 000 km2. Therefore, they may qualify as threatened based on the B1 EOO thresholds, which demonstrates the importance of the recent increase of knowledge regarding the taxonomy, systematics and genetics of the group.

Phylomitogenomics provides new perspectives on the Euphasmatodea radiation (Insecta: Phasmatodea)

Phasmatodea species diversity lies almost entirely within its suborder Euphasmatodea, which exhibits a pantropical distribution and is considered to derive from a recent and rapid evolutionary radiation. To shed light on Euphasmatodea origins and diversification, we assembled the mitogenomes of 17 species from transcriptomic sequencing data and analysed them along with 22 already available Phasmatodea mitogenomes and 33 mitogenomes representing most of the Polyneoptera lineages. Maximum Likelihood and Bayesian Inference approaches retrieved consistent topologies, both showing the widespread conflict between phylogenetic approaches and traditional systematics. We performed a divergence time analysis leveraging ten fossil specimens representative of most polyneopteran lineages: the time tree obtained supports an older radiation of the clade with respect to previous hypotheses. Euphasmatodea diversification is inferred to have started ~ 187 million years ago, suggesting that the Triassic-Jurassic mass extinction and the breakup of Pangea could have contributed to the process. We also investigated Euphasmatodea mitogenomes patterns of dN, dS and dN/dS ratio throughout our time-tree, trying to characterize the selective regime which may have shaped the clade evolution.

Phylotranscriptomics resolves interspecific relationships and indicates multiple historical out-of-North America dispersals through the Bering Land Bridge for the genus Picea (Pinaceae)

A robust phylogeny is prerequisite to understand the evolution and biogeography of organisms. However, ancient and recent evolutionary radiations occurred in many plant lineages, which pose great challenges for phylogenetic analysis, especially for conifers characterized by large effective population sizes and long generation times. Picea is an important component of the dark coniferous forests in the Northern Hemisphere. Previous studies improved our understanding of its evolutionary history, but its interspecific relationships and biogeographic history remain largely unresolved. In the present study, we reconstructed a well-resolved phylogeny of Picea by comparative transcriptomic analysis based on a complete species sampling. The phylogenetic analysis, together with molecular dating and ancestral area reconstruction, further supports the North American origin hypothesis for Picea, and indicates that this genus experienced multiple out-of-North America dispersals by the Bering Land Bridge. We also found that spruces in the Japanese Archipelago have multiple origins, and P. morrisonicola from the Taiwan Island has a close relationship with species from the Qinghai-Tibetan Plateau and adjacent regions. Our study provides the first complete phylogeny of Picea at the genomic level, which is important for future studies of this genus.

NGS-Indel Coder: A pipeline to code indel characters in phylogenomic data with an example of its application in milkweeds (Asclepias)

Targeted genome sequencing approaches allow characterization of evolutionary relationships using a considerable number of nuclear genes and informative characters. However, most phylogenomic analyses only utilize single nucleotide polymorphisms (SNPs). Studies at the species level, especially in groups that have recently radiated, often recover low amounts of phylogenetically informative variation in coding regions, and require non-coding sequences, which are richer in indels, to resolve gene trees. Here, NGS-Indel Coder, a pipeline to detect and omit false positive indels inferred from assemblies of short read sequence data, was developed to resolve the relationships among and within major clades of the American milkweeds (Asclepias), which are the result of a rapid and recent evolutionary radiation, and whose phylogeny has been difficult to resolve. This pipeline was applied to a Hyb-Seq data set of 768 loci including targeted exons and flanking intron regions from 33 milkweed species. Robust species tree inference was improved by excluding small alignment partitions (<100 bp) that increased gene tree ambiguity and incongruence. To further investigate the robustness of indel coding, data sets that included small and large indels were explored, and species trees derived from concatenated loci versus coalescent methods based on gene trees were compared. The phylogeny of Asclepias obtained using nuclear data was well resolved, and phylogenetic information from indels improved resolution of specific nodes. The Temperate North American, Mexican Highland, and Incarnatae clades were well supported as monophyletic. Asclepias coulteri, which has been considered part of the Sonoran Desert clade based on plastome analyses, was placed as sister to all the other milkweed species studied here, rather than as a member of that clade. Two groups within the Temperate North American and Mexican clades were not resolved, and the inferred relationships strongly conflicted when comparing results based on data sets that did or did not include indel characters. This new pipeline represents a step forward in making maximal use of the information content in phylogenomic data sets.

