Interpreting an Archaean paleoenvironment through 3D imagery of microbialites.

While stromatolites, and to a lesser extent thrombolites, have been extensively studied in order to unravel Precambrian (>539 Ma) biological evolution, studies of clastic-dominated microbially induced sedimentary structures (MISS) are relatively scarce. The lack of a consolidated record of clastic microbialites creates questions about how much (and what) information on depositional and taphonomic settings can be gleaned from these fossils. We used µCT scanning, a non-destructive X-ray-based 3D imaging method, to reconstruct morphologies of ancient MISS and mat textures in two previously described coastal Archaean samples from the ~3.48 Ga Dresser Formation, Pilbara, Western Australia. The aim of this study was to test the ability of µCT scanning to visualize and make 3D measurements that can be used to interpret the biotic-environmental interactions. Fossil MISS including mat laminae with carpet-like textures in one sample and mat rip-up chips in the second sample were investigated. Compiled δ13C and δ34S analyses of specimens from the Dresser Fm. are consistent with a taxonomically diverse community that could be capable of forming such MISS. 3D measurements of fossil microbial mat chips indicate significant biostabilization and suggest formation in flow velocities >25 cm s-1. Given the stratigraphic location of these chips in a low-flow lagoonal layer, we conclude that these chips formed due to tidal influence, as these assumed velocities are consistent with recent modeling of Archaean tides. The success of µCT scanning in documenting these microbialite features validates this technique both as a first step analysis for rare samples prior to the use of more destructive techniques and as a valuable tool for gaining insight into microbialite taphonomy.

Reconstruction of an enigmatic Pennsylvanian cone reveals a relationship to Sphenophyllales.

PREMISE: We studied the 3D morphology of a small, well-preserved cone from the Pennsylvanian Mazon Creek Lagerstätte to characterize its structure and determine its systematic affinity. Previously tentatively assigned to the enigmatic Tetraphyllostrobus, we show that it differs in key respects from that genus as described. METHODS: We systematically compared the new fossil with relevant Paleozoic cone genera and employed advanced imaging techniques, including scanning electron microscopy, Airyscan confocal super-resolution microscopy, optical microscopy, and X-ray microcomputed tomography to visualize and reconstruct the fossil cone in 3D. RESULTS: The analyses demonstrate unequivocally that the sporophylls of the new Mazon Creek cone are arranged in whorls of six and have characters typical of Sphenophyllales, including epidermal cells with undulatory margins and in situ spores assignable to Columinisporites. The combination of characters, including sporophyll arrangement, anatomy, and spore type, supports the establishment of Hexaphyllostrobus kostorhysii gen. et sp. nov. within Sphenophyllales. Furthermore, we show that Tetraphyllostrobus, although originally described as possessing smooth monolete spores, actually possesses Columinisporites-type spores, indicating that it, too, was most likely a sphenophyll. CONCLUSIONS: The recognition of Hexaphyllostrobus contributes to our knowledge of Pennsylvanian sphenophyll diversity, and in particular increases the number of species with in situ Columinisporites-type spores. Attribution of Hexaphyllostrobus to Sphenophyllales calls into question current interpretations of Tetraphyllostrobus suggesting that future research on better-preserved macrofossil material may demonstrate a sphenophyllalean relationship.

Comparing Methodologies for Stomatal Analyses in the Context of Elevated Modern CO2.

Leaf stomata facilitate the exchange of water and CO2 during photosynthetic gas exchange. The shape, size, and density of leaf pores have not been constant over geologic time, and each morphological trait has potentially been impacted by changing environmental and climatic conditions, especially by changes in the concentration of atmospheric carbon dioxide. As such, stomatal parameters have been used in simple regressions to reconstruct ancient carbon dioxide, as well as incorporated into more complex gas-exchange models that also leverage plant carbon isotope ecology. Most of these proxy relationships are measured on chemically cleared leaves, although newer techniques such as creating stomatal impressions are being increasingly employed. Additionally, many of the proxy relationships use angiosperms with broad leaves, which have been increasingly abundant in the last 130 million years but are absent from the fossil record before this. We focus on the methodology to define stomatal parameters for paleo-CO2 studies using two separate methodologies (one corrosive, one non-destructive) to prepare leaves on both scale- and broad-leaves collected from herbaria with known global atmospheric CO2 levels. We find that the corrosive and non-corrosive methodologies give similar values for stomatal density, but that measurements of stomatal sizes, particularly guard cell width (GCW), for the two methodologies are not comparable. Using those measurements to reconstruct CO2 via the gas exchange model, we found that reconstructed CO2 based on stomatal impressions (due to inaccurate measurements in GCW) far exceeded measured CO2 for modern plants. This bias was observed in both coniferous (scale-shaped) and angiosperm (broad) leaves. Thus, we advise that applications of gas exchange models use cleared leaves rather than impressions.

