Beyond the usual climate? Factors determining flowering and fruiting phenology across a genus over 117 years.

PREMISE: Although changes in plant phenology are largely attributed to changes in climate, the roles of other factors such as genetic constraints, competition, and self-compatibility are underexplored. METHODS: We compiled >900 herbarium records spanning 117 years for all eight nominal species of the winter-annual genus Leavenworthia (Brassicaceae). We used linear regression to determine the rate of phenological change across years and phenological sensitivity to climate. Using a variance partitioning analysis, we assessed the relative influence of climatic and nonclimatic factors (self-compatibility, range overlap, latitude, and year) on Leavenworthia reproductive phenology. RESULTS: Flowering advanced by ~2.0 days and fruiting by ~1.3 days per decade. For every 1°C increase in spring temperature, flowering advanced ~2.3 days and fruiting ~3.3 days. For every 100 mm decrease in spring precipitation, each advanced ~6-7 days. The best models explained 35.4% of flowering variance and 33.9% of fruiting. Spring precipitation accounted for 51.3% of explained variance in flowering date and 44.6% in fruiting. Mean spring temperature accounted for 10.6% and 19.3%, respectively. Year accounted for 16.6% of flowering variance and 5.4% of fruiting, and latitude for 2.3% and 15.1%, respectively. Nonclimatic variables combined accounted for <11% of the variance across phenophases. CONCLUSIONS: Spring precipitation and other climate-related factors were dominant predictors of phenological variance. Our results emphasize the strong effect of precipitation on phenology, especially in the moisture-limited habitats preferred by Leavenworthia. Among the many factors that determine phenology, climate is the dominant influence, indicating that the effects of climate change on phenology are expected to increase.

Goldenrod herbariomics: Hybrid-sequence capture reveals the phylogeny of Solidago.

PREMISE: The phylogenetic relationships among the ca. 138 species of goldenrods (Solidago; Asteraceae) have been difficult to infer due to species richness, and shallow interspecific genetic divergences. This study aims to overcome these obstacles by combining extensive sampling of goldenrod herbarium specimens with the use of a custom Solidago hybrid-sequence capture probe set. METHODS: A set of tissues from herbarium samples comprising ca. 90% of Solidago species was assembled and DNA was extracted. A custom hybrid-sequence capture probe set was designed, and data from 854 nuclear regions were obtained and analyzed from 209 specimens. Maximum likelihood and coalescent approaches were used to estimate the genus phylogeny for 157 diploid samples. RESULTS: Although DNAs from older specimens were both more fragmented and produced fewer sequencing reads, there was no relationship between specimen age and our ability to obtain sufficient data at the target loci. The Solidago phylogeny was generally well-supported, with 88/155 (57%) nodes receiving ≥95% bootstrap support. Solidago was supported as monophyletic, with Chrysoma pauciflosculosa identified as sister. A clade comprising Solidago ericameriodes, Solidago odora, and Solidago chapmanii was identified as the earliest diverging Solidago lineage. The previously segregated genera Brintonia and Oligoneuron were identified as placed well within Solidago. These and other phylogenetic results were used to establish four subgenera and fifteen sections within the genus. CONCLUSIONS: The combination of expansive herbarium sampling and hybrid-sequence capture data allowed us to quickly and rigorously establish the evolutionary relationships within this difficult, species-rich group.

Two cytotype niche shifts are of different magnitude in Solidago gigantea.

PREMISE: Polyploidy may serve to contribute to range size if autopolyploid cytotypes are adapted to differing ecological conditions. This study aims to establish the geographic distribution of cytotypes within the giant goldenrod (Solidago gigantea), and to assess whether cytotypes exhibit differing ecological tolerances and morphology. METHODS: A range-wide set of 629 Solidago gigantea individuals was obtained through field collecting, sampling from herbarium specimens, and incorporating existing chromosome counts. Cytotype of each unknown sample was estimated by observing allele numbers at twelve microsatellite loci, a strategy that was assessed by comparing estimated to known cytotype in 20 chromosome-counted samples. Abiotic ecological differentiation was assessed for two transitions: diploid-tetraploid and tetraploid-hexaploid. Morphological differentiation among cytotypes was assessed. RESULTS: Microsatellite repeat variation accurately estimated cytotype in 85% of samples for which ploidy was known. Applying this approach to samples of unknown ploidy established that the three cytotypes are non-randomly distributed. Although niche modeling and MANOVA approaches identified significant differences in macro-climatic conditions for both cytotype transitions, the tetraploid to hexaploid transition was more substantial. Leaf length and width did not differ among cytotypes. Although leaf vestiture exhibited strong trends, no absolute differences were observed among cytotypes. CONCLUSIONS: With the largest such study to date, we established niche transitions among giant goldenrod cytotypes of differing magnitudes. Collectively, this suggests that whole-genome duplication has contributed to Solidago gigantea's large range.

