Back to the future: Using herbarium specimens to isolate nodule-associated bacteria.

Herbarium specimens are increasingly being used as sources of information to understand the ecology and evolution of plants and their associated microbes. Most studies have used specimens as a source of genetic material using culture-independent approaches. We demonstrate that herbarium specimens can also be used to culture nodule-associated bacteria, opening the possibility of using specimens to understand plant-microbe interactions at new spatiotemporal scales. We used historic and contemporary nodules of a common legume, Medicago lupulina, to create a culture collection. We were able to recover historic bacteria in 15 genera from three specimens (collected in 1950, 2004, and 2015). This work is the first of its kind to isolate historic bacteria from herbarium specimens. Future work should include inoculating plants with historic strains to see if they produce nodules and if they affect plant phenotype and fitness. Although we were unable to recover any Ensifer, the main symbiont of Medicago lupulina, we recovered some other potential nodulating species, as well as many putative growth-promoting bacteria.

Dataset about Mexico's forest diversity: Site locations of tree species, wood densities, and geographic database of forest-vegetation provinces.

A dataset about three topics is provided, as a follow-up to the article "Mexico's forest diversity: common tree species and proposed forest-vegetation provinces" by Ricker et al. [1]. Firstly, 6927 site locations are provided for 22,532 trees of 1452 species. Secondly, measurements of basic wood-densities are reported for 779 tree species, obtained from 5256 trunk-core samples from Mexico's national forest inventory, and ranging from 0.05 to 0.93 g/cm3. Third, the data and maps of the forest-vegetation provinces from [1] were updated with the new cartography of Mexico's vegetation and land use (base year 2018). The maps are available now in an adjusted presentation as a shapefile-set for ArcGIS, as well as map-package and image files.

Resurrected seeds from herbarium specimens reveal rapid evolution of drought resistance in a selfing annual.

PREMISE: Increased aridity and drought associated with climate change are exerting unprecedented selection pressures on plant populations. Whether populations can rapidly adapt, and which life history traits might confer increased fitness under drought, remain outstanding questions. METHODS: We utilized a resurrection ecology approach, leveraging dormant seeds from herbarium collections to assess whether populations of Plantago patagonica from the semi-arid Colorado Plateau have rapidly evolved in response to approximately ten years of intense drought in the region. We quantified multiple traits associated with drought escape and drought resistance and assessed the survival of ancestors and descendants under simulated drought. RESULTS: Descendant populations displayed a significant shift in resource allocation, in which they invested less in reproductive tissues and relatively more in both above- and below-ground vegetative tissues. Plants with greater leaf biomass survived longer under terminal drought; moreover, even after accounting for the effect of increased leaf biomass, descendant seedlings survived drought longer than their ancestors. CONCLUSIONS: Our results document rapid adaptive evolution in response to climate change in a selfing annual and suggest that shifts in tissue allocation strategies may underlie adaptive responses to drought in arid or semi-arid environments. This work also illustrates a novel approach, documenting that under specific circumstances, seeds from herbarium specimens may provide an untapped source of dormant propagules for future resurrection experiments.

Phenotypic divergence of traits that mediate antagonistic and mutualistic interactions between island and continental populations of the tropical plant, Tribulus cistoides (Zygophyllaceae).

Island systems have long served as a model for evolutionary processes due to their unique species interactions. Many studies of the evolution of species interactions on islands have focused on endemic taxa. Fewer studies have focused on how antagonistic and mutualistic interactions shape the phenotypic divergence of widespread nonendemic species living on islands. We used the widespread plant Tribulus cistoides (Zygophyllaceae) to study phenotypic divergence in traits that mediate antagonistic interactions with vertebrate granivores (birds) and mutualistic interactions with pollinators, including how this is explained by bioclimatic variables. We used both herbarium specimens and field-collected samples to compare phenotypic divergence between continental and island populations. Fruits from island populations were larger than on continents, but the presence of lower spines on mericarps was less frequent on islands. The presence of spines was largely explained by environmental variation among islands. Petal length was on average 9% smaller on island than continental populations, an effect that was especially accentuated on the Galápagos Islands. Our results show that Tribulus cistoides exhibits phenotypic divergence between island and continental habitats for antagonistic traits (seed defense) and mutualistic traits (floral traits). Furthermore, the evolution of phenotypic traits that mediate antagonistic and mutualistic interactions partially depended on the abiotic characteristics of specific islands. This study shows the potential of using a combination of herbarium and field samples for comparative studies on a globally distributed species to study phenotypic divergence on island habitats.

