Two new genera and three new subterranean species of Hydrobiidae (Caenogastropoda: Truncatelloidea) from Tunisia

The aquatic biodiversity of springs and groundwater systems of North Africa remains largely unexplored. In an earlier field survey of Tunisian springs, a new gastropod genus, Bullaregia, was discovered as a phylogenetically independent lineage of uncertain position within the family Hydrobiidae. Here, we provide taxonomic and phylogenetic assignments for three newly collected populations of hydrobiids from springs in northern Tunisia based on morphological, anatomical and genetic (mtCOI and 18S) data. Among these and specimens of Bullaregia, major differences were observed in male and female genitalia as well as in mtCOI sequences (divergence 8.0–9.1%). Based on these findings, we describe two new genera and three new species: Belgrandiellopsis chorfensis gen. et sp. nov., Belgrandiellopsis secunda gen. et sp. nov. and Biserta putealis gen. et sp. nov. In all our phylogenetic analyses, these three new species were well resolved as a monophyletic group together with Bullaregia tunisiensis. Unexpectedly, this clade emerged as sister to the European valvatiform genera Corbellaria and Kerkia and not to the recently discovered clade of groundwater, conchologically similar, species living in Bulgaria (Balkan Peninsula). These Tunisian species are each locally endemic and form part of a newly discovered clade which in future systematic studies could eventually be identified as a distinct hydrobiid subfamily.

these favourable conditions, northern African ground waters are currently under considerable stress due to climate change and/or anthropogenic factors such as land use or water pollution (Ouedraogo et al. 2016). In Tunisia, human populations have long used groundwater resurgences (springs and wells) as drinking and irrigation water. As a result, wells and captured springs can be found across the country, and these are also home to highly adapted, subterranean organisms. While more than 100 stygobiont (i.e., obligate subterranean) species have been discovered in North Africa (Gibert & Culver 2009), information on the subterranean biodiversity of Tunisia, including those species living in springs of underground waters, is still very limited (García et al. 2010). The restricted geographic distribution of these species, often of difficult access, coupled to their small body size and convergent structures (Culver 2012) make biodiversity inventories in these ecosystems a real challenge.
Molluscs are an important component of spring and groundwater systems of North Africa (García et al. 2010). For more than a decade now, surveys of continental underground ecosystems (e.g., streams, springs and wells) in Tunisia have revealed that the most abundant and frequent mollusc species belong to the gastropod family Hydrobiidae Stimpson, 1865 (Khalloufi & Boumaiza 2007;Khalloufi et al. 2017). Even when applying the sensu stricto definition based on molecular evidence (Wilke et al. 2001(Wilke et al. , 2013, hydrobiids are generally considered one of the most species-rich families of freshwater molluscs, and have been described as a hotspot of diversity in Northern Africa (Strong et al. 2008;Miller et al. 2018). However, the status of most Tunisian hydrobiid species is unknown, and the species richness picture of this family in Tunisia is still incomplete. Minute-sized snails from this region were previously discovered mainly from surface fresh-and brackish-waters. Based on shell morphology, most species were tentatively assigned to the non-hydrobiid genera Amnicola Gould & Haldeman, 1840, Bythinella Moquin-Tandon, 1856 and Paludestrina D'Orbigny, 1840 (Bourguignat 1860(Bourguignat , 1864Debeaux 1863;Issel 1880;Letourneux & Bourguignat 1887;Pallary 1921Pallary , 1923Seurat 1921Seurat , 1934. Later on, according to new anatomical evidence, Boeters (1976) and Glöer et al. (2010) transferred some of these taxa to the hydrobiid genera Pseudamnicola Paulucci, 1878, Mercuria Boeters, 1971 and Hydrobia Hartmann, 1821. In the latter study (Glöer et al. 2010), new species of Pseudamnicola and Mercuria from Tunisia were also identified based on samples from museum collections. Recent molecular phylogenies (Delicado et al. 2015 have also unveiled unknown lineages within Pseudamnicola which may represent new species to science. relationships and position within Hydrobiidae, we also add genetic (mitochondrial and nuclear) data for these and other recently discovered subterranean hydrobiid species from Europe (Rysiewska et al. 2016;Osikowski et al. 2017) to the previous molecular phylogeny (Khalloufi et al. 2017). This work forms part of a series of upcoming papers that will describe hydrobiid diversity in neglected and highly stressed aquatic ecosystems in Tunisia.