Extending beyond Gondwana: Cretaceous Cunoniaceae from western North America.

Cunoniaceae are important elements of rainforests across the Southern Hemisphere. Many of these flowering plants are considered Paleo-Antarctic Rainforest Lineages that had a Gondwanan distribution since the Paleocene. Fossils of several modern genera within the family, such as Ceratopetalum, have indicated biogeographical connections between South America and Australia in the Cenozoic. Here, we report a dramatic geographical range extension for Ceratopetalum, and Cunoniaceae as a whole, based on two exceptionally preserved fossil winged fruits from Campanian (c. 82-80 Ma old) deposits on Sucia Island, Washington, USA. The fossils were studied using physical sectioning, light microscopy, micro-computed tomography scanning and multiple phylogenetic analyses. The fossil fruits share diagnostic characters with Ceratopetalum such as the presence of four to five persistent calyx lobes, a prominent nectary disk, persistent stamens, a semi-inferior ovary and two persistent styles. Based on morphological comparisons with fruits of extant species and support from phylogenetic analyses, the fossils are assigned to a new species Ceratopetalum suciensis. These fossils are the first unequivocal evidence of crown Cunoniaceae from the Cretaceous of North America, indicating a more complicated biogeographical history for this important Gondwanan family.

Fossil palm reading: using fruits to reveal the deep roots of palm diversity.

PREMISE: Fossils are essential for understanding evolutionary history because they provide direct evidence of past diversity and geographic distributions. However, resolving systematic relationships between fossils and extant taxa, an essential step for many macroevolutionary studies, requires extensive comparative work on morphology and anatomy. While palms (Arecaceae) have an excellent fossil record that includes numerous fossil fruits, many are difficult to identify due in part to limited comparative data on modern fruit structure. METHODS: We studied fruits of 207 palm species, representing nearly every modern genus, using X-ray microcomputed tomography. We then developed a morphological data set to test whether the fossil record of fruits can improve our understanding of palm diversity in the deep past. To evaluate the accuracy with which this data set recovers systematic relationships, we performed phylogenetic pseudofossilization analyses. We then used the data set to investigate the phylogenetic relationships of five previously published fossil palm fruits. RESULTS: Phylogenetic analyses of fossils and pseudofossilization of extant taxa show that fossils can be placed accurately to the tribe and subtribe level with this data set, but node support must be considered. The phylogenetic relationships of the fossils suggest origins of many modern lineages in the Cretaceous and early Paleogene. Three of these fossils are suitable as new node calibrations for palms. CONCLUSIONS: This work improves our knowledge of fruit structure in palms, lays a foundation for applying fossil fruits to macroevolutionary studies, and provides new insights into the evolutionary history and early diversification of Arecaceae.

C3 plant carbon isotope discrimination does not respond to CO2 concentration on decadal to centennial timescales.

Plant carbon isotope discrimination is complex, and could be driven by climate, evolution and/or edaphic factors. We tested the climate drivers of carbon isotope discrimination in modern and historical plant chemistry, and focus in particular on the relationship between rising [CO2 ] over Industrialization and carbon isotope discrimination. We generated temporal records of plant carbon isotopes from museum specimens collected over a climo-sequence to test plant responses to climate and atmospheric change over the past 200 yr (including Pinus strobus, Platycladus orientalis, Populus tremuloides, Thuja koraiensis, Thuja occidentalis, Thuja plicata, Thuja standishii and Thuja sutchuenensis). We aggregated our results with a meta-analysis of a wide range of C3 plants to make a comprehensive study of the distribution of carbon isotope discrimination and values among different plant types. We show that climate variables (e.g. mean annual precipitation, temperature and, key to this study, CO2 in the atmosphere) do not drive carbon isotope discrimination. Plant isotope discrimination is intrinsic to each taxon, and could link phylogenetic relationships and adaptation to climate quantitatively and over ecological to geological time scales.

