Lignin Analysis by HPLC and FTIR: Spectra Deconvolution and S/G Ratio Determination.

Fourier transform infrared spectroscopy (FTIR) is a simple nondestructive technique that allows the user to obtain quick and accurate information about the structure of the constituents of wood. Spectra deconvolution is a computational technique, complementary to FTIR analysis, which improves the resolution of overlapped or unobserved bands in the raw spectra. High performance liquid chromatography (HPLC) is an analytical technique useful to determine the ratio of the lignin monomers obtained by the alkaline nitrobenzene oxidation method. Furthermore, lignin content has been commonly determined by wet chemical methods; Klason lignin determination is a quick and accessible method. Here, we detail the procedures for chemical analysis of the wood lignin using these techniques. Additionally, the deconvolution process of FTIR spectra for the determination of the S/G ratio, in lignin isolated by this or other methods, is explained in detail.

Tree species differ in plant economic spectrum traits in the tropical dry forest of Mexico.

In tropical dry forests, studies on wood anatomical traits have concentrated mainly on variations in vessel diameter and frequency. Recent research suggests that parenchyma and fibers also play an important role in water conduction and in xylem hydraulic safety. However, these relationships are not fully understood, and wood trait variation among different functional profiles as well as their variation under different water availability scenarios have been little studied. In this work, we aim to (1) characterize a set of wood anatomical traits among six selected tree species that represent the economic spectrum of tropical dry forests, (2) assess the variation in these traits under three different rainfall regimes, and (3) determine the relationships between wood anatomical traits and possible functional trade-offs. Differences among species and sites in wood traits were explored. Linear mixed models were fitted, and model comparison was performed. Most variation occurred among species along the economic spectrum. Obligate deciduous, low wood density species were characterized by wood with wide vessels and low frequency, suggesting high water transport capacity but sensitivity to drought. Moreover, high cell fractions of carbon and water storage were also found in these tree species related to the occurrence of abundant parenchyma or septate fibers. Contrary to what most studies show, Cochlospermum vitifolium, a succulent tree species, presented the greatest variation in wood traits. Facultative deciduous, high wood density species were characterized by a sturdy vascular system that may favor resistance to cavitation and low reserve storage. Contrary to our expectations, variation among the rainfall regimes was generally low in all species and was mostly related to vessel traits, while fiber and parenchyma traits presented little variation among species. Strong functional associations between wood anatomical traits and functional trade-offs were found for the six tree species studied along the economic spectrum of tropical dry forests.

Ecological implications of stomatal density and stomatal index in the adult stage of Mimosa L. (Leguminosae, Caesalpinioideae).

Differences in stomatal density (SD) and stomatal index (SI) are associated with the conditions of the environment in which they are distributed. Mimosa species are important elements in different plant communities, yet knowledge of the ecological implications of its stomatal characteristics is scarce. For this reason, SD and SI were determined in seven Mimosa species from different environments in this study. Five individuals per species were selected, and a sample of leaflets was obtained from each. Fifteen mature leaflets per individual were then extracted and observed by optical microscopy. SD, SI, epidermal cell density (ECD), and guard cell length (GCL) values were obtained. Differences between species were analyzed through a balanced analysis of variance test, and the correspondence between the stomatal characteristics and 21 climate variables was determined by canonical correspondence analysis. The species differed in all evaluated characteristics. It should be noted that only M. affinis showed differences between the leaflet surfaces. Both DE and ECD were negatively associated with altitude and solar radiation and positively with temperature and precipitation. SI was explained by temperature and seasonality of precipitation, and GCL by temperature oscillation and seasonality of precipitation. The results suggest that the stomatal characteristics of the leaflets confer resistance in the species to alterations in environmental conditions.

How Important Are Functional and Developmental Constraints on Phenotypic Evolution? An Empirical Test with the Stomatal Anatomy of Flowering Plants.

