An in-depth investigation of cryptic taxonomic diversity in the rare endemic mustard Draba maguirei.

PREMISE: Previously published evidence suggests that Draba maguirei, a mustard endemic to a few localities in the Bear River, Wellsville, and Wasatch Mountains of northern Utah, may represent a cryptic species complex rather than a single species. Conservation concerns prompted an in-depth systematic study of this taxon and its putative relatives. METHODS: Sampling most known populations of D. maguirei s.l. (D. maguirei var. maguirei and D. maguirei var. burkei), we integrate data from geography, ecology, morphology, cytogenetics and pollen, enzyme electrophoresis, and the phylogenetic analysis of nuclear internal transcribed spacer sequences to explore potential taxonomic diversity in the species complex. RESULTS: Draba maguirei var. burkei is shown here to be a distinct species (D. burkei) most closely related to D. globosa, rather than to D. maguirei. Within D. maguirei s.s., the northern (high elevation) and southern (low elevation) population clusters are genetically isolated and morphologically distinguishable, leading to the recognition here of the southern taxon as D. maguirei subsp. stonei. CONCLUSIONS: Our study reveals that plants traditionally assigned to D. maguirei comprise three genetically divergent lineages (D. burkei and two newly recognized subspecies of D. maguirei), each exhibiting a different chromosome number and occupying a discrete portion of the geographic range. Although previously overlooked and underappreciated taxonomically, the three taxa are morphologically recognizable based on the distribution and types of trichomes present on the leaves, stems, and fruit. Our clarification of the diversity and distribution of these taxa provides an improved framework for conservation efforts.

A Hyb-Seq phylogeny of Boechera and related genera using a combination of Angiosperms353 and Brassicaceae-specific bait sets.

PREMISE: Although Boechera (Boechereae, Brassicaceae) has become a plant model system for both ecological genomics and evolutionary biology, all previous phylogenetic studies have had limited success in resolving species relationships within the genus. The recent effective application of sequence data from target enrichment approaches to resolve the evolutionary relationships of several other challenging plant groups prompted us to investigate their usefulness in Boechera and Boechereae. METHODS: To resolve the phylogeny of Boechera and closely related genera, we utilized the Hybpiper pipeline to analyze two combined bait sets: Angiosperms353, with broad applicability across flowering plants; and a Brassicaceae-specific bait set designed for use in the mustard family. Relationships for 101 samples representing 81 currently recognized species were inferred from a total of 1114 low-copy nuclear genes using both supermatrix and species coalescence methods. RESULTS: Our analyses resulted in a well-resolved and highly supported phylogeny of the tribe Boechereae. Boechereae is divided into two major clades, one comprising all western North American species of Boechera, the other encompassing the eight other genera of the tribe. Our understanding of relationships within Boechera is enhanced by the recognition of three core clades that are further subdivided into robust regional species complexes. CONCLUSIONS: This study presents the first broadly sampled, well-resolved phylogeny for most known sexual diploid Boechera. This effort provides the foundation for a new phylogenetically informed taxonomy of Boechera that is crucial for its continued use as a model system.

Once more unto the breach, dear friends: Resolving the origins and relationships of the Pellaea wrightiana hybrid complex.

PREMISE: The taxonomic status of Wright's cliff brake fern, Pellaea wrightiana, has been in dispute ever since it was first described by Hooker in 1858. Previously published evidence suggested that this "taxon" may represent a polyploid complex rather than a single discrete species, a hypothesis tested here using a multifaceted analytical approach. METHODS: Data derived from cytogenetics, spore analyses, leaf morphometrics, enzyme electrophoresis, and phylogenetic analyses of plastid and nuclear DNA sequences are used to elucidate the origin, relationships, and taxonomic circumscription of P. wrightiana. RESULTS: Plants traditionally assigned to this taxon represent three distinct polyploids. The most widespread, P. wrightiana, is a fertile allotetraploid that arose through hybridization between two divergent diploid species, P. truncata and P. ternifolia. Sterile triploids commonly identified as P. wrightiana, were found to be backcross hybrids between this fertile tetraploid and diploid P. truncata. Relatively common across Arizona and New Mexico, they are here assigned to P. ×wagneri hyb. nov. In addition, occasional sterile tetraploid plants assigned to P. wrightiana are shown here to be hybrids between the fertile allotetraploid and the tetraploid P. ternifolia subsp. arizonica. These tetraploid hybrids originated independently in two regions of parental sympatry (southern Arizona and west Texas) and are here assigned to P. ×gooddingii hyb. nov. CONCLUSIONS: Weaving together data from a diversity of taxonomic approaches, we show that plants identified as P. wrightiana represent three morphologically distinguishable polyploids that have arisen through repeated hybridization events involving the divergent sexual taxa P. ternifolia and P. truncata.