A bi-organellar phylogenomic study of Pandanales: inference of higher-order relationships and unusual rate-variation patterns.

We used a bi-organellar phylogenomic approach to address higher-order relationships in Pandanales, including the first molecular phylogenetic study of the panama-hat family, Cyclanthaceae. Our genus-level study of plastid and mitochondrial gene sets includes a comprehensive sampling of photosynthetic lineages across the order, and provides a framework for investigating clade ages, biogeographic hypotheses and organellar molecular evolution. Using multiple inference methods and both organellar genomes, we recovered mostly congruent and strongly supported relationships within and between families, including the placement of fully mycoheterotrophic Triuridaceae. Cyclanthaceae and Pandanaceae plastomes have slow substitution rates, contributing to weakly supported plastid-based relationships in Cyclanthaceae. While generally slowly evolving, mitochondrial genomes exhibit sporadic rate elevation across the order. However, we infer well-supported relationships even for slower evolving mitochondrial lineages in Cyclanthaceae. Clade age estimates across photosynthetic lineages are largely consistent with previous studies, are well correlated between the two organellar genomes (with slightly younger inferences from mitochondrial data), and support several biogeographic hypotheses. We show that rapidly evolving non-photosynthetic lineages may bias age estimates upwards at neighbouring photosynthetic nodes, even using a relaxed clock model. Finally, we uncovered new genome structural variants in photosynthetic taxa at plastid inverted repeat boundaries that show promise as interfamilial phylogenetic markers.

Correction: Reinvestigating an enigmatic Late Cretaceous monocot: morphology, taxonomy, and biogeography of Viracarpon.

[This corrects the article DOI: 10.7717/peerj.4580.].

Cornalean affinities, phylogenetic significance, and biogeographic implications of Operculifructus infructescences from the Late Cretaceous (Campanian) of Mexico.

IN ENGLISH: Premise of the Study Cretaceous Cornales provide a crucial record of the early history of asterids. Most lineages of the order are well represented in the fossil record, but South African families of Curtisiaceae and Grubbiaceae remain poorly understood. Seventy-three specimens of a fossil infructescence belonging to the genus Operculifructus Estrada-Ruiz & Cevallos-Ferriz emend. Hayes & Smith from the Late Cretaceous (Campanian) El Gallo Formation, Baja California, Mexico bear previously undescribed characters that suggest a relationship to Grubbiaceae. Methods Microstructures of the fossils were examined through light microscopy and x-ray microcomputed tomography (microCT) scanning. Modern Grubbia tomentosa (Thunb.) Harms fruits were scanned for comparison to the fossil material. Phylogenetic analyses using the 77 fruit characters of Atkinson () were performed to test relationships of the fossil to major lineages of the order. Several analyses applied topological constraints to the extant taxa, based on various genetically supported hypotheses of relationship within Cornales. Key Results Novel structures of Operculifructus newly observed here include (1) anatropous ovules, (2) drupaceous fruits, (3) an epigynous disc, (4) and a stylar canal in the center of the disc aligned with the micropylar protrusion of the seed. Phylogenetic analysis consistently resolves Operculifructus as sister to Grubbiaceae. Conclusions Operculifructus provides direct evidence for the occurrence of Grubbiaceae in the Late Cretaceous, much older than previous Eocene evidence. The phylogeny of Atkinson () indicates that the new phylogenetic position recovered for Operculifructus also establishes the presence of the most basal drupaceous cornalean fruits in North America by the Campanian. RESUMEN EN ESPAÑOL Hipótesis de la Investigación Cornales cretácicos representan un registro esencial en la historia de los astéridos. Casi todos los linajes del orden están bien representados en el registro fósil, pero las familias africanas sureñas Curtisiaceae y Grubbiaceae permanecen pobremente entendidas. Setenta y tres ejemplares de una infrutescencia fósil perteneciente al género Operculifructus Estrada-Ruiz & Cevallos-Ferriz emend. Hayes & Smith de la formación campaniana (Cretácico Tardío) El Gallo, Baja California, México, poseen caracteres no descritos previamente y sugieren una relación con Grubbiaceae. Metodología Microestructuras de los fósiles fueron examinadas con microscopio de luz y microtomografía computarizada (micro-CT) de rayos X. Frutos actuales de Grubbia tomentosa (Thunb.) Harms fueron escaneados para su comparación con el material fósil. Se realizaron análisis filogenéticos usando los 77 caracteres de frutos de Atkinson () para probar las relaciones de los fósiles con los linajes principales del orden. En algunos análisis se aplicaron restricciones topológicas a los taxa actuales basándose en varias hipótesis, soportadas genéticamente, de las relaciones dentro de Cornales. Resultados Centrales (Cruciales) Las estructuras novedosas de Operculifructus, recientemente observadas aquí, incluyen (1) óvulos anátropos; (2) frutos drupáceos; (3) disco epígino; y (4) un canal estilar en el centro del disco alineado con la protuberancia micropilar de la semilla. Los análisis filogenéticos consistentemente dan como resultado que Operculifructus es hermano de Grubbiaceae. Conclusiones Operculifructus proporciona evidencia directa de la presencia de Grubbiaceae en el Cretácico Tardío, mucho antes que la previa evidencia en el Eoceno. La filogenia de Atkinson () indica que la nueva posición filogenética recuperada para Operculifructus también resalta la presencia de frutos drupáceos cornaleanos más basales en Norteamérica durante el Campaniano.