AbstractQuantifying the relative contribution of functional and developmental constraints on phenotypic variation is a long-standing goal of macroevolution, but it is often difficult to distinguish different types of constraints. Alternatively, selection can limit phenotypic (co)variation if some trait combinations are generally maladaptive. The anatomy of leaves with stomata on both surfaces (amphistomatous) present a unique opportunity to test the importance of functional and developmental constraints on phenotypic evolution. The key insight is that stomata on each leaf surface encounter the same functional and developmental constraints but potentially different selective pressures because of leaf asymmetry in light capture, gas exchange, and other features. Independent evolution of stomatal traits on each surface imply that functional and developmental constraints alone likely do not explain trait covariance. Packing limits on how many stomata can fit into a finite epidermis and cell size-mediated developmental integration are hypothesized to constrain variation in stomatal anatomy. The simple geometry of the planar leaf surface and knowledge of stomatal development make it possible to derive equations for phenotypic (co)variance caused by these constraints and compare them with data. We analyzed evolutionary covariance between stomatal density and length in amphistomatous leaves from 236 phylogenetically independent contrasts using a robust Bayesian model. Stomatal anatomy on each surface diverges partially independently, meaning that packing limits and developmental integration are not sufficient to explain phenotypic (co)variation. Hence, (co)variation in ecologically important traits like stomata arises in part because there is a limited range of evolutionary optima. We show how it is possible to evaluate the contribution of different constraints by deriving expected patterns of (co)variance and testing them using similar but separate tissues, organs, or sexes.

Combining exposed tree roots and UAV imagery to quantify land denudation in central Mexico.

Approximately 42 % of Mexico is affected by soil denudation resulting from moderate to severe sheet erosion and gullying processes. At Huasca de Ocampo (central Mexico), soil degradation has been linked to intense land use dating back to pre-Hispanic times as well as to unfavorable geological, geomorphic, and climatic conditions. Here, we quantify erosion rates with high precision at annual to multi-decadal timescales by combining, for the first time, dendrogeomorphic reconstructions and UAV-based remote sensing. To assess rates of sheet erosion and gullying processes over the longer-term erosion rates (10-60 yrs), we assessed the age and first exposure of 159 roots to determine sheet erosion rates and gullying processes. At shorter timescales (<3 yrs), we employed an Unmanned Aerial Vehicle (UAV) to develop digital surface models (DSMs) for February 2020 and September 2022. Exposed roots provided evidence of sheet erosion ranging between 2.8 and 43.6 mm yr-1 and channel widening ranging between 11 and 270 mm yr-1, with highest erosion rates found along gully slopes. The UAV-based approach pointed to intense gully headcut retreat with rates between 164.8 and 870.4 mm yr-1; within gullies, channel widening rates ranged between 88.7 and 213.6 mm yr-1 and gully incision rates were between 11.8 and 109.8 mm yr-1. The two approaches yielded very comparable results regarding gully erosion and channel widening; this underlines the potential of using exposed roots to quantifying soil degradation processes retrospectively and considerably beyond the period covered by UAV imagery.

Leaf habit determines the hydraulic and resource-use strategies in tree saplings from the Sonoran Desert.

