Adaptation of stomatal conductance, photosynthesis and water-use efficiency at shoot and canopy scales in adjacent stands of Dacrycarpus dacrydioides and Podocarpus totara.

We tested an approach to estimate daily canopy net photosynthesis, A, based on estimates of transpiration, E, using measurements of sap flow and water-use efficiency, ω, by measuring δ13C in CO2 respired from shoots in the canopies of two conifers (Podocarpaceae) native to New Zealand. The trees were planted in adjacent 20-year-old stands with the same soil and environmental conditions. Leaf area index was lower for Dacrycarpus dacrydioides D.Don in Lamb (1.34 m2 m-2) than for Podocarpus totara G.Benn. ex D.Don var. totara (2.01 m2 m-2), but mean (± standard error) stem diameters were the same at 152 ± 21 mm for D. dacrydioides and 154 ± 25 mm for P. totara. Over a 28-day period, daily A (per unit ground area) ranged almost five-fold but there were no significant differences between species (mean 2.73 ± 1.02 gC m-2 day-1). This was attributable to higher daily values of E (2.63 ± 0.83 mm day-1) and lower ω (1.35 ± 0.53 gC kg H2O-1) for D. dacrydioides compared with lower E (1.82 ± 0.72 mm day-1) and higher ω (1.90 ± 0.77 gC kg H2O-1) for P. totara. We attributed this to higher nitrogen availability and nitrogen concentration per unit foliage area, Na, and greater exposure to irradiance in the D. dacrydioides canopy compared with P. totara. Our findings support earlier observations that D. dacrydioides is more adapted to sites with poor drainage. In contrast, the high retention of leaf area and maintaining low rates of transpiration by P. totara, resulting in higher water-use efficiency, is an adaptive response to survival in dry conditions. Our findings show that physiological adjustments for two species adapted to different environments led to similar canopy photosynthesis rates when the trees were grown in the same conditions. We demonstrated consistency between whole-tree and more intensive shoot-scale measurements, confirming that integrated approaches are appropriate for comparative estimates of carbon uptake in stands with different species.

A new fossil Acmopyle with accessory transfusion tissue and potential reproductive buds: Direct evidence for ever-wet rainforests in Eocene Patagonia.

PREMISE: Acmopyle (Podocarpaceae) comprises two extant species from Oceania that are physiologically restricted to ever-wet rainforests, a confirmed fossil record based on leaf adpressions and cuticles in Australia since the Paleocene, and a few uncertain reports from New Zealand, Antarctica, and South America. We investigated fossil specimens with Acmopyle affinities from the early Eocene Laguna del Hunco site in Patagonia, Argentina. METHODS: We studied 42 adpression leafy-shoot fossils and included them in a total evidence phylogenetic analysis. RESULTS: Acmopyle grayae sp. nov. is based on heterophyllous leafy shoots with three distinct leaf types. Among these, bilaterally flattened leaves uniquely preserve subparallel, linear features that we interpret as accessory transfusion tissue (ATT, an extra-venous water-conducting tissue). Some apical morphologies of A. grayae shoots are compatible with the early stages of ovuliferous cone development. Our phylogenetic analysis recovers the new species in a polytomy with the two extant Acmopyle species. We report several types of insect-herbivory damage. We also transfer Acmopyle engelhardti from the middle Eocene Río Pichileufú flora to Dacrycarpus engelhardti comb. nov. CONCLUSIONS: We confirm the biogeographically significant presence of the endangered West Pacific genus Acmopyle in Eocene Patagonia. Acmopyle is one of the most drought-intolerant genera in Podocarpaceae, possibly due to the high collapse risk of the ATT, and thus the new fossil species provides physiological evidence for the presence of an ever-wet rainforest environment at Laguna del Hunco during the Early Eocene Climatic Optimum.

Plastome sequencing of South American Podocarpus species reveals low rearrangement rates despite ancient gondwanan disjunctions.

