As anthropogenically-forced ocean temperatures continue to rise, the physiological response of marine macrophytes becomes exceedingly relevant. The Red Sea is a semi-isolated sea- the warmest in the world (SST up to 34°C) - already exhibiting signs of rapid warming rates exceeding those of other tropical oceans. This will have profound effects on the physiology of marine organisms, specifically marine macrophytes, which have direct influence on the dynamic carbonate system of the Red Sea. The aim of this paper is to define the physiological capability and thermal optima and limits of six ecologically important Red Sea macrophytes- ranging from seagrasses to calcifying and non-calcifying algae- and to describe the effects of increasing thermal stress on the performance and limits of each macrophyte in terms of activation energy. Of the species considered, Halophila stipulacae, Halimeda optunia, Halimeda monile and Padina pavonica thrive in thermal extremes and may be more successful in future Red Sea warming scenarios. Specifically, Halimeda opuntia increased productivity and calcification rates up to 38°C, making it the most thermally resilient macrophyte. Halophila stipulacae is the most productive seagrass, and hence has the greatest positive effect on Omega saturation state and offers chemical buffer capacity to future ocean acidification.

Seagrass, mangrove, saltmarshes and macroalgae - the coastal vegetated habitats, offer a promising nature-based solution to climate change mitigation, as they sequester carbon in their living biomass and in marine sediments. Estimation of the macrophyte organic carbon contribution to coastal sediments is key for understanding the sources of blue carbon sequestration, and for establishing adequate conservation strategies. Nevertheless,identification of marine macrophytes has been challenging and current estimations are uncertain. In this dissertation, time- and cost-efficient DNA-based methods were used to fingerprint marine macrophytes and estimate their contribution to the organic pool accumulated in blue carbon habitats. First, a suitable short-length DNA barcode from the universal 18S gene was chosen among six barcoding regions tested, as it successfully recovered degraded DNA from sediment samples and fingerprinted marine macrophyte taxa. Second, an experiment was performed to test whether the abundance of eDNA represents the content of organic carbon within the macrophytes; results supported this notion, indicating a positive correlation (R2 = 0.85) between eDNA and organic carbon. Third, using the chosen barcode, eDNA of marine macrophyte was identified from sediments of seagrass meadows and mangrove forests in the Arabian Red Sea, to further estimate contributions to the organic carbon pools. Estimations based on eDNA were compared against estimations of organic carbon based on stable isotope analyses from the same sediments; results from both methods were similar. In addition, this research provided the first quantitative evidence of the contribution of macroalgae to coastal and oceanic carbon pools. Hitherto, macroalgae have been ignored in blue carbon assessments because their fingerprinting was challenging and there was no evidence of their carbon export. The results of this dissertation demonstrate that eDNA offers an unprecedent taxonomic discrimination, and resolve the contribution of marine macrophytes to the organic pools in blue carbon sediments.

Las angiospermas marinas son organismos esenciales para los ecosistemas costeros de zonas templadas. Sostienen una amplia diversidad biológica, regulan la dinámica sedimentaria costera y capturan grandes cantidades de carbono atmosférico. Sin embargo, a pesar de los servicios ecosistémicos que proporcionan, se encuentran amenazadas por actividades humanas y, especialmente, por la contaminación marina. Para conservar las praderas de angiospermas marinas es necesario desarrollar herramientas de seguimiento a todos los niveles de organización biológica que permitan detectar el estrés fisiológico para prevenir la regresión de estos valiosos hábitats, o de determinar la magnitud de estos procesos a gran escala. Es por ello que se plantea esta tesis con el objetivo de determinar el grado de afección de los vertidos de salmuera procedente de plantas desaladoras, además de su interacción con otros estresores ambientales, desde la escala molecular (expresión de genes) hasta la poblacional (cobertura de las praderas). Estos análisis podrán permitir la selección de biomarcadores específicos que sirvan como indicadores de alerta temprana de estrés y puedan permitir la toma de medidas de gestión para conservar. Los capítulos 2 y 3 de la tesis mostraron, por un lado, una estabilidad general de las praderas someras de la