Material and methods
Live specimens were collected from three localities in northern Tunisia: Chorfa Spring, Province Béja,36.865918º N,9.363313º E,430 m a.s.l.;Ettoute Spring,Province Bizerte,36.883898º N,9.505894º E, 415 m a.s.l.; and Soudene Well (Menzel Bourguiba city), Province Bizerte, 37.09046º N, 9.777997º E, 115 m a.s.l. (Fig. 1). This was done within the substrate with a 300 μm mesh cloth. Samples were preserved in 70% ethanol in the field and sorted in the laboratory under a MOTIC ST-39 series stereo microscope. Anatomical structures were studied under an Olympus SZX12 stereo microscope and photographed using a Keyence VHX 2000 3D Digital Microscope in combination with the program VHX-2000 Communication software ver. 2.3.5.0 (Keyence Corporation 2009-2012. The radula was extracted from the buccal mass according to the first step of a CTAB protocol for DNA isolation (Wilke et al. 2006). Then, after drying, radulae were mounted on metallic stubs and sputter-coated with gold (Balter Sputter Coater SCD004) for 50 s so that photographs could be captured with a field emission scanning electron microscope (FESEM) DSM982 Gemini (Carl Zeiss GmbH, Germany). Anatomical characteristics (genitalia, stomach, radula and gill) were described, following the standard terminology of Hershler & Ponder (1998). Shell measurements were made with an eye-piece micrometer under the stereo microscope and consisted of length and width of the entire shell, body whorl and aperture, aperture high and width of antepenultimate and penultimate whorls (see abbreviations below). The number of specimens subjected to morphometry is indicated in the corresponding sections of the text. The 11 variables for shell dimensions and number of body whorls followed a normal distribution according to Kolmogorov-Smirnov tests and have equal variances among groups as indicated by Levene's test of homogeneity of variance. Consequently, an ANOVA could be used to test for statistical differences among these variables in the three species. These statistical calculations have been performed with the R 3.6.1 statistical environment (R Development Core Team 2019) and the R package car (Fox & Weisberg 2019). The sex of the holotype was not examined to keep the shell intact. The holotype and some of the paratypes were deposited in the Collection of Molluscs of the Museo Nacional de Ciencias Naturales de Madrid (MNCN), Spain; the other paratypes and DNA samples were deposited in the collection of the University of Giessen Systematics and Biodiversity (UGSB) (Diehl et al. 2018) in Germany and the collection of N. Khalloufi at the Faculty of Sciences of Bizerte, University of Carthage, in Tunisia.
We genetically analysed one to two specimens from each sample. GenBank sequences from another 32 hydrobiid species (Table 1) resembling the newly discovered Tunisian populations in shell shape and/or with evidence of their close relationship to them (Khalloufi et al. 2017) were added to the genetic data matrices to assess the phylogenetic status and position of these snails within the Hydrobiidae. Genomic DNA was extracted from whole specimens using the CTAB protocol of Wilke et al. (2006). Regions of the mitochondrial cytochrome c oxidase subunit I (COI) and nuclear ribosomal 18S rRNA (18S) were amplified and sequenced using the primer pairs LCO1490 (Folmer et al. 1994) and COR722b (Davis et al. 1998) for COI, and the universal metazoan 18S primers (Holland et al. 1991) for 18S. Amplification conditions for COI and 18S gene fragments were those of Delicado et al. (2012) and Schreiber et al. (2012), respectively. The amplified PCR product was sequenced in an ABI 3730 XL sequencer (Life Technologies, Carlsbad, CA, USA) using a Big Dye Terminator kit ver. 3.1 (Life Technologies). New sequences were deposited in GenBank (Table 1).
Forward and reverse sequences were assembled and edited in Sequencher 4.6 (Gene Codes, Ann Arbor, MI). Sequences obtained de novo were compiled in two datasets (one per gene partition), together with sequences of other selected hydrobiid species (Table 1). Protein-coding COI sequences were unambiguously aligned in MEGA 7.0.14 (Kumar et al. 2016). The 18S fragments were aligned using MAFFT 7.402 (Katoh et al. 2002) with default settings for gap penalties [gap opening penalty (GOP) = 1.53]. Sequence divergences (uncorrected p-distances) were calculated in MEGA. Wilke et al. (2013) found little saturation within the Hydrobiidae for the COI and 18S regions and we did not, therefore, perform saturation tests. The substitution models TIM2 (Kimura 1981) +I (invariable sites) +G (rate variation among sites) and TrNef (Tamura-Nei model with equal base frequencies; Tamura & Nei 1993) +I +G were selected as best fitting the COI and 18S datasets, respectively, in jModelTest 2.1.7 (Darriba et al. 2012) with the corrected Akaike's information criterion (AICc; Akaike 1974;Sugiura 1978;Hurvich & Tsai 1989).
Phylogenetic relationships of the hydrobiid species examined were estimated under maximum likelihood (ML) and Bayesian inference (BI) approaches. Maximum likelihood analysis was performed using RAxML-HPC2 8 (Stamatakis 2014) on the computer cluster of the Cyber Infrastructure for the Phylogenetic Research project (CIPRES; www.phylo.org) using the GTR (Tavaré 1986) +I +G model for   (Ronquist et al. 2012) using the substitution models selected through jModelTest. Markov chain Monte Carlo (MCMC) methods were run with random starting trees and 4 chains for 5 000 000 generations, sampling one generation every 1000 sampling points. After verifying chain convergence (i.e., average standard deviations of split frequencies < 0.01), the first 10% of the samples was discarded as burn-in. The robustness of the inferred trees was evaluated by Bayesian posterior probabilities (BPPs). Tree topologies and branch supports were visualized in FigTree 1.4.3 (Rambaut 2010).