Improving species delimitation for effective conservation: a case study in the endemic maple-leaf oak (Quercus acerifolia).

Species delimitation is challenging in lineages that exhibit both high plasticity and introgression. This challenge can be compounded by collection biases, which may downweight specimens morphologically intermediate between traditional species. Additionally, mismatch between named species and observable phenotypes can compromise species conservation. We studied the species boundaries of Quercus acerifolia, a tree endemic to Arkansas, U.S. We performed morphometric analyses of leaves and acorns from 527 field and 138 herbarium samples of Q. acerifolia and its close relatives, Q. shumardii and Q. rubra. We employed two novel approaches: sampling ex situ collections to detect phenotypic plasticity caused by environmental variation and comparing random field samples with historical herbarium samples to identify collection biases that might undermine species delimitation. To provide genetic evidence, we also performed molecular analyses on genome-wide SNPs. Quercus acerifolia shows distinctive morphological, ecological, and genomic characteristics, rejecting the hypothesis that Q. acerifolia is a phenotypic variant of Q. shumardii. We found mismatches between traditional taxonomy and phenotypic clusters. We detected underrepresentation of morphological intermediates in herbarium collections, which may bias species discovery and recognition. Rare species conservation requires considering and addressing taxonomic problems related to phenotypic plasticity, mismatch between taxonomy and morphological clusters, and collection biases.

A taxonomic dataset of preserved specimen occurrences of Theobroma and Herrania (Malvaceae, Byttnerioideae) stored in 2020.

Background: Species from the "cacao group" are traditionally allocated into two genera, Theobroma and Herrania (Malvaceae, Byttnerioideae), both groups of Neotropical species economically relevant, such as the cacao tree (Theobromacacao), which forms the source of chocolate. This study aimed at compiling and describing a dataset of preserved specimen collections available in the Global Biodiversity Information Facility repository (GBIF) for Tropical Americas. Data were exhaustively revisited and analysed in terms of taxonomic identity, conditions of collection and georeferencing, all of which should enable downstream taxonomic, geographic and evolutionary analyses. New information: Our dataset compiles 7975 records of preserved specimen collections found at herbaria. Records are from 18 species of Theobroma and 14 of Herrania, occurring in 60 countries or major territories, with two species endemic to a single country (H.kofanorum from Ecuador and H.laciniifolium from Colombia). Occurrence records are mostly restricted to the Amazon rainforest and species with more occurrence records are cupuí, T.subincanum (1535 records), followed by the cacao tree, T.cacao (1500 records), the latter having cultivated specimens in Africa, Asia and Oceania. In the case of the genus Herrania, H.nitida and H.purpurea are the species with the majority of occurrences (respectively, 431 and 273 records). Most of the botanical samples from these genera are found in American, Brazilian and Colombian collections, with a particular strength for American herbaria. We describe how occurrence records are spread spatially and temporally and highlight key field expeditions responsible for enhancing most of the knowledge of cacao and its wild relatives, especially in countries where they prevail, such as Colombia (with 29 species), Ecuador (23 species), Brazil (18 species) and Peru (15 species). Specifically, expeditions in these countries were led by American and European initiatives in conjunction with local funding in the mid-20th century. We emphasise how initiatives of such kind seems to have weakened in the 21st century and most of the collections of Theobroma and Herrania made afterwards are from various collectors that seek to resample specimens in already explored sites.

