The isolation, characterization, and application of WRKY genes as useful molecular markers in tropical trees

The improvement of tropical tree crops using conventional breeding methods faces challenges due to the length of time involved. Thus, like most crops, there is an effort to utilize molecular genetic markers in breeding programs to select for desirable agronomic traits. Known as marker assisted breeding or marker assisted selection, genetic markers associated with a phenotype of interest are used to screen and select material reducing the time necessary to evaluate candidates. As the focus of this research was improving disease resistance in tropical trees, the usefulness of the WRKY gene superfamily was investigated as candidates for generating useful molecular genetic markers. WRKY genes encode plant-specific transcriptional factors associated with regulating plants' responses to both biotic and abiotic stress. ^ One pair of degenerate primers amplified 48 WRKY gene fragments from three taxonomically distinct, economically important, tropical tree crop species: 18 from Theobroma cacao L., 21 from Cocos nucifera L. and 9 from Persea americana Mill. Several loci from each species were polymorphic because of single nucleotide substitutions present within a putative non-coding region of the loci. Capillary array electrophoresis-single strand conformational polymorphism (CAE-SSCP) mapped four WRKY loci onto a genetic linkage map of a T. cacao F2 population segregating for resistance to witches' broom disease. Additionally, PCR primers specific for four T. cacao loci successfully amplified WRKY loci from 15 members of the Byttneriae tribe. A method was devised to allow the reliable discrimination of alleles by CAE-SSCP using only the mobility assigned to the sample peaks. Once this method was validated, the diversity of three WRKY loci was evaluated in a germplasm collection of T. cacao . One locus displayed high diversity in the collection, with at least 18 alleles detected from mobility differences of the product peaks. The number of WRKY loci available within the genome, ease of isolation by degenerate PCR, codominant segregation demonstrated in the F2 population, and usefulness for screening germplasm collections and closely related wild species demonstrates that the WRKY superfamily of genes are excellent candidates for developing a number of genetic molecular markers for breeding purposes in tropical trees. ^

Irradiance and spectral quality affect Asian tropical rain forest tree seedling development

Iridescent blue leaf coloration in four Malaysian rain forest understory plants, Diplazium tomentosum Bl. (Athyriaceae), Lindsaea lucida Bi. (Lindsaeaceae), Begonia pavonina Ridl. (Begoniaceae), and Phyllagathis rotundifolia Bl. (Melastoma- taceae) is caused by a physical effect, constructive interference of reflected blue light. The ultrastructural basis for this in D. tomentosum and L. lucida is multiple layers of cellulose microfibrils in the uppermost cell walls of the adaxial epidermis. The helicoidal arrangement of these fibrils is analogous to that which produces a similar color in arthropods. In B. pavonina and P. rotundifolia the blue-green coloration is caused by parallel lamellae in specialized plastids adjacent to the abaxial wall of the adaxial epidermis. The selective advantage of this color production, if any, is unknown.

Climatic Controls on Phytoplankton Biomass in a Sub-tropical Estuary, Florida Bay, USA

The extraction of climatic signals from time series of biogeochemical data is further complicated in estuarine regions because of the dynamic interaction of land, ocean, and atmosphere. We explored the behavior of potential global and regional climatic stressors to isolate specific shifts or trends, which could have a forcing role on the behavior of biogeochemical descriptors of water quality and phytoplankton biomass from Florida Bay, as an example of a sub-tropical estuary. We performed statistical analysis and subdivided the bay into six zones having unique biogeochemical characteristics. Significant shifts in the drivers were identified in all the chlorophyll a time series. Chlorophyll a concentrations closely follow global forcing and display a generalized declining trend on which seasonal oscillations are superimposed, and it is only interrupted by events of sudden increase triggered by storms which are followed by a relatively rapid return to pre-event conditions trailing again the long-term trend.

Forest Resource Islands in a Sub-tropical Marsh: Soil–Site Relationships in Everglades Hardwood Hammocks

