A new genus and species of uncertain phylogenetic position within the family Hydrobiidae ( Caenogastropoda , Truncatelloidea ) discovered in Tunisian springs

The species richness of the aquatic (non-marine) gastropod family Hydrobiidae Stimpson, 1865 reaches its peak in the European region partly because other areas, such as North Africa, remain to be extensively surveyed. Most of the hydrobiid species described in North Africa have been ascribed to the subfamilies Pseudamnicolinae, Hydrobiinae and the genus Mercuria. Little is known about the presence of other hydrobiid subfamilies. This study examines several specimens of gastropods collected from two springs in Tunisia. Based on a comprehensive literature review and rigorous anatomical and molecular comparisons with known species from North Africa, Europe and Asia Minor of similar morphology, we here describe a new genus, Bullaregia gen. nov., and new species Bullaregia tunisiensis gen. et sp. nov. Although the shell shape of this Tunisian species resembles that of European hydrobiid genera such as Belgrandiella, it differs in other anatomical structures (i.e., penis with a glandular strap-like lobe, and two seminal receptacles, SR1 pedunculate, SR2 sessile and less developed). Using maximum likelihood and Bayesian approaches, phylogenetic relationships inferred from mtCOI sequences point to an independent evolutionary lineage for this new taxon outside Belgrandiella and other known hydrobiid subfamilies.


Introduction
The aquatic (non-marine) gastropod family Hydrobiidae Stimpson, 1865 (sensu Wilke et al. 2013) occurs mainly in the Western Palaearctic, Nearctic, northern Neotropic and South Africa.In Europe, a high species richness has been described for this family.However, this is likely the consequence of an underreporting of the hydrobiid fauna living in other regions such as North Africa.Based only on shell characters, Van Damme (1984) and Kristensen (1985) reported the presence in North Africa of seven European genera of Hydrobiidae sensu lato (i.e., Belgrandia Bourguignat, 1869;Bythinella Moquin-Tandon, 1855;Hadziella Kuscer, 1932;Horatia Bourguignat, 1887;Hydrobia Hartmann, 1821;Peringia Paladilhe, 1874 andPseudamnicola Paulucci, 1878) and one endemic genus (i.e., Heideella Backhuys and Boeters, 1974).More recently, Ghamizi et al. (1999) and Bodon et al. (1999) added other species and genera to the Moroccan Hydrobiidae list, mostly stygobiont species.However, despite these important discoveries, most studies have provided mere descriptions of shell morphology without focusing on anatomical structures.In prior work, we confirmed that by adding anatomical and molecular data to these shell-based identifications, the reliability of generic classification could be much improved (Delicado et al. 2016).
In Tunisia, a few Hydrobiidae species have been shell-determined over the past centuries (Bourguignat 1860(Bourguignat , 1864;;Issel 1880;Debeaux 1863;Letourneux & Bourguignat 1887;Pallary 1921Pallary , 1923;;Seurat 1921Seurat , 1934)).So far, the most relevant study of this family in Tunisia was that by Boeters (1976) who listed 28 species belonging to three genera: Pseudamnicola, Hydrobia and Mercuria Boeters, 1971.Based on these descriptions, an extensive survey in the past ten years of freshwater superficial and subterranean malacofauna has revealed Hydrobiidae to be among the most species-and population rich families in the country.In contrast, Tunisia's subterranean aquatic fauna has recently received much less attention, although this region (especially NW Tunisia) has numerous wells and springs, and some caves.Early studies examining the general subterranean and spring fauna of Tunisia were those by Seurat (1921Seurat ( , 1934) ) and Debeaux (1863), who discovered only a few gastropod and two isopod species in these habitats.One example of Hydrobiidae is the species 'Hydrobia' nana (Terver, 1939) found by Debeaux (1863) in springs of Algeria and two localities of northern Tunisia.The few more recent surveys have detected new records and new species of phreatic microgastropods (such as Pseudamnicola) as well as other stygobiont or eustygophilous taxa such as worms (Charni et al. 2004;Harrath et al. 2001Harrath et al. , 2004) ) and crustaceans (Toumi et al. 2013).However, to our knowledge true stygobiont species of Hydrobiidae inhabiting Tunisian groundwaters have been rarely reported.In consequence, there are no appropriate references for the straightforward identification of Tunisian groundwater species.
In summary, we could argue that the picture of hydrobiid species richness in North Africa is still very incomplete, and that only surface-water genera of the subfamilies Pseudamnicolinae, Hydrobiinae and the genus Mercuria have been more intensively studied.This situation prompted a new survey of ground-and surface waters to search for possible endemic forms in Tunisia.The collection of specimens obtained contains some unknown Hydrobiidae species.We here describe one of these species and assign it to the genus Bullaregia gen.nov.As the shell shape of this new genus resembles that of the European Belgrandiella (subfamily Belgrandiellinae), we conducted a systematic review using both molecular and morphological methods.Our results indicate this taxon is a distinct hydrobiid genus characterized by a particular set of anatomical characters (not known for other subfamilies) and suggest its uncertain position within the Hydrobiidae phylogeny.

