Anatomical features of Fagaceae wood statistically extracted by computer vision approaches: Some relationships with evolution.

The anatomical structure of wood is complex and contains considerable information about its specific species, physical properties, growth environment, and other factors. While conventional wood anatomy has been established by systematizing the xylem anatomical features, which enables wood identification generally up to genus level, it is difficult to describe all the information comprehensively. This study apply two computer vision approaches to optical micrographs: the scale-invariant feature transform algorithm and connected-component labelling. They extract the shape and pore size information, respectively, statistically from the whole micrographs. Both approaches enable the efficient detection of specific features of 18 species from the family Fagaceae. Although the methods ignore the positional information, which is important for the conventional wood anatomy, the simple information on the shape or size of the elements is enough to describe the species-specificity of wood. In addition, according to the dendrograms calculated from the numerical distances of the features, the closeness of some taxonomic groups is inconsistent with the types of porosity, which is one of the typical classification systems for wood anatomy, but consistent with the evolution based on molecular phylogeny; for example, ring-porous group Cerris and radial-porous group Ilex are nested in the same cluster. We analyse which part of the wood structure gave the taxon-specific information, indicating that the latewood zone of group Cerris is similar to the whole zone of group Ilex. Computer vision approaches provide statistical information that uncovers new aspects of wood anatomy that have been overlooked by conventional visual inspection.

Spatiotemporal Coupling of Vessel Cavitation and Discharge of Stored Xylem Water in a Tree Sapling.

Water discharge from stem internal storage compartments is thought to minimize the risk of vessel cavitation. Based on this concept, one would expect that water storage compartments involved in the buffering of xylem tensions empty before the onset of vessel cavitation under drought stress, and potentially refill after soil saturation. However, scant in vivo data exist that elucidate this localized spatiotemporal coupling. In this study on intact saplings of American chestnut (Castanea dentata), x-ray computed microtomography (microCT) showed that the xylem matrix surrounding vessels releases stored water and becomes air-filled either concurrent to or after vessel cavitation under progressive drought stress. Among annual growth rings, the xylem matrix of the current year remained largely water-filled even under severe drought stress. In comparison, microtomography images collected on excised stems showed that applied pressures of much greater than 0 MPa were required to induce water release from the xylem matrix. Viability staining highlighted that water release from the xylem matrix was associated primarily with emptying of dead fibers. Refilling of the xylem matrix and vessels was detected in intact saplings when the canopy was bagged and stem water potential was close to 0 MPa, and in leafless saplings over the winter period. In conclusion, this study indicates that the bulk of water stored in the xylem matrix is released after the onset of vessel cavitation, and suggests that capillary water contributes to overall stem water storage under drought but is not used primarily for the prevention of drought-induced vessel cavitation in this species.

Synchrotron X-ray imaging of a dichasium cupule of Castanopsis from Eocene Baltic amber.

PREMISE OF THE STUDY: The Eocene Baltic amber deposit represents the largest accumulation of fossil resin worldwide, and hundreds of thousands of entrapped arthropods have been recovered. Although Baltic amber preserves delicate plant structures in high fidelity, angiosperms of the "Baltic amber forest" remain poorly studied. We describe a pistillate partial inflorescence of Castanopsis (Fagaceae), expanding the knowledge of Fagaceae diversity from Baltic amber. METHODS: The amber specimen was investigated using light microscopy and synchrotron-radiation-based X-ray micro-computed tomography (SRµCT). KEY RESULTS: The partial inflorescence is a cymule, consisting of an involucre of scales that surround all four pistillate flowers, indicating a dichasium cupule. Subtending bracts are basally covered with peltate trichomes. Flowers possess an urecolate perianth of six nearly free lobes, 12 staminodia hidden by the perianth, and a tri-locular ovary that is convex-triangular in cross section. The exceptional three-dimensional preservation suggests that the fossil belongs to the extant East Asian genus Castanopsis. The amber inclusion represents the first record of Castanopsis from Baltic amber and the first pistillate inflorescence of Fagaceae from Eurasia. CONCLUSIONS: The partial female inflorescence reported here provides an important addition to acorns of Castanopsis described from middle Eocene strata of Europe. Furthermore, the intercontinental distribution of Castanopsis in the Eocene is confirmed. The amber fossil also broadens the picture of the Baltic amber source area, indicating oligotrophic, sandy, bog-like habitats. Finally, this study underscores the great benefit of SRµCT as a powerful tool to investigate plant inclusions from amber in a nondestructive way.

Associated morphometric and geospatial differentiation among 98 species of stone oaks (Lithocarpus).