The drought susceptibility of woody saplings may explain their low survival in arid environments. Therefore, it is critical to determine which morphological and physiological traits are more responsive to drought among young plants. This study tested whether plant responses to experimental drought differ between two plant functional groups: the deciduous and evergreen species. We predicted that deciduous species would present a tighter stomatal control under drought, coupled with fast carbon fixation under no stress, tending toward isohydry and faster growth rates than the evergreen species. Using 1-year-old saplings from three evergreen and four deciduous Sonoran Desert tree species, we evaluated their hydraulic and gas exchange traits under three experimental irrigation conditions: high, intermediate and low water availability. We measured CO2 assimilation rates (A), stomatal conductance (gs), the level of iso-anisohydry (as the plant's ability to maintain constant their water potential) and seven morphological and growth-related traits throughout 2 months. Under high water availability, saplings reached their maximum values of A and gs, which were significantly higher for deciduous than evergreen species. Correlations among hydroscape area (HA) and leaf traits positioned species along the iso/anisohydric continuum. Deciduous species presented isohydric characteristics, including low HA, high gs, A and Huber values (HVs), and traits indicative of a faster use of resources, such as low stem-specific density (SSD) and low leaf mass per area (LMA). By contrast, evergreen species showed traits that indicate slow resource use and anisohydric behavior, such as high HA, SSD and LMA, and low gs, A and HVs. Deciduous species drastically reduced gas exchange rates in response to drought, while evergreen maintained low rates independently of drought intensity. Overall, desert saplings showed strategies concordant with the iso-anisohydric continuum and the fast-slow use of resources.

Cellulose in Secondary Xylem of Cactaceae: Crystalline Composition and Anatomical Distribution.

Cellulose is the main polymer that gives strength to the cell wall and is located in the primary and secondary cell walls of plants. In Cactaceae, there are no studies on the composition of cellulose. The objective of this work was to analyze the crystallinity composition and anatomical distribution of cellulose in Cactaceae vascular tissue. Twenty-five species of Cactaceae were collected, dried, and milled. Cellulose was purified and analyzed with Fourier transform infrared spectroscopy, the crystallinity indexes were calculated, and statistical analyzes were performed. Stem sections were fixed, cut, and stained with safranin O/fast green, for observation with epifluorescence microscopy. The crystalline cellulose ratios had statistical differences between Echinocereus pectinatus and Coryphantha pallida. All cacti species presented a higher proportion of crystalline cellulose. The fluorescence emission of the cellulose was red in color and distributed in the primary wall of non-fibrous species; while in the fibrous species, the distribution was in the pits. The high percentages of crystalline cellulose may be related to its distribution in the non-lignified parenchyma and primary walls of tracheary elements with helical or annular thickenings of non-fibrous species, possibly offering structural rigidity and forming part of the defense system against pathogens.

Fluorescence Microscopy Methods for the Analysis and Characterization of Lignin.

Lignin is one of the most studied and analyzed materials due to its importance in cell structure and in lignocellulosic biomass. Because lignin exhibits autofluorescence, methods have been developed that allow it to be analyzed and characterized directly in plant tissue and in samples of lignocellulose fibers. Compared to destructive and costly analytical techniques, fluorescence microscopy presents suitable alternatives for the analysis of lignin autofluorescence. Therefore, this review article analyzes the different methods that exist and that have focused specifically on the study of lignin because with the revised methods, lignin is characterized efficiently and in a short time. The existing qualitative methods are Epifluorescence and Confocal Laser Scanning Microscopy; however, other semi-qualitative methods have been developed that allow fluorescence measurements and to quantify the differences in the structural composition of lignin. The methods are fluorescence lifetime spectroscopy, two-photon microscopy, Föster resonance energy transfer, fluorescence recovery after photobleaching, total internal reflection fluorescence, and stimulated emission depletion. With these methods, it is possible to analyze the transport and polymerization of lignin monomers, distribution of lignin of the syringyl or guaiacyl type in the tissues of various plant species, and changes in the degradation of wood by pulping and biopulping treatments as well as identify the purity of cellulose nanofibers though lignocellulosic biomass.

The role of wood anatomical traits in the coexistence of oak species along an environmental gradient.