Recent diversification in an ancient lineage of Notothenioid fishes (Bovichtus: Notothenioidei)

Notothenioids are among the most intensively studied lineages of marine fishes. However, notothenioid research is predominately focused on the approximately 100 species of Antarctic cryonotothenioids. Far less attention is devoted to the non-Antarctic lineages Bovichtidae, Pseudaphritis urvillii, and Eleginops maclovinus, all of which originated prior to the diversification of cryonotothenioid species. Here we utilize DNA sequence data from mitochondrial and nuclear genes, as well as meristic trait morphology to investigate the evolutionary history of Bovichtidae. Bovichtus is the only polytypic lineage of early diverging non-Antarctic notothenioids providing a unique opportunity to contextualize the diversification dynamics of cryonotothenioids with their non-Antarctic relatives. We find strong evidence that species of Bovichtus represent a recent evolutionary radiation with divergence times similar to those estimated among the most closely related species of cryonotothenioids. The divergence in traditional meristic trait morphology among species of Bovichtus is consistent with their phylogenetic relationships. The phylogeny of Bovichtus implies the wide geographic distribution of species in the clade is likely the result of West Wind drift-driven dispersal. The phylogeny and divergence time estimates results reject a hypothesis that species persistence in Bovichtus reflects long periods of evolutionary stasis. Instead, we hypothesize that patterns of extinction and diversification in Bovichtus closely mirror those observed in their Antarctic relatives.

Divergent trait and environment relationships among parallel radiations in Pelargonium (Geraniaceae): a role for evolutionary legacy?

Functional traits in closely related lineages are expected to vary similarly along common environmental gradients as a result of shared evolutionary and biogeographic history, or legacy effects, and as a result of biophysical tradeoffs in construction. We test these predictions in Pelargonium, a relatively recent evolutionary radiation. Bayesian phylogenetic mixed effects models assessed, at the subclade level, associations between plant height, leaf area, leaf nitrogen content and leaf mass per area (LMA), and five environmental variables capturing temperature and rainfall gradients across the Greater Cape Floristic Region of South Africa. Trait-trait integration was assessed via pairwise correlations within subclades. Of 20 trait-environment associations, 17 differed among subclades. Signs of regression coefficients diverged for height, leaf area and leaf nitrogen content, but not for LMA. Subclades also differed in trait-trait relationships and these differences were modulated by rainfall seasonality. Leave-one-out cross-validation revealed that whether trait variation was better predicted by environmental predictors or trait-trait integration depended on the clade and trait in question. Legacy signals in trait-environment and trait-trait relationships were apparently lost during the earliest diversification of Pelargonium, but then retained during subsequent subclade evolution. Overall, we demonstrate that global-scale patterns are poor predictors of patterns of trait variation at finer geographic and taxonomic scales.

Phylogenomic evidence for a recent and rapid radiation of lizards in the Patagonian Liolaemus fitzingerii species group

Rapid evolutionary radiations are difficult to resolve because divergence events are nearly synchronous and gene flow among nascent species can be high, resulting in a phylogenetic “bush”. Large datasets composed of sequence loci from across the genome can potentially help resolve some of these difficult phylogenetic problems. A suitable test case is the Liolaemus fitzingerii species group of lizards, which includes twelve species that are broadly distributed in Argentinean Patagonia. The species in the group have had a complex evolutionary history that has led to high morphological variation and unstable taxonomy. We generated a sequence capture dataset for 28 ingroup individuals of 580 nuclear loci, alongside a mitogenomic dataset, to infer phylogenetic relationships among species in this group. Relationships among species were generally weakly supported with the nuclear data, and along with an inferred age of ∼2.6 million years old, indicate either rapid evolution, hybridization, incomplete lineage sorting, non-informative data, or a combination thereof. We inferred a signal of mito-nuclear discordance, indicating potential hybridization between L. melanops and L. martorii, and phylogenetic network analyses provided support for 5 reticulation events among species. Phasing the nuclear loci did not provide additional insight into relationships or suspected patterns of hybridization. Only one clade, composed of L. camarones, L. fitzingerii, and L. xanthoviridis was recovered across all analyses. Genomic datasets provide molecular systematists with new opportunities to resolve difficult phylogenetic problems, yet the lack of phylogenetic resolution in Patagonian Liolaemus is biologically meaningful and indicative of a recent and rapid evolutionary radiation. The phylogenetic relationships of the Liolaemus fitzingerii group may be best modeled as a reticulated network instead of a bifurcating phylogeny.