Abbreviations
Shell measurements AH = aperture height AL = aperture length AW = aperture width LBW = length of the body whorl NSW = number of spire whorls SL = shell length SW = shell width WAW = width of antepenultimate whorl WBW = width of body whorl WPW = width of penultimate whorl

Results
The combined alignment of the COI (648 bp) and 18S (468 bp) sequences yielded 1116 bp. Average base frequencies for COI were 30.8% A, 13.0% C, 11.2% G and 45% T. Based on the combined dataset, ML and BI trees agreed in depicting a well-supported (BS = 100%, BPP = 1) monophyletic clade, informally referred to herein as the 'Tunisian clade', comprising the Tunisian populations examined here and Bullaregia tunisiensis. Each of the Tunisian populations formed a well-resolved subclade within this clade (Fig. 2). Uncorrected pairwise sequence divergences among representatives of this clade ranged from 7.9-9.3% for COI and were 0% for 18S. These results are similar to those observed among other genera of our phylogenetic study (e.g., 6.2-10% COI and 0% 18S among the subterranean genera from Bulgaria). Phylogenetic relationships within the Tunisian clade were well supported between populations from Chorfa and Ettoute Springs (BS = 86%, BPP = 0.96). The remaining relationships within this clade were weakly supported. BI analysis resolved (BPP = 1) the Tunisian clade as sister to the species Corbellaria celtiberica Girardi & Boeters, 2012  Genus Belgrandiellopsis gen. nov. urn:lsid:zoobank.org:act:6073FD1F-91C0-4C93-B1CB-09AA54B1FABD

Diagnosis
Shell small (maximum length slightly exceeding 2 mm), ovate-conic to elongate-conic, with obtuse and planispiral protoconch; whorls slightly convex, with weak sutures. Penultimate whorl tall relative to remaining whorls. Aperture pyriform to ovate, complete, angled on top and often fused to the body whorl. Umbilicus closed by inner lip. Operculum corneous, whitish to yellowish, thin, pliable, elongateellipsoidal, spiral, paucispiral, with submarginal nucleus. One pair of small triangular basal cusps on radular central tooth. Ctenidium well developed. Stomach with a small gastric (posterior) caecum. Presence of bursa copulatrix; unpigmented renal oviduct with a spherical pouch at the end of the coiled section; two seminal receptacles. Penis attached to the neck behind the right eye with two glandular lobes distantly positioned, each of them bearing a terminal papilla.

Etymology
Refers to the shell similarities with the hydrobiid genus Belgrandiella; gender feminine.