Two fruit types can be distinguished among stone oaks (Lithocarpus) species: the 'acorn' (AC) and the 'enclosed receptacle' (ER) types. Our morphometric analysis of 595 nuts from 98 species (one third of all Lithocarpus spp.) found substantial transition in mechanical protection of the seed between two woody fruit tissues (exocarp and receptacle) of two fruit types. AC fruits were smaller in seed and fruit size and the thin brittle exocarp largely enclosed the seed, whereas ER fruits were larger and the seed was mostly enclosed by thick woody receptacle tissue. The differences in these two tissues were considerably greater between compared to within fruit type and species. Geospatial distribution showed that seed size of all examined species increased with elevation and decreased with latitude, the physical defense increased with both elevation and latitude, and ER-fruit species were more common at higher elevation. The two fruit types represent distinct suites of associated traits that respond differently to the various biotic and abiotic factors associated with geographic variation, profoundly impacting the evolution of the two fruit types. The co-occurrence of two fruit types in the same forest could be a consequence of distinct fruit and animal interactions.

Estructura poblacional y distribución espacial del roble negro (Colombobalanus excelsa, Fagaceae), especie endémica, en fragmentos de bosque andino colombiano / Population structure and spatial distribution of black oak (Colombobalanus excelsa, Fagaceae), endemic species, in Colombian Andean forest fragments

Resumen Los bosques andinos en Colombia son reconocidos por su alta biodiversidad y endemismo, sin embargo han experimentado un severo proceso de fragmentación debido a procesos antrópicos. Como resultado de esto, muchas especies vulnerables han sido particularmente afectadas. Este es el caso del roble negro (Colombobalanus excelsa (Lozano, Hern. Cam., & Henao) Nixon & Crepet), especie endémica y poco estudiada de los Andes colombianos, cuya distribución está restringida a cuatro localidades en el país, una de ellas el sector suroriental del departamento del Huila. Con el fin de estudiar las poblaciones de roble negro en la Serrania de Peñas Blancas (Huila), localidad más sur de distribución; en 2006 se establecieron 16 parcelas de 50 x 20 m (1.6 ha) en fragmentos de bosque, donde se determinó la estructura etaria y la distribución espacial de esta especie. En total se registraron 1 228 individuos de roble negro entre adultos, juveniles y plántulas; los cuáles presentaron patrones de distribución Gamma y Weibull. Con estos resultados se pudo confirmar que la mayoría de los individuos se encuentran en las primeras clases diamétricas. Asimismo, sus poblaciones presentaron un patrón de distribución agregado debido al proceso de extracción de madera. Estos resultados indican que las poblaciones de roble negro se encuentran en una fase de recuperación despúes de una fuerte intervención antrópica. El conocimiento del actual estado de los bosques de roble negro, puede contribuir para el diseño de efectivos planes de conservacion de la especie en Colombia.

Abstract The Colombian Andean forests are recognized for its great biodiversity and endemism; however, they have undergone a severe process of fragmentation due to anthropic factors. As a result, many vulnerable species have been especially affected. That is the case of the black oak (Colombobalanus excelsa (Lozano, Hern. Cam., & Henao) Nixon & Crepet), an endemic and poorly studied species of the Colombian Andes, which has a distribution restricted to only four areas in the country, one of which is the Southeastern Huila region. To examine black oak populations in Serranía Peñas Blancas (Huila), in 2006 we studied the age structure and spatial distribution in plots of 50 x 20 m (1.6 ha) in forest fragments, in southern distribution. In total, 1 228 black oak species were registered among saplings, juvenile and adults, and they have distribution patterns Gamma and Weibull. These results confirm that the majority of the individuals are in the first diametric class. Also, these populations show an added distribution pattern due to the wood extraction process. These results indicate that black oak populations are in a recovery phase after forceful anthropic intervention. Knowing the current status of black oak forests can contribute to the design of effective conservation plans of this species in Colombia. Rev. Biol. Trop. 66(2): 486-494. Epub 2018 June 01.

Xylem vulnerability to cavitation can be accurately characterised in species with long vessels using a centrifuge method.

Vulnerability to cavitation curves describe the decrease in xylem hydraulic conductivity as xylem pressure declines. Several techniques for constructing vulnerability curves use centrifugal force to induce negative xylem pressure in stem or root segments. Centrifuge vulnerability curves constructed for long-vesselled species have been hypothesised to overestimate xylem vulnerability to cavitation due to increased vulnerability of vessels cut open at stem ends that extend to the middle or entirely through segments. We tested two key predictions of this hypothesis: (i) centrifugation induces greater embolism than dehydration in long-vesselled species, and (ii) the proportion of open vessels changes centrifuge vulnerability curves. Centrifuge and dehydration vulnerability curves were compared for a long- and short-vesselled species. The effect of open vessels was tested in four species by comparing centrifuge vulnerability curves for stems of two lengths. Centrifuge and dehydration vulnerability curves agreed well for the long- and short-vesselled species. Centrifuge vulnerability curves constructed using two stem lengths were similar. Also, the distribution of embolism along the length of centrifuged stems matched the theoretical pressure profile induced by centrifugation. We conclude that vulnerability to cavitation can be accurately characterised with vulnerability curves constructed using a centrifuge technique, even in long-vesselled species.

