ACTA BOT. CROAT. 79 (1), 2020 3 Acta Bot. Croat. 79 (1), 3–14, 2020 CODEN: ABCRA 25 DOI: 10.37427/botcro-2020-001 ISSN 0365-0588 eISSN 1847-8476 The effect of salinity gradient and heavy metal pollution on arbuscular mycorrhizal fungal community structure in some Algerian wetlands Warda Sidhoum1,2*, Kheira Bahi2,3, Zohra Fortas1 1 Laboratory of Microorganisms Biology and Biotechnology, University of Oran 1 Ahmed Ben Bella, Oran, Algeria 2 Abdelhamid Ibn Badis University, Mostaganem, Algeria 3 Department of Biology, Faculty of Natural Science and Life, University of Oran 1 Ahmed Ben Bella, Oran, Algeria Abstract – Algerian natural wetlands suffer from anthropogenic disturbances due to industrial development and urbanization. This study was designed to draw attention to arbuscular mycorrhizal fungi (AMF) distribu- tion and community assemblages following heavy metal and salinity concentrations in two wetlands subjected to domestic and industrial effluents. Rhizospheric soil and roots of 18 plant species were collected in two wet- lands along a decreasing salinity gradient. The results showed that 72.72% of plant species exhibit an association within arbuscular mycorrhizas (AM), and 36.36% a dual association between AM and dark septate endophytes (DSE). A total of 33 AMF morphospecies were distinguished on the basis of morphological criteria dominated by taxa belonging to Glomeraceae and Acaulosporaceae. Soil contamination was investigated by determining metallic trace elements (MTE) (Cd, Cu, Ni, Pb, Cr and Zn) using an atomic absorption spectrophotometer. Val- ues of the pollution index revealed wetlands that were particularly polluted by lead. Two-way ANOVA showed significant variations in metal content among sampling locations and transects. Principal component analysis showed that species richness, and mycorrhizal frequency were slightly affected by MTE. This opens possibilities for their utilization in polluted soil remediation. Keywords: Dark septate endophytes, metallic trace elements, mycorrhizal association, saline wetlands, soil pollu- tion * Corresponding author e-mail: sidhoumwarda@yahoo.fr Introduction Oran is located in the north-west of Algeria, and con- stitutes a wetland complex of eight zones, four of which are classified as of International Importance. Great Sebkha and Macta (since 2001), Telamine Lake (LT) and les Salines d’Arzew (since 2004), while others, though not be classi- fied as Ramsar, have nonetheless received attention from the Ramsar Convention (Chenchouni and Si Bachir 2010). These wetlands are ecologically important ecosystems, pro- viding important winter grounds for several world popu- lations of endangered bird species. In particular, species belonging to the Anas and Tadorna orders, overwinter in significant numbers in these areas (Boucheker et al. 2011, Samraoui et al. 2015). The wetland soils are mostly Solonchak types, contain- ing large amounts of exchangeable sodium and soluble salts (Benziane 2013). These habitats are characterized by the presence of flooded or water saturated soils for at least part of the growing season. These natural hydrosystems have halophylic plants, such as Amaranthaceae (Ghodbani and Amokrane 2013, Megharbi et al. 2016). However, these eco- systems can be modified by various factors, among them aridity causing changes in soil properties and trace element pollution. This is due to human activities, including, ur- banization processes, domestic sewage discharges, livestock wastewater and industrial effluent (Bouldjedri et al. 2011, Domínguez-Beisiegel et al. 2016). Several studies have shown that arbuscular mycorrhizal fungi (AMF) exist in the roots of wetland plants and woody species grown on flooded soils (D’Souza and Rodrigues 2013), in aquatic macrophytes, freshwater wetland plant communities and salt marshes (Xu et al. 2016). It has also been shown that AM fungal diversity in wetlands is com- https://www.scirp.org/journal/articles.aspx?searchcode=Laboratory+of+Biology+of+Development+and+Differanciation%2c+Department+of+Biology%2c+Faculty+of+Natural+Science+and+Life%2c+University+of+Oran+1+(Ahmed+Benbella)%2c+Oran%2c+Algeria&searchfield=affs&page=1&skid=0 SIDHOUM W, BAHI K, FORTAS Z 4 ACTA BOT. CROAT. 79 (1), 2020 parable to that of most terrestrial ecosystems and for the growth and development of wetland plant species, and thus AM fungi are functionally essential (Tuheteru et al. 2015). The primary abiotic factors: soil flooding, nutrient, oxy- gen availability, salinity, and high levels of heavy metals in soil strongly affect the abundance and distribution of AM fungi in aquatic ecosystems (Millar and Bennett 2016). As previously reported, the intraradical and extraradical my- celia of metallic stress adapted AMF isolates are capable of sequestering heavy metals and alleviating metal toxicity to plants (Cabral et al. 2015). This indicates that these fungi have evolved a tolerance to metallic trace elements (MTE), and play a role in the phytoremediation of metal polluted sites, even in polluted aquatic and semi-aquatic habitats (Wężowicz et al. 2015). A number of surveys on AMF associated with wetland plants have been performed in order to investigate their di- versity and colonization potential (D’Souza and Rodrigues 2013, Kumar and Muthukumar 2014) along a soil hydrologi- cal gradient (de Marins et al. 2009, Miller and Bever 1999, Turner et al. 2000), salinity (Roda et al. 2008, Saint-Etienne et al. 2006, Yang et al. 2010), or