Oaks (Quercus) are a dominant woody plant genus in the northern hemisphere, which occupy a wide range of habitats and are ecologically diverse. We analysed the wood anatomical traits, the variables derived and the relative hydraulic conductivity of 21 oak species to identify their performance according to abiotic factors, leaf phenological patterns and phylogenetic restrictions by analysing the interspecific variation along an environmental gradient. First, we determine the causes of anatomical trait variation in the oaks, analysing the functional trade-offs related to distribution along the environmental gradient. We measure the phenotypic plasticity of the anatomical traits to determine the role of environment and geographic distance in the range of phenotypic plasticity. Second, we examined if oaks co-occurred along the environmental gradient. Then we analysed if wood anatomical traits reflect differences among their phylogenetic section, leaf habit and a phylogenetic section/leaf habit category. Last, we tested the phylogenetic signal. Our results showed that vessel diameter, vessel frequency, wood density and relative hydraulic conductivity are the main axes of trait variation in the species analysed among leaf habit categories. The aridity index and seasonal precipitation drive the variation in the analysed traits. Higher environmental distance resulted in a higher relative distance plasticity index among traits. Co-occurrence of oak species with different leaf habits and phylogenetic trajectories may promote complementary resource acquisition. The phylogenetic signal in the oak species studied was low, which implies labile wood traits.

Distribution and Chemical Composition of Lignin in Secondary Xylem of Cactaceae.

Cactaceae family has heterogeneity in the accumulation of lignocellulose due to the diversity of shapes and anatomy of the wood. Most studies focus on fibrous and dimorphic species; but the non-fibrous species are poorly studied. The aims of this work were to analyze the syringyl/guaiacyl ratio of lignin and its distribution in secondary xylem, especially in non-fibrous species. The syringyl/guaiacyl (S/G) ratio was quantified from 34 species of cacti by nitrobenzene oxidation of free-extractive wood. The distribution of lignocellulose in wood sections stained with safranin O/fast green was determined with epifluorescence microscopy. The S/G ratio was heterogeneous; most of the non-fibrous species had a higher percentage of syringyl, while the fibrous ones accumulate guaiacyl. Fluorescence emission showed that vessel elements and wide-band tracheids had similar tonalities. It is hypothesized that the presence of a higher percentage of syringyl in most cacti is part of the defense mechanism against pathogens, which together with the succulence of the stem represent adaptations that contribute to survival in their hostile environments.

A new Oligocene-Miocene tree from Panama and historical Anacardium migration patterns.

Migration of Boreotropical megathermal taxa during the Oligocene and Miocene played a key role in assembling diversity in tropical regions. Despite scattered fossil reports, the cashew genus Anacardium offers an excellent example of such migration. The fossil woods described here come from localities in Veraguas, Panama mapped as Oligocene-Miocene. We studied, described, and identified two well-preserved specimens using wood anatomical characteristics and completed extensive comparisons between fossil and extant material. The studied fossil woods share several diagnostic features with the modern Anacardium genus, including large solitary vessels, large intervessel-pitting, a simple vessel-ray pitting pattern, and mostly 1-3 seriate rays with large rhomboidal solitary crystals. We propose a new fossil species named Anacardium gassonii sp. nov., that adds an essential piece to the understanding of the historical biogeography of the genus. In addition, our findings confirm previous interpretations of this species' migration from Europe to North America and its crossing through Panama, leading to subsequent diversification in South America. This discovery provides an important link to the historical migration patterns of the genus, supporting the notion of an Eocene migration to the Neotropics via Boreotropical bridges, as well as an Oligocene-Miocene crossing of Central America followed by diversification in South America.

Leaf water relations reflect canopy phenology rather than leaf life span in Sonoran Desert trees.