Network Analysis of Protein Adaptation: Modeling the Functional Impact of Multiple Mutations

The evolution of new biochemical activities frequently involves complex dependencies between mutations and rapid evolutionary radiation. Mutation co-occurrence and covariation have previously been used to identify compensating mutations that are the result of physical contacts and preserve protein function and fold. Here, we model pairwise functional dependencies and higher order interactions that enable evolution of new protein functions. We use a network model to find complex dependencies between mutations resulting from evolutionary trade-offs and pleiotropic effects. We present a method to construct these networks and to identify functionally interacting mutations in both extant and reconstructed ancestral sequences (Network Analysis of Protein Adaptation). The time ordering of mutations can be incorporated into the networks through phylogenetic reconstruction. We apply NAPA to three distantly homologous β-lactamase protein clusters (TEM, CTX-M-3, and OXA-51), each of which has experienced recent evolutionary radiation under substantially different selective pressures. By analyzing the network properties of each protein cluster, we identify key adaptive mutations, positive pairwise interactions, different adaptive solutions to the same selective pressure, and complex evolutionary trajectories likely to increase protein fitness. We also present evidence that incorporating information from phylogenetic reconstruction and ancestral sequence inference can reduce the number of spurious links in the network, whereas preserving overall network community structure. The analysis does not require structural or biochemical data. In contrast to function-preserving mutation dependencies, which are frequently from structural contacts, gain-of-function mutation dependencies are most commonly between residues distal in protein structure.

Integrating genomic and phenotypic data to evaluate alternative phylogenetic and species delimitation hypotheses in a recent evolutionary radiation of grasshoppers.

Although resolving phylogenetic relationships and establishing species limits are primary goals of systematics, these tasks remain challenging at both conceptual and analytical levels. Here, we integrated genomic and phenotypic data and employed a comprehensive suite of coalescent-based analyses to develop and evaluate competing phylogenetic and species delimitation hypotheses in a recent evolutionary radiation of grasshoppers (Chorthippus binotatus group) composed of two species and eight putative subspecies. To resolve the evolutionary relationships within this complex, we first evaluated alternative phylogenetic hypotheses arising from multiple schemes of genomic data processing and contrasted genetic-based inferences with different sources of phenotypic information. Second, we examined the importance of number of loci, demographic priors, number and kind of phenotypic characters and sex-based trait variation for developing alternative species delimitation hypotheses. The best-supported topology was largely compatible with phenotypic data and showed the presence of two clades corresponding to the nominative species groups, one including three well-resolved lineages and the other comprising a four-lineage polytomy and a well-differentiated sister taxon. Integrative species delimitation analyses indicated that the number of employed loci had little impact on the obtained inferences but revealed the higher power provided by an increasing number of phenotypic characters and the usefulness of assessing their phylogenetic information content and differences between sexes in among-taxa trait variation. Overall, our study highlights the importance of integrating multiple sources of information to test competing phylogenetic hypotheses and elucidate the evolutionary history of species complexes representing early stages of divergence where conflicting inferences are more prone to appear.

Resolving Recent Plant Radiations: Power and Robustness of Genotyping-by-Sequencing.