Vascular Plants from the Journey through Portugal (1797-1801) by Hoffmannsegg and Link at the Herbarium of the Real Jardín Botánico of Madrid.

During the journey through Portugal by Hoffmannsegg and Link (1797-1801), these authors collected an appreciable number of specimens, most of which have been lost. Their collections are relevant since they were used by themselves or by other authors to describe numerous species. In the herbarium of the Real Jardín Botánico of Madrid, 70 specimens from this journey have been located. In the archive of this institution the letters that Hoffmannsegg and Link sent to Cavanilles accompanying these plants have also been located. The analysis of these letters, the herbarium labels and of the protologues has permitted to establish that 15 specimens are original material, four of which had already been proposed as lectotypes by other authors (Airochloa caudata Link, Silene fuscata Link ex Brot., Silene micrantha Link ex Otth and Silene pernoctans Link). The designation of a neotype for Stipa gigantea Link should be superseded, because an original material has been found. Thus, a lectotype for this taxon is proposed.

Ferns at the digital herbarium of the Central Siberian Botanical Garden SB RAS.

BACKGROUND: According to the data in Index Herbariorum as of 1 December 2020, there are 3426 active herbaria in the world, containing 396,204,891 specimens and 124 herbaria in Russia with more than 16,175,000 specimens. The Central Siberian Botanical Garden of the Siberian Branch of the Russian Academy of Sciences (CSBG SB RAS, Novosibirsk), founded in 1946, historically has two herbarium collections (NS and NSK). Currently these collections contain about 800,000 herbarium specimens comprising vascular plants, mosses, lichens and fungi gathered from all over the world. Digitisation of the NSK type specimens of vascular plants began in 2014 by using the special scanner Herbscan. In 2018, we started digitisation of the NS and NSK collections by using ObjectScan 1600.Pteridophytes (ferns, lycophytes and their extinct free-sporing relatives) are a diverse group of plants that today comprises approximately 12,900 species and plays a major role in terrestrial ecosystems. All herbarium specimens of ferns, collected over 170 years between 1851 and 2021 and stored in the NS and NSK collections, were digitised in 2021, placed at the CSBG SB RAS digital Herbarium (http://herb.csbg.nsc.ru:8081) and published through GBIF. Twenty families of Polypodiopsida, but not Equisetaceae, were included in this dataset. Family Ophioglossaceae was digitised and published in GBIF as a separate dataset. NEW INFORMATION: By August 2021, more than 62,600 specimens with good quality images and fully-captured label transcriptions had been placed at CSBG SB RAS Digital Herbarium. A total of 7,758 records of fern occurrences of 363 taxa in the world with 92% geolocations including 5100 records from Russia with 98.7% geolocations that are new for GBIF.org in 2021 were entered. In the dataset specimens from 43 countries of Europe, Asia, America, Africa and Australia (Oceania), 89% of them from Russia, are presented.

Allopatry, hybridization, and reproductive isolation in Arctostaphylos.

PREMISE: Hybridization plays a key role in introgressive adaptation, speciation, and adaptive radiation as a source of evolutionary innovation. Hybridization is considered common in Arctostaphylos, yet species boundaries are retained in stands containing multiple species. Arctostaphylos contains diploids and tetraploids, and recent phylogenies indicate two clades; we hypothesize combinations of these traits limit or promote hybridization rates. METHODS: We statistically analyzed co-occurrence patterns of species by clade membership and ploidy level from 87 random 0.1 ha plots. We sampled multiple sites to analyze for percent hybridization based on morphology. Finally, phenophases were analyzed by scoring herbarium sheets for a large number of taxa from both clades as well as tetraploids, and second, surveying three field sites over two years for divergence in phenological stages between co-occurring taxa. RESULTS: Most taxa in Arctostaphylos are allopatric relative to other congenerics. When two taxa co-occur, the patterns are a diploid with a tetraploid, or two diploids from different clades. When three taxa co-occur, the pattern is two diploids from different clades and a tetraploid. Field and herbarium data both indicate flowering phenology is displaced between diploids from the two clades; one of the diploid clades and tetraploids overlap considerably. CONCLUSIONS: The two deep clades in Arctostaphylos are genetically distant, with hybrids rare or non-existent when taxa co-occur. Reproductive isolation between clades is enhanced by displaced flowering phenology for co-occurring species. Within clades, taxa appear to have few reproductive barriers other than an allopatric distribution or different ploidy levels.