When it only takes one to tango: assessing the impact of apomixis in the fern genus Pteris.

PREMISE: Apomixis (asexual reproduction by seed, spore, or egg) has evolved repeatedly across the tree of life. Studies of animals and angiosperms show that apomictic lineages are often evolutionarily short-lived and frequently exhibit different distributions than their sexual relatives. However, apomixis is rare in these groups. Less is known about the role of apomixis in the evolution and biogeography of ferns, in which ~10% of species are apomictic. Apomixis is especially common in the fern genus Pteris (34-39% of species); however, because of the limited taxonomic and geographic sampling of previous studies, the true frequency of apomixis and its associations with geography and phylogeny in this lineage remain unclear. METHODS: We used spore analyses of herbarium specimens to determine reproductive mode for 127 previously unsampled Pteris species. Then we leveraged biogeographic and phylogenetic analyses to estimate the global distribution and evolution of apomixis in Pteris. RESULTS: Among all Pteris species examined, we found that 21% are exclusively apomictic, 71% are exclusively sexual, and 8% have conflicting reports. Apomixis is unevenly distributed across the range of the genus, with the Paleotropics exhibiting the highest frequency, and has evolved numerous times across the Pteris phylogeny, with predominantly East Asian and South Asian clades containing the most apomictic species. CONCLUSIONS: Apomixis arises frequently in Pteris, but apomictic species do not appear to diversify. Species that encompass both apomictic and sexual populations have wider ranges than exclusively sexual or apomictic species, which suggests that sexual and apomictic ferns could occupy separate ecological niches.

A drought-driven model for the evolution of obligate apomixis in ferns: evidence from pellaeids (Pteridaceae).

PREMISE: Xeric environments impose major constraints on the fern life cycle, yet many lineages overcome these limitations by evolving apomixis. Here, we synthesize studies of apomixis in ferns and present an evidence-based model for the evolution and establishment of this reproductive strategy, focusing on genetic and environmental factors associated with its two defining traits: the production of "unreduced" spores (n = 2n) and the initiation of sporophytes from gametophyte tissue (i.e., diplospory and apogamy, respectively). METHODS: We evaluated existing literature in light of the hypothesis that abiotic characteristics of desert environments (e.g., extreme diurnal temperature fluctuations, high light intensity, and water limitation) drive the evolution of obligate apomixis. Pellaeid ferns (Cheilanthoideae: Pteridaceae) were examined in detail, as an illustrative example. We reconstructed a plastid (rbcL, trnG-trnR, atpA) phylogeny for the clade and mapped reproductive mode (sexual versus apomictic) and ploidy across the resulting tree. RESULTS: Our six-stage model for the evolution of obligate apomixis in ferns emphasizes the role played by drought and associated abiotic conditions in the establishment of this reproductive approach. Furthermore, our updated phylogeny of pellaeid ferns reveals repeated origins of obligate apomixis and shows an increase in the frequency of apomixis, and rarity of sexual reproduction, among taxa inhabiting increasingly dry North American deserts. CONCLUSIONS: Our findings reinforce aspects of other evolutionary, physiological, developmental, and omics-based studies, indicating a strong association between abiotic factors and the establishment of obligate apomixis in ferns. Water limitation, in particular, appears critical to establishment of this reproductive mode.

Infraspecific diversification of the star cloak fern (Notholaena standleyi) in the deserts of the United States and Mexico.