BACKGROUND: Historical reconstructions within Podocarpaceae have provided valuable information to disentangle biogeographic scenarios that begun 65 Mya. However, early molecular phylogenies of Podocarpaceae failed to agree on the intergeneric relationships within the family. The aims of this study were to test whether plastome organization is stable within the genus Podocarpus, to estimate the selective regimes affecting plastome protein-coding genes, and to strengthen our understanding of the phylogenetic relationships and biogeographic history. METHODS AND RESULTS: We sequenced the plastomes of four South American species from Patagonia, southern Yungas, and Brazilian subtropical forests. We compared their plastomes to those published from Brazil, Africa, New Zealand, and Southeast Asia, along with representatives from other genera within Podocarpaceae as outgroups. The four newly sequenced plastomes ranged in size between 133,791 bp and 133,991 bp. Gene content and order among chloroplasts from South American, African and Asian Podocarpus were conserved and different from the plastome of P. totara, from New Zealand. Most genes showed substitution patterns consistent with a conservative selective regime. Phylogenies inferred from either complete sequences or protein coding regions were mostly congruent with previous studies, but showed earlier branching of P. salignus, P. totara and P. sellowii. CONCLUSIONS: Highly similar and conserved plastomes of African, South American and Asian species suggest that P. totara plastome should be revised and compared to other species from Oceanic distribution. Furthermore, given such structural conservation, we suggest plastome sequencing is not useful to test whether genomic order can be climatically or geologically structured.

Demographic modelling helps track the rapid and recent divergence of a conifer species pair from Central Mexico.

Secondary contact of recently diverged species may have several outcomes, ranging from rampant hybridization to reinforced reproductive isolation. In plants, selfing tolerance and disjunct reproductive phenology may lead to reproductive isolation at contact zones. However, they may also evolve under both allopatric or parapatric frameworks and originate from adaptive and/or neutral forces. Inferring the historical demography of diverging taxa is thus a crucial step to identify factors that may have led to putative reproductive isolation. We explored various competing demographypotheses to account for the rapid divergence of a fir species complex (Abies flinckii-A. religiosa) distributed in "sky-islands" across central Mexico (i.e., along the Trans-Mexican Volcanic Belt; TMVB). Despite co-occurring in two independent sympatric regions (west and centre), these taxa rarely interbreed because of disjunct reproductive phenologies. We genotyped 1147 single nucleotide polymorphisms, generated by GBS (genotyping by sequencing), across 23 populations, and compared multiple scenarios based on the geological history of the TMVB. The best-fitting model revealed one of the most rapid and complete speciation cases for a conifer species-pair, dating back to ~1.2 million years ago. Coupled with the lack of support for stepwise colonization, our coalescent inferences point to an early cessation of interspecific gene flow under parapatric speciation; ancestral gene flow during divergence was asymmetrical (mostly from western firs into A. religiosa) and exclusive to the most ancient (i.e., central) contact zone. Factors promoting rapid reproductive isolation should be explored in other slowly evolving species complexes as they may account for the large tropical and subtropical diversity.

Proteomic Analysis of S-Nitrosation Sites During Somatic Embryogenesis in Brazilian Pine, Araucaria angustifolia (Bertol.) Kuntze.

Cysteine S-nitrosation is a redox-based post-translational modification that mediates nitric oxide (NO) regulation of various aspects of plant growth, development and stress responses. Despite its importance, studies exploring protein signaling pathways that are regulated by S-nitrosation during somatic embryogenesis have not been performed. In the present study, endogenous cysteine S-nitrosation site and S-nitrosated proteins were identified by iodo-TMT labeling during somatic embryogenesis in Brazilian pine, an endangered native conifer of South America. In addition, endogenous -S-nitrosothiol (SNO) levels and S-nitrosoglutathione reductase (GSNOR) activity were determined in cell lines with contrasting embryogenic potential. Overall, we identified an array of proteins associated with a large variety of biological processes and molecular functions with some of them already described as important for somatic embryogenesis (Class IV chitinase, pyruvate dehydrogenase E1 and dehydroascorbate reductase). In total, our S-nitrosoproteome analyses identified 18 endogenously S-nitrosated proteins and 50 in vitro S-nitrosated proteins (after GSNO treatment) during cell culture proliferation and embryo development. Furthermore, SNO levels and GSNOR activity were increased during embryo formation. These findings expand our understanding of the Brazilian pine proteome and shed novel insights into the potential use of pharmacological manipulation of NO levels by using NO inhibitors and donors during somatic embryogenesis.