Material and methods
The material was found at two localities in northwestern Tunisia: Aïn Gnaa (Jendouba, northwestern Tunisia), 36.5583 N,8.74667 E,alt. 206 m a.s.l.;Djebba (Beja,northwestern Tunisia),36.47125 N,9.0997223 E, alt.342 m a.s.l.(Fig. 1).Live specimens were collected within the river substrate with a sieve of 300 μm mesh cloth.The samples were preserved in 70% ethanol and sorted in the laboratory under an optical stereo microscope (MOTIC-ST-39 Series).Anatomical characteristics (genitalia, radula, stomach and gill) were studied under the stereo microscope and compared with other described forms from North Africa, Europe and Asia Minor.Morphological characters were described based on the standard terminology of Hershler & Ponder (1998).Shell measurements were made with an eye-piece micrometer under the stereo microscope.The sex of the holotype was not examined in order to keep the shell intact.Photographs were taken with a camera system and a digital adapter.The type material is deposited in the Collection of Molluscs of the Museo Nacional de Ciencias Naturales de Madrid (MNCN).
Our phylogenetic analyses included one specimen of Bullaregia tunisiensis gen.et sp.nov.from a spring in Djebba (type locality) and 22 hydrobiid representatives of other European genera whose shell morphology resembles the new African hydrobiid here discovered (Table 1).Genomic DNA was extracted using the CTAB protocol of Wilke et al. (2006).The mitochondrial fragment of cytochrome c oxidase subunit I (COI) was amplified with the primers LCO1490 (Folmer et al. 1994) and COR722b (Wilke & Davis 2000).The cycling sequence conditions were as described in Schreiber et al. (2012).Final PCR products were sequenced in an ABI 3730 XL sequencer (Life Technologies, Carlsbad, CA, USA) using a Big Dye Terminator kit v. 3.1 (Life Technologies).New sequences were deposited in GenBank (Table 1).Amplified sequences were edited in Sequencher 4.6 (Gene Codes, Ann Arbor, MI).The protein-coding mitochondrial COI sequence was unambiguously aligned in PAUP* 4.0a123 (Swofford 2002) together with sequences of other hydrobiid species obtained from GenBank (Table 1).As Wilke et al. (2013) found little saturation among COI sequences of hydrobiids and other sister families, we did not test for saturation in our alignment.Prior to the species tree reconstruction, we searched for closely related taxa on GenBank using BLASTN 2.3.1 (Zhang et al. 2000) against the National Center for Biotechnology Information (NCBI) nucleotide database.As no high sequence identity with other European and/or African hydrobiid was identified (one hundred contigs showed BLASTN % identity ≤ 86%, most of them corresponding to species of the American hydrobiid genus Pyrgulopsis Call & Pilsbry, 1886), we compared our fragments to sequences from similar species which potentially may belong to the same genus or subfamily.
Phylogenetic relationships were inferred by Bayesian-inference (BI) and Maximum Likelihood (ML).For these methods, Partition Finder 1.1.1.(Lanfear et al. 2012) was used to select the best-fit model of nucleotide evolution by codon position within our COI dataset, implementing corrected Akaike's information criterion (AICc; Akaike 1974;Sugiura 1978;Hurvich & Tsai 1989) for model selection and RAxML-HPC v.8 (Stamatakis 2014) for likelihood estimation.BI was performed through two independent runs of four Metropolis-coupled Markov chains in MrBayes 3.1.2(Huelsenbeck 2000;Huelsenbeck & Ronquist 2001), using 2.5 million generations each and a sample frequency of 1000.The analysis was terminated when the average standard deviation of split frequencies was less than 0.01, indicating that the runs reached convergence.The first 10% sampled trees were discarded as burn-in.Partitioned ML analysis was undertaken using RAxML-HPC v.8 on the computer cluster of the Cyber-Infrastructure for Phylogenetic Research project (CIPRES, www.phylo.org).Clade support for the ML phylogeny was assessed by nonparametric bootstrapping (Felsenstein 1985) using 100 pseudoreplicates, and by posterior probabilities (BPPs) for the BI.