PREMISE: Not all ferns grow in moist and shaded habitats. One well-known example is Notholaena standleyi, a species that thrives in deserts of the southwestern United States and Mexico. This species exhibits several "chemotypes" that differ in farina (flavonoid exudates) color and chemistry. By integrating data from molecular phylogenetics, cytology, biochemistry, and biogeography, we circumscribed the major evolutionary lineages within N. standleyi and reconstructed their diversification histories. METHODS: Forty-eight samples were selected from across the geographic distribution of N. standleyi. Phylogenetic relationships were inferred using four plastid and five nuclear markers. Ploidy levels were inferred using spore sizes calibrated by chromosome counts, and farina chemistry was compared using thin-layer chromatography. RESULTS: Four clades are recognized, three of which roughly correspond to previously recognized chemotypes. The diploid clades G and Y are found in the Sonoran and Chihuahuan deserts, respectively; they are estimated to have diverged in the Pleistocene, congruent with the postulated timing of climatological events separating these two deserts. Clade P/YG is tetraploid and partially overlaps the distribution of clade Y in the eastern Chihuahuan Desert. It is apparently confined to limestone, a geologic substrate rarely occupied by members of the other clades. The cryptic (C) clade, a diploid group known only from southern Mexico and highly disjunct from the other three clades, is newly recognized here. CONCLUSIONS: Our results reveal a complex intraspecific diversification history of N. standleyi, traceable to a variety of evolutionary drivers including classic allopatry, parapatry with or without changes in geologic substrate, and sympatric divergence through polyploidization.

Low-copy nuclear sequence data confirm complex patterns of farina evolution in notholaenid ferns (Pteridaceae).

Notholaenids are an unusual group of ferns that have adapted to, and diversified within, the deserts of Mexico and the southwestern United States. With approximately 40 species, this group is noted for being desiccation-tolerant and having "farina"-powdery exudates of lipophilic flavonoid aglycones-that occur on both the gametophytic and sporophytic phases of their life cycle. The most recent circumscription of notholaenids based on plastid markers surprisingly suggests that several morphological characters, including the expression of farina, are homoplasious. In a striking case of convergence, Notholaena standleyi appears to be distantly related to core Notholaena, with several taxa not before associated with Notholaena nested between them. Such conflicts can be due to morphological homoplasy resulting from adaptive convergence or, alternatively, the plastid phylogeny itself might be misleading, diverging from the true species tree due to incomplete lineage sorting, hybridization, or other factors. In this study, we present a species phylogeny for notholaenid ferns, using four low-copy nuclear loci and concatenated data from three plastid loci. A total of 61 individuals (49 notholaenids and 12 outgroup taxa) were sampled, including 31 out of 37 recognized notholaenid species. The homeologous/allelic nuclear sequences were retrieved using PacBio sequencing and the PURC bioinformatics pipeline. Each dataset was first analyzed individually using maximum likelihood and Bayesian inference, and the species phylogeny was inferred using *BEAST. Although we observed several incongruences between the nuclear and plastid phylogenies, our principal results are broadly congruent with previous inferences based on plastid data. By mapping the presence of farina and their biochemical constitutions on our consensus phylogenetic tree, we confirmed that the characters are indeed homoplastic and have complex evolutionary histories. Hybridization among recognized species of the notholaenid clade appears to be relatively rare compared to that observed in other well-studied fern genera.

Admixture, evolution, and variation in reproductive isolation in the Boechera puberula clade.

BACKGROUND: Hybridization is very common in plants, and the incorporation of new alleles into existing lineages (i.e. admixture) can blur species boundaries. However, admixture also has the potential to increase standing genetic variation. With new sequencing methods, we can now study admixture and reproductive isolation at a much finer scale than in the past. The genus Boechera is an extraordinary example of admixture, with over 400 hybrid derivates of varying ploidy levels. Yet, few studies have assessed admixture in this genus on a genomic scale. RESULTS: In this study, we used Genotyping-by-Sequencing (GBS) to clarify the evolution of the Boechera puberula clade, whose six members are scattered across the western United States. We further assessed patterns of admixture and reproductive isolation within the group, including two additional species (B. stricta and B. retrofracta) that are widespread across North America. Based on 14,815 common genetic variants, we found evidence for some cases of hybridization. We find evidence of both recent and more ancient admixture, and that levels of admixture vary across species. CONCLUSIONS: We present evidence for a monophyletic origin of the B. puberula group, and a split of B. puberula into two subspecies. Further, when inferring reproductive isolation on the basis of presence and absence of admixture, we found that the accumulation of reproductive isolation between species does not seem to occur linearly with time since divergence in this system. We discuss our results in the context of sexuality and asexuality in Boechera.