Coalescent-based species delimitation in North American pinyon pines using low-copy nuclear genes and plastomes.

PREMISE: Accurate species delimitation is essential for evolutionary biology, conservation, and biodiversity management. We studied species delimitation in North American pinyon pines, Pinus subsection Cembroides, a natural group with high levels of incomplete lineage sorting. METHODS: We used coalescent-based methods and multivariate analyses of low-copy number nuclear genes and nearly complete high-copy number plastomes generated with the Hyb-Seq method. The three coalescent-based species delimitation methods evaluated were the Generalized Mixed Yule Coalescent (GMYC), Poisson Tree Process (PTP), and Trinomial Distribution of Triplets (Tr2). We also measured admixture in populations with possible introgression. RESULTS: Our results show inconsistencies among GMYC, PTP, and Tr2. The single-locus based GMYC analysis of plastid DNA recovered a higher number of species (up to 24 entities, including singleton lineages and clusters) than PTP and the multi-locus coalescent approach. The PTP analysis identified 10 species whereas Tr2 recovered 13, which agreed closely with taxonomic treatments. CONCLUSIONS: We found that PTP and GMYC identified species with low levels of ILS and high morphological divergence (P. maximartinezii, P. pinceana, and P. rzedowskii). However, GMYC method oversplit species by identification of more divergent samples as singletons. Moreover, both PTP and GMYC were incapable of identifying some species that are readily identified morphologically. We suggest that the divergence times between lineages within North American pinyon pines are so disparate that GMYC results are unreliable. Results of the Tr2 method coincided well with previous delimitations based on morphology, DNA, geography, and secondary chemistry.

Building an embryo: An auxin gene toolkit for zygotic and somatic embryogenesis in Brazilian pine.

Many studies in the model species Arabidopsis thaliana characterized genes involved in embryo formation. However, much remains to be learned about the portfolio of genes that are involved in signal transduction and transcriptional regulation during plant embryo development in other species, particularly in an evolutionary context, especially considering that some genes involved in embryo patterning are not exclusive of land plants. This study, used a combination of domain architecture phylostratigraphy and phylogenetic reconstruction to investigate the evolutionary history of embryo patterning and auxin metabolism (EPAM) genes in Viridiplantae. This approach shed light on the co-optation of auxin metabolism and other molecular mechanisms that contributed to the radiation of land plants, and specifically to embryo formation. These results have potential to assist conservation programs, by directing the development of tools for obtaining somatic embryos. In this context, we employed this methodology with critically endangered and non-model species Araucaria angustifolia, the Brazilian pine, which is current focus of conservation efforts using somatic embryogenesis. So far, this approach had little success since somatic embryos fail to completely develop. By profiling the expression of genes that we identified as necessary for the emergence of land-plant embryos, we found striking differences between zygotic and somatic embryos that might explain the developmental arrest and be used to improve A. angustifolia somatic culture.

A Talk between Flavonoids and Hormones to Reorient the Growth of Gymnosperms.

Plants reorient the growth of affected organs in response to the loss of gravity vector. In trees, this phenomenon has received special attention due to its importance for the forestry industry of conifer species. Sustainable management is a key factor in improving wood quality. It is of paramount importance to understand the molecular and genetic mechanisms underlying wood formation, together with the hormonal and environmental factors that affect wood formation and quality. Hormones are related to the modulation of vertical growth rectification. Many studies have resulted in a model that proposes differential growth in the stem due to unequal auxin and jasmonate allocation. Furthermore, many studies have suggested that in auxin distribution, flavonoids act as molecular controllers. It is well known that flavonoids affect auxin flux, and this is a new area of study to understand the intracellular concentrations and how these compounds can control the gravitropic response. In this review, we focused on different molecular aspects related to the hormonal role in flavonoid homeostasis and what has been done in conifer trees to identify molecular players that could take part during the gravitropic response and reduce low-quality wood formation.