Building the monocot tree of death: Progress and challenges emerging from the macrofossil-rich Zingiberales.

PREMISE OF THE STUDY: Inclusion of fossils in phylogenetic analyses is necessary in order to construct a comprehensive "tree of death" and elucidate evolutionary history of taxa; however, such incorporation of fossils in phylogenetic reconstruction is dependent on the availability and interpretation of extensive morphological data. Here, the Zingiberales, whose familial relationships have been difficult to resolve with high support, are used as a case study to illustrate the importance of including fossil taxa in systematic studies. METHODS: Eight fossil taxa and 43 extant Zingiberales were coded for 39 morphological seed characters, and these data were concatenated with previously published molecular sequence data for analysis in the program MrBayes. KEY RESULTS: Ensete oregonense is confirmed to be part of Musaceae, and the other seven fossils group with Zingiberaceae. There is strong support for Spirematospermum friedrichii, Spirematospermum sp. 'Goth', S. wetzleri, and Striatornata sanantoniensis in crown Zingiberaceae while "Musa" cardiosperma, Spirematospermum chandlerae, and Tricostatocarpon silvapinedae are best considered stem Zingiberaceae. Inclusion of fossils explains how different topologies from morphological and molecular data sets is due to shared plesiomorphic characters shared by Musaceae, Zingiberaceae, and Costaceae, and most of the fossils. CONCLUSIONS: Inclusion of eight fossil taxa expands the Zingiberales tree and helps explain the difficulty in resolving relationships. Inclusion of fossils was possible in part due to a large morphological data set built using nondestructive microcomputed tomography data. Collaboration between paleo- and neobotanists and technology such as microcomputed tomography will help to build the tree of death and ultimately improve our understanding of the evolutionary history of monocots.

Reinvestigating an enigmatic Late Cretaceous monocot: morphology, taxonomy, and biogeography of Viracarpon.

Angiosperm-dominated floras of the Late Cretaceous are essential for understanding the evolutionary, ecological, and geographic radiation of flowering plants. The Late Cretaceous-early Paleogene Deccan Intertrappean Beds of India contain angiosperm-dominated plant fossil assemblages known from multiple localities in central India. Numerous monocots have been documented from these assemblages, providing a window into an important but poorly understood time in their diversification. One component of the Deccan monocot diversity is the genus Viracarpon, known from anatomically preserved infructescences. Viracarpon was first collected over a century ago and has been the subject of numerous studies. However, resolution of its three-dimensional (3D) morphology and anatomy, as well as its taxonomic affinities, has remained elusive. In this study we investigated the morphology and taxonomy of genus Viracarpon, combining traditional paleobotanical techniques and X-ray micro-computed tomography (µCT). Re-examination of type and figured specimens, 3D reconstructions of fruits, and characterization of structures in multiple planes of section using µCT data allowed us to resolve conflicting interpretations of fruit morphology and identify additional characters useful in refining potential taxonomic affinities. Among the four Viracarpon species previously recognized, we consider two to be valid (Viracarponhexaspermum and Viracarponelongatum), and the other two to be synonyms of these. Furthermore, we found that permineralized infructescences of Coahuilocarpon phytolaccoides from the late Campanian of Mexico correspond closely in morphology to V. hexaspermum. We argue that Viracarpon and Coahuilocarpon are congeneric and provide the new combination, Viracarpon phytolaccoides (Cevallos-Ferriz, Estrada-Ruiz & Perez-Hernandez) Matsunaga, S.Y. Smith, & Manchester comb. nov. The significant geographic disjunction between these two occurrences indicates that the genus Viracarpon was widespread and may be present in other Late Cretaceous assemblages. Viracarpon exhibits character combinations not present in any extant taxa and its affinities remain unresolved, possibly representing an extinct member of Alismatales. The character mosaic observed in Viracarpon and the broad distribution of the genus provide new data relevant to understanding early monocot evolution and suggest that the (thus far) largely invisible Late Cretaceous monocot diversification was characterized by enigmatic and/or stem taxa.

