DNA Barcoding, GIS-Facilitated Seed Germination and Pilot Cultivation of Teucrium luteum subsp. gabesianum (Lamiaceae), a Tunisian Local Endemic with Potential Medicinal and Ornamental Value.

In the context of plant conservation and sustainable use of unique neglected and underutilized phytogenetic resources, this study focused on the Tunisian local endemic Teucrium luteum subsp. gabesianum (Lamiaceae). Using Geographical Information Systems and online databases, detailed taxon-specific ecological profiling was produced for the first time, which illustrated the temperature and climate conditions in its wild habitats and facilitated the investigation of how temperature affects its seed germination, thus making its cultivation in anthropogenic environments possible. Following the seed propagation first reported herein (77.5−81.25% at temperatures between 15 and 25 °C), species-specific in situ and ex situ conservation efforts or sustainable exploitation strategies can be enabled. This study also reported for the first time how chemical and integrated nutrient management (INM) fertilizers affect the growth and pilot cultivation of its seedlings (INM more advantageous). The firstly-reported herein DNA barcoding may enable its traceability, allowing future product design. The multidisciplinary approach followed has paved the way to bridge important research gaps hindering conservation efforts and/or the sustainable exploitation of this local Tunisian endemic plant to date. Based on the aforementioned results, the feasibility and readiness timescale for its sustainable exploitation was overviewed and re-evaluated herein, upgrading (>two-fold) its potential value for the medicinal-cosmetic, agro-alimentary, and ornamental-horticultural sectors.

Bioactivities and phenolic composition of Limonium boitardii Maire and L. cercinense Brullo & Erben (Plumbaginaceae): two Tunisian strict endemic plants.

Limonium genus is traditionally used in North Africa for disease treatment and in cosmetic. This study investigates for the first time the antioxidant, anti-inflammatory and enzyme inhibitory activities of two Tunisian endemic plants, L. boitardii and L. cercinense. Analysis of phenolic compounds was carried out by using RP-HPLC. Total phenolic and flavonoid contents, antioxidant activity (antiradical, reducing and total antioxidant activities), tyrosinase and α-glucosidase, collagenase inhibition activities were determined. Inhibiting of NO release in LPS-stimulated macrophages was assessed. L. cercinense exhibited strong antioxidant and anti-inflammatory effects. In the tyrosinase and α-glucosidase inhibitor activity tests, the two species were highly active, especially L. cercinense. High total phenolic and flavonoid contents were recorded in L. cercinense and myricitrin, myricetin, myricetin 3-O-ß-D-galatopyranoside, luteolin 7-O-glucoside and rutin were the main phenolics in both species. The results obtained render L. boitardii and L. cercinense as valuable new natural sources for cosmetic and pharmacological applications.

Medicinal-Cosmetic Potential of the Local Endemic Plants of Crete (Greece), Northern Morocco and Tunisia: Priorities for Conservation and Sustainable Exploitation of Neglected and Underutilized Phytogenetic Resources.

Medicinal-aromatic plants (MAPs) are important sources for the development of new valuable products of interest to human and animal health, and are also used as ornamentals for the horticulture industry. However, the increased global demand and the uncontrolled exploitation of these plants constitute a threat to their sustainability. To date, few scientific investigations have focused on MAPs valorization and their domestication. The purpose of this study was to evaluate for the first time the medicinal-cosmetic potential of 399 local endemic Mediterranean plants confined to Crete (223 taxa), the Mediterranean coast-Rif of Morocco (94), and Tunisia (82). The new methodological scheme was developed by experts through three multidisciplinary co-creative workshops and was adjusted by end-users to point-scoring of nine attributes evaluating the potential of the targeted neglected and underutilized plants (NUPs) in the medicinal-cosmetic sector. The results were demonstrated as percentage of the maximum possible score. These assessments were further linked and discussed with respect to feasibility and readiness timescale evaluations for sustainable exploitation of the focal NUPs. A great diversity of local endemic NUPs (30 taxa, 11 families) were associated with interesting medicinal-cosmetic properties (>35% up to 94.44%). Among them, 8 taxa showed the highest medicinal-cosmetic potential (>55% of maximum possible score), half of which are threatened with extinction. Although ex-situ conservation efforts and applied research work are needed to safeguard and unlock the full potential of the local endemic NUPs evaluated herein, the proposed multifaceted evaluation scheme revealed that some local endemic NUPs of the studied regions can be sustainably exploited in short- or medium-term, following successful examples of Cretan NUPs e.g., Origanum dictramnus. The sustainable exploitation of high scored taxa of the studied regions can be fastened through targeted species-specific research bridging extant research gaps and facilitating conservation and stakeholder attraction.