Are buffalograss (Buchloë dactyloides) cytotypes spatially and ecologically differentiated?

PREMISE: Although autopolyploidy is common among dominant Great Plains grasses, the distribution of cytotypes within a given species is typically poorly understood. This study aims to establish the geographic distribution of cytotypes within buffalograss (Buchloë dactyloides) and to assess whether individual cytotypes have differing ecological tolerances. METHODS: A range-wide set of 578 B. dactyloides individuals was obtained through field collecting and sampling from herbarium specimens. The cytotype of each sample was estimated by determining allele numbers at 13 simple sequence repeat loci, a strategy that was assessed by comparing estimated to known cytotype in 79 chromosome-counted samples. Ecological differentiation between the dominant tetraploid and hexaploid cytotypes was assessed with analyses of macroclimatic variables. RESULTS: Simple sequence repeat variation accurately estimated cytotype in 89% of samples from which a chromosome count had been obtained. Applying this approach to samples of unknown ploidy established that diploids and pentaploids are rare, with the common tetraploid and hexaploid cytotypes generally occurring in sites to the north/west (tetraploid) or south/east (hexaploid) portions of the species range. Both MANOVA and niche modeling approaches identified significant but subtle differences in macroclimatic conditions at the set of locations occupied by these two dominant cytotypes. CONCLUSIONS: Incorporating chromosome count vouchers and cytotype-estimated herbarium records allowed us to perform the largest study of cytotype niche differentiation to date. Buffalograss cytotypes differ greatly in frequency, the common tetraploid and hexaploid cytotypes are non-randomly distributed, and these two cytotypes are subtly ecologically differentiated.

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.

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.

A step-by-step protocol for meiotic chromosome counts in flowering plants: A powerful and economical technique revisited.

PREMISE: Counting chromosomes is a fundamental botanical technique, yet it is often intimidating and increasingly sidestepped. Once mastered, the basic protocol can be applied to a broad range of taxa and research questions. It also reveals an aspect of the plant genome that is accessible with only the most basic of resources-access to a microscope with 1000× magnification is the most limiting factor. METHODS AND RESULTS: Here we provide a detailed protocol for choosing, staining, and squashing angiosperm pollen mother cells. The protocol is supplemented by figures and two demonstration videos. CONCLUSIONS: The protocol we provide will hopefully demystify and reinvigorate a powerful and once commonplace botanical technique that is available to researchers regardless of their location and resources.

A basic ddRADseq two-enzyme protocol performs well with herbarium and silica-dried tissues across four genera.

PREMISE: The ability to sequence genome-scale data from herbarium specimens would allow for the economical development of data sets with broad taxonomic and geographic sampling that would otherwise not be possible. Here, we evaluate the utility of a basic double-digest restriction site-associated DNA sequencing (ddRADseq) protocol using DNAs from four genera extracted from both silica-dried and herbarium tissue. METHODS: DNAs from Draba, Boechera, Solidago, and Ilex were processed with a ddRADseq protocol. The effects of DNA degradation, taxon, and specimen age were assessed. RESULTS: Although taxon, preservation method, and specimen age affected data recovery, large phylogenetically informative data sets were obtained from the majority of samples. DISCUSSION: These results suggest that herbarium samples can be incorporated into ddRADseq project designs, and that specimen age can be used as a rapid on-site guide for sample choice. The detailed protocol we provide will allow users to pursue herbarium-based ddRADseq projects that minimize the expenses associated with fieldwork and sample evaluation.

Native range genetic variation in Arabidopsis thaliana is strongly geographically structured and reflects Pleistocene glacial dynamics.

Despite Arabidopsis thaliana's pre-eminence as a model organism, major questions remain regarding the geographic structure of its genetic variation due to the geographically incomplete sample set available for previous studies. Many of these questions are addressed here with an analysis of genome-wide variation at 10 loci in 475 individuals from 167 globally distributed populations, including many from critical but previously un-sampled regions. Rooted haplotype networks at three loci suggest that A. thaliana arose in the Caucasus region. Identification of large-scale metapopulations indicates clear east-west genetic structure, both within proposed Pleistocene refugia and post-Pleistocene colonized regions. The refugia themselves are genetically differentiated from one another and display elevated levels of within-population genetic diversity relative to recolonized areas. The timing of an inferred demographic expansion coincides with the Eemian interglacial (approximately 120,000 years ago). Taken together, these patterns are strongly suggestive of Pleistocene range dynamics. Spatial autocorrelation analyses indicate that isolation by distance is pervasive at all hierarchical levels, but that it is reduced in portions of Europe.