Crowds Replicate Performance of Scientific Experts Scoring Phylogenetic Matrices of Phenotypes.

Scientists building the Tree of Life face an overwhelming challenge to categorize phenotypes (e.g., anatomy, physiology) from millions of living and fossil species. This biodiversity challenge far outstrips the capacities of trained scientific experts. Here we explore whether crowdsourcing can be used to collect matrix data on a large scale with the participation of nonexpert students, or "citizen scientists." Crowdsourcing, or data collection by nonexperts, frequently via the internet, has enabled scientists to tackle some large-scale data collection challenges too massive for individuals or scientific teams alone. The quality of work by nonexpert crowds is, however, often questioned and little data have been collected on how such crowds perform on complex tasks such as phylogenetic character coding. We studied a crowd of over 600 nonexperts and found that they could use images to identify anatomical similarity (hypotheses of homology) with an average accuracy of 82% compared with scores provided by experts in the field. This performance pattern held across the Tree of Life, from protists to vertebrates. We introduce a procedure that predicts the difficulty of each character and that can be used to assign harder characters to experts and easier characters to a nonexpert crowd for scoring. We test this procedure in a controlled experiment comparing crowd scores to those of experts and show that crowds can produce matrices with over 90% of cells scored correctly while reducing the number of cells to be scored by experts by 50%. Preparation time, including image collection and processing, for a crowdsourcing experiment is significant, and does not currently save time of scientific experts overall. However, if innovations in automation or robotics can reduce such effort, then large-scale implementation of our method could greatly increase the collective scientific knowledge of species phenotypes for phylogenetic tree building. For the field of crowdsourcing, we provide a rare study with ground truth, or an experimental control that many studies lack, and contribute new methods on how to coordinate the work of experts and nonexperts. We show that there are important instances in which crowd consensus is not a good proxy for correctness.

Open data and digital morphology.

Over the past two decades, the development of methods for visualizing and analysing specimens digitally, in three and even four dimensions, has transformed the study of living and fossil organisms. However, the initial promise that the widespread application of such methods would facilitate access to the underlying digital data has not been fully achieved. The underlying datasets for many published studies are not readily or freely available, introducing a barrier to verification and reproducibility, and the reuse of data. There is no current agreement or policy on the amount and type of data that should be made available alongside studies that use, and in some cases are wholly reliant on, digital morphology. Here, we propose a set of recommendations for minimum standards and additional best practice for three-dimensional digital data publication, and review the issues around data storage, management and accessibility.

Species diversity driven by morphological and ecological disparity: a case study of comparative seed morphology and anatomy across a large monocot order.

Phenotypic variation can be attributed to genetic heritability as well as biotic and abiotic factors. Across Zingiberales, there is a high variation in the number of species per clade and in phenotypic diversity. Factors contributing to this phenotypic variation have never been studied in a phylogenetic or ecological context. Seeds of 166 species from all eight families in Zingiberales were analyzed for 51 characters using synchrotron based 3D X-ray tomographic microscopy to determine phylogenetically informative characters and to understand the distribution of morphological disparity within the order. All families are distinguishable based on seed characters. Non-metric multidimensional scaling analyses show Zingiberaceae occupy the largest seed morphospace relative to the other families, and environmental analyses demonstrate that Zingiberaceae inhabit both temperate and tropical regions, while other Zingiberales are almost exclusively tropical. Temperate species do not cluster in morphospace nor do they share a common suite of character states. This suggests that the diversity seen is not driven by adaptation to temperate niches; rather, the morphological disparity seen likely reflects an underlying genetic plasticity that allowed Zingiberaceae to repeatedly colonize temperate environments. The notable morphoanatomical variety in Zingiberaceae seeds may account for their extraordinary ecological success and high species diversity as compared to other Zingiberales.