Interacciones potenciales parásito-hospedero entre el escarabajo Dendroctonus (Coleoptera: Scolytidae) y Pinus (Pinaceae) en México / Potential parasite-host interactions between Dendroctonus (Coleoptera: Scolytidae) beetle and Pinus (Pinaceae) in Mexico

Resumen Introducción: La relación parásito-hospedero entre Dendroctonus y Pinus es reconocida como una amenaza importante para los bosques de coníferas, debido a que puede causar una alta mortalidad de árboles y consecuentemente una pérdida de cobertura forestal. Objetivo: Identificar potenciales interacciones parásito-huésped entre descortezadores y pinos, así como la contribución de las condiciones ambientales para el establecimiento de estas interacciones ecológicas. Métodos: Se realizaron modelos de nicho ecológico y análisis de redes ecológicas con la plataforma web de minería de datos espaciales SPECIES (http://species.conabio.gob.mx/). Se incluyeron siete especies de descortezadores, 52 de pinos y 19 variables bioclimáticas del portal WorldClim. Resultados: Se identificaron 140 interacciones potenciales, de las cuales el 42 % no han sido registradas previamente. El análisis de redes ecológicas nos permitió caracterizar las relaciones parásito-hospedero en generalistas y especialistas. En cuanto al análisis de nicho ecológico, se determinó la distribución potencial de los descortezadores combinando las distribuciones de Pinus y variables climáticas como predictores. Se observó que las variables climáticas contribuyen de forma negativa para la mayoría de las especies de Dendroctonus, es decir, estas variables son restrictivas en casi toda su distribución, por el contrario, las variables bióticas (Pinus) fueron positivas e informativas sobre la mayoría de las distribuciones. Conclusiones: Los patrones de coexistencia de descortezadores y pinos nos proporcionan información sobre las interacciones que se pueden establecer entre estas especies y que hasta hoy pueden ser desconocidas. Además, la integración de factores bióticos (hospederos) y abióticos (clima), permite obtener modelos de distribución geográfica que caracteriza las regiones con condiciones favorables para la presencia de las especies, así como las especies de hospederos con los que podrían estar interactuando en dichas regiones. Considerando el riesgo fitosanitario por descortezadores, nuestros resultados brindan escenarios geográficos y ecológicos de riesgo donde infestaciones por descortezadores podrían darse, así como la posible emergencia de nuevas interacciones parásito-hospedero desconocidas hasta hoy. Estos modelos son una herramienta que en el futuro pueden dirigir esfuerzos de trabajo de campo para validar y complementar nuestros resultados.

Abstract Introduction: The parasite-host relationship between bark beetles of the genus Dendroctonus and species of the genus Pinus has been recognized as important threat to coniferous forests because it can cause high tree mortality and therefore a loss of forests. Objectives: We aimed to identify the potential unknown parasite-host relationships among bark beetles and pine trees species, as well as the contribution of environmental conditions as driver of these ecological interactions. Methods: We carried out ecological niche models and ecological network analysis using the spatial datamining platform SPECIES (http://species.conabio.gob.mx/). Our study included seven species of bark beetles, 52 species of the genus Pinus, and 19 bioclimatic variables from Worldclim dataset. Results: We identified 140 potential interactions between Dendroctonus and Pinus species, of which 42 % have not been previously registered. Complex inference network analysis allowed us to characterize ecological parasite-host interactions in generalists and specialists. Regarding the ecological niche analysis, we determined potential bark beetle distributions by combining Pinus species ranges and climatic variables as predictors. We noted that climatic variables contributed negatively to Dendroctonus distribution for the most species, i.e. these abiotic variables are restrictive in almost the entire distribution of the beetles; conversely, biotic variables (i.e. Pinus species) were positive and informative on the most of species ranges. Conclusions: Coexistence patterns of bark beetles and Pinus provide us information on the interactions that are able to establish among these species and that until now can be unknown. In addition, integrating biotic (hosts) and abiotic (climate) factors, allows us to obtain geographic distribution models that characterize the regions with favorable conditions for the presence of bark beetles, as well as the host species with which they could be interacting in those regions. Considering the phytosanitary risk due to bark beetles, our results provide geographic and ecological scenarios where bark beetles infestations may occur, as well as the possible emergence of new parasite-host interactions. These models can be a tool to address future fieldwork efforts to validate and complement our results.