Plants from arid environments display covarying traits to survive or resist drought. Plant drought resistance and ability to survive long periods of low soil water availability should involve leaf phenology coordination with leaf and stem functional traits related to water status. This study tested correlations between phenology and functional traits involved in plant water status regulation in 10 Sonoran Desert tree species with contrasting phenology. Species seasonal variation in plant water status was defined by calculating their relative positions along the iso/anisohydric regulation continuum based on their hydroscape areas (HA)-a metric derived from the relationship between predawn and midday water potentials-and stomatal and hydraulic traits. Additionally, functional traits associated with plant water status regulation, including lamina vessel hydraulic diameter (DHL), stem-specific density (SSD) and leaf mass per area (LMA) were quantified per species. To characterize leaf phenology, leaf longevity (LL) and canopy foliage duration (FD) were determined. Hydroscape area was strongly correlated with FD but not with leaf longevity (LL); HA was significantly associated with SSD and leaf hydraulic traits (DHL, LMA) but not with stem hydraulic traits (vulnerability index, relative conductivity); and FD was strongly correlated with LMA and SSD. Leaf physiological characteristics affected leaf phenology when it was described as canopy FD better than when described as LL. Stem and leaf structure and hydraulic functions were not only relevant for categorizing species along the iso/anisohydric continuum but also allowed identifying different strategies of desert trees within the 'fast-slow' plant economics spectrum. The results in this study pinpoint the set of evolutionary pressures that shape the Sonoran Desert Scrub physiognomy.

Xochiquetzallia (Asparagaceae, Brodiaeoideae), a new genus segregated from the paraphyletic Dandya.

A new genus, Xochiquetzallia, for the Brodiaeoideae, Asparagaceae family is here proposed. A taxonomic analysis based on morphology highlights its synapomorphies. The characters that distinguish Xochiquetzallia are the absence of a pith in the gynophore and the presence of an entire stigma. The recognition of Dandya purpusii as a monotypic genus is supported by the development of a short floral tube (< 2 mm) and a pith in the gynophore, as well as a divided stigma shared with the other genera of the Milla clade, Bessera, Jaimehintonia, Petronymphe and Milla. A key to its taxonomic determination is given for both the Xochiquetzallia species and the Milla clade genera.

Functional anatomy and xylem cavitation resistance of three species of monocotyledons grown on flooded substrates.

There are few investigations that analyze the xylem functional anatomy of monocotyledons, as the methods have been developed for woody plants. This study describes the root, rhizome and aerial stem xylem anatomy and functional anatomy of Canna indica, Cyperus papyrus and Phragmites communis grown on flooded substrates; and it aims to evaluate the relationship between the xylem anatomy and its cavitation resistance. To calculate the indexes of vulnerability, mesomorphy, collapse and relative hydraulic conductivity in the three organs mentioned, the diameter, number of vessels per mm2 , thickness of the walls and the length of the tracheal elements were recorded. In addition, the xylem specific conductivity of the aerial stem was measured with the pipette method, and its resistance to cavitation was determined experimentally by the air injection technique. The protoxylem is xeromorphic, it has longer vessel elements, smaller diameters, thin walls and scalariform perforation plates, whereas the metaxylem is mesomorphic, with shorter vessel elements, larger diameters, thicker walls and simple perforation plates. Both present low collapse resistance but have a high relative hydraulic conductivity. P. communis recorded the highest cavitation resistance, and the number of vessels per mm2 was related to xylem cavitation resistance in Canna indica and Cyperus papyrus. The experimental results of this investigation match partially the anatomical indexes and showed that the xylem of these species has a low specific conductivity and is more vulnerable to cavitation than that of other monocots.

Active pollination drives selection for reduced pollen-ovule ratios.

PREMISE: Variation in pollen-ovule ratios is thought to reflect the degree of pollen transfer efficiency-the more efficient the process, the fewer pollen grains needed. Few studies have directly examined the relationship between pollen-ovule ratio and pollen transfer efficiency. For active pollination in the pollination brood mutualisms of yuccas and yucca moths, figs and fig wasps, senita and senita moths, and leafflowers and leafflower moths, pollinators purposefully collect pollen and place it directly on the stigmatic surface of conspecific flowers. The tight coupling of insect reproductive interests with pollination of the flowers in which larvae develop ensures that pollination is highly efficient. METHODS: We used the multiple evolutionary transitions between passive pollination and more efficient active pollination to test if increased pollen transfer efficiency leads to reduced pollen-ovule ratios. We collected pollen and ovule data from a suite of plant species from each of the pollination brood mutualisms and used phylogenetically controlled tests and sister-group comparisons to examine whether the shift to active pollination resulted in reduced pollen-ovule ratios. RESULTS: Across all transitions between passive and active pollination in plants, actively pollinated plants had significantly lower pollen-ovule ratios than closely related passively pollinated taxa. Phylogenetically corrected comparisons demonstrated that actively pollinated plant species had an average 76% reduction in the pollen-ovule ratio. CONCLUSIONS: The results for active pollination systems support the general utility of pollen-ovule ratios as indicators of pollination efficiency and the central importance of pollen transfer efficiency in the evolution of pollen-ovule ratio.