nutrient content (Cornwell et al. 2001, Jayachandran and Shetty 2003). There has been a small amount of research focused on AMF communities in MTE polluted wetlands (Carrasco et al. 2006, Ban et al. 2017), but there has been no report of the simultaneous ef- fect of salinity and heavy metals on AMF distribution in wetland habitats. The present study, therefore, was aimed at evaluating the AM fungal diversity in heavy metals pol- luted saline wetlands and at adding to knowledge on these populated areas with heterogeneous plant species that have rarely been considered mycorrhizal, as well as at exploring the impact of soil salinity gradient and trace element pollu- tion on AMF community structures. Materials and methods Study area This survey was carried out in two wetlands located in Oran city (western region of Algeria), namely Telamine Lake (LT) (35°42’50”N 0°23’30’’W) located in the district of Gdyel (eastern Oran) at 7 km from Hassi Amer industrial Zone II. Dayet Morsli (DM) (35°39’58”N 0°36’27”W) located in Es- Sénia district in the south of Oran at a distance of 2 km north of the industrial Zone I of Es-Sénia. The altitude ranged be- tween 50 and 87 m a.s.l. The regional climate is of the semi- arid Mediterranean type characterized by a cold and rainy winter followed by a hot dry summer spread over 4 to 6 con- secutive months where the average temperature varies be- tween 14.1 °C and 22.5 °C and precipitation varies between 250 and 400 mm per year. Sampling The investigations were conducted at each site (LT and DM) along four transects 200 m long and 10 m wide. Start- ing from the wetland water edge to the periphery. Each tran- sect was divided into three plots according to the salinity gradient and plant distribution: from 0 m (where no vegeta- tion was present) to 30 m, and electrical conductivity (EC) varying between 6 to 9.5 dSm–1, and 30 m to 60 m (2 dSm–1 1indicates that the soil is polluted. AMF DIVERSITY IN WETLAND STRESSES ACTA BOT. CROAT. 79 (1), 2020 5 Assessment of AMF and DSE colonization Young roots (with root tips) were washed in tap water to remove soil particles, and then fixed in FAA formalin, glacial acetic acid, and ethanol (1:1:18, v/v/v). Roots were washed several times in tap water, cleared in 10% (w/v) KOH while heating to approximately 90 °C for 1h, acidified in 10% lac- tic acid, and stained with 0.1% trypan blue in lactophenol at 90 °C for 1h, according to a modified method of Phillips and Hayman (1970). For each root system, AMF colonization was estimated by optical microscopy (Olympus CX22) from 50 root fragments of approximately 1 cm in length. Mycor- rhizal development was evaluated according to the meth- od of Trouvelot et al. (1986), and expressed as mycorrhizal frequency (f%), intensity of colonization (M%), mycorrhi- zal intensity of colonized root fragments (m%), arbuscules abundance (A%) and arbuscules abundance of colonized root fragments (a%). In the case of DSE colonization, mi- crosclerotia and hyphae were scored collectively, and the fre- quency of DSE occurrence in roots (DSE%) was calculated as the ratio between colonized root fragments by DSE and the total number of examined root fragments. Isolation and taxonomic identification of AM fungal spores The plants were carefully dug out from the soil and the majority of bulk soil was manually removed from the roots. Only the soil closely attached to the root system was analyzed. AM fungal spores were isolated via wet sieving (Gerdemann and Nicolson 1963) followed by soil centrifugation (1398 × g during 10 minutes at room temperature) in 50% sucrose solution and filtration (mesh size 40 µm) (Brundrett et al. 1996). Intact, noncollapsed spores, with cytoplasmic content were separated into groups according to general morpho- logical similarities recorded under a stereomicroscope (Lei- ca EZ4HD), and the diameter of spores was measured. Also, permanent slides of all spores were prepared with the use of a drop of polyvinyl alcohol/lactic acid/glycerol (PVLG) mixed with Melzer’s reagent (1:1, v/v) on a slide. Species identifica- tion was based on the examination of spore morphological and subcellular characteristics. Obtained results were com- pared to the descriptions of Oehl et al. (2011), and Redecker et al. (2013); other available descriptions are found on the sites www.agro.ar.szczecin.pl/~jblaszkowski/ and https://in- vam.wvu.edu/, while the nomenclature employed follows that used by the Mycobank (www.mycobank.org). Statistical analysis Ecological measures of diversity used to describe the structure of AMF communities included spore density (number of spores in 100 g of soil), species richness (num- ber of identified AMF species per soil sample), relative abun- dance (RA), isolation frequency (IF), Shannon’s diversity in- dex (H’), evenness (E), Simpson’s index of dominance (D), and Sorensen’s coefficient (Cs) (Simpson 1949). The relative abundance refers to how common or rare a species is relative to other, and it was calculated from the formula: RA = spore number of a species genus total number of identifie ( ) dd spore samples ×10 Isolation frequency was calculated following the formula: IF = number of soil samples where a species genus total number ( ) of soil samples The Shannon diversity index is a mathematical measure of species diversity in a community. H’ value was calculated according to the formula: H’ = -∑Pi ln Pi, where Pi is the rel- ative abundance of each identified species per sampling