Eocene Araucaria Sect. Eutacta from Patagonia and floristic turnover during the initial isolation of South America.

PREMISE: Eocene floras of Patagonia document biotic response to the final separation of Gondwana. The conifer genus Araucaria, distributed worldwide during the Mesozoic, has a disjunct extant distribution between South America and Australasia. Fossils assigned to Australasian Araucaria Sect. Eutacta usually are represented by isolated organs, making diagnosis difficult. Araucaria pichileufensis E.W. Berry, from the middle Eocene Río Pichileufú (RP) site in Argentine Patagonia, was originally placed in Sect. Eutacta and later reported from the early Eocene Laguna del Hunco (LH) locality. However, the relationship of A. pichileufensis to Sect. Eutacta and the conspecificity of the Araucaria material among these Patagonian floras have not been tested using modern methods. METHODS: We review the type material of A. pichileufensis alongside large (n = 192) new fossil collections of Araucaria from LH and RP, including multi-organ preservation of leafy branches, ovuliferous complexes, and pollen cones. We use a total evidence phylogenetic analysis to analyze relationships of the fossils to Sect. Eutacta. RESULTS: We describe Araucaria huncoensis sp. nov. from LH and improve the whole-plant concept for Araucaria pichileufensis from RP. The two species respectively resolve in the crown and stem of Sect. Eutacta. CONCLUSIONS: Our results confirm the presence and indicate the survival of Sect. Eutacta in South America during early Antarctic separation. The exceptionally complete fossils significantly predate several molecular age estimates for crown Eutacta. The differentiation of two Araucaria species demonstrates conifer turnover during climate change and initial South American isolation from the early to middle Eocene.

Updating embryonic ontogenesis in Araucaria angustifolia: from Burlingame (1915) to the present.

This study addresses gaps in our understanding of pre-fertilization and archegonia development and reinterprets embryonic ontogenesis from Burlingame (Bot Gaz 59:1-39, 1915) to the present based on timescale and structural features allowing us to determine functionally and developmentally accurate terminology for all these stages in A. angustifolia. Different from previous reports, only after pollination, pre-fertilization tissue development occurs (0-13 months after pollination (MAP)) and gives rise to a mature megagametophyte. During all this period, pollen is in a dormant state at the microphyla, and pollen tube germination in nucellus tissue is only observed at the stage of archegonia formation (13 MAP) and not at the free nuclei stage as reported before. For the first time, 14 months after pollination, a fertilization window was indicated, and at 15 MAP, the polyzygotic polyembryony from different archegonia was also seen. After that, subordinated proembryo regression occurs and at least three embryonic developmental stages of dominant embryo were characterized: proembryogenic, early embryogenic, and late embryogenic (15-23 MAP). Along these stages, histochemical and ultrastructural analyses suggest the occurrence of cell death in suspensor and in cap cells of dominant embryo that was not previously reported. The differentiation of meristems, procambium, pith, and cortex tissues in late embryogenic stage was detailed. The morphohistological characterization of pre-fertilization and embryonic stages, together with the timescale of megastrobili development, warranted a referential map of female reproductive structure in this species.

Growth, wood anatomy and stable isotopes show species-specific couplings in three Mexican conifers inhabiting drought-prone areas.

An improved understanding of how tree species will respond to warmer conditions and longer droughts requires comparing their responses across different environmental settings and considering a multi-proxy approach. We used several traits (tree-ring width, formation of intra-annual density fluctuations - IADFs, wood anatomy, Δ13C and δ18O records) to retrospectively quantify these responses in three conifers inhabiting drought-prone areas in northwestern Mexico. A fir species (Abies durangensis) was studied in a higher altitude and slightly rainier site and two pine species were sampled in a nearby, lower drier site (Pinus engelmannii, Pinus cembroides). Tree-ring-width indices (TRWi) of the studied species showed a very similar year-to-year variability likely indicating a common climatic signal. Wood anatomy analyses done over 3.5 million measured cells, showed that P. cembroides lumen area was much smaller than in the other two species and it remained constant along all the studied period (over 64 years). Instead, cell wall thickness was widest in P. engelmannii and this species presented the highest amount of intra-annual density fluctuations. Climate and wood anatomy correlations pointed out that lumen area was positively affected by winter precipitation for all studied species, while cell-wall thickness was negatively affected by this season's precipitation in all species but P. cembroides. Stable isotope analysis showed significantly lower values of Δ13C for P. cembroides and no significant δ18O differences between the three species, although they shared a common decreasing trend. With very distinct wood anatomical traits (smaller cells, compact morphology), P. cembroides stood out as the better adapted species in its current environment and could be less affected by future drier climate. P. engelmannii and A. durangensis showed high plasticity at wood anatomical level, allowing them to promptly respond to seasonal water availability but likely gives few advantages on future climate scenarios with longer and frequent drought spells.