Differences in the Structural Chemical Composition of the Primary Xylem of Cactaceae: A Topochemical Perspective.

The xylem of Cactaceae is a complex system with different types of cells whose main function is to conduct and store water, mostly during the development of primary xylem, which has vessel elements and wide-band tracheids. The anatomy of primary xylem of Cactaceae has been widely studied, but little is known about its chemical composition. The aim of this study was to determine the structural chemical composition of the primary xylem of Cactaceae and to compare it with the anatomy in the group. Seeds from eight cacti species were used, representing the Pereskioideae, Opuntioideae, and Cactoideae subfamilies. Seeds were germinated and grown for 8 months. Subsequently, only the stem of the seedling was selected, dried, milled, and processed following the TAPPI T-222 om-02 norm; lignin was quantified using the Klason method and cellulose with the Kurshner-Höffer method. Using Fourier transform infrared spectroscopy, the percentage of syringyl and guaiacyl in lignin was calculated. Seedlings of each species were fixed, sectioned, and stained for their anatomical description and fluorescence microscopy analysis for the topochemistry of the primary xylem. The results showed that there were significant differences between species (p < 0.05), except in the hemicelluloses. Through a principal component analysis, it was found that the amount of extractive-free stem and hot water-soluble extractives were the variables that separated the species, followed by cellulose and hemicelluloses since the seedlings developed mainly parenchyma cells and the conductive tissue showed vessel elements and wide-band tracheids, both with annular and helical thickenings in secondary walls. The type of lignin with the highest percentage was guaiacyl-type, which is accumulated mainly in the vessels, providing rigidity. Whereas in the wide-band tracheids from metaxylem, syringyl lignin accumulated in the secondary walls S2 and S3, which permits an efficient flow of water and gives the plant the ability to endure difficult conditions during seedling development. Only one species can be considered to have paedomorphosis since the conductive elements had a similar chemistry in primary and secondary xylem.

Variability and composition of calcium oxalate crystals in embryos-seedlings-adult plants of the globose cacti Mammillaria uncinata.

The morphology and elemental composition of crystals in embryos, seedlings and adult plants of the globose cacti Mammillaria uncinata were studied. Samples of mature fruits and adult plants were collected. To obtain embryos and seedlings, seeds from mature fruits were germinated under laboratory conditions. Both embryos and seedlings as well as stem and root of the adult plants were processed by conventional microtechniques and tissue macerations to isolate the individual crystals. The crystal morphology was evaluated by light and scanning electron microscopy while its chemical composition was analyzed by energy dispersive spectroscopy, IR and Raman spectroscopy. The results demonstrated the occurrence of calcium oxalate crystals in the three stages of plant growth. Solitary and small crystals are present in embryos and seedlings in contrast crystals are aggregates or conglomerates in the adult plants. The compositional analysis showed that seedling crystals contain carbon (50.37%), oxygen (45.29%) and calcium (3.36%) while in adult plants the percentage has changed to carbon (12.54%), oxygen (53.06%) and calcium (34.38%). In the IR spectrum, the vibration bands around 1321 and 1621 cm-1 are attributed to the calcium oxalate in the dihydrate state (weddellite), the Raman peak at 1475 cm-1 shows also that crystals correspond to the pure state of calcium oxalate dihydrate state. The crystal size was also different for seedlings and adult plants, mean values varied from 12.11 to 13.38 µm for width and length, respectively in seedlings and from 65.10 to 73.90 µm, in adult plants. It is concluded that the elemental composition, size and morphology of crystals in M. uncinata depend on the growth stage, as it happens in other plant species.