Remarks
Belgrandiellopsis gen. nov. differs from the conchologically similar genus Belgrandiella in having a strap-like penis with two glandular lobes, two seminal receptacles and one pair of basal cusps on the central radular teeth (for comparison see Szarowska 2006); from the closely related genus Bullaregia according to the presence of two lobes on the inner edge of the penis, smaller gastric caecum, shorter bursal duct, larger bursa copulatrix and in having generally a smaller number of cusps on radular teeth (see Khalloufi et al. 2017); and from Biserta gen. nov. according to its smaller shell umbilicus, longer penis with a larger distance between penial lobes, slightly smaller bursa copulatrix and longer bursal duct (Figs 4, 6, 8). Belgrandiellopsis gen. nov. differs in its mean COI divergence by 9.3% and 8.4% from the closely related Bullaregia and Biserta gen. nov., respectively, and by 17.6% from Belgrandiella.  shell. Elongate-conic, brown to yellowish, with 4-4.5 whorls (Fig. 3A-D; Table 2). Protoconch (Fig. 3G) obtuse, near planispiral, about 1.2 whorls, diameter about 350 µm; protoconch microsculpture granulated. Teleoconch whorls weakly convex, separated by deep sutures; body whorl occupying ⅔ of total shell length. Aperture about 40% of total shell length, pyriform, strongly angled on the top and narrowly disjunct from parietal wall of body whorl; inner lip thin but thicker than outer lip; peristome margin ( Fig. 3B) straight. Umbilicus narrow and closed by inner lip.
Male genitalia. Penis cylindrical (Fig. 4D-E), medium sized, unpigmented, slender, slightly folded; filament short, tapering; basal portion intermediate in width, shorter than distal portion, attached centrally to the head well behind the eyes; penis having two developed lobes located relatively far from one another on opposite sides of penis; both lobes with distal papilla, showing by transparency tubular glands distal; basal lobe larger, thumb-like, not pedunculated; distal lobe smaller, pyriform, pedunculated. Prostate gland small, close, bean-shaped, longer than wide (0.2 mm / 0.10 mm). Posterior vas deferens developed in its median portion with several loops (about 4 mm in length and 0.01 mm in section); seminal vesicle yellowish. Pallial vas deferens straight, narrow, whitish, unciliated, entering the posteroventral portion of gland and exiting from its anterior end.  (Fig. 4F). Bursa copulatrix ovoid to pyriform, having a duct as long as bursal length. Renal oviduct unpigmented, coiled, having a spherical pouch at the end of the coiled section. Two seminal receptacles; SR1 medium sized, elongate, pedunculate, joining renal oviduct just above the insertion point with bursal duct; SR2 small, globular, sessile, located on renal oviduct near loop before pouch (Fig. 4G-H).

Ecology and distribution
Belgrandiellopsis chorfensis gen. et sp. nov. was found in a small spring near a rural settlement called Chorfa between the cities of Mateur and Beja, in northern Tunisia. It is known only from the type locality and some springs in this region (e.g., Ain Elgaid Spring). The specimens were found on the bottom and attached to the underside (algae-side) of gravel and stones. Density decreased drastically downstream from the water resurgence. Pseudamnicola sp. was the only co-occurring gastropod species.

Remarks
Differentiated from its congener according to its smaller shell size ( Diagnosis Shell 1.7 to 2.1 mm high, elongate-conic, brown-yellowish; central radular tooth formula (6)5-C-5(6)/1-1; bursa copulatrix ovoid with short duct; two seminal receptacles, SR1 more developed and pedunculated, SR2 very small, globular, located near loop before the pouch; prostate gland bean shaped, ovate in section, folded; penis cylindrical, slender, distal end tapered, unpigmented, with two lobes relatively distant positioned; basal lobe more developed and middle positioned. Distinguished from other closely related species by >8.4% for COI.
Male genitalia. Penis cylindrical (Fig. 6D), medium sized, unpigmented, slender, slightly folded; filament short, tapering; basal portion intermediate in width, longer than distal portion, attached centrally to the head well behind the eyes; penis having two developed lobes, both with distal papilla, showing by transparency tubular glands; lobes located relatively distant from one another on opposite sides of penis; basal lobe large, conic, sessile, positioned centrally along penis; distal lobe smaller, pyriform, pedunculated. Prostate gland small, close, bean-shaped, longer than wide (0.2 mm / 0.10 mm). Posterior vas deferens whitish, forming the seminal vesicle and developed in its median portion with several loops (about 3.5 mm in length and 0.05 mm in section). Pallial vas deferens straight, narrow, whitish, unciliated, entering the posteroventral portion of gland and exiting from its anterior end.
FeMale genitalia. Ovary simple, with stalked lobes, occupying near 40% of visceral coil. Distal female genitalia occupying near 50% of pallial roof; albumen gland less developed than capsule gland (Fig. 6E). Bursa copulatrix ovoid to pyriform, having a duct slightly shorter than bursal length. Renal oviduct unpigmented, coiled, having a very small, ovoid to spherical pouch at the end of the coiled section. Two seminal receptacles; SR1 medium sized, elongate, pedunculate, joining renal oviduct just above the insertion point with bursal duct; SR2 small, globular, sessile, located on renal oviduct near loop before pouch (Fig. 6F-G).