GIS-Facilitated Seed Germination and Multifaceted Evaluation of the Endangered Abies marocana Trab. (Pinaceae) Enabling Conservation and Sustainable Exploitation.

In the frame of the sustainable use of neglected and underutilized phytogenetic resources, and along with numerous studies in Abies spp. due to the innate conservation value of fir forests, this research focused on the Moroccan endemic fir, Abies marocana. The aim was triple-fold: to assess its potential and dynamics in economic sectors for sustainable exploitation; to determine the ecological conditions in which the species naturally thrives; and to find the appropriate requirements for its successful seed germination. We sourced multifaceted evaluations for three economic sectors performed in three levels, using 48 attributes and eight criteria from previous studies of our own, and the relevant species-specific assessments are overviewed herein in detail. The species' ecological profile was constructed using Geographical Information Systems (GIS) and open access data (Worldclim). Seed germination trials were performed to examine the effect of cold stratification (non-stratified, one- and two-months stratified seeds), the influence of four temperatures (10 °C, 15 °C, 20 °C, and 25 °C), and interactions thereof in relation to germination percentage (GP) and mean germination time (MGT). The experiments showed that the interaction of cold stratification and germination temperature has a strong effect on the GP and MGT of A. marocana seeds. A detailed GIS-derived ecological profile of the focal species was created in terms of precipitation and temperature natural regimes, enabling the interpretation of the seed germination results. The multifaceted evaluations reveal an interesting potential of the Moroccan fir in different economic sectors, which is mainly compromised due to extant research gaps, unfavorable conditions, and low stakeholder attraction. The findings of this study fill in extant research gaps, contribute to in situ and ex situ conservation strategies, and can facilitate the sustainable exploitation of this emblematic local endemic plant of northern Morocco.

Agro-Alimentary Potential of the Neglected and Underutilized Local Endemic Plants of Crete (Greece), Rif-Mediterranean Coast of Morocco and Tunisia: Perspectives and Challenges.

The neglected and underutilized plants (NUPs) could become alternative food sources in the agro-alimentary sector, enriching human and animal diets, offering the opportunity for sustainable exploitation, resilience to climate change, and production with resistance to pests and diseases. In the Mediterranean countries, these valuable resources are threatened by climate change, overexploitation, and/or monoculture. In this framework, we evaluated 399 local endemic NUPs of Crete (Greece), the Mediterranean coast, Rif of Morocco, and Tunisia, regarding their agro-alimentary potential, and assessed their feasibility and readiness timescale for sustainable exploitation with own previously published methodology. The methodological scheme was developed by experts in co-creative workshops, using point-scoring of seven attributes to evaluate the potential of the targeted NUPs in the agro-alimentary. Our results showed a diversity of promising local endemic NUPs of different families in the studied regions (Lamiaceae members are prominent), and we outlined the cases of 13 taxa with the highest optimum scores of agro-alimentary potential (>70%). Despite the diversity or the promising potential and current ex-situ conservation efforts to bridge gaps, our study indicated that only a few cases of Cretan local endemic NUPs can be sustainably exploited in the short-term. However, it is argued that many more local endemic NUPs can easily follow sustainable exploitation schemes if specific research gaps are bridged. Since NUPs can help to increased diversification of food production systems by adding new nutritional/beneficial species to human and animal diets, basic and applied research, as well as market and stakeholder attraction, is suggested as prerequisite to unlock the full potential of the focal endemic NUPs in the agro-alimentary sector.

Eco-Geographical, Morphological and Molecular Characterization of a Collection of the Perennial Endemic Species Medicago tunetana (Murb.) A.W. Hill (Fabaceae) from Tunisia.