site and calculated by the following formula Pi = ni/N, where ni is the number of spores of a species and N is the total num- ber of all identified spores. The evenness also called “equita- bility”, refers to the homogeneity of the species, 0 50% and the RA > 5%. All statistical analyses were performed using the SPSS software package (version 23.0). The data were analyzed by two-way ANOVA with site (geographical location of the wetlands) and plots (the position in the transect) as main factors. Multiple mean comparisons were performed using Tukey’s HSD post hoc test at the 0.05 level of probability. A principal component analysis (PCA) was performed in or- der to verify the environmental variables that best explain AMF community structure. Because the variables were mea- sured in different units, a correlation test was used (all vari- ables were normalized using division by their standard de- viations). Results Species composition and abundance The floristic survey on sites reveals the presence of a vari- able phytodiversity comprising 46 vascular plant species (34 in DM, and 28 in LT) belonged to 44 genera and 21 fami- lies, thus indicating that Dayet Morsli was the most diversi- fied site (Tab. 1). Moreover, flora was characterized by the dominance of taxa belonging to the families Amaranthaceae (26.47% in DM and 25% in LT) and Asteraceae (23.52% in DM and 17.85% LT), followed in DM by Poaceae (17.64%), Solanaceae (8.82%), Malvaceae, Liliaceae (5.88%), while 2.94% of the remaining species belonged to other families. https://invam.wvu.edu/ https://invam.wvu.edu/ SIDHOUM W, BAHI K, FORTAS Z 6 ACTA BOT. CROAT. 79 (1), 2020 On the other hand, in LT the Asteraceae were followed by Poaceae and Apiaceae (each represented with 7.14% of the species), and the rest of surveyed plant families (each repre- sented with 3.57% of the species). Soil chemical properties Results of soil physicochemical analyzes (Tab. 2) revealed an average EC value 4.72 dSm–1 in LT (2.07–7.98 dSm–1) and 4.42 in DM (0.2–9.5 dSm–1), in addition to a slightly alka- line pH varying from 7.3 to 8.5 in DM and from 7.33 to 8.31 in LT. Overall, the results showed higher levels of heavy met- als in DM topsoils than those found in LT. These values are greater than the maximum concentration foreseen in the en- vironmental soil quality guidelines AFNOR NFU 44–041. A high level of Pb was found at both sites, while Cd and Cr were present in DM only. The pollution index in DM is considerably greater with values exceeding 1, indicating that DM is a moderately polluted site. Two-way ANOVA showed significant differences be- tween the investigated sites along transects in salinity de- gree (EC) (P < 0.001), Cr (P < 0.001), as well as Zn (P < 0.05) and Cu concentrations (P < 0.01), which significantly and steadily decreased from sea to inland regions. Also, significant differences were noticed between wet- land sites for all MTE concentrations (except Zn), pollu- tion index, and salinity (Ni, Cr, Cu and PI at P < 0.001, Pb (P < 0.01), Cd and EC (P < 0.05)). No significant effect was observed in the interaction (site*plot) on Zn and Ni. The obtained results highlight the effect of distance from water which has a very important role in soil MTE concentration. Tab. 1. Abundances of plant species in the wetlands Dayet Morsli and Telamine Lake, Algeria, and their accession numbers at the Plant Ecology Laboratory Herbarium of the University Oran 1 Ahmed Ben Bella, Oran, Algeria. Abundance classes: I – very rare, II – rare, III – occasional, IV – frequent, V – abundant (Bradai et al. 2015). / = no accession number in the herbarium. Family Plant species Herbarium accession no. Dayet Morsli Telamine Lake Aizoaceae Mesembryanthemum crystallinum L. 00790 II I Amaranthaceae Chenopodium album L. 00739 II I Amaranthaceae Arthrocnemum macrostachyum (Monic.) K.Koch 00745 III III Amaranthaceae Atriplex halimus L. 00727 IV III Amaranthaceae Atriplex canescens (Pursh) Nutt. / . III Amaranthaceae Sarcocornia perennis (Mill.) A.J.Scott 00747 III III Amaranthaceae Salicornia patula Duval-Jouve 00746 II III Amaranthaceae Suaeda vera Forssk. ex J.F.Gmel. 00767 III IV Amaranthaceae Atriplex prostrata DC. 00720 I . Amaranthaceae Beta macrocarpa Gus. 00732 I . Apiaceae Daucus carota subsp. carota (L.) Thell. 01924 . III Apiaceae Smyrnium olusatrum L. 01993 . II Arecaceae Chamaerops humilis L. 00449 I I Asteraceae Scolymus maculatus L. 03033 I I Asteraceae Calendula stellata Cav. 02824 I II Asteraceae Centaurea pullata L. 02966 I II Asteraceae Glebionis coronaria (L.) Spach 02878 I . Asteraceae Limbarda crithmoides subsp. longifolia (Arcang.) Greuter 02764 . I Asteraceae Dittrichia viscosa (L.) Greuter. 02763 I . Asteraceae Silybum marianum (L.) Gaertn. 02941 II . Asteraceae Taraxacum erythrospermum Andrz. ex Besser 03096 . II Brassicaceae Sinapis arvensis L. subsp. arvensis 01146 I I Cucurbitaceae Ecballium elaterium (L.) A.Rich. 02682 I . Cynomoriaceae Cynomorium coccineum  (L.) subsp. coccineum 01873 . I Fabaceae Trifolium tomentosum L. 01446 I . Joncaceae Juncus maritimus Lamk. 00461 . I Lamiaceae Marrubium vulgare L. 02346 I II Liliaceae Asphodelus tenuifolius Cav. 00496 I . Liliaceae Asphodelus ramosus L. 00493 . II Malvaceae Malva sylvestris L. 01819 II I Malvaceae Lavatera multiflora (Cav.) Soldano et al. 01826 I . Oxalidaceae Oxalis pes-caprae L. 01683 . II Plantaginaceae Plantago lagopus L. 02583 I . Poaceae Avena sterilis L. 00252 I . Poaceae Hordeum murinum L. subsp. Glaucum (Steud.) Tzvelev 00386 II II Poaceae Anisantha madritensis (L.) Nevski 00345 II . Poaceae Hordeum maritimum With. subsp. Maritimum / I Poaceae Phalaris canariensis L. 00143 II . Poaceae Phragmites australis subsp. altissima (Benth.) 