Elemental composition and nutritional value of Araucaria angustifolia seeds from subtropical Brazil.

Consumed by populations in South America, Araucaria angustifolia seeds have received little study regarding elemental composition and nutritional value. Thirty-five seed sites from subtropical Brazil were sampled and seed concentrations of C, N, K, Ca, Mg, P, Fe, Zn, Mn, Cu, Mo, Ni, Co, Cr, Ba, and Cd were determined. The highest concentration of N was observed in samples from regions with Cfa climate (humid subtropical, oceanic climate, without dry season with hot summer) and igneous rock, which was superior to regions with Cfb climate (humid subtropical, oceanic climate, without dry season with temperate summer) and metamorphic rock. Seeds can be a source of nutrients: K (11.8 g kg-1), P (4.1 g kg-1), Mn (9.1 mg kg-1), Cu (7.2 mg kg-1), Mo (0.93 mg kg-1), and Cr (0.65 mg kg-1). Values for Ba (0.93 mg kg-1) and Cd (0.19 mg kg-1) indicated no risk to human health. This study expands knowledge regarding the elemental composition of A. angustifolia. Results indicate that these seeds have nutritional value, and their consumption can be a good strategy to improve overall human nutrition in this region of South America.

Review of the Nearctic (north of Mexico) species of Elacatis Pascoe (Coleoptera: Salpingidae: Othniinae).

The North American (north of Mexico) species of Elacatis were revised, based on external and genitalic structures of adults. Seven species are recognized, though the historical inclusion of E. fasciatus Bland among Nearctic species is very likely based on an erroneous collecting locality. Two new species are described, with type localities (counties only) in parentheses: E. larsoni (Nebraska: Box Butte County) and E. stephani (Arizona: Cochise County). The following new synonym is proposed: Othnius umbrosus LeConte 1861 = Othnius lugubris Horn 1868; therefore, only E. umbrosus (LeConte) is associated with dead/dying conifers in western North America. Larval E. umbrosus are thought to be xylophagous, while adults are very likely predaceous. Elacatis senecionis (Champion) and E. immaculatus (Champion) are recorded from north of Mexico for the first time. A lectotype is designated for Elacatis longicornis Horn. A key to the seven species in Canada and the United States is provided, supplemented with photographic images of habiti and selected structural features. Maps of known distributions, based on geo-referenced locality lists, are provided.

Single-provenance mature conifers show higher non-structural carbohydrate storage and reduced growth in a drier location.

Since growth is more sensitive to drought than photosynthesis, trees inhabiting dry regions are expected to exhibit higher carbohydrate storage and less growth than their conspecifics from more humid regions. However, the same pattern can be the result of different genotypes inhabiting contrasting humidity conditions. To test if reduced growth and high carbohydrate storage are environmentally driven by drought, we examined the growth and non-structural carbohydrate (NSC) concentrations in single-provenance stands of mature trees of Pinus contorta Douglas and Pinus ponderosa Douglas ex C. Lawson planted at contrasting humidity conditions (900 versus 300 mm of annual precipitation) in Patagonia, Chile. Individual tree growth was measured for each species and at each location as mean basal area increment of the last 10 years (BAI10), annual shoot elongation for the period 2011-14, and needle length for 2013 and 2014 cohorts. Additionally, needle, branch, stem sapwood and roots were collected from each sampled tree to determine soluble sugars, starch and total NSC concentrations. The two species showed lower mean BAI10 and 2013 needle length in the dry site; P. ponderosa also had lower annual shoot extension for 2011 and 2014, and lower 2014 needle length, in the dry than in the mesic site. By contrast, NSC concentrations of all woody tissues for both species were either similar or higher in the dry site when compared with the mesic site. Patterns of starch and sugars were substantially different: starch concentrations were similar between sites except for roots of P. ponderosa, which were higher in the dry site, while sugar concentrations of all woody tissues in both species were higher in the dry site. Overall, our study provides evidence that reduced growth along with carbon (C) accumulation is an environmentally driven response to drought. Furthermore, the significant accumulation of low-molecular weight sugars in the dry site is compatible with a prioritized C allocation for osmoregulation. However, since this accumulation did not come at the expense of reduced starch, it is unlikely that growth was limited by C supply in the dry site.