In order to characterize and conserve the endemic pastoral species Medicago tunetana, many prospecting missions were carried out in mountainous regions of the Tunisian ridge. Twenty-seven eco-geographical and morphological traits were studied for six M. tunetana accessions and followed by molecular analysis using seven Simple Sequence Repeat (SSR). Only five markers were polymorphic and reproductible in the six M. tunetana populations. A total of 54 alleles were observed with an average of 10.8 bands/primer/genotype. Mean Polymorphism Information Content (PIC), Nei gene diversity (h) Shannon's information index (I) indicated the high level of polymorphism. The generated dendrogram with hierarchical UPGMA cluster analysis grouped accessions into two main groups with various degree of subclustring. All the studied accessions shared 57% of genetic similarity. Analysis of variance showed high significant difference between morphological traits among M. tunetana populations where MT3 from Kesra showed different morphological patterns regarding leaf, pod and seeds traits. Canonical correspondence analysis (CCA) showed two principal groups of M. tunetana populations based on potassium, total and active lime contents in soil. Our results suggest that SSR markers developed in M. truncatula could be a valuable tool to detect polymorphism in M. tunetana. Furthermore, the studied morphological markers showed a large genetic diversity among M. tunetana populations. This approach may be applicable for the analysis of intra specific variability in M. tunetana accessions. Our study could help in the implementation of an effective and integrated conservation programs of perennial endemic Medicago.

Ethnobotanical Study on Plant Used by Semi-Nomad Descendants' Community in Ouled Dabbeb-Southern Tunisia.

Thanks to its geographic location between two bioclimatic belts (arid and Saharan) and the ancestral nomadic roots of its inhabitants, the sector of Ouled Dabbeb (Southern Tunisia) represents a rich source of plant biodiversity and wide ranging of ethnobotanical knowledge. This work aims to (1) explore and compile the unique diversity of floristic and ethnobotanical information on different folk use of plants in this sector and (2) provide a novel insight into the degree of knowledge transmission between the current population and their semi-nomadic forefathers. Ethnobotanical interviews and vegetation inventories were undertaken during 2014-2019. Thirty informants aged from 27 to 84 were interviewed. The ethnobotanical study revealed that the local community of Ouled Dabbeb perceived the use of 70 plant species belonging to 59 genera from 31 families for therapeutic (83%), food (49%), domestic (15%), ethnoveterinary (12%), cosmetic (5%), and ritual purposes (3%). Moreover, they were knowledgeable about the toxicity of eight taxa. Nearly 73% of reported ethnospecies were freely gathered from the wild. The most commonly used plant parts were leaves (41%) followed by flowers and inflorescence (16%). We reported the use and collection of non-renewable parts (underground storage organs and roots) for 20 ethnospecies. Interestingly, a comparison with the available literature in Tunisia and neighboring countries reveals 13 new useful plants as well as 17 plants with new uses and demonstrates an important reservoir of traditional ethnobotanical heritage that is still sustained by respondents stemming from the semi-nomadic lifestyle of their ancestors (74% of cited taxa). These data could set a basis for further phytochemical and pharmacological research and conservative approach of the most relevant plant species including endemic overused and endangered taxa.

A re-circulating horizontal flow constructed wetland for the treatment of synthetic azo dye at high concentrations.

A re-circulating horizontal flow constructed wetland (RHFCW) system was developed in a greenhouse. This system was operated with Typha domingensis to study the phytoremediation capacity of this macrophyte species in different developing stages for synthetic textile wastewater with the pollutant type, the amaranth (AM) azo dye. Experiments were applied with a fixed flow rate Q = 10 L/h corresponding to a theoretical residence time of 3 h. The synthetic feeding to the RHFCW container was re-circulated back until the required water quality was achieved. The performance of this pilot-scale system was compared to an unplanted RHFCW. The effect of the initial dye concentration was studied using four dye concentrations (10, 15, 20, and 25 mg/L). The following parameters pH, color, COD, BOD5, NO3-, NO2-, and NH4+ were monitored during treatment. The maximum efficiencies obtained for discoloration, COD, NO3-, and NH4+ were 92 ± 0.14%, 56 ± 1.12%, 92 ± 0.34%, and 97 ± 0.17% respectively. Experiences demonstrate a decrease of removal efficiencies of studied parameters with the increase of dye concentrations, leading to an increase of the duration of treatment. Changes in activities of antioxidant enzymes (superoxide dismutase (SOD), guaiacol peroxidase (GPX), catalase (CAT), ascorbic peroxidase (APX), and glutathione reductase (GR)) and their relation to plant defense system against stress were studied. Enzymes were evaluated in leaves of T. domingensis during the remediation of the azo dye (amaranth). During treatment, an increase of enzymes activities was observed in accordance with the high removal efficiency.

Use of ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry system as valuable tool for an untargeted metabolomic profiling of Rumex tunetanus flowers and stems and contribution to the antioxidant activity.