X-rays and virtual taphonomy resolve the first Cissus (Vitaceae) macrofossils from Africa as early-diverging members of the genus.

PREMISE OF THE STUDY: Fossilized seeds similar to Cissus (Vitaceae) have been recognized from the Miocene of Kenya, though some were previously assigned to the Menispermaceae. We undertook a comparative survey of extant African Cissus seeds to identify the fossils and consider their implications for the evolution and biogeography of Cissus and for African early Miocene paleoenvironments. METHODS: Micro-computed tomography (µCT) and synchrotron-based X-ray tomographic microscopy (SRXTM) were used to study seed morphology and anatomy. Virtual taphonomy, using SRXTM data sets, produced digital fossils to elucidate seed taphonomy. Phylogenetic relationships within Cissus were reconstructed using existing and newly produced DNA sequences for African species. Paleobiology and paleoecology were inferred from African nearest living relatives. KEY RESULTS: The fossils were assigned to four new Cissus species, related to four modern clades. The fossil plants were interpreted as climbers inhabiting a mosaic of riverine woodland and forest to more open habitats. Virtual taphonomy explained how complex mineral infill processes concealed key seed features, causing the previous taxonomic misidentification. Newly sampled African species, with seeds most similar to the fossils, belong to four clades within core Cissus, two of which are early diverging. CONCLUSIONS: Virtual taphonomy, combined with X-ray imaging, has enabled recognition of the first fossil Cissus and Vitaceae from Africa. Early-divergent members of the core Cissus clade were present in Africa by at least the early Miocene, with an African origin suggested for the Cissus sciaphila clade. The fossils provide supporting evidence for mosaic paleoenvironments inhabited by early Miocene hominoids.

Revisiting the Zingiberales: using multiplexed exon capture to resolve ancient and recent phylogenetic splits in a charismatic plant lineage.

The Zingiberales are an iconic order of monocotyledonous plants comprising eight families with distinctive and diverse floral morphologies and representing an important ecological element of tropical and subtropical forests. While the eight families are demonstrated to be monophyletic, phylogenetic relationships among these families remain unresolved. Neither combined morphological and molecular studies nor recent attempts to resolve family relationships using sequence data from whole plastomes has resulted in a well-supported, family-level phylogenetic hypothesis of relationships. Here we approach this challenge by leveraging the complete genome of one member of the order, Musa acuminata, together with transcriptome information from each of the other seven families to design a set of nuclear loci that can be enriched from highly divergent taxa with a single array-based capture of indexed genomic DNA. A total of 494 exons from 418 nuclear genes were captured for 53 ingroup taxa. The entire plastid genome was also captured for the same 53 taxa. Of the total genes captured, 308 nuclear and 68 plastid genes were used for phylogenetic estimation. The concatenated plastid and nuclear dataset supports the position of Musaceae as sister to the remaining seven families. Moreover, the combined dataset recovers known intra- and inter-family phylogenetic relationships with generally high bootstrap support. This is a flexible and cost effective method that gives the broader plant biology community a tool for generating phylogenomic scale sequence data in non-model systems at varying evolutionary depths.

Seed morphology and anatomy and its utility in recognizing subfamilies and tribes of Zingiberaceae.

PREMISE OF THE STUDY: Recent phylogenetic analyses based on molecular data suggested that the monocot family Zingiberaceae be separated into four subfamilies and four tribes. Robust morphological characters to support these clades are lacking. Seeds were analyzed in a phylogenetic context to test independently the circumscription of clades and to better understand evolution of seed characters within Zingiberaceae. METHODS: Seventy-five species from three of the four subfamilies were analyzed using synchrotron based x-ray tomographic microscopy (SRXTM) and scored for 39 morphoanatomical characters. KEY RESULTS: Zingiberaceae seeds are some of the most structurally complex seeds in angiosperms. No single seed character was found to distinguish each subfamily, but combinations of characters were found to differentiate between the subfamilies. Recognition of the tribes based on seeds was possible for Globbeae, but not for Alpinieae, Riedelieae, or Zingibereae, due to considerable variation. CONCLUSIONS: SRXTM is an excellent, nondestructive tool to capture morphoanatomical variation of seeds and allows for the study of taxa with limited material available. Alpinioideae, Siphonochiloideae, Tamijioideae, and Zingiberoideae are well supported based on both molecular and morphological data, including multiple seed characters. Globbeae are well supported as a distinctive tribe within the Zingiberoideae, but no other tribe could be differentiated using seeds due to considerable homoplasy when compared with currently accepted relationships based on molecular data. Novel seed characters suggest tribal affinities for two currently unplaced Zingiberaceae taxa: Siliquamomum may be related to Riedelieae and Monolophus to Zingibereae, but further work is needed before formal revision of the family.