The fossil flip-leaves (Retrophyllum, Podocarpaceae) of southern South America.

PREMISE OF THE STUDY: The flip-leaved podocarp Retrophyllum has a disjunct extant distribution in South American and Australasian tropical rainforests and a Gondwanic fossil record since the Eocene. Evolutionary, biogeographic, and paleoecological insights from previously described fossils are limited because they preserve little foliar variation and no reproductive structures. METHODS: We investigated new Retrophyllum material from the terminal Cretaceous Lefipán, the early Eocene Laguna del Hunco, and the early/middle Eocene Río Pichileufú floras of Patagonian Argentina. We also reviewed type material of historical Eocene fossils from southern Chile. KEY RESULTS: Cretaceous Retrophyllum superstes sp. nov. is described from a leafy twig, while Eocene R. spiralifolium sp. nov. includes several foliage forms and a peduncle with 13 pollen cones. Both species preserve extensive damage from sap-feeding insects associated with foliar transfusion tissue. The Eocene species exhibits a suite of characters linking it to both Neotropical and West Pacific Retrophyllum, along with several novel features. Retrophyllum araucoensis (Berry) comb. nov. stabilizes the nomenclature for the Chilean fossils. CONCLUSIONS: Retrophyllum is considerably older than previously thought and is a survivor of the end-Cretaceous extinction. Much of the characteristic foliar variation and pollen-cone morphology of the genus evolved by the early Eocene. The mixed biogeographic signal of R. spiralifolium supports vicariance and represents a rare Neotropical connection for terminal-Gondwanan Patagonia, which is predominantly linked to extant Australasian floras due to South American extinctions. The leaf morphology of the fossils suggests significant drought vulnerability as in living Retrophyllum, indicating humid paleoenvironments.

Evaluación de la regeneración natural de tres especies coníferas en áreas de distribución natural en el altiplano occidental de Guatemala / Evaluation of natural regeneration of coniferous species in areas of natural distribution in the western highlands of Guatemala

Los bosques de coníferas del altiplano occidental de Guatemala han sido perturbados por intervenciones humanasy fenómenos naturales, lo cual ha propiciado la ocurrencia del fenómeno de regeneración natural. En esteescenario se evaluó la densidad de la regeneración natural de tres especies coníferas (Pinus oocarpa Schiede, P.pseudostrobus Lindl. y P. tecunumanii Eguiluz & Perry), en 60 sitios distribuidos en espacios de borde y claro. Las variables evaluadas fueron: fuente de disturbio que originó su establecimiento, variación de la densidad respecto de la fuente de semilla y estimación de la distancia de alcance de dispersión de regeneración adecuada, utilizando unidades de muestreo de 25 m2 para regeneración natural y 500 m2 para los árboles fuente de semilla. Las fuentes de disturbio identificadas fueron: extracción forestal 55%, incendios forestales 24%, plagas 8%, agricultura, alud con 5% cada una y huracán 3%. La variación de la densidad de regeneración natural respecto de la fuente de semilla,corresponde a los modelos de J invertida para bordes, y de variable de subpoblaciones para claros. Las distancias de dispersión de regeneración natural establecida para P. oocarpa fue 65 m en borde y 160 m en claro; para P.pseudostrobus fue 75 m en borde y 175 m en claro; y para P. tecunumanii fue 70 m en borde y 170 m en claro.Las distancias de dispersión con densidades aceptables según Instituto Nacional de Bosques-Programa RegionalForestal para Centroamérica, para las tres especies varían de 65-175 m.