Rumex tunetanus is an untapped resource with no available information about their chemical composition. That is why, the present study has conducted comprehensive metabolites profiling of the hydromethanol extracts of the Rumex tunetanus flowers and stems using RP-UHPLC-ESI-QTOF-MS. As results, 60 compounds were characterized for the first time in Rumex tunetanus between them, 18 photochemical were firstly identified in the Polygonaceae family in negative ionization mode. In this regard, quercetin-3-O-glucuronide and (-)-epicatechin gallate were the most abundant phenolic compounds in flowers and stems, respectively. Moreover, positive correlations were found between the antioxidant activity measured by DPPH and FRAP assays with the total phenolic compounds (r = 0.98; r = 0.99, respectively) and the abundance of some phenolic subfamilies such as hydroxycinnamic acids, hydroxybenzoic acids, flavonols and flavones with r > 0.86. The compounds displaying significant (P < 0.01) and good correlations with the antioxidant activity (r > 0.93) were hydroxybenzoic acid, rutin, quercetin-3-O-glucuronide, quercetin-3-O-glucoside, quercetin and luteolin-7-O-rutinoside. In conclusion, Rumex tunetanus flowers and stems showed different bioactive compound profiles and significant antioxidant properties and could be further used in food and nutraceutical industries.

Genetic diversity and population structure of six species of Capparis in Tunisia using AFLP markers.

In order to study the genetic diversity, the phylogeographic pattern and hybridization between six Tunisian Capparis species, 213 accessions of Caper were genotyped with three primer combinations of amplified fragment length polymorphism (AFLP) markers. Out of 750 fragments generated, 636 were polymorphic and 407 of them were restricted to a single species. STRUCTURE and PCoA analyses clearly separated morphologically different populations into six distinct genetic ones. The UPGMA analysis grouped the species into three main clusters: G1 grouped C. spinosa subsp. spinosa var. spinosa and C. sicula subsp. sicula; G2 grouped C. ovata subsp. ovata and C. orientalis and G3 clustered C. zoharyi and C. aegyptia. Populations from G1, G2 and G3 were mainly distributed in arid, subhumid, and semi-arid bioclimates, respectively. Additional genetic studies on Capparis could help to identify genes underlying speciation events and local adaptation to geographic areas leading to the development of breeding programs.

Phenolic contents and antioxidant activity of ethanolic extract of Capparis spinosa.

Caper plant (Capparis spinosa) extracts have been associated with diverse biological activities including anti-oxidant properties. In this work, we characterized the hydro-ethanolic extract obtained from C. spinosa leaves [hydroethanolic extract of C. spinosa (HECS)] by analyzing the content in anti-oxidant compounds such as polyphenols, flavonoids and anthocyanins. Further, we evaluated HECS antioxidant activities in vitro using bleaching of 1,1-diphenyl-2-picrylhydrazyl radical and ABTS test as well as by pretreatment of HeLa cells exposed to Fe(2+) or H2O2. Our findings indicate that HECS contains high amount of total phenolic compounds and high levels of flavonoids and anthocyanins. Furthermore, HECS exhibited antioxidant activity in both chemical and biological tests. Specially, pretreatment of HeLa cells with different concentrations of the extract conferred protection against lipid peroxidation and modulated activities of two antioxidant enzymes, SOD and catalase. These results revealed HECS antioxidant effects and suggest that C. spinosa leaves are a potential source of natural antioxidant molecules with possible applications in industry and medicine.

Spatio-temporal variability of faunal and floral assemblages in Mediterranean temporary wetlands.

Six temporary wetlands in the region of Sejenane (Mogods, NW Tunisia) were studied in order to characterize the aquatic flora and fauna and to quantify their spatio-temporal variability. Samplings of aquatic fauna, phytosociological relevés, and measurements of the physicochemical parameters of water were taken during four different field visits carried out during the four seasons of the year (November 2009-July 2010). Despite the strong anthropic pressures on them, these temporary wetlands are home to rich and diversified biodiversity, including rare and endangered species. Spatial and temporal variations affect fauna and flora differently, as temporal variability influences the fauna rather more than the plants, which are relatively more dependent on spatial factors. These results demonstrate the interest of small water bodies for maintaining biodiversity at the regional level, and thus underscore the conservation issues of Mediterranean temporary wetlands that are declining on an ongoing basis currently.

High genetic diversity and distinctiveness of rear-edge climate relicts maintained by ancient tetraploidisation for Alnus glutinosa.