angiosperma marina Posidonia oceanica en la provincia de Alicante y, por otro, una grave regresión de la pradera en la bahía de la ciudad de Alicante. Esto se debe a la coexistencia de diversos impactos ambientales en esa zona que son responsables de esta pérdida de cobertura (619 hectáreas desde 1984), a pesar de la estabilidad general de estas praderas a mayor escala. De esta forma se puede determinar que los procesos de declive de estos ecosistemas no se produce de forma global y generalizada sino a escala local y, por tanto, las herramientas de gestión deben enfocarse en una menor escala espacial y atendiendo a los estresores específicos de cada zona. En el capítulo 4 se sometió a la angiosperma marina Zostera chilensis (endémica del Pacífico sudamericano) a incrementos de salinidad (+3 y +6 practical salinity units, psu) con sales artificiales para determinar su respuesta y tolerancia a potencial vertido de salmuera. La planta mostró una reducción fotoquímica una producción de peróxido de hidrógeno y una sobreexpresón de genes relativos a la osmorregulación y el estrés oxidativo. La respuesta etabólica fue similar a ambos incrementos de salinidad, pero la mayor producción de peróxido y de enfriamiento no fotoquímico a +6 psu parecen indicar que esta salinidad está por encima del umbral de tolerancia de la planta, y por tanto que podría ser vulnerable a un in. En los capítulos 5, 6 y 7 se aplicaron distintos incrementos de salinidad a P. oceanica (desde +2 hasta +6 psu) con salmuera real de una planta desaladora, tanto en condiciones de laboratorio como de terreno (frente a vertido de una planta desaladora) y midiendo respuestas metabólicas tanto en hojas como en meristemos apicales. La salmuera mostró incrementar ciertos indicadores de estrés comparado con sales artificiales y la producción de peróxido de hidrógeno, la peroxidación de lípidos y la expresión de genes relacionados con el estrés osmótico y oxidativo fueron superiores en condiciones naturales (terreno). Además las respuestas fueron más claras en los meristemos apicales en comparación con las hojas. Todos estos resultados indican que los vertidos de salmuera en interacción con los factores naturales ambientales pueden genera un mayor estrés metabólico y fisiológico, que además se puede medir más claramente en los meritemos que en las hojas. El uso de biomarcadores moleculares y bioquímicos en praderas expuestas a vertidos de salmuera, puede permitir la detección temprana de estrés y la potencial interacción con otros factores ambientales (naturales o antrópicos) que puedan comprometer la fisiología y la supervivencia de la planta. Por tanto estos indicadores tienen el potencial de usarse, en combinación con indicadores fenológicos o poblacionales, en planes de seguimiento para medir el grado de afección de un estresor. En esta tesis se proponen una serie de biomarcadores específicos al estrés generado por la salmuera con el fin de desarrollar medidas de gestión costera específicas a escala local que puedan detectar el impacto ambiental y prevenir la degradación de estos ecosistemas marinos. / Tesis financiada por la convocatoria UAFPU98 del programa propio del Vicerrectorado de Investigación de la Universidad de Alicante.

Seagrass meadows are a crucial foraging habitat for marine megaherbivores. The Red Sea hosts two megaherbivore species, the green turtle Chelonia mydas and the dugong Dugong dugon, along with twelve seagrass species. Seagrass habitats in the Saudi Arabian coast of the Red Sea are currently under pressure from large scale coastal developments. I used multiple methods to assess seagrass and megaherbivore densities in Al-Wajh lagoon, a unique semi-enclosed bay on the northeastern coast of the Red Sea that is currently targeted for development. Seagrasses were assessed using quadrat surveys while megaherbivores censuses were conducted by both unmanned aerial vehicles (UAV) and boat-based observers. Both seagrass and sea turtles were patchily distributed throughout the bay. Dugongs were never encountered during surveys but feeding trails and off-effort encounters suggest at least occasional use of the area. While there were some qualitative patterns between seagrass composition and feeding trail/sea turtle abundance, there was not enough data to achieve statistical significance. The UAV generally outperformed boat-based surveys, but the inability of the drone to distinguish turtles through dense seagrass coverage is a methodological limitation that should be overcome by using hyperspectral cameras in future work. Overall, my results highlight the importance of ground truthing habitat maps, reveal interesting questions for additional study, and provide some direction for future research and conservation efforts within Al-Wajh Lagoon.

