**2. Materials and methods**

#### **2.1. Study site**

This study focuses on wetlands that consist of permanent or temporary areas of fresh or brackish water and adjacent lands. They include all wadis, chotts, lagoons, hill lakes, sebkhas and dams. The majority of these areas, several of which were of international importance, were found in the north, particularly near the coast. In this study, 117 stations namely *lagoons* (a stretch of salt water partially or completely separated from the open ocean by barriers of sand or coral distributed along the Tunisian coasts), *lakes* (a body of relatively still freshwater of considerable size, localized in a basin that was surrounded by land and most of them were fed and drained by rivers and streams), *sebkhas* (North African vernacular name for a shallow, salty depression. It was a common wetland type especially in semi-arid and arid climate), *wadis* (a natural stream of water of fairly large size flowing in a definite course or channel or series of diverging and converging channels), *hill lakes* (distinguished by a height >10 m and a volume >1 million m<sup>3</sup> ) and *dams* (characterized by a reservoir volume more than 3 million m<sup>3</sup> and a height of 15 m) were prospected (**Table 1**).

#### **2.2. Sampling methods and laboratory procedures**

Quantitative samples of amphipods were taken in spring of 2008, 2009 and 2010 in the early morning hours using quadrates method [36, 37]. In the bank of each site, eight quadrates of 50 × 50 cm<sup>2</sup> were randomly placed. The content of each quadrat (7 cm depth) was placed in an


physicochemical components (granulometry, temperature, humidity, etc.) of wetlands enable them to perform many ecological functions such as shoreline stabilization and water purification. Lacaze [2] mentioned that lagoon wetlands harbour a diverse fauna, but were threatened by intense anthropogenic exploitation and pollution. As they receive continental freshwater from their catchment area, many lagoons have been subjected to severe degradation of water quality caused by pollution and*/*or eutrophication [3]. In Tunisia, semi-closed shallow lagoons

Among wetlands, sandy beaches were more studied and characterized by the presence of a large number of invertebrates. Talitridae amphipods were among the most dominant invertebrates living on wetlands [6]. These talitrids play an important role as decomposers of organic matter and were considered as potential bio-indicators of sandy beaches quality [7, 8]. This role was estimated using genetic approach, behavioural approach as well as reproduction and

In Tunisia, amphipod communities inhabiting wetlands bank, other than sandy beaches [21– 23, 31–35] have not received much attention. Through this study, we propose a description as exhaustive as possible of the biodiversity of these communities taking into account geographical, climatic and edaphic specificities. More specifically, we addressed the following questions: (1) Does the diversity of Talitridae amphipods follow a north-south cline? (2) Is the

This study focuses on wetlands that consist of permanent or temporary areas of fresh or brackish water and adjacent lands. They include all wadis, chotts, lagoons, hill lakes, sebkhas and dams. The majority of these areas, several of which were of international importance, were found in the north, particularly near the coast. In this study, 117 stations namely *lagoons* (a stretch of salt water partially or completely separated from the open ocean by barriers of sand or coral distributed along the Tunisian coasts), *lakes* (a body of relatively still freshwater of considerable size, localized in a basin that was surrounded by land and most of them were fed and drained by rivers and streams), *sebkhas* (North African vernacular name for a shallow, salty depression. It was a common wetland type especially in semi-arid and arid climate), *wadis* (a natural stream of water of fairly large size flowing in a definite course or channel or series of diverging and converging channels), *hill lakes* (distinguished by a height >10 m and a

Quantitative samples of amphipods were taken in spring of 2008, 2009 and 2010 in the early morning hours using quadrates method [36, 37]. In the bank of each site, eight quadrates of

were randomly placed. The content of each quadrat (7 cm depth) was placed in an

) and *dams* (characterized by a reservoir volume more than 3 million m<sup>3</sup>

were among the most sensitive areas to environmental stresses [4, 5].

correlation between specific diversity and wetlands type is significant?

spatio-temporal distribution studies [9–32].

**2. Materials and methods**

**2.1. Study site**

102 Selected Studies in Biodiversity

volume >1 million m<sup>3</sup>

50 × 50 cm<sup>2</sup>

and a height of 15 m) were prospected (**Table 1**).