Ecological differentiation facilitates fine-scale coexistence of sexual and asexual Boechera.

PREMISE OF THE STUDY: Ecological differentiation (ED) between sexual and asexual organisms may permit the maintenance of reproductive polymorphism. Several studies of sexual/asexual ED in plants have shown that the geographic ranges of asexuals extend beyond those of sexuals, often in areas of higher latitude or elevation. But very little is known about ED at fine scales, wherein coexistence of sexuals and asexuals may be permitted by differential niche occupation. METHODS: We used 149 populations of sexual and apomictic lineages in the genus Boechera (rock cress) collected across a portion of this mustard's vast range. We characterized reproductive mode, ploidy, and species identity or hybrid parentage of each individual, and then used a multipronged statistical approach to (1) identify ED between sexuals and asexuals; (2) investigate the impacts of two confounding factors, polyploidy and hybridization, on ED; and (3) determine the environmental variables underlying ED. KEY RESULTS: We found that sexuals and asexuals are significantly ecologically differentiated across the landscape, despite fine-scale interdigitation of these two reproductive forms. Asexual reproduction was strongly associated with greater disturbance, reduced slope, and greater environmental variability. Although ploidy had little effect on the patterns observed, hybridization has a unique impact on the relationships between asexual reproduction and specific environmental variables. CONCLUSIONS: Ecological differentiation along the axes of disturbance, slope, and climatic variability, as well as the effects of heterozygosity, may contribute to the maintenance of sexuality and asexuality across the landscape, ultimately impacting the establishment and spread of asexual lineages.

Can asexuality confer a short-term advantage? Investigating apparent biogeographic success in the apomictic triploid fern Myriopteris gracilis.

PREMISE OF THE STUDY: Although asexual taxa are generally seen as evolutionary dead ends, asexuality appears to provide a short-term benefit in some taxa, including a wider geographic distribution compared to sexual relatives. However, this may be an illusion created by multiple, morphologically cryptic, asexual lineages, each occupying a relatively small area. In this study we investigate the role of multiple lineages in the biogeography of Myriopteris gracilis Fée (Pteridaceae), a North American apomictic triploid fern species with a particularly large range. METHODS: Range-wide asexuality was assessed by counting spores/sporangium in 606 Myriopteris gracilis specimens from across the species range, and lineage structure was assessed with both plastid DNA sequence and Genotyping By Sequencing (GBS) SNP datasets. KEY RESULTS: Spore counting of >600 specimens identified no sexual populations, establishing that Myriopteris gracilis is exclusively asexual. The plastid data estimated the crown age of M. gracilis at ca. 2.5 mya and identified two lineages, each largely confined to the eastern or western portions of the range. These groups were further subdivided by the GBS data, revealing at least seven asexual lineages of varying geographic distributions, each occupying a relatively small portion of the total range of M. gracilis. CONCLUSIONS: Although maintained exclusively through asexual reproduction, the broad distribution of Myriopteris gracilis is a compilation of numerous, independently formed asexual lineages. Since no single asexual lineage occupies the full extent of the species distribution, recurrent lineage formation should be considered when evaluating the short-term benefit of asexuality in this taxon and others.

Horizontal transfer of an adaptive chimeric photoreceptor from bryophytes to ferns.

Ferns are well known for their shade-dwelling habits. Their ability to thrive under low-light conditions has been linked to the evolution of a novel chimeric photoreceptor--neochrome--that fuses red-sensing phytochrome and blue-sensing phototropin modules into a single gene, thereby optimizing phototropic responses. Despite being implicated in facilitating the diversification of modern ferns, the origin of neochrome has remained a mystery. We present evidence for neochrome in hornworts (a bryophyte lineage) and demonstrate that ferns acquired neochrome from hornworts via horizontal gene transfer (HGT). Fern neochromes are nested within hornwort neochromes in our large-scale phylogenetic reconstructions of phototropin and phytochrome gene families. Divergence date estimates further support the HGT hypothesis, with fern and hornwort neochromes diverging 179 Mya, long after the split between the two plant lineages (at least 400 Mya). By analyzing the draft genome of the hornwort Anthoceros punctatus, we also discovered a previously unidentified phototropin gene that likely represents the ancestral lineage of the neochrome phototropin module. Thus, a neochrome originating in hornworts was transferred horizontally to ferns, where it may have played a significant role in the diversification of modern ferns.