Styleworts under the microscope: a taxonomic account of Levenhookia (Stylidiaceae).

A taxonomic revision of the Australian endemic genus Levenhookia R.Br. (Stylidiaceae) recognises 12 species, of which L. aestiva Wege, sp. nov. from south-western Australia is newly described. Levenhookia preissii (Sond.) F.Muell. is lectotypified and recircumscribed as a Swan Coastal Plain endemic, resulting in its addition to the Threatened and Priority Flora List for Western Australia. Lectotypes are also selected for L. dubia Sond., L. leptantha Benth., L. sonderi (F.Muell.) F.Muell. and L. stipitata (Benth.) F.Muell. ex Benth. Verification of herbarium records has expanded the known distribution of L. murfetii Lowrie & Conran and L. pulcherrima Carlquist and has confirmed the widespread distribution of L. dubia across southern Australia including Tasmania, where it is currently listed as extinct-surveys based on information gleaned from historical collections may lead to its rediscovery in this State. Descriptions, distribution maps and photographs for all species are provided along with a key to species.

Metagenomic analysis of historical herbarium specimens reveals a postmortem microbial community.

Advances in DNA extraction and next-generation sequencing have made a vast number of historical herbarium specimens available for genomic investigation. These specimens contain not only genomic information from the individual plants themselves, but also from associated microorganisms such as bacteria and fungi. These microorganisms may have colonized the living plant (e.g., pathogens or host-associated commensal taxa) or may result from postmortem colonization that may include decomposition processes or contamination during sample handling. Here we characterize the metagenomic profile from shotgun sequencing data from herbarium specimens of two widespread plant species (Ambrosia artemisiifolia and Arabidopsis thaliana) collected up to 180 years ago. We used blast searching in combination with megan and were able to infer the metagenomic community even from the oldest herbarium sample. Through comparison with contemporary plant collections, we identify three microbial species that are nearly exclusive to herbarium specimens, including the fungus Alternaria alternata, which can comprise up to 7% of the total sequencing reads. This species probably colonizes the herbarium specimens during preparation for mounting or during storage. By removing the probable contaminating taxa, we observe a temporal shift in the metagenomic composition of the invasive weed Am. artemisiifolia. Our findings demonstrate that it is generally possible to use herbarium specimens for metagenomic analyses, but that the results should be treated with caution, as some of the identified species may be herbarium contaminants rather than representing the natural metagenomic community of the host plant.

Changes in Foliar Functional Traits of S. pyrenaicus subsp. carpetanus under the Ongoing Climate Change: A Retrospective Survey.

The sensitivity of stomatal behavior and patterning (i.e., distribution, density, size) to environmental stimuli, renders them crucial for defining the physiological performance of leaves. Thus, assessing long-term modifications in stomatal traits in conserved specimens arises as a valuable eco-physiological approach to predict how the rising trend of warmer, drier summers could affect plant fitness; particularly in mountain areas already experiencing climate aggravation and lacking the related monitoring schemes like Mediterranean high-mountains. Variations in foliar and stomatal traits were studied in conserved specimens of Senecio pyrenaicus subsp. carpetanus from Sierra de Guadarrama over the past 71 years. Our findings revealed decreasing trends in leaf width, stomatal size, and increasing tendency in stomatal density, all correlated with the recent 30-year climate exacerbation in these mountains. This evidenced a positive selection favoring traits that allow safeguarding plant performance under drier, hotter weather conditions. The significant relation between stomatal traits and climatic variables upholds the role of stomatal patterning in sensing environmental cues in this species, feasibly optimizing physiological responses involved in the growth-water loss trade-off. The transition to smaller, densely packed stomata observed in recent decades could indicate local-adaptive plasticity in this species, enhancing stomatal response, as coarser environmental conditions take place in Sierra de Guadarrama.