Reading the leaves: A comparison of leaf rank and automated areole measurement for quantifying aspects of leaf venation.

The reticulate venation that is characteristic of a dicot leaf has excited interest from systematists for more than a century, and from physiological and developmental botanists for decades. The tools of digital image acquisition and computer image analysis, however, are only now approaching the sophistication needed to quantify aspects of the venation network found in real leaves quickly, easily, accurately, and reliably enough to produce biologically meaningful data. In this paper, we examine 120 leaves distributed across vascular plants (representing 118 genera and 80 families) using two approaches: a semiquantitative scoring system called "leaf ranking," devised by the late Leo Hickey, and an automated image-analysis protocol. In the process of comparing these approaches, we review some methodological issues that arise in trying to quantify a vein network, and discuss the strengths and weaknesses of automatic data collection and human pattern recognition. We conclude that subjective leaf rank provides a relatively consistent, semiquantitative measure of areole size among other variables; that modal areole size is generally consistent across large sections of a leaf lamina; and that both approaches-semiquantitative, subjective scoring; and fully quantitative, automated measurement-have appropriate places in the study of leaf venation.

Virtual taphonomy using synchrotron tomographic microscopy reveals cryptic features and internal structure of modern and fossil plants.

While more commonly applied in zoology, synchrotron radiation X-ray tomographic microscopy (SRXTM) is well-suited to nondestructive study of the morphology and anatomy of both fossil and modern plants. SRXTM uses hard X-rays and a monochromatic light source to provide high-resolution data with little beam-hardening, resulting in slice data with clear boundaries between materials. Anatomy is readily visualized, including various planes of section from a single specimen, as clear as in traditional histological sectioning at low magnifications. Thus, digital sectioning of rare or difficult material is possible. Differential X-ray attenuation allows visualization of different layers or chemistries to enable virtual 3-dimensional (3D) dissections of material. Virtual potential fossils can be visualized and digital tissue removal reveals cryptic underlying morphology. This is essential for fossil identification and for comparisons between assemblages where fossils are preserved by different means. SRXTM is a powerful approach for botanical studies using morphology and anatomy. The ability to gain search images in both 2D and 3D for potential fossils gives paleobotanists a tool--virtual taphonomy--to improve our understanding of plant evolution and paleobiogeography.

Scanning electron microscopy and synchrotron radiation x-ray tomographic microscopy of 330 million year old charcoalified seed fern fertile organs.

Abundant charcoalified seed fern (pteridosperm) pollen organs and ovules have been recovered from Late Viséan (Mississippian 330 Ma) limestones from Kingswood, Fife, Scotland. To overcome limitations of data collection from these tiny, sometimes unique, fossils, we have combined low vacuum scanning electron microscopy on uncoated specimens with backscatter detector and synchrotron radiation X-ray tomographic microscopy utilizing the Materials Science and TOMCAT beamlines at the Swiss Light Source of the Paul Scherrer Institut. In combination these techniques improve upon traditional cellulose acetate peel sectioning because they enable study of external morphology and internal anatomy in multiple planes of section on a single specimen that is retained intact. The pollen organ Melissiotheca shows a basal parenchymatous cushion bearing more than 100 sporangia on the distal face. Digital sections show the occurrence of pollen in some sporangia. The described ovule is new and has eight integumentary lobes that are covered in spirally arranged glandular hairs. Virtual longitudinal sections reveal the lobes are free above the pollen chamber. Results are applied in taxonomy and will subsequently contribute to our understanding of the former diversity and evolution of ovules, seeds, and pollen organs in the seed ferns, the first seed-bearing plants to conquer the land.