Deciphering the sexual diploid members of the Boechera suffrutescens complex (Brassicaceae, Boechereae).

Boechera is a model genus that is of particular interest for understanding apomixis due to the presence of numerous apomictic diploid lineages that are tightly correlated with hybridisation events. Boechera includes many narrowly distributed endemics and apomictic hybrid lineages that obscure morphological boundaries amongst taxa. In this study, we focus on the Boechera suffrutescens complex, a phylogenetically well-supported but taxonomically complex north-western United States clade whose diploid species currently include the widespread B. suffrutescens and two narrowly distributed serpentine endemics, B. constancei and B. rollei. Using a 15-locus microsatellite dataset, we infer ploidy and sexual vs. apomictic reproduction for all individuals and then assess species limits for all sexual diploid samples. Our results support the recognition of B. rollei and B. constancei as distinct species and reveal three divergent sexual diploid lineages within B. suffrutescens sensu lato. The latter three lineages exhibit geographic, genetic and morphological coherence and consequently warrant recognition at the species rank. These include Boechera suffrutescens s.s., which is restricted to Idaho and eastern Oregon, Boechera botulifructa, a newly described species distributed along the Cascade Mountain Province from Lassen County, California north to Deschutes County, Oregon and the heretofore dismissed species Boechera duriuscula (basionym ≡ Arabis duriuscula), which occurs along the Sierra Nevada Province from Plumas County southwards to Fresno County, California. Our data also reveal substructure in B. constancei that is likely attributable to the highly fragmented distribution of its serpentine habitat. This refined taxonomic framework for the B. suffrutescens complex enhances Boechera as a model system, adds to our knowledge of speciation in edaphically extreme environments and provides information on ongoing conservation efforts for these taxa.

Boechera microsatellite website: an online portal for species identification and determination of hybrid parentage.

Boechera (Brassicaceae) has many features to recommend it as a model genus for ecological and evolutionary research, including species richness, ecological diversity, experimental tractability and close phylogenetic proximity to Arabidopsis . However, efforts to realize the full potential of this model system have been thwarted by the frequent inability of researchers to identify their samples and place them in a broader evolutionary context. Here we present the Boechera Microsatellite Website (BMW), a portal that archives over 55 000 microsatellite allele calls from 4471 specimens (including 133 nomenclatural types). The portal includes analytical tools that utilize data from 15 microsatellite loci as a highly effective DNA barcoding system. The BMW facilitates the accurate identification of Boechera samples and the investigation of reticulate evolution among the ±83 sexual diploid taxa in the genus, thereby greatly enhancing Boechera 's potential as a model system. Database URL: http://sites.biology.duke.edu/windhamlab/.

Development of microsatellite markers for buffalograss (Buchloë dactyloides; Poaceae), a drought-tolerant turfgrass alternative.

PREMISE OF THE STUDY: Buchloë dactyloides (Poaceae) is an important component of Great Plains prairies and a popular drought-tolerant turfgrass alternative in North America. This species comprises an autopolyploid series, and microsatellite primers were developed to understand the distribution of genetic variation among cytotypes and across its large geographic range. METHODS AND RESULTS: Fifteen microsatellite loci were designed and successfully amplified in six B. dactyloides populations. Within-population genetic diversity was comparatively high, consistent with B. dactyloides' life history. Allelic variation at 13 loci was consistent with the cytotype established in chromosome-counted samples. CONCLUSIONS: This variable, interpretable set of loci allows for the determination of multilocus genotype in B. dactyloides individuals of varying cytotype. Data such as these from a range-wide sample set can provide important insights for germplasm conservation and crop improvement in this ecologically and economically important species.

Next-generation sampling: Pairing genomics with herbarium specimens provides species-level signal in Solidago (Asteraceae).

PREMISE OF THE STUDY: The ability to conduct species delimitation and phylogeny reconstruction with genomic data sets obtained exclusively from herbarium specimens would rapidly enhance our knowledge of large, taxonomically contentious plant genera. In this study, the utility of genotyping by sequencing is assessed in the notoriously difficult genus Solidago (Asteraceae) by attempting to obtain an informative single-nucleotide polymorphism data set from a set of specimens collected between 1970 and 2010. METHODS: Reduced representation libraries were prepared and Illumina-sequenced from 95 Solidago herbarium specimen DNAs, and resulting reads were processed with the nonreference Universal Network-Enabled Analysis Kit (UNEAK) pipeline. Multidimensional clustering was used to assess the correspondence between genetic groups and morphologically defined species. RESULTS: Library construction and sequencing were successful in 93 of 95 samples. The UNEAK pipeline identified 8470 single-nucleotide polymorphisms, and a filtered data set was analyzed for each of three Solidago subsections. Although results varied, clustering identified genomic groups that often corresponded to currently recognized species or groups of closely related species. DISCUSSION: These results suggest that genotyping by sequencing is broadly applicable to DNAs obtained from herbarium specimens. The data obtained and their biological signal suggest that pairing genomics with large-scale herbarium sampling is a promising strategy in species-rich plant groups.