00227 I . Primulaceae Lysimachia arvensis (L.) U. Manns & Andreb. 02112 I . Rhamnaceae Ziziphus lotus (L.) Desf. 01802 . II Solanaceae Datura stramonium L. 02437 I . Solanaceae Nicotiana glauca Graham 02426 I . Solanaceae Solanum indicum L. / I . Tamaricaceae Tamarix sp. / . I Urticaceae Urtica pilulifera L. 00698 I I http://www.tela-botanica.org/bdtfx-nn-49948-synthese?referentiel=bdtfx&niveau=2&module=fiche&action=fiche&num_nom=101080&type_nom=nom_scientifique&nom=Plantaginaceae AMF DIVERSITY IN WETLAND STRESSES ACTA BOT. CROAT. 79 (1), 2020 7 Fungal root colonization Arbuscular mycorrhizae were observed in 72.72% of plant species since the dual association between AM fungi and DSE was found in 36.36% of plant species. One species (Asphodelus tenuifolius) showed AM, DSE, and ectomycorrhizae (Tab. 3). No AM was noted in roots of some species: Mesembryanthemum crystallinum, Arthrocnemum macro- stachyum, Salicornia patula and Tamarix gallica. The mean AM frequen- cy (f%) varied with particular species, ranging from 10% (Beta macrocar- pa and Sarcocornia perennis) to 100% (Calendula stellata and Marrubium vulgare). AM colonization intensity (M%) varied from 0.69% in Sarco- cornia perennis to 67.62% in Centaurea pullata. The mean arbuscule richness (A%) was also diverse and being lowest in all Amaranthaceae, in particular, Atriplex canescens (0.93%) and highest in Asteraceae, i.e. in Centaurea pullata (60.61%) (Tab. 3). Arum type morphology was pres- ent in 76.47% of mycorrhized plants, 11.76% in Paris type morphology and the rest were not identified as AM morphology, their association be- ing generally characterized by hyphal proliferation with few arbuscules. DSE was found in 54.54% of collected plants, although the DSE oc- currence frequency in roots was high in the case of several taxa (Tab. 3). Several forms of mycelia were encountered in both the outer cortex and the rhizoderm. Here, brownish, thick mycelium (4-5 μm) was developed into microsclerotia around the central cylinder or thin mycelium (2 μm) stained with trypan blue-lacking dark pigmentations. They subsequently formed hyphal complexes in cortical cells or aggregates to form a puzzle or brain-like microsclerotium and thick-walled mycelium for plate lobed and unlobed hyaline mycelium. Other fungal endophytes were noted: the remaining Olpidium spp. (Chytridiomycota) spores found in Asphodelus tenuifolius, Beta macro- carpa, and Atriplex halimus rhizodermis and outer cortex. Chlamydo- spore-like structures in Beta macrocarpa roots and fine hyphae with fan- shaped branches and Glomus tenue swellings (Glomeromycota) were discovered in A. halimus roots. AMF community composition and diversity On the basis of morphological criteria, 33 morphospecies were dis- tinguished from analyzed site samples (Tab. 4). AM community was characterized by the presence of several taxa belonging to: Glomerace- ae (16 taxa) represented by five genera, Glomus (5 spp.), Rhizoglomus (4 spp.), Funneliformis (3 spp.), Sclerocystis (2 spp.) and Septoglomus (1 sp.), Acaulosporaceae (Acaulospora 10 spp.), Diversisporaceae (Diversispora 2 spp. and Tricispora 1 sp.), Claroideoglomeraceae (Claroideoglomus 2 spp.), Archaeosporaceae (Archaeospora 1 sp.), Paraglomeraceae (Para- glomus 1 sp.), and Ambisporaceae (Ambispora 1 sp.). However, fourteen morphospecies remained unidentified since their morphological char- acteristics did not fit with any description of known species. Species richness of the sampled plant species varied in the rhizo- spheric soils, with maximum levels in DM (28 species) followed by LT (20 species). Fourteen species are classified as general and were found at all sites. A total of 12 species were classified as exclusive. There were ten only in DM (Acaulospora cf. collicosa, A. alpina, A. rehmii, Diversispora sp. 1, Divercispora tortuosa, Tricispora nevadensis, Glomus macrocarpum, G. diaphanum, Sclerocystis rubiformis and Rhizoglomus sp. 3) and 2 AMF species that were restricted to LT (Acaulospora sp. 3 and G. lamellosum). The highest number of spores in both sites belonged to Acaulospo- raceae (Tab. 4), followed by Archaeosporaceae and Glomeraceae. In DM the Glomeraceae were dominated by Rhizoglomus (6.74%), Glo-Ta b. 2 . V ar ia tio n in tr ac e el em en t c on ce nt ra tio ns (p pm ) a nd so il pa ra m et er s m ea su re d in th e w et la nd s D ay et M or sl i a nd T el am in e La ke a t d iff er en t d is ta nc es fr om th e w at er ’s ed ge . E C – e le ct ri ca l c on - du ct iv ity . C SQ – c ri te ri a in so il qu al ity a cc or di ng to q ua lit y N or m es A FN O R N FU 4 4- 04 1. D at a ar e m ea n va lu es ± S D , n = 4 . M ea ns in th e sa m e co lu m n w ith d iff er en t l et te rs a re si gn ifi ca nt ly d iff er en t fr om e ac h ot he r ( P < 0. 05 ) a cc or di ng to th e Tu ke y– te st (n s = n ot si gn ifi ca nt , * P < 0 .0 5, * * P < 0. 01 , * ** P < 0 .0 01 ). T he n um be rs fo r s ite , p lo t, an d si te *p lo t o n th e ta bl e bo tt om a re th e va lu es o f t w o- w ay A N O VA F -s ta tis tic s. Pl ot s – th e di st an ce fr om sa m pl in g ar ea a nd th e w at er e dg e (0 -3 0 m , 3 0- 60 m , a nd > 60 m ). Si te Pl ot s pH EC (d Sm –1 ) Pb N i C d C r C u Z n Po llu tio n in de x D ay et M or sl i 0- 30 8. 