Genetic transformation of European chestnut somatic embryos with a native thaumatin-like protein (CsTL1) gene isolated from Castanea sativa seeds.

The availability of a system for direct transfer of antifungal candidate genes into European chestnut (Castanea sativa Mill.) would offer an alternative approach to conventional breeding for production of chestnut trees tolerant to ink disease caused by Phytophthora spp. For the first time, a chestnut thaumatin-like protein gene (CsTL1), isolated from chestnut cotyledons, has been overexpressed in three chestnut somatic embryogenic lines. Transformation experiments have been performed using an Agrobacterium tumefaciens Smith and Townsend vector harboring the neomycin phosphotransferase (NPTII) selectable and the green fluorescent protein (EGFP) reporter genes. The transformation efficiency, determined on the basis of the fluorescence of surviving explants, was clearly genotype dependent and ranged from 32.5% in the CI-9 line to 7.1% in the CI-3 line. A total of 126 independent transformed lines were obtained. The presence and integration of chestnut CsTL1 in genomic DNA was confirmed by polymerase chain reaction (PCR) and Southern blot analyses. Quantitative real-time PCR revealed that CsTL1 expression was up to 13.5-fold higher in a transgenic line compared with its corresponding untransformed line. In only one of the 11 transformed lines tested, expression of the CsTL1 was lower than the control. The remaining 115 transformed lines were successfully subjected to cryopreservation. Embryo proliferation was achieved in all of the transgenic lines regenerated and the transformed lines showed a higher mean number of cotyledonary stage embryos and total number of embryos per embryo clump than their corresponding untransformed lines. Transgenic plants were regenerated after maturation and germination of transformed somatic embryos. Furthermore, due to the low plantlet conversion achieved, axillary shoot proliferation cultures were established from partially germinated embryos (only shoot development), which were multiplied and rooted according to procedures already established. Transgenic plants were acclimatized and grown in a greenhouse. No phenotypic differences were found with control plants, suggesting no potential cytotoxic effects of the green fluorescent protein. The results reported in the present work could be considered as a first step toward the production of fungal-disease tolerant cisgenic chestnut plants.

Scaling of tree vascular transport systems along gradients of nutrient supply and altitude.

A recent metabolic scaling theory predicts that plants minimize resistance to hydraulic conduction in the bulk transport network by narrowing the diameter of xylem conduits distally. We hypothesized that trees growing at high altitude or on nutrient-depleted soils would prioritize survival over minimizing hydraulic resistance, and that their vascular systems would be structured differently from those of trees growing under more benign conditions. In fact, conduits were observed to narrow towards the periphery of vascular system within all 45 trees of three species we investigated, and scaling relationships were indistinguishable across a range of environments. Thus, conduit tapering relationships appear to be invariant with respect to environmental conditions.

Earlywood vessels of Castanea sativa record temperature before their formation.

The aim of this study was to identify the climatic signal contained in the earlywood vessel size of the ring-porous chestnut (Castanea sativa) and the physiological processes involved in the underlying mechanisms. In order to assign the encoded signal to a specific physiological process, bud phenology and vessel formation were monitored along an elevation transect and chronologies of the size of the first row of earlywood vessels were retrospectively correlated with 40 yr of early spring temperatures. The first vessels appeared in late April to early May, after encoding both a negative temperature signal in February-March (during tree quiescence) and a positive temperature signal in early April (at the time of resumption of shoot growth). We hypothesize that February and March temperatures affect cambial sensitivity to auxin, preconditioning tree responses later in the season. Furthermore, April temperature is related to tree activation whereby new hormone production fosters vessel expansion.

American Chestnut [Castanea dentata (Marsh.) Borkh].

The key to successful transformation of American chestnut is having the correct combination of explant tissue, selectable and scorable markers, and a reliable regeneration system. Rapidly dividing somatic embryos, growing as proembryogenic masses, are a suitable tissue; the bar gene is a suitable selectable marker in conjunction with 1.0 to 10 mg/L phosphirothricin (PPT); and the mgfp5-ER gene is an effective nondestructive scorable marker. We have also found that the more gently the somatic embryos are treated during the inoculation and co-cultivation steps, the higher the transformation efficiency. The average transformation efficiency that can be expected using the described protocol is approx 20 stable and embryogenic transformation events/g of somatic embryo tissue. Cell line and batch-to-batch deviations both upward and downward should be expected. Finally, somatic embryos can be induced to form shoots, which can then be micropropagated and acclimatized.

Selecting earlywood vessels to maximize their environmental signal.