Genus-wide microsatellite primers for the goldenrods (Solidago; Asteraceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for studies of polyploid evolution, ecological genetics, conservation genetics, and species delimitation in the genus Solidago. • METHODS AND RESULTS: Illumina sequencing of a shotgun library from S. gigantea identified ca. 1900 putative single-copy loci. Fourteen loci were subsequently shown to be amplifiable, single-copy, and variable in a broad range of Solidago species. • CONCLUSIONS: The utility of these markers both across the genus and in herbarium specimens of a wide age range will facilitate numerous inter- and intraspecific studies in the ca. 120 Solidago species.

Does hybridization drive the transition to asexuality in diploid Boechera?

Gametophytic apomixis is a common form of asexual reproduction in plants. Virtually all gametophytic apomicts are polyploids, and some view polyploidy as a prerequisite for the transition to apomixis. However, any causal link between apomixis and polyploidy is complicated by the fact that most apomictic polyploids are allopolyploids, leading some to speculate that hybridization, rather than polyploidy, enables apomixis. Diploid apomixis presents a rare opportunity to isolate the role of hybridization, and a number of diploid apomicts have been documented in the genus Boechera (Brassicaceae). Here, we present the results of a microsatellite study of 1393 morphologically and geographically diverse diploid individuals, evaluating the hypothesis that diploid Boechera apomicts are hybrids. This genus-wide dataset was made possible by the applicability of a core set of microsatellite loci in 69 of the 70 diploid Boechera species and by our ability to successfully genotype herbarium specimens of widely varying ages. With few exceptions, diploid apomicts exhibited markedly high levels of heterozygosity resulting from the combination of disparate genomes. This strongly suggests that most apomictic diploid Boechera lineages are of hybrid origin, and that the genomic consequences of hybridization allow for the transition to gametophytic apomixis in this genus.

Do asexual polyploid lineages lead short evolutionary lives? A case study from the fern genus Astrolepis.

A life-history transition to asexuality is typically viewed as leading to a heightened extinction risk, and a number of studies have evaluated this claim by examining the relative ages of asexual versus closely related sexual lineages. Surprisingly, a rigorous assessment of the age of an asexual plant lineage has never been published, although asexuality is extraordinarily common among plants. Here, we estimate the ages of sexual diploids and asexual polyploids in the fern genus Astrolepis using a well-supported plastid phylogeny and a relaxed-clock dating approach. The 50 asexual polyploid samples we included were conservatively estimated to comprise 19 distinct lineages, including a variety of auto- and allopolyploid genomic combinations. All were either the same age or younger than the crown group comprising their maternal sexual-diploid parents based simply on their phylogenetic position. Node ages estimated with the relaxed-clock approach indicated that the average maximum age of asexual lineages was 0.4 My, and individual lineages were on average 7 to 47 times younger than the crown- and total-ages of their sexual parents. Although the confounding association between asexuality and polyploidy precludes definite conclusions regarding the effect of asexuality, our results suggest that asexuality limits evolutionary potential in Astrolepis.

Further insights into the phylogeny of Arabidopsis (Brassicaceae) from nuclear Atmyb2 flanking sequence.

Arabidopsis thaliana is the preeminent plant model organism. However, significant advances in evolution and ecology are being made by expanding the scope of research beyond this single species into the broader genus Arabidopsis. Surprisingly, few studies have rigorously investigated phylogenetic relationships between the nine Arabidopsis species, and this study evaluates both these and hypotheses related to two instances of intra-generic hybridization. DNA sequences from the 5' flanking region of the nuclear Atmyb2 gene from 12 of the 14 Arabidopsis taxa were used to reconstruct the generic phylogeny. The strict consensus tree was highly concordant with previous studies, identifying lineages corresponding to widespread species but exhibiting a large basal polytomy. Our data indicates that the paternal parent of the allopolyploid A. suecica is A. neglecta rather than A. arenosa s.l., although the need for a detailed phylogeographical study of these three species is noted. Finally, our data provided additional phylogenetic evidence of hybridization between Arabidopsis lyrata s.l. and A. halleri s.l. Taken together, the well-defined lineages within the genus and the potential for hybridization between them highlight Arabidopsis as a promising group for comparative and experimental studies of hybridization.