24 ±0 .2 8a 7. 36 ±0 .3 8a 72 1. 97 ±0 .0 0a 3 2. 07 ±0 .0 0a 5 .6 1± 0. 00 a 50 8. 87 ±0 .0 0a 1 18 .0 4± 0. 00 a 1 88 .0 ±0 .0 0a 2 .6 3± 0. 00 ab 30 -6 0 7. 79 ±0 .4 2a 5. 12 ±0 .6 5b 82 0. 01 ±4 5. 31 a 3 8. 02 ±3 .1 7a 8 .0 1± 1. 25 ab 29 8. 60 ±1 7. 55 b 1 18 .5 5± 6. 57 a 18 2. 00 ±7 .4 6a 2. 79 ±0 .2 0a >6 0 8. 00 ±0 .2 7a 0. 42 ±0 .1 7c 27 0. 32 ±4 39 .3 a 2 8. 96 ±1 0. 81 a 1 .8 4± 3. 59 b 9 7. 14 ±9 0. 44 c 42 .0 4± 46 .9 2b 9 8. 07 ±9 2. 30 a 0. 95 ±1 .2 2b Te la m in e La ke 0- 30 7. 45 ±0 .2 4a 7. 29 ±0 .7 5a 6 5. 91 ±5 4. 59 a 1 5. 60 ±5 .3 5a 1 .7 9± 3. 87 a 4 4. 65 ±2 5. 66 a 11 .3 3± 6. 29 a 14 9. 13 ±3 9. 85 a 0. 45 ±0 .3 0a 30 -6 0 7. 80 ±0 .3 1a b 2. 98 ±0 .9 3b 1 01 .4 ±1 63 .0 5a 1 5. 22 ±4 .2 7a 0 .4 2± 0. 48 a 2 4. 41 ±6 .8 3a 14 .1 5± 6. 63 a 16 6. 91 ±5 0. 26 a 0. 40 ±0 .2 5a >6 0 8. 01 ±0 .0 6b 0. 76 ±0 .4 6c 27 8. 23 ±3 91 .9 7a 7 .9 9± 8. 45 a 3 .1 7± 3. 45 a 1 5. 01 ±3 .5 4a 5 .6 ±1 .5 5a 5 3. 75 ±7 6. 01 b 0. 90 ±0 .2 8a C SQ 10 0 50 2 15 0 10 0 30 0 1 Si te 4. 21 n s 5 .1 5 * 4. 22 * * 45 .0 9 ** * 7. 7 * 16 1. 65 ** * 57 .0 8* ** 1. 59 n s 27 .4 0 ** * Pl ot 0. 94 n s 19 2. 5 ** * 0. 88 n s 2. 40 n s 0. 65 n s 33 .9 7 ** * 5. 96 * * 5. 56 * 1. 92 n s Si te *p lo t 4. 87 * 8 .0 4 ** 3. 91 * 0. 42 n s 4. 32 * 25 .3 2 ** * 4. 09 * 0. 12 n s 5. 91 * * SIDHOUM W, BAHI K, FORTAS Z 8 ACTA BOT. CROAT. 79 (1), 2020 mus (1.66%), Funnelifomis (1.21%), Septoglomus (0.71%), and Sclerocystis (0.34 %), while Funnelifomis (5.98%), Septoglomus (3.45%), Glomus (0.37%), Rhizoglomus (1.12%), and Sclero- cystis (0.45%) dominated in LT. The other families were not equally present in the studied sites. In DM, Claroideoglom- eraceae (2.29%) were more dominant than Ambisporaceae (1.94%) and Diversisporaceae (1.6%), but in LT, we found Paraglomaceae (1.02%) then Ambisporaceae (0.6%). Spore density is higher in LT (1077.09/100 g of soil) than that seen in DM (640/100 g of soil). Based on relative abundance and isolation frequency, six species were dominant in DM (Acau- lospora thomii, A. cf. morrowae, A. rehmii, A. leavis, A. melea and Archaeospora undulata), whereas only three dominated in LT: A. thomii, A. leavis and Ar. undulata (Tab. 4). On the other hand, a few AMF species had low relative abundances but high isolation frequency, such as the spe- cies in DM: A. leavis (6.96% RA, 100% IF), A. melea (6.18% RA 100% and 80% IF), and Tricispora nevadensis (1.23% RA and 71.42% IF). RA of Claroideoglomus claroideum is 2.29 % in DM and 2.75 in LT, while IF is 85.71% in DM and 60% LT. In addition, RA and IF of some other species in LT are: F. mosseae (0.75%, 60%), Se. constrictum (3.45%, 60%), and A. cf. morrowae (4.34%, 60%). Two-way ANOVA showed (Table 4) the existence of significant negative effects in AM frequency (P < 0.001), species richness (P < 0.01), and spore density (P < 0.01) under increasing gradient salinity (tran- sect from water to inland), while DSE was affected positively (P < 0.001). Moreover, only spore density was significantly different (P < 0.01) among wetland sites. No significant effect was noted of the interaction (site*plot) on species richness. In general, the mean values of the Shannon index showed that DM was slightly more diversified (1.69 ± 0.33) than LT (1.32 ± 0.29). Calculation of the AM fungal commu- nity evenness indicates a more equitable distribution among AM fungal communities found in DM (0.73 ± 0.13) than that found in LT (0.60 ± 0.12). Simpson’s index was higher in LT (0.24 ± 0.09) than DM (0.19 ± 0.06) reflecting a slight AM fungal species dominance among AM fungal communities. Sorensen’s coefficient indicated 58% of similarity in AMF community composition between wetland sites. No signifi- cant differences were noted in diversity indices affected by sites or the (site*plot) interaction except for dominance in- dex (F = 4.25, P < 0.05) which was significantly increased with salinity gradient (Tab.5). Multivariate analysis Eigenvalues of the two first principal components ex- plained a large proportion (Component 1 43.58% and Com- ponent 2 22.53%) of the total variance (Fig. 1). Component 1 was strongly correlated with heavy metals values and DSE frequency, which showed positive correlation coefficient val- ues (0.52 to 0.96). These metal concentrations were highly correlated with each other except for Zn (0.66 ≤ r ≤ 0.96; P < 0.001). The component 2 was more correlated with pH (0.54), f% (0.67), EC (0.77), spore density (–0.54), spe- cies richness (–0.75), and Zn (–0.63). Tab. 3. Fungal root endophyte colonization of plant species in the wetlands Dayet Morsli (DM) and Telamine Lake (LT). Arbuscular my- corrhizal types: P – Paris-type, A – Arum-type, -: not determined, NM – non mycorrhizal, E – ectomycorrhizal, Mycorrhizal frequency f% – the ratio between colonized root fragments by Arbuscular mycorrhizal fungi and the total number of examined root fragments, m% – mycorrhizal intensity of colonized root fragments, M% – the colonization intensity in all root system, a% – arbuscule abundance of colonized root fragments, A% – arbuscule abundance of all root system, DSE% – the ratio between colonized root fragments by Dark septate endophytes and the total number of examined root fragments. Family Plant species Location AM type Mycorrhizal frequency f% M% m% a% A% DSE % Aizoaceae Mesembryanthemum crystallinum DM NM 0 0 0 0 0 70 Amaranthaceae Arthrocnemum macrostachyum DM NM 0 0 0 0 0 0 Atriplex canescens LT A 48 7.14 14.88 