Marine phytomyxids (Cercozoa: Phytomyxea) represent a group of obligate biotrophic eukaryotes known for infecting several algae and oomycetes and counted among three taxa generally reported to behave as seagrass-pathogens. Due to their low-key nature, they are observed only sporadically and very limited information about their diversity, life cycles or distribution is available. In order to extend the knowledge of this enigmatic group of marine protists, an extensive research on a phytomyxid found in the invasive seagrass Halophila stipulacea was carried out. In situ observations, light and scanning electron microscopy and molecular methods were used to approach the species' ecology, geographical range and phylogenetic placement within the class Phytomyxea. The organism was confirmed to be present in the Red, the Mediterreanean and the Caribbean Sea, suggesting a potential case of a host-parasite comigration on a global scale. Phylogenetically it represents the first and so far the only described member of the "TAGIRI-5 environmental clade" - possibly a whole new order within the Phytomyxea, outside the already established Plasmodiophorida and Phagomyxida. The infection rates and times of occurrence are specified for all the finding-sites, indicating an interesting seasonal pattern in the...

Many recent studies have sought to monitor health characteristics of seagrasses, including changes in aerial extent, biomass and fish community structure. While these studies have provided important information on the ecology of seagrass communities on southeast Australia, little attempt has been made to subject these sampling procedures to rigorous experimental testing and review. This study employed commonly-used standard methods for sampling seagrass community characteristics in two sites in New South Wales. Where possible, sampling protocols were tested for accuracy and efficiency at a range of temporal and spatial scales. The ARCView Geographic Information System was used to construct vegetation polygons of seagrass distribution on the Tweed River, and in the Ukerebagh Channel annually over a 5-year period. For one year (2000), distributions from identical photographs were mapped twice to identify procedural errors. In general, errors relating to incorrect boundary identification were low compared to inter-annual variability. Inter-annual variability in seagrass beds was higher than for adjacent mangrove and saltmarsh. Estimates of biomass were derived from standard replicate 0.25m x 0.25m quadrats. The experiment contrasted two sites of similar geomorphic setting. Ukerebagh Channel on the Tweed River, and Woolooware Bay with Botany Bay are both shallow, sandy marine deltaic settings supporting stands of Zostera capricorni. Significant differences were found in the degree of replication required to identify significant changes in seagrass biomass at the two sites. Ukerebagh Channel supported relatively dense stands of Z. capricorni with low intra-site variability. Here 8 replicates were sufficient to detect 10 percent change. Towra Point presented a contrast, in which 15 replicates were required to detect a similar level of change. Woolooware Bay at Towra Point has suffered from increased sedimentation relating to alterations in current velocities at Towra Point, and the result highlights the greater degree of replication required to determine significance changes in disturbed systems. The fish populations in the seagrass at Towra Point were sampled using buoyant pop nets. Fish communities differed significantly from those sampled in adjacent mangrove and saltmarsh. Differences in fish assemblages between spring high tides, neap high tides and low tides are attributed to movements of fish between seagrass and adjacent mangrove and saltmarsh. This mosaic of habitats is utilized by a number of species over a tidal cycle, with seagrass providing an important low-tide refuge for many species utilizing mangrove and saltmarsh at high tide. Limitations in the efficiency of buoyant pop nets were exposed in a novel experiment which demonstrated differences in escape rates between species. Flat-tailed mullet (Liza argenta) are likely to be under-represented in experiments using this technique. Recommendations are made regarding optimal sampling protocols for monitoring seagrass in the region. All techniques tested are suitable, though some require modification. Some texts have under-estimated the degree of replication required to appropriately monitor changes in seagrass biomass in disturbed systems, where density is lower and intra-site variability higher. The buoyant pop-nets may require modification in open-water seagrass situations where escape by Liza argenta and Acanthopagrus australis were at unacceptable levels.