Disentangling species boundaries and phylogenetic relationships within recent evolutionary radiations is a challenge due to the poor morphological differentiation and low genetic divergence between species, frequently accompanied by phenotypic convergence, interspecific gene flow and incomplete lineage sorting. Here we employed a genotyping-by-sequencing (GBS) approach, in combination with morphometric analyses, to investigate a small western Mediterranean clade in the flowering plant genus Linaria that radiated in the Quaternary. After confirming the morphological and genetic distinctness of eight species, we evaluated the relative performances of concatenation and coalescent methods to resolve phylogenetic relationships. Specifically, we focused on assessing the robustness of both approaches to variations in the parameter used to estimate sequence homology (clustering threshold). Concatenation analyses suffered from strong systematic bias, as revealed by the high statistical support for multiple alternative topologies depending on clustering threshold values. By contrast, topologies produced by two coalescent-based methods (NJ$_{\mathrm{st}}$, SVDquartets) were robust to variations in the clustering threshold. Reticulate evolution may partly explain incongruences between NJ$_{\mathrm{st}}$, SVDquartets and concatenated trees. Integration of morphometric and coalescent-based phylogenetic results revealed (i) extensive morphological divergence associated with recent splits between geographically close or sympatric sister species and (ii) morphological convergence in geographically disjunct species. These patterns are particularly true for floral traits related to pollinator specialization, including nectar spur length, tube width and corolla color, suggesting pollinator-driven diversification. Given its relatively simple and inexpensive implementation, GBS is a promising technique for the phylogenetic and systematic study of recent radiations, but care must be taken to evaluate the robustness of results to variation of data assembly parameters.

A pilot study applying the plant Anchored Hybrid Enrichment method to New World sages (Salvia subgenus Calosphace; Lamiaceae)

We conducted a pilot study using Anchored Hybrid Enrichment to resolve relationships among a mostly Neotropical sage lineage that may have undergone a recent evolutionary radiation. Conventional markers (ITS, trnL-trnF and trnH-psbA) have not been able to resolve the relationships among species nor within portions of the backbone of the lineage. We sampled 12 representative species of subgenus Calosphace and included one species of Salvia’s s.l. closest relative, Lepechinia, as outgroup. Hybrid enrichment and sequencing were successful, yielding 448 alignments of individual loci with an average length of 704bp. The performance of the phylogenomic data in phylogenetic reconstruction was superior to that of conventional markers, increasing both support and resolution. Because the captured loci vary in the amount of net phylogenetic informativeness at different phylogenetic depths, these data are promising in phylogenetic reconstruction of this group and likely other lineages within Lamiales. However, special attention should be placed on the amount of phylogenetic noise that the data could potentially contain. A prior exploration step using phylogenetic informativeness profiles to detect loci with sites with disproportionately high substitution rates (showing “phantom” spikes) and, if required, the ensuing filtering of the problematic data is recommended. In our dataset, filtering resulted in increased support and resolution for the shallow nodes in maximum likelihood phylogenetic trees resulting from concatenated analyses of all the loci. Additionally, it is expected that an increase in sampling (loci and taxa) will aid in resolving weakly supported, short deep internal branches.

Anchored phylogenomics improves the resolution of evolutionary relationships in the rapid radiation of Protea L.

Estimating phylogenetic relationships in relatively recent evolutionary radiations is challenging, especially if short branches associated with recent divergence result in multiple gene tree histories. We combine anchored enrichment next-generation sequencing with species tree analyses to produce a robust estimate of phylogenetic relationships in the genus Protea (Proteaceae), an iconic radiation in South Africa. We sampled multiple individuals within 59 out of 112 species of Protea and 6 outgroup species for a total of 163 individuals, and obtained sequences for 498 low-copy, orthologous nuclear loci using anchored phylogenomics. We compare several approaches for building species trees, and explore gene tree-species tree discrepancies to determine whether poor phylogenetic resolution reflects a lack of informative sites, incomplete lineage sorting, or hybridization. Phylogenetic estimates from species tree approaches are similar to one another and recover previously well-supported clades within Protea, in addition to providing well-supported phylogenetic hypotheses for previously poorly resolved intrageneric relationships. Individual gene trees are markedly different from one another and from species trees. Nonetheless, analyses indicate that differences among gene trees occur primarily concerning clades supported by short branches. Species tree methods using hundreds of nuclear loci provided strong support for many previously unresolved relationships in the radiation of the genus Protea. In cases where support for particular relationships remains low, these appear to arise from few informative sites and lack of information rather than strongly supported disagreement among gene trees.