13.07 0.93 8 Atriplex halimus LT A 58.49 18.74 32.03 11.54 2.16 13.2 Atriplex halimus DM A 57.25 21.63 38.44 10 2 12 Beta macrocarpa DM – 10 3.4 5.63 5.76 0.2 72 Salicornia patula DM NM 0 0 0 0 0 0 Sarcocornia perennis LT – 1.82 0.02 1 0 0 7.27 Sarcocornia perennis DM – 10 0.69 1.35 0 0 28 Suaeda vera LT A 36 8.16 22.67 21.69 1.77 4 Suaeda vera DM A 76.19 28.9 39.16 41.26 11.93 0 Asteraceae Silybum marianum DM A 93.55 8.53 8.93 34.9 2.92 0 Calendula stellata DM P 100 61.27 61.27 85.26 52.25 0 Centaurea pullata LT A 98 67.62 69 89.64 60.61 46 Dittrichia viscosa DM P 89.66 56.12 62.6 81.8 45.91 Limbarda crithmoides subsp. longifolia LT A 87.88 31.03 35.31 93.38 28.98 12 Scolymus maculates DM A 45 13 28.89 64.67 8.41 0 Juncaceae Juncus maritimus LT A 60 9.06 15.1 3.89 0.35 0 Lamiaceae Marrubium vulgare LT A 98.08 48.67 49.3 88.14 42.8 0 Liliaceae Marrubium vulgare DM A 100 52.21 52.21 70.76 36.94 0 Asphodelus tenuifolius DM E A 10 2.5 0 0 0 0 27.5 Tamaricaceae Tamarix sp. LT NM 0 0 0 0 0 30 AMF DIVERSITY IN WETLAND STRESSES ACTA BOT. CROAT. 79 (1), 2020 9 Tab. 4. The relative abundance of arbuscular mycorrhizal fungi (AMF) isolates in the wetlands Dayet Morsli (DM) and Telamine Lake (LT). Values are expressed as mean values ± standard de- viation, n = 12. RA – relative abundance (%), IF – isolation frequency (%), SR – species richness, Average SR – the mean of species number in all plant rhizospheres for each site. SD – Spore density. AMF species RA DM (%) IF (%) RA LT (%) IF (%) Acaulosporaceae 48.11 – 31.86 Acaulospora cf. collicosa 0.29±0.77 14.28 – – Acaulospora cf. alpina 0.09±0.24 14.28 – – Acaulospora thomii 12.23±5.51 100.00 15.94±3.96 100.00 Acaulospora leavis 6.96±4.20 100.00 6.09±5.74 60.00 Acaulospora melea 6.18±5.47 100.00 4.55±5.39 80.00 Acaulospora tortuosa 0.05±0.12 14.28 – – Acaulospora aff. morrowae 11.58±7.58 100.00 4.34±3.89 60.00 Acaulospora rehmii 10.88±9.04 85.71 – – Acaulospora sp. 1 – – 0.6±0.89 40.00 Acaulospora elegans – – 0.32±0.71 20.00 Ambisporaceae 1.94 – 0.6 Ambispora reticulata 1.94±4.59 28.57 0.6±1.34 20.00 Archaeosporaceae 15.67 – 18.39 Archaeospora undulata 15.67±15.62 71.42 18.39±15.63 80.00 Diversisporaceae 1.6 – – – Diversispora sp. 0.09±0.24 14.28 – – Diversispora tortuosa 0.28±0.65 28.57 – – Tricispora nevadensis 1.23±1.45 71.42 – – Claroideoglomeraceae 2.29 – 2.75 – Claroideoglomus claroideum 2.29±2.07 85.71 2.1±3.12 60.00 Claroideoglomus lamellosum – – 0.65±0.99 40.00 Glomeraceae 10.53 – 12.43 Funneliformis caledonium 0.38±1.00 14.28 1.36±2.31 40.00 Funneliformis geosporum 0.03±0.07 14.28 3.56±6.43 40.00 Funneliformis mosseae 0.80±1.24 42.85 1.06±1.46 60.00 Glomus achrum – – – – Glomus nanolumen 0.62±0.82 42.85 0.37±0.51 40.00 Glomus macrocarpum 0.64±1.70 14.28 – – Glomus diaphanum 0.25±0.67 14.28 – – Glomus sp. 1 0.15±0.40 14.28 – – Rhizoglomus microagregatum 0.23±0.42 28.57 0.2±0.27 40.00 Rhizoglomus sp. 1 2.91±7.27 28.57 0.42±0.93 20.00 Rhizoglomus sp. 2 3.42±9.07 14.28 0.50±1.13 20.00 Rhizoglomus sp. 3 0.18±0.49 14.28 – – Sclerocystis rubiformis 0.03±0.07 14.28 – – Sclerocystis sp.1 0.11±0.30 14.28 0.45±0.63 40.00 Septoglomus constrictum 0.71±1.49 28.57 3.45±5.30 60.00 Paraglomeraceae – – 1.02 – Paraglomus sp. – – 1.02±1.43 40.00 Unidentified 16.93±23.45 – 33.81±31.81 – Total 100 100 SR 28 20 Average SR 10.43±2.37 10.2±4.6 SD (100 g soil) 640.3±269.9 1077.9±364.16 It is worth noting that along the component 1, the vari- ables were clustered in three main groups. The ETM formed a distinct group within DM site and the nearest plots to the water (0–30 and 30–60). This group was negatively corre- lated to AMF spore density which recorded the highest level near to the water (plots 0–30 and 30–60) in Telamine Lake site. The third group gathering species richness and mycor- rhizal frequency in both sites and in plot (> 60) was strongly reduced by both salinity and Zinc concentration. Discussion In this study, we present a detailed report on fungal root endophyte occurrence in eighteen plant species. Heavy metal contents in soil and their effect on the fungal composition and root colonization was also investigated. The most represented families of the two study sites are Amaranthaceae and Asteraceae, whose dominance in wet- lands was reported by several studies (Megharbi et al. 2016, Neffar et al. 2013). Halophytic species of Amaranthaceae and Asteraceae are known to be naturally better adapted to survive environmental stresses, compared to salt-sensitive crop plants commonly chosen for phytoremediation pur- poses as described by Manousaki and Kalogerakis (2011). Research findings suggest them as ideal candidates for phy- toremediation of both saline and non-saline, heavy met- al polluted soils, such as Atriplex halimus (Mesnoua et al. 2016), Mesembryanthemum crystallinum (Lutts and Lefèvre SIDHOUM W, BAHI K, FORTAS Z 10 ACTA BOT. CROAT. 