Results
The alignment of COI sequences yielded 648 bp for which Partition Finder identified TrN (Tamura & Nei 1993) + I (invariable sites) + G (rate variation among sites), F81 (Felsenstein 1981) + I and GTR (Tavaré 1986) + I + G as the best-fit models of nucleotide substitution for the 1 st , 2 nd and 3 rd codon position, respectively.Average base frequencies were 31.2%A, 13.5% C, 12.1% G and 43.2% T. ML and BI gave rise to similar topologies and both showed Bullaregia gen.nov.outside the subfamilies to which this genus could belong (Fig. 2).These subfamilies, i.e., Belgrandiellinae, Belgrandiinae and Horatiinae on the one hand, and Islamiinae on the other, formed two different monophyletic groups well supported by ML and BI (bootstrap value = 70 and 72%, and BPP = 0.99 and 0.99, respectively).Though the exact phylogenetic position of Bullaregia gen.nov.remains unclear, both analyses showed a distant relationship between this and the European genus Belgrandiella (uncorrected pairwise distances of 17.1%).Morphological data also supported these distant relationships.Differences in shell and anatomical structures between Bullaregia gen.nov.and other hydrobiid genera mentioned above are described in the following section.Genus Bullaregia gen.nov.urn:lsid:zoobank.org:act:93209E6C-8DBC-4869-BAAD-FA4692D5FAB1

Diagnosis
Shell ovate-conic, penultimate whorl tall relative to remaining whorls; aperture pyriform, angled on top; umbilicus closed by inner lip.Operculum corneous, yellowish, thin, pliable, ellipsoidal, paucispiral with submarginal nucleus.One pair of basal cusps on radular central tooth.Ctenidium bearing welldeveloped gill filaments.Presence of bursa copulatrix; unpigmented renal oviduct with a pouch at the end of the coiled section; two seminal receptacles, SR1 elongate and pedunculate, SR2 globular, sessile and less developed.Strap-like penis attached to the neck behind the right eye, with one dorsal, strap-like glandular lobe.

Etymology
Referring to the archeological site Bulla Regia, the region where the genus was found; gender feminine.

Remarks
This genus differs from conchologically similar genera from Europe (e.g., Belgrandiella, Terranigra Radoman, 1978 andLitthabitella Boeters, 1970) and Central Asia (e.g., Chirgisia Glöer, Boeters & Pešić, 2015) in bearing a strap-like penis (tapered in Belgrandiella, Terranigra and Chirgisia, but broadly triangular in Litthabitella), with one dorsal glandular lobe (absent in all the aforementioned genera) (see Radoman 1983;Szarowska 2006;Glöer et al. 2014).Bullaregia gen.nov.resembles Terranigra and Litthabitella in having two seminal receptacles (none and one in Chirgisia and Belgrandiella, respectively), but they are distinguished by their globular shape (elongate in Terranigra and Litthabitella), and in the position and absence of the duct.Bullaregia gen.nov.conforms to Litthabitella in the radula features, but differs from Belgrandiella in the number of basal cusps on the radular central tooth (two pairs in the latter) (see Szarowska 2006).

Etymology
The species epithet tunisiensis is an adjectival toponym that refers to Tunisia, where the species was found.
OperculuM.With ca 2 whorls (Fig. 3B) and muscle attachment area oval and located near the nucleus.
Male gentialia.Penis strap-like with a well-developed glandular lobe on the outer edge, 33% total penis length; glandular lobe strap-like and with a short duct; distal end of penis tapered, dark pigmented (Figs 3C , 5A).The penis has distinct basal and distal portions.The distal portion is smooth, with tapered end and variable in length.The basal portion is cylindrical and folded.Prostate gland, close, bean-shaped in outline, oval in section, and about twice as long as wide (0.45 mm/0.25 mm).Posterior vas deferens forms the seminal vesicle with many loops.It is well-developed (about 4 mm in length and 0.1 mm in section) and strongly undulating, coiling immediately after it emerges from the testis and extending over the posterior and middle part of the stomach.The pallial vas deferens is a straight and narrow tube unciliated, entering postero-ventral portion of gland and exiting from its anterior end.
FeMale genitalia.Ovary simple with stalked lobes.Pallial oviduct bearing an ovoid bursa copulatrix with a duct longer than its length; unpigmented renal oviduct, with a pouch at the end of the coiled section; two seminal receptacles; SR1 elongate with a short duct, close to the insertion point of the bursal duct to the renal oviduct; SR2 globular, less developed and sessile, positioned near antero-ventral edge of bursa (Fig. 5B).