Coniferous forests of the western highlands of Guatemala have been disturbed by human intervention and natural phenomena, which has allowed the occurrence of the phenomenon of natural regeneration. In this scenario thedensity of natural regeneration of conifers three species evaluated (Pinus oocarpa Schiede, P. pseudostrobus Lindl.and P. tecunumanii Eguiluz & Perry) in 60 sites distributed in space and clear edge. The variables evaluated were thesource of disturbance that caused its establishment, density variation of the source of seed and distance estimationrange scattering adequate regeneration, using sampling units of 25 m2 for natural regeneration and 500 m2 seedsource trees. Disturbance sources identified were logging 55%, 24% forest fires, pests 8%, agriculture, avalanche5% each and hurricane 3%. The variation of the density of natural regeneration on the seed source, corresponds to the inverted J models for edges, and to clear variable subpopulations. Dispersal distances of natural regenerationwas established for P.oocarpa 65 m in edges, and 160 m in the clears; P. pseudostrobus 75 m in edges, and 175 min the clears; and P. tecunumanii was 70 m in edges, and 170 m in the clears. Dispersal distances with acceptabledensities according National Forestry Institute-Regional Forestry Programme for Central American, for the threespecies vary from 65-175 m.

A Modified Protocol for High-Quality RNA Extraction from Oleoresin-Producing Adult Pines.

RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes through high-throughput sequencing technologies, microarray, and also northern blots, RT-PCR, and RTqPCR. Even though many specific protocols designed for plants with high content of secondary metabolites have been developed, these are often expensive, time consuming, and not suitable for a wide range of tissues. Here we present a modification of the method previously described using the commercially available Concert™ Plant RNA Reagent (Invitrogen) buffer for field-grown adult pine trees with high oleoresin content.

Somatic Embryogenesis in Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae).

This chapter deals with the features of somatic embryogenesis (SE) in Araucaria angustifolia, an endangered and native conifer from south Brazil. In this species SE includes the induction and proliferation of embryogenic cultures composed of pro-embryogenic masses (PEMs), which precede somatic embryos development. A. angustifolia SE model encompasses induction, proliferation, pre-maturation, and maturation steps. Double-staining with acetocarmine and Evan's blue is useful to evaluate the embryonic somatic structures. In this chapter we describe A. angustifolia SE protocols and analyzes morphological features in the different SE developmental stages.

Toward establishing a morphological and ultrastructural characterization of proembryogenic masses and early somatic embryos of Araucaria angustifolia (Bert.) O. Kuntze.

Somatic embryogenesis is a morphogenetic route useful for the study of embryonic development, as well as the large-scale propagation of endangered species, such as the Brazilian pine (Araucaria angustifolia). In the present study, we investigated the morphological and ultrastructural organization of A. angustifolia somatic embryo development by means of optical and electron microscopy. The proembryogenic stage was characterized by the proliferation of proembryogenic masses (PEMs), which are cellular aggregates composed of embryogenic cells (ECs) attached to suspensor-like cells (SCs). PEMs proliferate through three developmental stages, PEM I, II, and III, by changes in the number of ECs and SCs. PEM III-to-early somatic embryo (SE) transition was characterized by compact clusters of ECs growing out of PEM III, albeit still connected to it by SCs. Early SEs showed a dense globular embryonic mass (EM) and suspensor region (SR) connected by embryonic tube cells (TCs). By comparison, early somatic and zygotic embryos showed similar morphology. ECs are round with a large nucleus, nucleoli, and many cytoplasmic organelles. In contrast, TCs and SCs are elongated and vacuolated with cellular dismantling which is associated with programmed cell death of SCs. Abundant starch grains were observed in the TCs and SCs, while proteins were more abundant in the ECs. Based on the results of this study, a fate map of SE development in A. angustifolia is, for the first time, proposed. Additionally, this study shows the cell biology of SE development of this primitive gymnosperm which may be useful in evolutionary studies in this area.