The quest to discover simple, sensitive and global bioindicators of nutrient enrichment and ecological health continues. The present study is the first to address this quest in Hervey Bay by investigating links between key physico-chemical water quality parameters and two diatom forms. Free floating in the water column as phytoplankton, and attached to seagrass leaves and artificial substrates as microalgal epiphytes, diatoms are assessed for their abundance and diversity along an environmental gradient. Relationships between Secchi depth (Zsd), light attenuation coefficient (Kz) and minimum seagrass compensation depth (Zc) are established for the recovery, growth and survival of aquatic plants following disturbance, with equations derived specifically for this region to allow conversions from Zsd to Kz. These relationships allow predictions to be made and provide a management tool to meet water quality targets and combat elevated nutrient and sediment loads that result from anthropogenic activity and affect all marine life, from microalgae to marine mammals. Environmental links, identified as drivers of change to biodiversity, focus research effort and provide predictive management tools. Consequently, catchment and coastal activities such as dredging, construction and canal development, with the potential to increase nutrient and sediment loads, can be managed and scheduled at times least likely to adversely impact near shore ecosystems and therefore improve the balance between growth and sustainability. Seasonal and zone differences were significant for many of the water quality parameters monitored in Hervey Bay during 1994. Water clarity measures consistently showed significant differences between near and offshore zones (Secchi depth; P<0.01, turbidity; P<0.05 and TSS; P<0.01) as well as oxygen (P<0.01), soluble reactive phosphate (SRP, P<0.05), oxides of nitrogen (NOx, P<0.01), chlorophyll a (Chl a, P<0.01) and pH (P<0.01). Some default trigger values for water quality targets listed by ANZECC/ARMCANZ (2000) for concentrations of key indicators, a threshold for risk of adverse biological effects, were exceeded in dry and flood periods during the 1993 to 1995 survey. The community structure of phytoplankton and seagrass epiphytes were examined by monitoring changes to and 35 seagrass epiphyte species coincident with changes in some key water quality parameters along a water quality gradient. Chl a, Secchi depth (Zsd), soluble reactive phosphate (SRP) and pH were found to be the best subset of water quality parameters to maximise the rank correlation with phytoplankton communities whereas NOx and temperature maximised the rank correlation with the Halophila ovalis seagrass epiphyte communities. These parameters statistically link key water quality parameters to changes in phytoplankton and seagrass epiphyte density and assemblage structure and are supported as significant drivers of change in biodiversity research. Evidence for nitrogen limitation was found in the post flood surveys for phytoplankton and the growth and assemblage structure for seagrass systems and their epiphytes. Seasonal and zone differences in phytoplankton assemblage structure were most apparent with some site influence detected. Cylindrotheca closterium dominated the phytoplankton assemblage structure at the near shore zone while Thalassionema nitzschioides and Chaetoceros sp. differentiated the river from the Waste Water Treatment Plant (WWTP) creek site. Rhizolsolenia and Guinardia sp. were found in significantly higher concentrations at offshore sites (P < 0.01) and Thalassionema nitzschioides (P < 0.01), Thalassiosira sp. (P < 0.01) and Pseudonitzschia sp. (P < 0.05) were found in significantly higher abundance in the near shore zone A Trichodesmium bloom was examined and post flood changes to the phytoplankton assemblage structure associated with increased nutrient loads, reduced water clarity (Secchi depth, TSS and turbidity) and changes to pH and salinity were assessed. The results of analyses of variance provided support to multivariate statistical analyses to identify phytoplankton as a useful and sensitive bioindicator of environmental change. Post flood phytoplankton cell density increased and species diversity rose from 10 to 38 species at the Mary River mouth however, the changes to cell density and assemblage structure were not reflected in Chl a concentrations. The Mary River mouth experienced growth of mainly small phytoplankton species (< 20 µm) while Pulgul Creek, a source of WWTP effluent, experienced a disproportional increase in the larger phytoplankton species (>20µm). The increase in phytoplankton cell density at