The anatomical features of earlywood vessels often reflect information about past climatic conditions. We examined the relationships between mean monthly temperature and mean vessel lumen area (MVA) in various categories of earlywood vessels. Subsets of earlywood vessels of chestnut (Castanea sativa Mill.) were selected from a previously reported dataset based on several progressive size-related procedures. To include all earlywood vessels, the minimum size considered was 10,000 microm2. Changes in the correlations between MVA and the mean air temperature in March are described and discussed. The results show that not all vessels embody the same information. The MVA of a proportion of the largest earlywood vessels in each annual ring was most closely related to March temperature, whereas MVA of the smallest earlywood vessels was better correlated with June temperature. This difference is probably a result of the vessels being formed at different times: early spring for the largest earlywood vessels and later in spring for the smallest earlywood vessels. Analyses combining large and small vessels yielded lower correlations between MVA and monthly temperature. The number, size and distribution of vessels can vary greatly from ring to ring. In making year-to-year comparisons, the best information is provided by observations on vessels of contemporaneous ontogenesis. Criteria for the selection and analysis of vessels in the assessment of temperature during the season of wood formation are proposed and discussed.

Shoot life span in relation to successional status in deciduous broad-leaved tree species in a temperate forest.

In trees, leaf life span is closely related to successional status. Although leaves are attached to shoots, shoot life span has been insufficiently studied in the context of ecological systems. Interspecific variation in shoot survivorship was investigated over 27 months in 15 temperate hardwood tree species. Relationships between shoot architecture and shoot survival were also investigated. Shoot life span was shortest in early successional species, and longest in late successional species, in each of the families Betulaceae and Fagaceae. In Salicaceae, all of which were early successional species, shoot life span was longer in mountainous than in riparian species. Early successional or riparian species distributed longer shoots densely, even in proximal positions on mother shoots, resulting in mutual shading and consequent early and massive shoot shedding. By contrast, late successional or mountainous species concentrated shoots in distal positions, allowing shoots to receive equally favorable light, resulting in a longer life span. These results reveal close relationships between shoot life span and environmental resource availability or successional status and suggest a causal relationship between shoot shedding and shoot architecture.

Geographic occurrence of intermediate type between Castanopsis sieboldii and C. cuspidata (Fagaceae) based on the structure of leaf epidermis.

The number of layers of epidermis in the leaves is used as a criterion to distinguish between Castanopsis sieboldii (two layers) and C. cuspidata (one layer). An intermediate type, which has one and two layers within a single leaf, is frequently seen in the field. The origin of the intermediate type has been supposed to be a hybrid between C. sieboldii and C. cuspidata. If the intermediate type is produced by hybridization, we expect that the F(1) seedlings of the intermediate type should occur in the co-occurrence area of those two species. To clarify the geographic occurrence of the intermediate type, we collected nuts of 443 mother trees from throughout the distribution area of the genus Castanopsis in Japan. A total of 7,260 seedlings germinated from these nuts were examined as to their leaf structure. The seedlings of the intermediate type occurred not only in the area where C. sieboldii and C. cuspidata coexisted, but also in the area where only C. sieboldii grows. The leaf structure of intermediate seedlings was independent of the traits of mother trees. These findings suggest that the intermediate seedlings that occurred in the area where only C. sieboldii grows are not hybrid between C. sieboldii and C. cuspidata but are a morphological variation of C. sieboldii. The difference in the structure of leaf epidermis is not always appropriate for the identification of the hybrid.

Combining and comparing morphometric shape descriptors with a molecular phylogeny: the case of fruit type evolution in Bornean Lithocarpus (Fagaceae).

Fruit type in the genus Lithocarpus (Fagaceae) includes both classic oak acorns and novel modifications. Bornean taxa with modified fruits can be separated into two sections (Synaedrys and Lithocarpus) based on subtle shape differences. By following strict criteria for homology and representation, this variation in shape can be captured and the sections distinguished by using elliptic Fourier or eigenshape analysis. Phenograms of fruit shape, constructed by using restricted maximum likelihood techniques and these morphometric descriptors, were incorporated into combined and comparative analyses with molecular sequence data from the internal transcribed spacer (ITS) region of the nuclear rDNA, using branch-weighted matrix representation. The combined analysis strongly suggested independent derivation of the novel fruit type in the two sections from different acornlike ancestors, while the comparative analysis indicated frequent decoupling between the molecular and morphological changes as inferred at well-supported nodes. The acorn fruit type has undergone little modification between ingroup and outgroup, despite large molecular distance. Greater morphological than molecular change was inferred at critical transitions between acorn and novel fruit types, particularly for section Lithocarpus. The combination of these two different types of data improved our understanding of the macroevolution of fruit type in this difficult group, and the comparative analysis highlighted the significant incongruities in evolutionary pattern between the two datasets.