79 (1), 2020 2015), Atriplex canescens, Suaeda vera (Ayyappan and Ra- vindran, 2014), Calendula officinalis L. (Hristozkova et al. 2016), Marrubium vulgare (Belabed et al. 2014), Limbarda crithmoides subsp. longifolia and Dittrichia viscosa (Turnau et al. 2010). The floristic survey showed that DM site has more diversified plant species than LT (34 plant species in DM, and 28 in LT), while the flora has not been affected, de- spite the high Pb, Cr and Cd concentrations in the soil, due to the presence of different native heavy metal accumulator plants. Soil salinity values were heterogeneous, and interest- ingly, salinity is the critical factor controlling the distribution of plant communities within the study sites, corroborating those of several Algerian wetland studies (Aliat et al. 2016, Chenchouni 2017). The current study shows that the levels of Pb (270–820 ppm) in DM and (101–278 ppm) in LT), Cd (1.84–8 ppm in DM and 1.79–3.17 in LT), Cu (118 ppm in DM) and Cr (97.14–508.87 ppm in DM) recorded exceeded AFNOR NFU 44–041 reference values (100, 2 and 150 ppm, respectively), and are higher than those reported by several authors of wetland soil studies (Teuchies et al. 2013, Belabed et al. 2017, Esmaeilzadeh et al. 2017). In addition, nickel and zinc concentrations in DM and LT soils in the present study are homogeneous and vary only slightly. They do not ex- ceed the reference values, but the highest values for Ni and Cu are recorded in DM and the highest Zn value is found in LT. Similar values were reported in Algerian wetlands near to industries, including El- Hadjar iron and steel complex (Belabed et al. 2017, Louhi et al. 2012). On the other hand, Tab. 5. Changes in fungal colonisation, fungal spore density, species richness and diversity indexes along transects at the Dayet Morsli and Telamine Lake wetland sites. Results are expressed as mean values ± standard deviation, n = 4. Means in the same column with different letters are significantly different from each other (P < 0.05) according to the Tukey–test (ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001). Plots – the distance from sampling area and the water edge (0-30 m, 30-60 m, and >60 m), DSE% – dark septate endophytes frequency, f% – mycorrhizal frequency, SR – species richness, SD – Spore density, H’ – Shannon index, D – dominance index, E – even- ness. The numbers for site, plot, and site*plot on the table bottom are F-statistics values of two-way ANOVA analysis of variance. Sites: Telamine Lake and Dayet Morsli. Site Plots DSE (%) f (%) SR SD H’ D E Dayet Morsli 0-30 70.00±0.00a 0.00±0.00a 9.00±0.00a 125.00±0.00a 1.50±0.30a 0.25±0.03a 0.69±0.13a 30-60 12.00±0.00b 47.08±17.39b 10.66±3.05ab 580.66±298.57a 1.45±0.18a 0.22±0.09a 0.61±0.04a >60 2.58±6.01c 96.63±4.79c 13.42±1.61b 542.85±240.17a 1.72±0.33a 0.18±0.05a 0.76±0.14a Telamine Lake 0-30 11.86±11.63a 50.49±17.40a 10.00±0.63a 1209.38±519.85a 1.50±0.28a 0.28±0.10a 0.72±0.07a 30-60 5.83±6.88a 62.02±18.22a 9.00±2.44a 1156.66±368.16a 1.41±0.43a 0.18±0.05a 0.57±0.14a >60 36.00±50.9a 54.00±62.22a 12.00±0.00a 480.00±28.284a 1.64±0.41a 0.14±0.02a 0.70±0.13a Site 3.27ns 0.85ns 0.84ns 12.77** 0.07ns 0.17ns 0.23ns Plot 11.96*** 12.20*** 6.65** 6.65** 1.00ns 4.25* 2.72ns Site*plot 21.19*** 10.27** 1.33ns 4.73* 0.02ns 0.56ns 0.29ns Fig. 1. Principal Component Analysis (PCA) ordination biplot of 24 soil samples in the wetlands Telamine Lake (LT) and Dayet Morsli (DM), Algeria, and the following variables: spore density (SD), f (mycorrhizal frequency), DSE (dark septate endophytes frequency), spe- cies richness (SR), pH, electrical conductivity (EC), pollution index (PI), and trace elements Cr, Cd, Cu, Ni, Pb, Zn. AMF DIVERSITY IN WETLAND STRESSES ACTA BOT. CROAT. 79 (1), 2020 11 PCA test showed a good correlation between the different total metal contents except for Zn, suggesting a common ori- gin as confirmed by Carrasco et al. (2006). The presence of these metals in stations located close to industrial areas con- firms their anthropogenic origin. According to Vardanyan et al. (2008), very high local concentrations of metals often occur as a result of a strong reducing environment coupled with industrial and municipal discharges. The variation of MTE between sites was significant at P < 0.001. However, their variation between plots was not sig- nificant, except for Cr, Cu, and Zn which decreased inverse- ly with salinity, indicating that they might arise from flood water. Elevated metal (Pb, Zn, and Cr) contents and toxic phosphate levels in DM are likely to impose toxic effects for plant establishment in addition to other constraints, but it is difficult to prove MTE toxicity because it depends on their soil availability which is generally reduced. Our data also showed higher mycorrhizal frequency in Asteraceae varied with particular species, that confirms several previous studies, showing high total colonization in Centaurea stoebe (Gucwa-Przepiora and Blaszkowski 2007) and Calendula officinalis (Hristozkova et al. 2016). These re- sults may explain the considerable effectiveness of Calendu- la officinalis and M. vulgare extensively mycorrhized in the phytoremediation of heavy metals (Hristozkova et al. 2016). As the genus Asphodelus is known to form AM (Cavagn- aro et al. 2001), we detected in our study the presence of AM, Ectomycorrhizae and DSE in A. tenuifolius roots. The microsclerotes (7.5%) and sporangia (27.5%) observed are reported here for the first time. This seems to be a kind of plant species adaptation to environmental stress caused by salinity, inundation and metal pollution. In this study, Tamarix sp. was not mycorrhized but Ben- cherif et al. (2015) reported 16 to 65% of AM frequency in Tamarix articulata Vahll in Algerian saline soils. No mycor- rhization was found in Salicornia patula and Arthrocnemum macrostachyum roots which confirm the results of Landwehr et al. (2002), in saline, sodic and gypsum soils. Arbuscule rates in Amaranthaceae were very low (A<12%), like those reported by Becerra et al. (2016). Plenchette and Dupon- nois (2005) hypothesized about the existence of a