Natural hybridization between genera that diverged from each other approximately 60 million years ago.

A fern from the French Pyrenees-×Cystocarpium roskamianum-is a recently formed intergeneric hybrid between parental lineages that diverged from each other approximately 60 million years ago (mya; 95% highest posterior density: 40.2-76.2 mya). This is an extraordinarily deep hybridization event, roughly akin to an elephant hybridizing with a manatee or a human with a lemur. In the context of other reported deep hybrids, this finding suggests that populations of ferns, and other plants with abiotically mediated fertilization, may evolve reproductive incompatibilities more slowly, perhaps because they lack many of the premating isolation mechanisms that characterize most other groups of organisms. This conclusion implies that major features of Earth's biodiversity-such as the relatively small number of species of ferns compared to those of angiosperms-may be, in part, an indirect by-product of this slower "speciation clock" rather than a direct consequence of adaptive innovations by the more diverse lineages.

Evidence for reciprocal origins in Polypodium hesperium (Polypodiaceae): a fern model system for investigating how multiple origins shape allopolyploid genomes.

UNLABELLED: • PREMISE OF THE STUDY: Many polyploid species are composed of distinct lineages originating from multiple, independent polyploidization events. In the case of allopolyploids, reciprocal crosses between the same progenitor species can yield lineages with different uniparentally inherited plastid genomes. While likely common, there are few well-documented examples of such reciprocal origins. Here we examine a case of reciprocal allopolyploid origins in the fern Polypodium hesperium and present it as a natural model system for investigating the evolutionary potential of duplicated genomes.• METHODS: Using a combination of uniparentally inherited plastid and biparentally inherited nuclear sequence data, we investigated the distributions and relative ages of reciprocally formed lineages in Polypodium hesperium, an allotetraploid fern that is broadly distributed in western North America.• KEY RESULTS: The reciprocally derived plastid haplotypes of Polypodium hesperium are allopatric, with populations north and south of 42°N latitude having different plastid genomes. Incorporating biogeographic information and previously estimated ages for the diversification of its diploid progenitors, we estimate middle to late Pleistocene origins of P. hesperium.• CONCLUSIONS: Several features of Polypodium hesperium make it a particularly promising system for investigating the evolutionary consequences of allopolyploidy. These include reciprocally derived lineages with disjunct geographic distributions, recent time of origin, and extant diploid progenitors.

A total evidence approach to understanding phylogenetic relationships and ecological diversity in Selaginella subg. Tetragonostachys.

PREMISE OF THE STUDY: Several members of Selaginella are renowned for their ability to survive extreme drought and "resurrect" when conditions improve. Many of these belong to subgenus Tetragonostachys, a group of ∼45 species primarily found in North and Central America, with substantial diversity in the Sonoran and Chihuahuan Deserts. We evaluated the monophyly and the age of subgenus Tetragonostachys and assess how drought tolerance contributed to the evolution of this clade. METHODS: Our study included most Tetragonostachys species, using plastid and nuclear sequences, fossil and herbarium records, and climate variables to describe the species diversity, phylogenetic relationships, divergence times, and climatic niche evolution in the subgenus. KEY RESULTS: We found that subgenus Tetragonostachys forms a monophyletic group sister to Selaginella lepidophylla and may have diverged from other Selaginella because of a Gondwanan-Laurasian vicariance event ca. 240 mya. The North American radiation of Tetragonostachys appears to be much more recent and to have occurred during the Early Cretaceous-late Paleocene interval. We identified two significant and nested ecological niche shifts during the evolution of Tetragonostachys associated with extreme drought tolerance and a more recent shift to cold climates. Our analyses suggest that drought tolerance evolved in the warm deserts of southwest North America and may have been advantageous for colonization of cold and dry boreal climates. CONCLUSIONS: Our investigation provides a foundation for future research addressing the genomics of ecological niche evolution and the potential role of reticulate evolution in Selaginella subgenus Tetragonostachys.

Global Brassicaceae phylogeny based on filtering of 1,000-gene dataset.