offshore sites occurred for both large and small species. Phytoplankton species dominance changed at each site during the postflood period. C. closterium remained the dominant species but increased from 34 to 648 cells/mL at Pulgul Creek however, C. similis, the second most dominant species was replaced by Pseudonitszchia sp., whose cell density rose from 2.0 to 320 cells/mL. A recent study also found that Cylindrotheca closterium, Skeletonema costatum, and Cyclotella choctawhatcheeana had strong positive relationships with coastal nutrients and suggested these species be used as potential reliable indicators of eutrophication (Toming and Jaanus, 2007). Some Pseudonitszchia sp. and dinoflagellate species have previously been noted as toxic and Chaetoceros sp. are associated with gill damage and fish kills. Rhizosolenia sp. and the Guinardia species G. flaccida and G. striatula, were indicators for the offshore relatively pristine sites, which may conversely, determine these species as indicators of environmental health in this waterway. Specificity of epiphyte attachment to particular seagrass species were detected and microalgal epiphyte assemblages on Halophila ovalis were identified as useful bioindicators of environmental gradients as this seagrass species is located at intertidal, shallow and deep water sites for a substantial part of the year. H. ovalis also recorded the highest average annual epiphyte loads (3873 ± 1882 cells/mm2) with the diatoms Diatoma vulgare and Cocconeis scutellum representing 19.6% and 17.2% of the total epiphyte cover. Highest cell density was recorded in autumn, followed by summer and winter: spring recorded the lowest epiphyte cover. Micro-algal epiphyte load was assessed as dry weight (g/m2), Chl a (µg/L) and cell density (cells/mm2) on artificial seagrass deployed along a water quality gradient. Equations were derived to describe percentage light transmission as a function of each measure of epiphyte load which allows transformation of data from one unit of expression to another and hence, allow comparison of past, current and future studies. Water column light attenuation was evaluated along a water quality gradient and linked to Secchi depths (Zsd) to derive equations that describe this relationship where for Hervey Bay, when Zsd < 4.26m, Kz = -1.2 ln Zsd + 1.74 and when Zsd ≥ 4.26m, Kz=1.37/Zsd. Species succession, diversity and seagrass growth were monitored for the first time during a recovery phase and limits for the seagrass compensation depth (Zc), were established on a seasonal basis for five seagrass species at four locations within intertidal, shallow and deep water zones in Hervey Bay and the Great Sandy Straits. Conceptual models for each site were constructed to describe habitat characteristics and include nutrient concentrations for SRP, NOx and ammonia (NH4+), Chl a, phytoplankton density, epiphyte density and light requirements measured as light attenuation, total suspended sediment, turbidity and Secchi depth at each location. The study provides a descriptive model for light attenuation and establishes (1) an annual and seasonal baseline water quality data set that characterises the waterways of Hervey Bay and the Great Sandy Straits (2) identification of phytoplankton species in Hervey Bay and their response to post flood changes in water quality (3) phytoplankton density and diversity along an environmental gradient with links established to Chl a, Secchi depth, SRP and pH (4) seagrass epiphyte assemblage structure along an environmental gradient with links established to NOx and temperature (5) conversion factors for epiphyte load expressed as dry weight, Chl a and cell density (6) functions to convert Secchi depth to light attenuation coeffiecients (Kz) (7) seagrass species succession at intertidal, shallow and deep water sites during a recovery growth phase in Hervey Bay (8) seagrass compensation depths (Zc) for five seagrass species at four locations and (9) habitat characterisation for seagrass recovery in Hervey Bay. Identification catalogues for phytoplankton and seagrass epiphytes were prepared with light and electron micrographs to assist future identification studies of diatoms in this region and for other similar biogeographical areas. The management of water quality to reach specific targets requires the capacity to predict seagrass compensation depth (Zc) as a function of water quality. Seasonal and annual light attenuation measurements are derived as a function of water quality at intertidal, shallow and deep water habitats during the colonisation of Zostera Capricornii, Halodule uninervis, Halophila ovalis, Halophila spinulosa and Halophila decipiens.