A phylogeny of the genus Encyclia (Orchidaceae: Laeliinae), with emphasis on the species of the Northern Hemisphere

AbstractEncyclia comprises over 150 species of orchids that occupy mainly seasonally dry habitats in tropical and subtropical America and are usually restricted to one or a few ecoregions. To determine whether species found in the same or neighboring ecoregions are closely related, we sampled more than 50 taxa, with an emphasis on species found in Mexico and Central America. We evaluated five DNA markers for their phylogenetic utility: the nuclear low copy gene PHYC, a plastid spacer region trnL‐F, a fragment of the plastid ycf1 gene, rpl32‐trnL, and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA; we sampled only the last three markers for the main analyses based on an assessment of phylogenetic informativeness. We analyzed plastid regions and ITS separately in phylogenetic inference using a variety of methods, as we found strong incongruence between them, according to the ILD test. We also estimated clade ages using a relaxed molecular clock with indirect calibrations based on a larger taxonomic sampling. Encyclia is a monophyletic genus that originated ca. 12.43 Ma (10.55–14.25 Ma) ago, and has undergone several recent evolutionary radiations. We found a strong association between phylogenetic relationships and the geographic distribution of species and clades in the Caribbean, Central American, and the Pacific coast of Megamexico. Several groups of Encyclia occur in the latter region, the most species‐rich being the E. meliosma complex, which is restricted to the Pacific slope of Mexico from north of the Isthmus of Tehuantepec up to Sonora.

Do mycorrhizal fungi drive speciation in Teagueia (Orchidaceae) in the upper Pastaza watershed of Ecuador?

The orchid genus Teagueia Luer (Orchidaceae, subtribe Pleurothallidinae) presents an extraordinary example of recent local evolutionary radiation. In principle, mutualisms might affect the origin of plant species via an effect on speciation. As orchids depend on mycorrhizal fungi for seed germination and early plantlet development we tested whether certain mycorrhizal fungi are acting as drivers of this radiation in Teagueia species. Sampling was carried out near Banos in east Andean Ecuador. Roots were collected from a total of 11 flowering individuals of eight morphospecies (referred to as Teagueia spp). The whole ITS1-5.8S- ITS2 nrDNA region and part of the 28S nrDNA were amplified, cloned and sequenced. Molecular phylogeny of the obtained sequences revealed four phylogenetic species of Tulasnellaceae and one of Atractiellales (Pucciniomycotina, Basidiomycota) associated with Teagueia spp. Tulasnelloid fungi were detected in all samples. Up to three different phylogenetic species of mycobionts were found associated with one Teagueia species. We found that co-occurring Teagueia species share mycobionts. All detected mycobionts had wide geographical distribution. Based on the available evidence we conclude that the extraordinary local radiation of Teagueia is most likely driven by other factors than by mycorrhizal fungi, but that mycorrhiza may be a key factor for the coexistence of so many closely related orchid species.

Gene prediction and annotation in Penstemon (Plantaginaceae): A workflow for marker development from extremely low-coverage genome sequencing.

• Premise of the study: Penstemon (Plantaginaceae) is a large and diverse genus endemic to North America. However, determining the phylogenetic relationships among its 280 species has been difficult due to its recent evolutionary radiation. The development of a large, multilocus data set can help to resolve this challenge.• Methods: Using both previously sequenced genomic libraries and our own low-coverage whole-genome shotgun sequencing libraries, we used the MAKER2 Annotation Pipeline to identify gene regions for the development of sequencing loci from six extremely low-coverage Penstemon genomes (∼0.005×–0.007×). We also compared this approach to BLAST searches, and conducted analyses to characterize sequence divergence across the species sequenced.• Results: Annotations and gene predictions were successfully added to more than 10,000 contigs for potential use in downstream primer design. Primers were then designed for chloroplast, mitochondrial, and nuclear loci from these annotated sequences. MAKER2 identified longer gene regions in all six Penstemon genomes when compared with BLASTN and BLASTX searches. The average level of sequence divergence among the six species was 7.14%.• Discussion: Combining bioinformatics tools into a workflow that produces annotations can be useful for creating potential phylogenetic markers from thousands of sequences even when genome coverage is extremely low and reference data are only available from distant relatives. Furthermore, the output from MAKER2 contains information about important gene features, such as exon boundaries, and can be easily integrated with visualization tools to facilitate the process of marker development.