Ancient Plant Genomics in Archaeology, Herbaria, and the Environment.

The ancient DNA revolution of the past 35 years has driven an explosion in the breadth, nuance, and diversity of questions that are approachable using ancient biomolecules, and plant research has been a constant, indispensable facet of these developments. Using archaeological, paleontological, and herbarium plant tissues, researchers have probed plant domestication and dispersal, plant evolution and ecology, paleoenvironmental composition and dynamics, and other topics across related disciplines. Here, we review the development of the ancient DNA discipline and the role of plant research in its progress and refinement. We summarize our understanding of long-term plant DNA preservation and the characteristics of degraded DNA. In addition, we discuss challenges in ancient DNA recovery and analysis and the laboratory and bioinformatic strategies used to mitigate them. Finally, we review recent applications of ancient plant genomic research.

New protocol for successful isolation and amplification of DNA from exiguous fractions of specimens: a tool to overcome the basic obstacle in molecular analyses of myxomycetes.

Herbarium collections provide an essential basis for a wide array of biological research and, with development of DNA-based methods, they have become an invaluable material for genetic analyses. Yet, the use of such material is hindered by technical limitations related to DNA degradation and to quantity of biological material. The latter is inherent for some biological groups, as best exemplified by myxomycetes which form minute sporophores. It is estimated that ca. two-thirds of myxomycete taxa are represented by extremely scanty material. As DNA isolation methods applied so far in myxomycete studies require destructive sampling of many sporophores, a large part of described diversity of the group remains unavailable for phylogenetic studies or barcoding. Here, we tested several procedures of DNA isolation and amplification to seek for an efficient and possibly non-destructive method of sampling. Tests were based on herbarium specimens of 19 species representing different taxonomic orders. We assayed several variants of isolation based on silica gel membrane columns, and a newly designed procedure using highly reduced amount of biological material (small portion of spores), based on fine disruption of spores and direct PCR. While the most frequently used column-based method led to PCR success in 89.5% of samples when a large amount of material was used, its performance dropped to 52% when based on single sporophores. Single sporophores provided amplicons in 89.5% of samples when using a kit dedicated to low-amount DNA samples. Our new procedure appeared the most effective (94.7%) while it used only a small fraction of spores, being nearly non-destructive; it was also the most cost-effective. We thus demonstrate that combination of adequate handling of spore micro-disruption coupled with application of direct PCR can be an efficient way to circumvent technical limitations for genetic studies in myxomycetes and thus can substantially improve taxon sampling for phylogeny and barcoding. Additionally, this approach gives a unique possibility to apply both molecular and morphological assays to the same structure (sporophore), which then can be further stored as documentation.

Isoetes dubsii and Isoetes santacruzensis, two new species from lowland areas in South America.

Isoetes dubsii sp. nov. and I. santacruzensis sp. nov., two new species from lowland areas in South America, are described, illustrated and compared to similar species. Isoetes dubsii can be distinguished from other species of the Brazilian Pantanal wetlands by a set of characters including leaves that are long, flexuous and trigonal in transverse section, tri-lobate stems, rudimentary velum, pustulate megaspores of 310‒390 µm diameter and laesurae of the megaspore at least four times wider than high. Isoetes santacruzensis has flexuous, filiform leaves, 0.4-0.8 mm wide at mid length and reaching up to 15 cm long, black or reddish-black sporangia, sclerified phyllopodia and sparsely verrucate megaspores of 320‒390 µm in diameter. We also include a key for species from the Brazilian Pantanal wetlands and Bolivia and spore images for all species that are discussed. Isoetes dubsii and I. santacruzensis are only known from their type localities and they may deserve special attention concerning their conservation status. However, based on our current knowledge on these species and according to IUCN Red List criteria, they are assessed here as data deficient (DD).