Spatial heterogeneity in soils is often characterized by the presence of resource-enriched patches ranging in size from a single shrub to wooded thickets. If the patches persist long enough, the primary constraint on production may transition from one limiting environmental factor to another. Tree islands that are scattered throughout the Florida Everglades basin comprise nutrient-enriched patches, or resource islands, in P-limited oligotrophic marshes. We used principal component analysis and multiple regressions to characterize the belowground environment (soil, hydrology) of one type of tree island, hardwood hammocks, and examined its relationship with the three structural variables (basal area, biomass, and canopy height) indicative of site productivity. Hardwood hammocks in the southern Everglades grow on two distinct soil types. The first, consisting of shallow, organic, relatively low-P soils, is common in the seasonally flooded Marl Prairie landscape. In contrast, hammocks on islands embedded in long hydroperiod marsh have deeper, alkaline, mineral soils with extremely high P concentrations. However, this edaphic variation does not translate simply into differences in forest structure and production. Relative water depth was unrelated to all measures of forest structure and so was soil P, but the non-carbonate component of the mineral soil fraction exhibited a strong positive relationship with canopy height. The development of P-enriched forest resource islands in the Everglades marsh is accompanied by the buildup of a mineral soil; however, limitations on growth in mature islands appear to differ substantively from those that dominate incipient stages in the transformation from marsh to forest. Key words: resource island; tree

Mapping Height and Biomass of Mangrove Forests in Everglades National Park with SRTM Elevation Data

We produced a landscape scale map of mean tree height in mangrove forests in Everglades National Park (ENP) using the elevation data from the Shuttle Radar Topography Mission (SRTM). The SRTM data was calibrated using airborne lidar data and a high resolution USGS digital elevation model (DEM). The resulting mangrove height map has a mean tree height error of 2.0 m (RMSE) over a pixel of 30 m. In addition, we used field data to derive a relationship between mean forest stand height and biomass in order to map the spatial distribution of standing biomass of mangroves for the entire National Park. The estimation showed that most of the mangrove standing biomass in the ENP resides in intermediate- height mangrove stands around 8 m. We estimated the total mangrove standing biomass in ENP to be 5.6 X 109 kg.

Monitoring of Tree Island Conditions in the Southern Everglades: The Effects of Hurricanes and Hydrology on the Status and Population Dynamics of Sixteen Tropical Hardwood Hammock Tree Islands

In 2005 we began a multi-year intensive monitoring and assessment study of tropical hardwood hammocks within two distinct hydrologic regions in Everglades National Park, under funding from the CERP Monitoring and Assessment Program. In serving as an Annual Report for 2010, this document, reports in detail on the population dynamics and status of tropical hardwood hammocks in Shark Slough and adjacent marl prairies during a 4-year period between 2005 and 2009. 2005-09 was a period that saw a marked drawdown in marsh water levels (July 2006 - July 2008), and an active hurricane season in 2005 with two hurricanes, Hurricane Katrina and Wilma, making landfall over south Florida. Thus much of our focus here is on the responses of these forests to annual variation in marsh water level, and on recovery from disturbance. Most of the data are from 16 rectangular permanent plots of 225-625 m2 , with all trees mapped and tagged, and bi-annual sampling of the tree, sapling, shrub, and herb layer in a nested design. At each visit, canopy photos were taken and later analyzed for determination of interannual variation in leaf area index and canopy openness. Three of the plots were sampled at 2-month intervals, in order to gain a better idea of seasonal dynamics in litterfall and litter turnover. Changes in canopy structure were monitored through a vertical line intercept method.

Human disturbance and forest diversity in the Tansa Valley, India

We assessed the diversity of woody plants at 15 forested sites in the Tansa Valley of Thane District, in Maharashtra, India. The fewest species (11) were seen at a degraded mangrove site near the river mouth, and the greatest number (150) in the rich semi-evergreen forest on Tungar Hill. For all sites there were 141 tree, 25 shrub and 15 liana species, a total of 181 species. Excluding the mangrove site, which had no species in common with the other 14 sites, we analyzed the species distributions in detail. 2 These sites ranged in area from 4 to 30 km each, had woody floras of 89 6 6 species, and varied in intensity of human impact. Despite a history of exploitation and substantial reduction in biomass from firewood collecting, set fires and illicit tree felling, considerable plant diversity remains in the area.We found a modest increase in species richness in transects away from two villages. We observed the exploitation of the forest by the principal users, primarily of the Warli Tribe. They exploited a wide variety of forest resources (92 species), for medicines, foods, construction materials, household goods, manure and other purposes. They collected 15 items for sale. By far the single most important item collected was firewood, which dramatically reduced forest biomass within 2 km of villages. The species distributions in these forest remnants are strongly nested, mostly due to varying degrees of disturbance at individual sites. The high species diversity on Tungar Hill is most likely a relict of the earlier character of forests throughout much of the valley. It merits the highest priorities for preservation, as a refuge for Western Ghat species at the northern limits of their distributions.