Ecology and distribution
This species was found in a spring and its outflow in the rural village Ettoute between the cities of Mateur and Beja, in northern Tunisia. Specimens were attached to the underside of gravel and stones covered by algae. In the small stream flowing out of the spring, the snails were found in the central section featuring clean and oxygenated waters and a silty substrate. The density of this species decreased drastically downstream from the water resurgence. Co-occurring gastropod species were Pseudamnicola sp., Galba truncatula (O.F. Müller, 1774), Bulinus truncatus (Audouin, 1827) and Physella acuta Draparnaud, 1805.

Diagnosis
Shell small (maximum length 2 mm), ovate-conic to elongate-conic, with obtuse and planispiral protoconch; whorls convex, with deep sutures. Penultimate whorl tall relative to remaining whorls. Aperture pyriform to ovate, complete, slightly angled on top and often fused to the body whorl. Umbilicus closed by inner lip. Operculum corneous, whitish to yellowish, thin, pliable, elongate-ellipsoidal, spiral, paucispiral, with submarginal nucleus. One pair of small triangular basal cusps on radular central tooth. Ctenidium well developed. Presence of bursa copulatrix; unpigmented renal oviduct with a spherical pouch at the end of the coiled section; two seminal receptacles. Penis attached to the neck behind the right eye with two glandular lobes closely positioned to one another, each of them bearing a terminal papilla.

Etymology
Biserta is one of the Latin names of Bizerte, name of the province where the genus was discovered; gender feminine.

Remarks
Biserta gen. nov. differs from the closely related genera Bullaregia and Belgrandiellopsis gen. nov. according to its larger shell umbilicus, two glandular lobes closely positioned to one another on the penis, slightly larger bursa copulatrix, shorter bursal duct and to its 8-8.9% COI sequence divergence. Diagnosis Shell 1.6 to 2.0 mm high, elongate-conic, brown-yellowish; central radular tooth formula (5)4-C-4(5)/1-1; bursa copulatrix pyriform, with a short duct; two seminal receptacles, SR1 more developed and pedunculated, SR2 small, globular, located near loop before the pouch; prostate gland bean shaped, ovate in section, folded; penis gradually tapering, unpigmented, with two sessile lobes closely positioned to one another. Distinguished from other closely related species by >8.0% for COI.

Etymology
The species epithet is from the Latin adjective 'putealis', meaning 'from a well'.
pigMentatiOn and anatOMy. Visceral coil brown-yellowish, showing by transparency the gonad; pallial coil with uniform and dense pigmentation on the roof, anteriorly to edge of mantle, foot and area between tentacles (Fig. 8C). Black small eyes surrounded by brownish pigment. Snout with medium distal lobation. Tentacles parallel-side, distal end nonexpanded. Ctenidium filaments about 13, small, taller than wide. Osphradium whitish, ellipsoidal, positioned centrally along ctenidium (Fig. 8A). Stomach slightly longer than wide with a small gastric (posterior) caecum (Fig. 8B); rectum wide, folded and S-shaped.
Male genitalia. Penis gradually tapering (Fig. 8D), medium sized, unpigmented, slightly folded; filament short, tapering; basal portion intermediate in width, shorter than distal portion, attached centrally to the head well behind the eyes; penis having two developed lobes, very close to one another on opposite sides of penis; both lobes having distal papilla and showing by transparency tubular glands; ventral lobe large, pyriform, pedunculated; dorsal lobe small, pyriform, sessile. Prostate gland small, close, beanshaped, longer than wide (0.25 mm / 0.05 mm). Posterior vas deferens with several loops, yellowish and developed in its median portion (about 2 mm in length and 0.05 mm in section). Pallial vas deferens straight, narrow, whitish, unciliated, entering the posteroventral portion of gland and exiting from its anterior end.
FeMale genitalia. Ovary simple, with stalked lobes, occupying near 40% of visceral coil. Distal female genitalia occupying near 50% of pallial roof; albumen gland approximately as long as capsule gland (Fig. 8E). Bursa copulatrix pyriform, having a shorter duct than bursal length. Renal oviduct unpigmented, coiled, having a spherical pouch at the end of the coiled section. Two seminal receptacles; SR1 medium sized, elongate, pedunculate, joining renal oviduct just above the insertion point with bursal duct; SR2 small, globular, sessile, located on renal oviduct near loop before pouch (Fig. 8F-G).