The mustard family (Brassicaceae) is a scientifically and economically important family, containing the model plant Arabidopsis thaliana and numerous crop species that feed billions worldwide. Despite its relevance, most phylogenetic trees of the family are incompletely sampled and often contain poorly supported branches. Here, we present the most complete Brassicaceae genus-level family phylogenies to date (Brassicaceae Tree of Life or BrassiToL) based on nuclear (1,081 genes, 319 of the 349 genera; 57 of the 58 tribes) and plastome (60 genes, 265 genera; all tribes) data. We found cytonuclear discordance between the two, which is likely a result of rampant hybridization among closely and more distantly related lineages. To evaluate the impact of such hybridization on the nuclear phylogeny reconstruction, we performed five different gene sampling routines, which increasingly removed putatively paralog genes. Our cleaned subset of 297 genes revealed high support for the tribes, whereas support for the main lineages (supertribes) was moderate. Calibration based on the 20 most clock-like nuclear genes suggests a late Eocene to late Oligocene origin of the family. Finally, our results strongly support a recently published new family classification, dividing the family into two subfamilies (one with five supertribes), together representing 58 tribes. This includes five recently described or re-established tribes, including Arabidopsideae, a monogeneric tribe accommodating Arabidopsis without any close relatives. With a worldwide community of thousands of researchers working on Brassicaceae and its diverse members, our new genus-level family phylogeny will be an indispensable tool for studies on biodiversity and plant biology.

Identifying multiple origins of polyploid taxa: a multilocus study of the hybrid cloak fern (Astrolepis integerrima; Pteridaceae).

PREMISE OF THE STUDY: Molecular studies have shown that multiple origins of polyploid taxa are the rule rather than the exception. To understand the distribution and ecology of polyploid species and the evolutionary significance of polyploidy in general, it is important to delineate these independently derived lineages as accurately as possible. Although gene flow among polyploid lineages and backcrossing to their diploid parents often confound this process, such post origin gene flow is very infrequent in asexual polyploids. In this study, we estimate the number of independent origins of the apomictic allopolyploid fern Astrolepis integerrima, a morphologically heterogeneous species most common in the southwestern United States and Mexico, with outlying populations in the southeastern United States and the Caribbean. METHODS: Plastid DNA sequence and AFLP data were obtained from 33 A. integerrima individuals. Phylogenetic analysis of the sequence data and multidimensional clustering of the AFLP data were used to identify independently derived lineages. KEY RESULTS: Analysis of the two datasets identified 10 genetic groups within the 33 analyzed samples. These groups suggest a minimum of 10 origins of A. integerrima in the northern portion of its range, with both putative parents functioning as maternal donors, both supplying unreduced gametes, and both contributing a significant portion of their genetic diversity to the hybrids. CONCLUSIONS: Our results highlight the extreme cryptic genetic diversity and systematic complexity that can underlie a single polyploid taxon.

Unique expression of a sporophytic character on the gametophytes of notholaenid ferns (Pteridaceae).

PREMISE OF THE STUDY: Not all ferns grow in moist, shaded habitats; some lineages thrive in exposed, seasonally dry environments. Notholaenids are a clade of xeric-adapted ferns commonly characterized by the presence of a waxy exudate, called farina, on the undersides of their leaves. Although some other lineages of cheilanthoid ferns also have farinose sporophytes, previous studies suggested that notholaenids are unique in also producing farina on their gametophytes. For this reason, consistent farina expression across life cycle phases has been proposed as a potential synapomorphy for the genus Notholaena. Recent phylogenetic studies have shown two species with nonfarinose sporophytes to be nested within Notholaena, with a third nonfarinose species well supported as sister to all other notholaenids. This finding raises the question: are the gametophytes of these three species farinose like those of their close relatives, or are they glabrous, consistent with their sporophytes? METHODS: We sowed spores of a diversity of cheilanthoid ferns onto culture media to observe and document whether their gametophytes produced farina. To place these species within a phylogenetic context, we extracted genomic DNA, then amplified and sequenced three plastid loci. The aligned data were analyzed using maximum likelihood to generate a phylogenetic tree. KEY RESULTS: Here we show that notholaenids lacking sporophytic farina also lack farina in the gametophytic phase, and notholaenids with sporophytic farina always display gametophytic farina (with a single exception). Outgroup taxa never displayed gametophytic farina, regardless of whether they displayed farina on their sporophytes. CONCLUSIONS: Notholaenids are unique among ferns in consistently expressing farina across both phases of the life cycle.