Factors affecting the nodulation and growth of tropical woody legume seedlings

This study surveys the occurrence of nodulation in woody legume species in Panamá and Costa Rica, describes nodule and root characteristics, and researches host-bacteria specificity, nodulation potential of soils, and the effects of light, added nitrogen, and rhizobia and VA mycorrhizal fungi inoculation on seedling growth. I examined 83 species in 37 genera and found 80% to be nodulated. Percent nodulated species in the Caesalpinioideae, Mimosoideae, and Papilionoideae was 17, 95, and 86, respectively, with no correlation between nodule morphology and tribal classification. Nodules formed mainly at root branch points which supports epidermal breaks as an important rhizobia infection route. More non-nodulated than nodulated species had root hairs. Several species emitted volatile sulfur-containing compounds, including the toxic compound ethylmercaptan, from roots, germinating seeds, and other tissues. These emissions may have an allelopathic action against pathogens, predators, or other plants. In contrast to the general non-specificity of most legumes for rhizobia, Mimosa pigra L. was highly specific and only nodulated in flooded soils. This species' specificity, combined with a limited occurrence of its root nodule bacteria may limit its natural distribution, but its spread as an invasive weed is facilitated when fill material from rivers is deposited in other areas. ^ An experimental light level of 1.5% of full sun completely inhibited seedling nodulation, as do similar naturally low levels in forest understory. In the forest, trees and seedlings were not nodulated. in some soils with suspected high N content. For six experimental species, added N progressively increased seedling growth while decreasing nodule biomass; at the highest level of added N nodulation was completely suppressed. Species and individuals showed variation in nodule biomass at high N applications which may indicate an opportunity for genetic selection for optimal N acquisition. Rhizobia inoculation had a small positive effect on seedling shoot growth, but VA mycorrhiza inoculation overwhelmingly increased seedling size, biomass, and leaf mineral concentration. In lowland tropical forest, VA mycorrhizal colonization appears indispensable for legume nodulation because of the fungus' ability to supply P in deficient soils. This requirement makes the legume-rhizobia-mycorrhiza association obligately tripartite. ^

The effects of climate on the growth and physiology of tropical rainforest trees

Tropical rainforests account for more than a third of global net primary production and contain more than half of the global forest carbon. Though these forests are a disproportionately important component of the global carbon cycle, the relationship between rainforest productivity and climate remains poorly understood. Understanding the link between current climate and rainforest tree stem diameter increment, a major constituent of forest productivity, will be crucial to efforts at modeling future climate and rainforest response to climate change. This work reports the physiological and stem growth responses to micrometeorological and phenological states of ten species of canopy trees in a Costa Rican wet tropical forest at sub-annual time intervals. I measured tree growth using band dendrometers and estimated leaf and reproductive phenological states monthly. Electronic data loggers recorded xylem sap flow (an indicator of photosynthetic rate) and weather at half-hour intervals. An analysis of xylem sap flow showed that physiological responses were independent of species, which allowed me to construct a general model of weather driven sap flow rates. This model predicted more than eighty percent of climate driven sap flow variation. Leaf phenology influenced growth in three of the ten species, with two of these species showing a link between leaf phenology and weather. A combination of rainfall, air temperature, and irradiance likely provided the cues that triggered leaf drop in Dipteryx panamensis and Lecythis ampla. Combining the results of the sap flow model, growth, and the climate measures showed tree growth was correlated to climate, though the majority of growth variation remained unexplained. Low variance in the environmental variables and growth rates likely contributed to the large amount of unexplained variation. A simple model that included previous growth increment and three meteorological variables explained from four to nearly fifty percent of the growth variation. Significant growth carryover existed in six of the ten species, and rainfall was positively correlated to growth in eight of the ten species. Minimum nighttime temperature was also correlated to higher growth rates in five of the species and irradiance in two species. These results indicate that tropical rainforest tree trunks could act as carbon sinks if future climate becomes wetter and slightly warmer. ^

Leaf morphology scales multi-annual trends in nutrient cycling and leaf, flower, and fruiting phenology among species in the sub-tropical hardwood forests of the northern Florida Keys

The subtropical hardwood forests of southern Florida are formed by 120 frost-sensitive, broadleaved angiosperm species that range throughout the Caribbean. Previous work on a series of small sized forest component patches of a 20 km2, forest preserve in northern Key Largo indicate that a shift in species composition was associated with a 100 year forest developmental sequence, and this shift was associated with an increasingly evergreen canopy. This document investigates the underlying differences of the biology of trees that live in this habitat, and is specifically focused on the impact of leaf morphology on changing nutrient cycling patterns. Measurements of the area, thickness, dry mass, nutrient content and longevity of several leaves from 3-4 individuals of ten species were conducted in combination with a two-year leaf litter collection and nutrient analysis to determine that species with thicker, denser leaves cycled scarce nutrients up to 2-3 times more efficiently than thin leaved tree species, and the leaf thickness/density index predicts role in forest development in a parallel direction as the index predicts nutrient cycling efficiency. A three year set of observations on the relative abundance of new leaves, flowers and fruits of the same tree species provides an opportunity to evaluate the consequences the leaf morphology/nutrient cycling/forest development relationship to forest habitat quality. Results of the three documents support a mechanistic link between forest development and nutrient cycling, and suggests that older forests are likely to be better habitats based on the availability of valuable forest products like new leaves, flowers, and fruits throughout the year.