Structural Characterization of Lignin in Four Cacti Wood: Implications of Lignification in the Growth Form and Succulence.

Wood lignin composition strongly depends on anatomical features and it has been used as a marker for characterizing major plant groups. Wood heterogeneity in Cactaceae is involved in evolutionary and adaptive processes within this group; moreover, it is highly correlated to the species growth form. Here we studied the lignin structure from different types of woods in four Cactaceae species with different stem morphologies (Pereskia lychnidiflora, tree/fibrous wood; Opuntia streptacantha and Pilosocereus chrysacanthus, tree/succulent fibrous wood; Ferocactus hamatacanthus, cylindrical stem/dimorphic wood) in order to determine their relationship with the wood anatomy in an evolutionary-adaptive context. Dioxane lignin was isolated and analyzed by pyrolysis coupled with gas chromatography and mass spectrometry (Py-GC/MS), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). The main linkages are the ß-O-4' ether (67-85%), the ß-ß' resinol (10-26%) and the ß-5' and α-O-4' linkages of the phenylcoumaran structures (≤7%). Spirodienone structures have a considerable abundance (5%) in the dimorphic wood of F. hamatacanthus. In addition, low contents (≤3%) of α,ß-diaryl ether, α-oxidized ß-O-4' ether and dibenzodioxocin structures were found. The sinapyl- and coniferyl acetates are not part of the wood lignin in any of the studied species. The low (≤5%) γ-acetylation in the F. hamatacanthus and P. chrysacanthus wood lignin is here interpreted as an evidence of a high specialization of the wood elements in the conduction/storage of water. The lignin of the studied Cactaceae is composed predominantly of guaiacyl and syringyl units (S/G: 0.9-16.4). High abundance of syringyl units (62-94%) in three of the four species is considered as a defense mechanism against oxidative agents, it is a very conspicuous trait in the most succulent species with dimorphic wood. Furthermore, it is also associated with ferulates and the herein called γ-acetylated guaiacyl-syringaresinol complexes acting as nucleation sites for lignification and as cross-links between lignin and carbohydrates at the wide-band tracheid-fiber junctions.

Incorporating fossils into the Pinaceae tree of life.

PREMISE OF THE STUDY: Pinaceae have a rich but enigmatic early fossil record, much of which is represented by permineralized seed cones. Our incomplete knowledge of morphology and anatomy in living and extinct species poses an important barrier to understanding their phylogenetic relationships and timing of diversification. METHODS: We expanded a morphology matrix to 46 fossil and 31 extant Pinaceae species, mainly adding characters from stem and leaf anatomy and seed cones. Using parsimony and Bayesian inference, we compared phylogenetic relationships for extant taxa with and without fossils from the morphology matrix combined with an alignment of plastid gene sequences. KEY RESULTS: Combined analysis of morphological and molecular characters resulted in a phylogeny of extant Pinaceae that was robust at all nodes except those relating to the interrelationships of Pinus, Picea, and Cathaya and the position of Cedrus. Simultaneous analysis of all fossil and extant species did not result in changes in the relationships among the extant species but did greatly reduce branch support. We found that the placement of most fossils was sensitive to the method of phylogenetic reconstruction when analyzing them singly with the extant species. CONCLUSIONS: A robust phylogenetic hypothesis for the main lineages of Pinaceae is emerging. Most Early Cretaceous fossils are stem or crown lineages of Pinus, but close relationships also were found between fossils and several other extant genera. The phylogenetic position of fossils broadly supports the existence of extant genera in the Lower Cretaceous.