Populations located at the rear-edge of a species' distribution may have disproportionate ecological and evolutionary importance for biodiversity conservation in a changing global environment. Yet genetic studies of such populations remain rare. This study investigates the evolutionary history of North-African low latitude marginal populations of Alnus glutinosa Gaertn., a European tree species that plays a significant ecological role as a keystone of riparian ecosystems. We genotyped 551 adults from 19 populations located across North Africa at 12 microsatellite loci and applied a coalescent-based simulation approach to reconstruct the demographic and evolutionary history of these populations. Surprisingly, Moroccan trees were tetraploids demonstrating a strong distinctiveness of these populations within a species otherwise known as diploid. Best-fitting models of demographic reconstruction revealed the relict nature of Moroccan populations that were found to have withstood past climate change events and to be much older than Algerian and Tunisian populations. This study highlights the complex demographic history that can be encountered in rear-edge distribution margins that here consist of both old stable climate relict and more recent populations, distinctively diverse genetically both quantitatively and qualitatively. We emphasize the high evolutionary and conservation value of marginal rear-edge populations of a keystone riparian species in the context of on-going climate change in the Mediterranean region.

Conservation of Mediterranean wetlands: interest of historical approach.

The wetlands of North Africa are an endangered and invaluable ecological heritage. Some of these wetlands are now protected by various conservation statutes; which actual impact has not yet been reliably evaluated. This article aims to assess the conservation management (Nature Reserve and Ramsar site) of a protected Tunisian lake, Majen Chitane, by using palaeoecological, historical and modern data, and by comparing it with the unprotected lake Majen Choucha. While located in similar environments, these lakes are today home to very different flora. Baseline conditions reconstructed from literature indicate that both lakes were very similar until the 1950s, and comparable to the current state of Majen Choucha, housing rich oligotrophic plant communities. In the 1960s, at the time that cultivation of the adjacent peatland began, Majen Chitane underwent strong ecological changes as the initial oligotrophic plant, diatom and zooplankton communities were replaced by eutrophication-tolerant ones. Eutrophication led to the local extinction of 40-55% of the hydrophytic and temporary-pool plant species, including those characteristic of the Isoetion. Given the damages and despite the recent conservation status of the site, it's unlikely that Majen Chitane will undergo any natural regeneration. Restoring it would start with completely protecting the complex lake-peatland and re-introducing the locally extinct species from Majen Choucha. This work exemplifies the usefulness of connecting palaeoecological, historical and modern data for the conservation of Mediterranean wetlands.

[Vegetation structures and conservation of Mediterranean temporary wetlands: Mogods region (northern Tunisia)]. / Structures de végétation et conservation des zones humides temporaires méditerranéennes : la région des Mogods (Tunisie septentrionale).

Floristic surveys and phytoecological relevés were conducted on 36 temporary wetlands of Mogods region. Multivariate analyses (CA, AHC) performed on these data reveal the high specific and biocoenotic diversity of Mogods wetlands, which appear controlled by substrate nature and hydrology. Among the 128 hydrophytic species inventoried, 38 are presently in precarious status and 6 are presumed extinct. The Mogods region harbours, moreover, very rare habitats (peatlands and semi-permanent lakes), and a vast plain so-called Garâa Sejenane, exceptionally rich in temporary wetlands. These results underline the urgency of an adapted conservatory management, based on the development of scientific studies dealing with structure and functioning of hydrophytic communities of the region.

Recent discovery of the small pillwort (Pilularia minuta Durieu, Marsileaceae) in Tunisia: hope for an endangered emblematic species of Mediterranean temporary pools?

This article presents a synthesis of all localities where Pilularia minuta, an endangered Pteridophyte endemic of acidic Mediterranean temporary pools, was observed since its discovery in 1835. It aims at analysing the implications of its recent discovery in Tunisia, based on the comparison between new and previously published floristic surveys. The obtained data confirm the heliophilous pioneer character of P. minuta and reveal a disturbance-favoured behaviour in Tunisia. The small pillwort, which occurs in 16 locations around the Mediterranean basin, could be characterised by a good dispersion. However, it experienced the extinction of a quarter of its known populations over the last century. This decline seems to have affected only small populations (Maritime-Alps, France; Lazio, Italy) and highly disturbed areas (Algeria), while large ones (Sardinia, Italy; Corsica and Hérault, France; Andalusia, Spain; western Morocco), presumably more stable, survived. In a context of metapopulation, these large populations could be considered as 'source populations', and should be taken more especially in account in conservation strategies. Further investigations are needed for improving the conservation of the rare plants of Mediterranean temporary pools as well as contributing to a better knowledge of the mechanisms controlling their distribution and their present-day status.