Surface mean flow and turbulence structure in tropical cyclone winds

Hurricanes are one of the deadliest and costliest natural hazards affecting the Gulf coast and Atlantic coast areas of the United States. An effective way to minimize hurricane damage is to strengthen structures and buildings. The investigation of surface level hurricane wind behavior and the resultant wind loads on structures is aimed at providing structural engineers with information on hurricane wind characteristics required for the design of safe structures. Information on mean wind profiles, gust factors, turbulence intensity, integral scale, and turbulence spectra and co-spectra is essential for developing realistic models of wind pressure and wind loads on structures. The research performed for this study was motivated by the fact that considerably fewer data and validated models are available for tropical than for extratropical storms. ^ Using the surface wind measurements collected by the Florida Coastal Monitoring Program (FCMP) during hurricane passages over coastal areas, this study presents comparisons of surface roughness length estimates obtained by using several estimation methods, and estimates of the mean wind and turbulence structure of hurricane winds over coastal areas under neutral stratification conditions. In addition, a program has been developed and tested to systematically analyze Wall of Wind (WoW) data, that will make it possible to perform analyses of baseline characteristics of flow obtained in the WoW. This program can be used in future research to compare WoW data with FCMP data, as gust and turbulence generator systems and other flow management devices will be used to create WoW flows that match as closely as possible real hurricane wind conditions. ^ Hurricanes are defined as tropical cyclones for which the maximum 1-minute sustained surface wind speeds exceed 74 mph. FCMP data include data for tropical cyclones with lower sustained speeds. However, for the winds analyzed in this study the speeds were sufficiently high to assure that neutral stratification prevailed. This assures that the characteristics of those winds are similar to those prevailing in hurricanes. For this reason in this study the terms tropical cyclones and hurricanes are used interchangeably. ^

The Influence of Phosphorus on Periphyton Mats from the Everglades and Three Tropical Karstic Wetlands

The distinctive karstic, freshwater wetlands of the northern Caribbean and Central American region support the prolific growth of calcite-rich periphyton mats. Aside from the Everglades, very little research has been conducted in these karstic wetlands, which are increasingly threatened by eutrophication. This study sought to (i) test the hypothesis that water depth and periphyton total phosphorus (TP) content are both drivers of periphyton biomass in karstic wetland habitats in Belize, Mexico and Jamaica, (ii) provide a taxonomic inventory of the periphytic diatom species in these wetlands and (iii) examine the relationship between periphyton mat TP concentration and diatom assemblage at Everglades and Caribbean locations. ^ Periphyton biomass, nutrient and diatom assemblage data were generated from periphyton mat samples collected from shallow, marl-based wetlands in Belize, Mexico and Jamaica. These data were compared to a larger dataset collected from comparable sites within Everglades National Park. A diatom taxonomic inventory was conducted on the Caribbean samples and a combination of ordination and weighted-averaging modeling techniques were used to compare relationships between periphyton TP concentration, periphyton biomass and diatom assemblage composition among the locations. ^ Within the Everglades, periphyton biomass showed a negative correlation with water depth and mat TP, while periphyton mat percent organic content was positively correlated with these two variables. These patterns were also exhibited within the Belize, Mexico and Jamaica locations, suggesting that water depth and periphyton TP content are both drivers of periphyton biomass in karstic wetland systems within the northern Caribbean region. ^ A total of 146 diatom species representing 39 genera were recorded from the three Caribbean locations, including a distinct core group of species that may be endemic to this habitat type. Weighted averaging models were produced that effectively predicted mat TP concentration from diatom assemblages for both Everglades (R2=0.56) and Caribbean (R2=0.85) locations. There were, however, significant differences among Everglades and Caribbean locations with respect to species TP optima and indicator species. This suggests that although diatoms are effective indicators of water quality in these wetlands, differences in species response to water quality changes can reduce the predictive power of these indices when applied across systems. ^

Using Florida Keys Reference Sites As a Standard for Restoration of Forest Structure in Everglades Tree Islands