**2.2. Sampling methods and laboratory procedures**


**Stations Wetland type Governorate GPS Sediment type** 70. El Kbir Wadi Siliana 36°07′11″/009°35′28″E Sandy-clay-silt 71. Bargou Wadi Siliana 36°05′25″N/009°33′48″E Loamy sand 72. Massouj Wadi Siliana 36°04′57″N/009°22′30″E Fine silt 73. Saboun Wadi Siliana 35°52′11″N/009°11′37″E Silty

Biodiversity of Amphipoda Talitridae in Tunisian Wetlands

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105

74. Zguifa Wadi Siliana 35°45′55″N/009°01′22″E Loamy sand 75. Raguey Wadi Jendouba 36°27′51″N/008°23′27″E Sandy loam 76. Mazbla Wadi Jendouba 36°29′11″N/008°18′28″E Sandy loam 77. El Maleh Wadi Ariana 36°58′41″N/010°09′55″E Sandy loam 78. Lanj Wadi Jendouba 36°34′46″N/008°30′25″E Sandy 79. Lahmam Wadi Jendouba 36°32′55″N/008°26′53″E Loamy sand 80. Soufi Wadi Jendouba 36°29′20″N/008°23′49″E Sandy loam 81. Menzel Tmim Wadi Nabeul 36°42′26″N/010°43′27″E Sandy loam 82. El Widyen Wadi Nabeul 36°47′03″N/010°53′39″E Sandy 83. Sliman Wadi Nabeul 36°41′36″N/010°28′53″E Loamy sand 84. Lebna Wadi Nabeul 36°39′13″N/010°54′31″E Sandy loam 85. Houith Hill lake Bizerte 37°4′59″N/009°58′5″E Loamy sand 86. Morra Hill lake Bizerte 37°05′53″N/009°59′08″E Sandy

87. Bnt Liba Hill lake Bizerte 37°05′52″N/009°59′08″E Sandy-clay-silt

88. Ghar Ettine Hill lake Bizerte 37°04′02″N/009°15′53″E Sandy 89. Sidi Daoued Hill lake Bizerte 37°03′14″N/009°23′47″E Sandy 90. Khelifa wadi Hill lake Zaghouan 36°13′40″N/009°47′13″E Sandy 91. Jetta Hill lake Siliana 35°59′44″N/009°26′48″E Sandy 92. Ain Ben Ali Hill lake Siliana 36°03′47″N/009°17′35″E Sandy 93. Zrab wadi Hill lake Siliana 36°02′8″N/009°16′54″E Sandy 94. Khalsi Hill lake Siliana 35°57′10″N/009°10′32″E Sandy 95. Jdaïda wadi Hill lake Siliana 35°53′53″N/009°11′12″E Loamy sand 96. Ettal wadi Hill lake Siliana 35°53′20″N/009°10′54″E Sandy loam 97. Ksayir Hamdoun Hill lake Siliana 35°48′10″N/009°03′57″E Sandy 98. Ouled Ali Hill lake Siliana 35°50′58″N/009°09′31″E Sandy 99. Zraybiya Hill lake Jendouba 36°28′25″N/008°21′29″E Loamy sand

100. At 5km d'El Kssour Hill lake Kef 35°52′05″N/008°55′52″E Clay 101. Bni Mtir Dam Jendouba 36°44′47″N/008°44′19″E Sandy loam 102. Sidi Barrak Dam Béja 37°00′52″N/009°06′12″E Sandy 103. El Hma Dam Ben Arous 36°35′16″N/010°18′24″E Sandy clay 104. Bakbaka Dam Ben Arous 36°34′35″N/010°20′17″E Loamy sand


**Stations Wetland type Governorate GPS Sediment type** 36. Thrayaa Sebkha Gabès 34°10′10″N/010°00′47″E Sandy silt loam 37. Gataaya Sebkha Kébili 33°41′44″N/008°53′44″E Sandy clay 38. Jemna Sebkha Kébili 33°34′48″N/009°00′15″E Clay