Herbarium data: Global biodiversity and societal botanical needs for novel research.

Building on centuries of research based on herbarium specimens gathered through time and around the globe, a new era of discovery, synthesis, and prediction using digitized collections data has begun. This paper provides an overview of how aggregated, open access botanical and associated biological, environmental, and ecological data sets, from genes to the ecosystem, can be used to document the impacts of global change on communities, organisms, and society; predict future impacts; and help to drive the remediation of change. Advocacy for botanical collections and their expansion is needed, including ongoing digitization and online publishing. The addition of non-traditional digitized data fields, user annotation capability, and born-digital field data collection enables the rapid access of rich, digitally available data sets for research, education, informed decision-making, and other scholarly and creative activities. Researchers are receiving enormous benefits from data aggregators including the Global Biodiversity Information Facility (GBIF), Integrated Digitized Biocollections (iDigBio), the Atlas of Living Australia (ALA), and the Biodiversity Heritage Library (BHL), but effective collaboration around data infrastructures is needed when working with large and disparate data sets. Tools for data discovery, visualization, analysis, and skills training are increasingly important for inspiring novel research that improves the intrinsic value of physical and digital botanical collections.

The Promise of Molecular and Genomic Techniques for Biodiversity Research and DNA Barcoding of the Arabian Peninsula Flora.

The Arabian Peninsula is known to have a comprehensive and rich endowment of unique and genetically diverse plant genetic resources. Analysis and conservation of biological diversity is a crucial issue to the whole Arabian Peninsula. The rapid and accurate delimitation and identification of a species is crucial to genetic diversity analysis and the first critical step in the assessment of distribution, population abundance and threats related to a particular target species. During the last two decades, classical strategies of evaluating genetic variability, such as morphology and physiology, have been greatly complemented by phylogenetic, taxonomic, genetic diversity and breeding research molecular studies. At present, initiatives are taking place around the world to generate DNA barcode libraries for vascular plant flora and to make these data available in order to better understand, conserve and utilize biodiversity. The number of herbarium collection-based plant evolutionary genetics and genomics studies being conducted has been increasing worldwide. The herbaria provide a rich resource of already preserved and identified material, and these as well as freshly collected samples from the wild can be used for creating a reference DNA barcode library for the vascular plant flora of a region. This review discusses the main molecular and genomic techniques used in plant identification and biodiversity analysis. Hence, we highlight studies emphasizing various molecular techniques undertaken during the last 10 years to study the plant biodiversity of the Arabian Peninsula. Special emphasis on the role of DNA barcoding as a powerful tool for plant biodiversity analysis is provided, along with the crucial role of herbaria in creating a DNA barcode library.

Endemism in the moss flora of North America.

PREMISE OF THE STUDY: Identifying regions of high endemism is a critical step toward understanding the mechanisms underlying diversification and establishing conservation priorities. Here, we identified regions of high moss endemism across North America. We also identified lineages that contribute disproportionately to endemism and document the progress of efforts to inventory the endemic flora. METHODS: To understand the documentation of endemic moss diversity in North America, we tabulated species publication dates to document the progress of species discovery across the continent. We analyzed herbarium specimen data and distribution data from the Flora of North America project to delineate major regions of moss endemism. Finally, we surveyed the literature to assess the importance of intercontinental vs. within-continent diversification for generating endemic species. KEY RESULTS: Three primary regions of endemism were identified and two of these were further divided into a total of nine subregions. Overall endemic richness has two peaks, one in northern California and the Pacific Northwest, and the other in the southern Appalachians. Description of new endemic species has risen steeply over the last few decades, especially in western North America. Among the few studies documenting sister species relationships of endemics, recent diversification appears to have played a larger role in western North America, than in the east. CONCLUSIONS: Our understanding of bryophyte endemism continues to grow rapidly. Large continent-wide data sets confirm early views on hotspots of endemic bryophyte richness and indicate a high rate of ongoing species discovery in North America.