Establishing the phylogenetic origin, history, and age of the narrow endemic Viola guadalupensis (Violaceae).

PREMISE OF THE STUDY: Climate change and shifts in land use are two major threats to biodiversity and are likely to disproportionately impact narrow endemics. Understanding their origins and the extent of their genetic diversity will enable land managers to better conserve these unique, highly localized gene pools. Viola guadalupensis is a narrow endemic of the Guadalupe Mountains (west Texas, USA). Its affinities within Viola section Chamaemelanium have been the subject of some debate. Furthermore, the polyploid and presumably reticulate relationships within this section remain largely unknown. METHODS: We counted chromosomes for V. guadalupensis. Phylogenies for the chloroplast trnL-F region and the low-copy nuclear gene GPI for 24 Viola taxa were generated and used to produce a polyploid phylogenetic network. Divergence dates were obtained by fossil calibration. KEY RESULTS: Meiotic chromosome counts revealed that V. guadalupensis is tetraploid (n = 12), and the presence of two GPI homoeologs further suggested allotetraploidy. Phylogenetic reconstructions showed that it originated through hybridization between unidentified members of subsection Canadenses (paternal parent) and subsection Nuttallianae (maternal parent). A fossil-calibrated relaxed clock dating analysis of GPI estimated the maximum age of V. guadalupensis to be 8.6 (5.7-11.6) Myr, suggesting the species evolved after the Guadalupe Mountains formed 12-13 Ma. CONCLUSIONS: Viola guadalupensis originated by intersubsectional hybridization followed by polyploidization. Within section Chamaemelanium, this phenomenon has occurred repeatedly in the last 9 Myr (at least for V. bakeri, V. douglasii, V. glabella, and V. sempervirens). Consequences for the systematics of the section are discussed.

The best of both worlds: Combining lineage-specific and universal bait sets in target-enrichment hybridization reactions.

PREMISE: Researchers adopting target-enrichment approaches often struggle with the decision of whether to use universal or lineage-specific probe sets. To circumvent this quandary, we investigate the efficacy of a simultaneous enrichment by combining universal probes and lineage-specific probes in a single hybridization reaction, to benefit from the qualities of both probe sets with little added cost or effort. METHODS AND RESULTS: Using 26 Brassicaceae libraries and standard enrichment protocols, we compare results from three independent data sets. A large average fraction of reads mapping to the Angiosperms353 (24-31%) and Brassicaceae (35-59%) targets resulted in a sizable reconstruction of loci for each target set (x̄ ≥ 70%). CONCLUSIONS: High levels of enrichment and locus reconstruction for the two target sets demonstrate that the sampling of genomic regions can be easily extended through the combination of probe sets in single enrichment reactions. We hope that these findings will facilitate the production of expanded data sets that answer individual research questions and simultaneously allow wider applications by the research community as a whole.

Using computer vision on herbarium specimen images to discriminate among closely related horsetails (Equisetum).

PREMISE: Equisetum is a distinctive vascular plant genus with 15 extant species worldwide. Species identification is complicated by morphological plasticity and frequent hybridization events, leading to a disproportionately high number of misidentified specimens. These may be correctly identified by applying appropriate computer vision tools. METHODS: We hypothesize that aerial stem nodes can provide enough information to distinguish among Equisetum hyemale, E. laevigatum, and E . ×ferrissii, the latter being a hybrid between the other two. An object detector was trained to find nodes on a given image and to distinguish E. hyemale nodes from those of E. laevigatum. A classifier then took statistics from the detection results and classified the given image into one of the three taxa. Both detector and classifier were trained and tested on expert manually annotated images. RESULTS: In our exploratory test set of 30 images, our detector/classifier combination identified all 10 E. laevigatum images correctly, as well as nine out of 10 E. hyemale images, and eight out of 10 E. ×ferrissii images, for a 90% classification accuracy. DISCUSSION: Our results support the notion that computer vision may help with the identification of herbarium specimens once enough manual annotations become available.