Ecology and distribution
This species was found only in a small well in southern Menzel Bourguiba city, northern Tunisia. The well is 1.5 m in diameter and 4 m deep. Specimens were very abundant on the rock walls and on the rocky and sandy bottom. Water salinity was about 1.5 ppt. Pseudamnicola sp. was the only co-occurring gastropod species.

Remarks
Some individuals of the collected population lack body pigmentation (Fig. 7C), which could indicate adaptation to subterranean conditions (Delicado 2018). Other anatomical structures resembled those of the pigmented individuals.

Discussion
The discovery of Bullaregia tunisiensis by Khalloufi et al. (2017) prompted our intense research on groundwater snails in an effort to gain a better understanding of the species richness, distribution and evolutionary patterns of hydrobiid clades in Tunisia. In prior work (Khalloufi et al. 2017), we detected an uncertain phylogenetic position of this genus within the Hydrobiidae. In the present study, we provide morphological and genetic data for previously unknown populations from Tunisian groundwater systems and evidence of three new taxa resolved in a monophyletic group together with B. tunisiensis. In our phylogenetic analyses (Fig. 2), no well-supported relationship was found between this group and the recently discovered clade of subterranean hydrobiid species from Bulgaria  ; instead, it formed a monophyletic group with Corbellaria celtiberica and Kerkia kusceri. The sister relationship between these two species of valvatiform snails was previously shown by Delicado et al. (2019). However, this grouping was not supported by morphological evidence as, for instance, the Tunisian snails feature elongate-conic shells and remarkable differences in female and male genitalia (see Girardi &Boeters 2012 andBodon et al. 2001 for morphological comparison with C. celtiberica and K. kusceri, respectively). We anticipate that additional genetic data from undiscovered populations of groundwater and spring snails living in unexplored areas such as Tunisia will change this phylogenetic picture and help to further unravel the phylogenetic position of this Tunisian clade within the Hydrobiidae.
The two new genera presented in this study raises the number of genera of Hydrobiidae sensu stricto in Tunisia to seven (Pseudamnicola, Hydrobia, Ecrobia Stimpson, 1865, Mercuria, Bullaregia, Belgrandiellopsis gen. nov., Biserta gen. nov.; Wilke et al. 2002;Glöer et al. 2010;Delicado et al. 2015;Khalloufi et al. 2017;Vandendorpe et al., 2019) and to eight for North Africa (García et al. 2010). The three subterranean genera recovered here as a monophyletic group (i.e., the Tunisian clade) are as genetically divergent (8.0-9.2% COI p-distances) as within other clades of hydrobiid subterranean genera (e.g., 7.8-11.8% COI p-distances; Osikowski et al. 2017). Although this genetic divergence falls into the range of intrageneric genetic variation reported for spring genera of Hydrobiidae (e.g., 5.39-11.15% COI p-distances within Corrosella Boeters, 1970;Delicado et al. 2012), we found sufficient morphological evidence to assign the three collected populations to two distinct new genera outside Bullaregia. The three genera comprising the Tunisian clade (i.e., Bullaregia, Belgrandiellopsis gen. nov. and Biserta gen. nov.) differ mainly in anatomical rather than in shell shape. For instance, Bullaregia presents a single medial lobe on the outer edge of the penis (two lobes on both edges of the penis in Belgrandiellopsis gen. nov. and Biserta gen. nov., more distally positioned in the former genus) and more cusps on the radular teeth.
These new genera and species show a limited distribution in Tunisia, as they are endemic to one or two localities. However, their distribution could extend to other localities in northern Tunisia and northeastern Algeria. Considering that many groundwater bodies have not yet been explored, the number of endemic taxa in Tunisia is likely larger. Thus, field surveys in these aquatic systems should be intensified to discover and inventory their faunal diversity. This point is crucial for the design of conservation measures before these local endemic species become extinct.