104 Selected Studies in Biodiversity

39. Blidette Sguira Sebkha Kébili 33°35′18″N/008°51′06″E Sandy silt loam 40. Blidette Kbira Sebkha Kébili 33°34′27″N/008°51′37″E Sandy silt loam 41. Guidma Sebkha Kébili 33°25′44″N/008°47′45″E Sandy clay 42. Golaa Sebkha Kébili 33°31′18″N/008°57′26″E Sandy clay 43. Zarzara Sebkha Kébili 33°31′07″N/008°56′30″E Clay

44. El Korsi Wadi Bizerte 37°11′12″N/009°46′52″E Sandy loam 45. Tinja Wadi Bizerte 37°10′10″N/009°45′26″E Loamy sand 46. Lebna wadi Estuary Wadi Nabeul 36°38′58″N/010°54′57″E Sandy loam 47. Khniss Wadi Monastir 35°43′13″N/010°48′57″E Sandy 48. Lakaarit Wadi Gabès 34°06′29″N/009°58′55″E Sandy

49. El Fared Wadi Gabès 33°44′59″N/010°12′31″E Sandy-clay-silt 50. Majerda Wadi Bizerte 37°05′03″N/010°08′17″E Loamy sand 51. Joumin Wadi Bizerte 37°0′37″N/009°41′59″E Sandy 52. Sidi Bou Ali Wadi Sousse 35°58′8″N/010°27′20″E Sandy 53. Hamdoun Wadi Monastir 35°46′51″N/010°40′48″E Sandy loam 54. Zerkine Wadi Gabès 33°41′22″N/010°15′12″E Sandy 55. Zigzaw Wadi Gabès 33°35′40″N/010°18′42″E Clay 56. Zas Wadi Medenine 33°30′53″N/010°20′28″E Clay 57. Koutine Wadi Medenine 33°26′34″N/010°23′9″E Silty 58. Hessi Amor Wadi Medenine 33°21′47″N/010°37′14″E Clay 59. Bouhamed Wadi Sidi Bouzid 33°18′6″N/010°44′5″E Silty

60. Demna Wadi Gabès 33°56′27″N/010°1′35″E Loamy sand 61. Maleh Wadi Gabès 34°0′2″N/009°59′57″E Loamy sand

62. Widran Wadi Sfax 34°31′7″N/010°4′17″E Clay 63. Zit Wadi Zaghouan 36°27′01″N/010°16′43″E Sandy 64. El Harat Wadi Zaghouan 36°21′50″N/010°18′34″E Sandy 65. Lassoued Wadi Siliana 36°24′20″N/010°12′37″E Sandy loam

66. Sidi Hmid Wadi Zaghouan 36°24′21″N/009°58′56″E Silty 67. Bouthiben Wadi Zaghouan 36°22′16″N/009°54′0″E Sandy 68. El Kbir wadi Wadi Siliana 36°13′26″N/009°44′49″E Loamy sand 69. Siliana Wadi Siliana 36°12′03″N/009°42′57″E Sandy-clay-silt


National Research Council Canada (NRCC) Canada) as unknowns along with the samples. Organic matter content was determined by weighing before and after ashing at 450°C for 3 h at

To compare the amphipod community structure among stations, different faunistic parameters were calculated using quantitative data such as species richness, relative species abundance, etc. Mean density of the amphipod community at each station and the mean density of

and evenness were calculated by the Shannon-Weaver index and Pielou's evenness index [40], respectively. The degree of similarity between sampling stations was evaluated using similarity cluster dendrograms. The analysis above was performed with the PRIMER software package [41]. Principal component analysis of amphipod distribution and site characteristics was

Temperature (°C) and humidity (%) were measured *in situ* in different wetland types. The mean values for these two parameters varied between 22.453 ± 2.797°C in dams, 27.387 ± 5.289°C in sebkhas, 51.243 ± 18.627% in sebkhas and 65.50 ± 12.388% in lagoons (**Figure 1A** and **B**).