third AM morphological type with no arbuscules in the Amarantha- ceae family. The high incidence (>50%) of DSE in the present study is in accordance with several reports on their great abun- dance in highly stressed ecosystems (Schmidt et al. 2008), as in wetlands (Kumar and Muthukumar 2014) and in heavily polluted soils (Čevnik et al. 2000). Additionally, the findings showed that DSE colonization is positively correlated with Cr and Cu concentration in soil, indicating that these fungal endophytes infect plant root systems occupying highly Cr and Cu stressed areas, or that plants might utilize them as an adaptation mechanism to resist abiotic stress in Cr and Cu polluted soil, due to their role in the accumulation of some trace elements, and the amelioration of the growth of some plants (Wang et al. 2016). DSE correlated negatively with mycorrhizal frequency in the investigated plant root systems, indicating the impor- tant competitive and antagonistic interaction between the two fungal endophytes, like those found by (Kandalepas et al. 2010), confirming our result. According to the same au- thors, DSE could be more resistant to adverse environmen- tal conditions than mycorrhizal fungi by being either more competitive in disturbed or moderately polluted soils or bet- ter equipped to survive. The distribution of AM fungal species of the current study showed a great AMF species richness, recorded in both DM and LT (28 and 20, respectively). These results are com- parable to those of Wang et al. (2011) (23 phylotypes) and de Marins et al. (2009) (27 morphotypes). The dominance of Glomeraceae and Acaulosporaceae was also reported by previous studies of wetlands (Sun et al. 2016). This domi- nance is owing to their ability to propagate via mycelial frag- ments, mycorrhizal root fragments and spore germination (Yang et al. 2015), and adaptation to stressful conditions of soil contamination with heavy metals compared with other AMF species metals (Lopes Leal et al. 2016). Septoglomus constrictum, Funneliformis mosseae and Funneliformis geos- porum isolated from study sites were also isolated in Algeri- an saline areas (Bencherif et al. 2015). These fungi and others encountered in our investigation (Cl. claroideum, A. mellea and Ar. undulata) were also recorded in saline or heavy met- al-polluted areas (Hammer et al. 2011). AM frequency cannot be affected by spore density, unlike species richness, and hence the infective AMF are not au- tomatically those producing a greater proportion of spores, but an increase in fungal colonization results from a greater AMF species diversity in root systems. These results are in line with those of de Marins et al. (2009) done in wetlands and hence they confirmed that spores occur both in the rhi- zosphere of macrophytes whose roots were internally colo- nized by AMF and in non-colonized macrophytes. However, mycorrhizal roots and extraradical mycelia in the soil also could be involved in plant root infection. On the other hand, salinity strongly affects AM plant status and species richness, indicating that mycorrhizal col- onization and diversity are indeed reduced with increas- ing salt levels, as with those previously found by Füzy et al. (2008). No correlation is observed between spore den- sity and salinity, because they are halotolerant and more adapted to soil salt levels in these areas. This result sug- gest that the soil salt concentration is not sufficiently high to affect negatively the sporulation in these AMF species and that probably they develop their own ability to survive and naturally occur in saline environments (Hammer et al. 2011). Inversely there are reports of low AMF spore densi- ty in saline areas (Barrow et al. 1997) while Aliasgharzad et al. (2001) suggest that higher fungal spore density in saline soils may be due to the fact that sporulation is stimulated under salt stress, and this means the possibility of produc- ing spores by AMF under low root-colonization levels in severe saline conditions. SIDHOUM W, BAHI K, FORTAS Z 12 ACTA BOT. CROAT. 79 (1), 2020 In addition, heavy metal pollution rates including Pb, Ni, Cu, and Cr concentrations disturb spore density but not my- corrhizal status, except for Cr which may inhibit root hyphal colonization. These results are in agreement with those of Vogel-Mikuš et al. (2005), indicating that the elevated con- centrations of As, Pb, Zn, Cd, and Cu exerted harmful ef- fects on AMF spore numbers in polluted plots. Heavy metals have been reported to reduce, delay, or even eliminate AMF colonization and spore density at heavy metal polluted sites (Wei et al. 2015). Salinity seems to affect AMF diversity and infectivity, but not sporulation, which is more influenced by heavy metal pollution. MTE may play a major role in the distribution of spore density. In sum, this is the first report on Algerian AM diversi- ty in saline and contaminated wetlands with heavy metals. It revealed a specific plant community and environmental stress, including high salinity as well as trace element pollu- tion. The salinity gradient and ETM pollution were found to be the dominant factors affecting AM community struc- tures. Both AMF diversity and infectivity decreased with the increase in soil salinity, while AM fungal sporulation rates were attenuated by MTE augmentation. This native flora is naturally adapted to soil stress and associated with AM and endophytic fungi. 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