Seagrasses are submerged marine plants that provide essential ecosystem functions, but are declining in abundance worldwide. As angiosperms, seagrasses are capable of sexual reproduction, but also propagate asexually through clonal rhizome growth. Clonal growth was traditionally considered the primary means for seagrass propagation. Recent developments in genetic techniques and an increasing number of studies examining seagrass population genetics, however, indicate that sexual reproduction is important for bed establishment and maintenance. Few studies have investigated the reproductive biology and ecology of sub-tropical seagrass species, although this information is necessary for effective management and restoration. This work investigates the influence of pore-water nutrients on flowering, water flow on seed dispersal, consumption on seed survival, and describes the reproductive phenology in Texas for the two dominant seagrass species in the Gulf of Mexico: turtle grass (Thalassia testudinum) and shoal grass (Halodule wrightii). These species exhibit distinctive reproductive seasons that span summertime months, but reproductive output varies spatially and temporally. Results of an in situ nutrient enrichment experiment indicate that turtle grass produces fewer flowers (but more somatic tissue) when exposed to high pore-water ammonium than when exposed to low pore-water ammonium, suggesting that nutrient loading has the potential to reduce seagrass reproductive output. Seed consumption may also limit reproduction and recruitment in some areas, as laboratory feeding experiments show that several local crustaceans consume shoal grass and turtle grass seeds and seedlings, which do not survive consumption. Dispersal experiments indicate that seed movement along the substrate depends on local water flow conditions, is greater for turtle grass than shoal grass, and is related to seed morphology. Under normal water flow conditions in Texas, turtle grass secondary seedling dispersal is relatively minimal (< 2.1 m d⁻¹) compared to primary dispersal, which can be on the order of kilometers, and shoal grass secondary seed dispersal can be up to 1.1 m d⁻¹, but seeds are likely retained in the parent meadow. Results from this work can be used when developing seagrass management, conservation and restoration actions and provide necessary information concerning a life history stage whose importance was historically under-recognized. / text

The first detailed investigation of Maldivian rim island development and reef-to-island connectivity is presented. Study sites were selected on windward and leeward rim aspects of Huvadhu Atoll, and analyses were undertaken at a millennial, contemporary and near-future temporal scales. At millennial temporal scales, contrasting models of island development were presented for the windward and leeward sites. Marked between-site differences were found in the timings of island initiation (2,800-2,000 cal. yr. B.P. and 4,200-3,600 cal. yr. B.P. at the windward and leeward sites respectively). Hence, sea-level does not represent the sole control upon island formation. The period of island initiation and heightened mobility occurred during the mid-Holocene sea-level highstand. Future sea-level rise may thus reactivate the process regime responsible for reef island initiation, potentially inducing further island building and/or heightened island mobilisation. Contemporary analyses highlighted the homogeneity of the sediment reservoir across marine, beach and island sediments. Specifically, sand-grade coral was dominant across all samples within both sites (>50%). The most likely source of sand-grade coral is excavator parrotfish, which was consistent with ecological survey-based estimates of sediment production (excavator parrotfish accounted for 72.8% and 68.2% of sediment production at the windward and leeward sites). The highest sediment production rates were found within the lagoonward environments (59.4% and 75.4% at the windward and leeward sites), which is consistent with the more recent lateral lagoonward mode of island building. With regard to near-future analyses, the apparent recent areal expansion of seagrass beds demonstrated the capacity of ecological changes to cause shifts in sediment production budgets (contributing an additional ~243 tonnes yr-1 of sediment on the leeward rim platform). In addition, significant increases in benthic sediment mobility were found at both study sites under sea-level rise scenarios. Increases in mobility were markedly larger in magnitude at the leeward site than at the windward site. A challenge for the adaptive capacity of atoll nations is thus to acknowledge this atoll-scale diversity in future management strategies.

Este trabalho trata da construção de mapas quantitativos de alvos subaquáticos a partir de imagens de satélite. Especificamente, trata da estimativa de biomassa de prados de capim-agulha (Halodue. Wrightii) no litoral de Pernambuco a partir de imagens Landsat5. A coluna d’água altera, de maneira distinta e exponencial os diferentes comprimentos de onda eletromagnéticos distorcendo as correlações existentes entre os mesmos. Devido a isso, torna-se imprescindível eliminar a influência da coluna d’água a fim de que se possa fazer qualquer tipo de análise quantitativa relacionando alvos subaquáticos com a resposta espectral do mesmo. O método testado neste trabalho baseia-se na construção de bandas invariantes em relação à coluna d’água. As bandas invariantes em relação à coluna d”água são feitas a partir de pares de bandas. O coeficiente de determinação (r2) encontrado foi de 0,582 para o par de bandas (2,4) e de 0,709 para o par de bandas( 3,4) o que demonstra a eficiência do método testado. / This work approaches the making of quantitative maps of underwater targets based on satellite images, specifically, the estimate of biomass in seagrass (Halodue Wrightii) fields on Pernambuco’s coast taken from images of Landsat5. The water column distinctly and exponentially alters the different electromagnetic wave lengths, distorting the existing correlation between them. Because of this, it is crucial to eliminate the water column’s effect so that we can carry out different kinds of quantitative analysis relating underwater targets with their spectral response. The method tested in this work is based on the construction of bands that do not vary in relation to the water column. These invariable bands are made from pairs of bands. The determination coefficient (r2) found was 0.582 for the pair of bands (2.4) and 0.709 for the pair of bands (3.4), which shows the efficiency of the abovementioned method.