The percentage of organic matter differs between and within wetland types (**Figure 1C**). The highest values were observed in the banks of Bizerte lagoon (9.46%), Majin Chitane (12.23%), Halk Menzel (16.13%), Bargou wadi (20.66%), Ouled Ali (17.62%) and Kasseb (12.64%)

An heterogeneity in grain size nature was observed between stations ranging from sandy substrates, loamy sand, sandy loam, sandy silt, sandy-clay, silty clay, clay-loam, sandy-clay-silt,

In the lagoon, the highest concentrations for the majority of heavy metals, vanadium, nickel, zinc, arsenic, cadmium, thallium and lead were recorded in the northern lagoon of Tunis. The bank of Bizerte lagoon was characterized by the highest concentrations of chromium (26.393 ppm) and manganese (281.748 ppm). While the highest copper content (39.098 ppm) was observed in El Bcherliya. The Korba lagoon revealed the highest concentration in cobalt and rubidium with 8.311 and 15.814 ppm, respectively. Bhiret El Biben was characterized by the highest concentration of lithium (29.087 ppm), strontium (2101.549 ppm) and tin (7.340 ppm). In addition, the lowest concentration for all the heavy metals studied was recorded in the bank of Sidi Ali Mekki lagoon. The different metals analysed in these lagoons do not exceed the maximum tolerate values [42] except lead that exceeds 100 ppm in the northern lagoon of Tunis (133.556 ppm).

. Species diversity

107

Biodiversity of Amphipoda Talitridae in Tunisian Wetlands

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each species at each station were expressed as number of individuals per m<sup>2</sup>

**3.1. Temperature, humidity, organic matter and grain size**

sandy-clay-loam, sandy-silt-loam, fine silt to clay substrates (**Table 1**).

the University of Salzburg.

performed using Xlstat software.

**2.4. Data analysis**

**3. Results**

(**Figure 1C**).

**3.2. Heavy metals**

**Table 1.** Localization of the studied stations.

individual bag, and then the animals were sorted by hand. Twenty minutes were devoted to each quadrat. Humidity and temperature of air and soil were measured *in situ* at each site. At the laboratory, amphipod specimens were preserved in 70% ethanol. Then, they were identified, counted and sexed. The identification of these species was carried out under Leica MS 5 binocular microscope, using the key of Ruffo [38].

#### **2.3. Soil analysis**

The particle size, organic matter and heavy metals of soil samples taken from 117 stations were analysed. Grain size distribution of these composite samples was analysed using different sieves in descending order (from 2 to 25 μm).

A subsample was brought to the inductively coupled plasma-mass spectrometry (ICP-MS) laboratory at University of Kiel and sieved to obtain the <250-μm grain size fraction which was then dried and milled [39]. Heavy metals were extracted from a 250-mg sample of powder with 10 mL 7 N nitric acid on a hot plate at 80°C (2.5 h). The solution was made up to 20 mL, centrifuged at 3500 rpm for 15 min, and the supernatant transferred to a 20-mL sample vial. The metals vanadium (V), chromium (Cr), manganese (Mn), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), cadmium (Cd), tin (Sn), thallium (Tl), lead (Pb), lithium (Li), rubidium (Rb) and strontium (Sr) were analysed by inductively coupled plasma-mass spectrometry (ICP-MS). Average analytical reproducibility was estimated from replicate analyses of some samples and was found to be better than 2% Relative Standard Deviation (RSD) (1 sigma relative standard deviation) for all elements. The accuracy of analytical results was monitored by analysing certified reference materials (CRM): GSMS-2 (marine sediment; Chinese Academy of Geological Sciences, PR China) and Reference material, coastal sediment (PACS-1) (coastal sediment; National Research Council Canada (NRCC) Canada) as unknowns along with the samples. Organic matter content was determined by weighing before and after ashing at 450°C for 3 h at the University of Salzburg.

#### **2.4. Data analysis**

To compare the amphipod community structure among stations, different faunistic parameters were calculated using quantitative data such as species richness, relative species abundance, etc. Mean density of the amphipod community at each station and the mean density of each species at each station were expressed as number of individuals per m<sup>2</sup> . Species diversity and evenness were calculated by the Shannon-Weaver index and Pielou's evenness index [40], respectively. The degree of similarity between sampling stations was evaluated using similarity cluster dendrograms. The analysis above was performed with the PRIMER software package [41]. Principal component analysis of amphipod distribution and site characteristics was performed using Xlstat software.