In south Florida, tropical hardwood forests (hammocks) occur in Everglades tree islands and as more extensive forests in coastal settings in the nearby Florida Keys. Keys hammocks have been less disturbed by humans, and many qualify as “old-growth,” while Everglades hammocks have received much heavier use. With improvement of tree island condition an important element in Everglades restoration efforts, we examined stand structure in 23 Keys hammocks and 69 Everglades tree islands. Based on Stand Density Index and tree diameter distributions, many Everglades hammocks were characterized by low stocking and under-representation in the smaller size classes. In contrast, most Keys forests had the dense canopies and open understories usually associated with old-growth hardwood hammocks. Subject to the same caveats that apply to off-site references elsewhere, structural information from mature Keys hammocks can be helpful in planning and implementing forest restoration in Everglades tree islands. In many of these islands, such restoration might involve supplementing tree stocking by planting native trees to produce more complete site utilization and a more open understory.

The effects of carbon dioxide fertilization on the ecology of tropical seagrass communities

Increasing atmospheric CO2 concentrations associated with climate change will likely influence a wide variety of ecosystems. Terrestrial research has examined the effects of increasing CO2 concentrations on the functionality of plant systems; with studies ranging in scale from the short-term responses of individual leaves, to long-term ecological responses of complete forests. While terrestrial plants have received much attention, studies on the responses of marine plants (seagrasses) to increased CO 2(aq) concentrations remain relatively sparse, with most research limited to small-scale, ex situ experimentation. Furthermore, few studies have attempted to address similarities between terrestrial and seagrass responses to increases in CO2(aq). The goals of this dissertation are to expand the scope of marine climate change research, and examine how the tropical seagrass, Thalassia testudinum responds to increasing CO 2(aq)concentrations over multiple spatial and temporal scales. ^ Manipulative laboratory and field experimentation reveal that, similar to terrestrial plants, seagrasses strongly respond to increases in CO 2(aq) concentrations. Using a novel field technique, in situ field manipulations show that over short time scales, seagrasses respond to elevated CO2(aq) by increasing leaf photosynthetic rates and the production of soluble carbohydrates. Declines in leaf nutrient (nitrogen and phosphorus) content were additionally detected, paralleling responses from terrestrial systems. Over long time scales, seagrasses increase total above- and belowground biomass with elevated CO2(aq), suggesting that, similar to terrestrial research, pervasive increases in atmospheric and oceanic CO2(aq) concentrations stand to influence the productivity and functionality of these systems. Furthermore, field experiments reveal that seagrass epiphytes, which comprise an important component of seagrass ecosystems, additionally respond to increased CO2(aq) with strong declines in calcified taxa and increases in fleshy taxa. ^ Together, this work demonstrates that increasing CO2(aq) concentrations will alter the functionality of seagrass ecosystems by increasing plant productivity and shifting the composition of the epiphyte community. These results have implications for future rates of carbon storage and sediment production within these widely distributed systems.^

Benthic diatom assemblages as indicators of water quality in the Everglades and three tropical karstic wetlands

Limestone-based (karstic) freshwater wetlands of the Everglades, Belize, Mexico, and Jamaica are distinctive in having a high biomass of CaCO3-rich periphyton mats. Diatoms are common components of these mats and show predictable responses to environmental variation, making them good candidates for assessing nutrient enrichment in these naturally ultraoligotrophic wetlands. However, aside from in the Everglades of southern Florida, very little research has been done to document the diatoms and their environmental preferences in karstic Caribbean wetlands, which are increasingly threatened by eutrophication. We identified diatoms in periphyton mats collected during wet and dry periods from the Everglades and similar freshwater karstic wetlands in Belize, Mexico, and Jamaica. We compared diatom assemblage composition and diversity among locations and periods, and the effect of the limiting nutrient, P, on species composition among locations. We used periphyton-mat total P (TP) as a metric of availability. A total of 176 diatom species in 45 genera were recorded from the 4 locations. Twenty-three of these species, including 9 that are considered indicative of Everglades diatom flora, were found in all 4 locations. In Everglades and Caribbean sites, we identified assemblages and indicator species associated with low and high periphyton-mat TP and calculated TP optima and tolerances for each indicator species. TP optima and tolerances of indicator species differed between the Everglades and the Caribbean, but weighted averaging models predicted periphyton-mat TP concentrations from diatom assemblages at Everglades (R2  =  0.56) and Caribbean (R2  =  0.85) locations. These results show that diatoms can be effective indicators of water quality in karstic wetlands of the Caribbean, but application of regionally generated transfer functions to distant sites provides less reliable estimates than locally developed functions.