Remarks
Populations of this species from the Aïn Gnaa and Djebba springs vary only scarcely in terms of morphology and anatomy.In specimens from the Aïn Gnaa spring, the shell is generally more slender and has deeper sutures and a sharper protoconch.The portion of the vas deferens, forming the seminal vesicle, is more coiled and more developed.Pigmentation in the Djebba spring specimens is more intense, especially in the mantle and distal portion of the penis.Individuals from these populations are conchologically differentiated from similar regional species, such as Pseudamnicola meluzzi Boeters, 1976 andP. conovula (Frauenfeld, 1863), by their smaller and slender shell, a taller penultimate whorl relative to remaining whorls and by an aperture pyriform, angled on top.
Representatives of these two populations featured a similar shell morphology as the specimens from Algeria deposited in the Muséum national d'Histoire naturelle, Paris (MNHN).These specimens were earlier identified as Amnicola seminium (Morelet, 1857) by Bourguignat.However, this species, illustrated by Morelet in the original description (Morelet 1857, pl.XII fig.10) differs from the Algerian specimens deposited in the MNHN.Based on Morelet's illustration (1857: pl.XII Fig. 10), the type of A. seminium has a larger body whorl, a shorter penultimate whorl and a rounder aperture (resembling Pseudamnicola) than the MNHN specimens.Thus, these Algerian snails could have been misidentified.Based on common shell features shared by these specimens and Bullaregia gen.nov., such as ovateconic shape, tall penultimate whorl, aperture angled on top and closed umbilicus, they could be assigned to this new genus.

Discussion
The freshwater gastropod diversity of some geographical regions including Europe, North America and Australasia, has been better explored than that of others (Strong et al. 2008).Our discovery here of Bullaregia gen.nov.calls for more field work in North Africa to further our understanding of global species richness, distribution and evolutionary patterns in hydrobiid clades.The new springsnail described shows a small distribution range.This means, it could be the second Hydrobiidae genus endemic to North Africa (after Heideella, see Bodon et al. 1999).Although the total number of African hydrobiid species remains unknown (Brown 1994), taxonomic revisions of the North African Hydrobiidae sensu lato have recognized 13 genera, including Bullaregia gen.nov.discovered herein.In total, 37 species belonging to these and other undiscovered hydrobiid genera are considered threatened in North Africa (Van Damme et al. 2010).Pollution, habitat loss and an ever drier climate are the factors thought to have most contributed to the decline of freshwater molluscs in North Africa.In addition, numerous Tunisian freshwater wells are presently at risk of salinization (Van Damme et al. 2010).
However, assignments of African taxa to European genera, often based on shell descriptions only, may be erroneous.For example, the presence of Belgrandiella in North Africa is still uncertain.Its first record in North Africa was detected through the synonymization of the African genus Maresia with Belgrandiella (suggested by Boeters 1988).Bourguignat (1877) described the genus Maresia from Algeria and designated Hydrobia dolichia Bourguignat, 1864 as the type species.Nonetheless, Kabat & Hershler (1993) stated that Bourguignat's Maresia is a junior homonym, and thus invalid as a mollusc.Additionally, some Maresia species, including the type species, M. dolichia, or M. nana (Bourguignat, 1864), have been ascribed to the genus Heideella (García et al. 2010).There is therefore no sound evidence confirming the presence of Belgrandiella in North Africa.
Despite their closely resembling shell shape, the high anatomical and molecular variation observed between Bullaregia gen.nov.and other European hydrobiids (e.g., Belgrandiella or Terranigra) reveals these genera as different entities.Our phylogenetic analyses, which included sequences of species of the subfamilies Belgrandiellinae, Islamiinae and Horatiinae, did not cluster Bullaregia gen.nov.within any of these groups (Fig. 2).Hence, pending more complete phylogenies for this family, Bullaregia gen.nov.could be a new hydrobiid subfamily distinguishable mainly by a dorsal glandular lobe on the penis and the joint presence of seminal receptacles and a pouch in the renal oviduct.Glandular ornaments on the penis have also been observed in males of the European genus Agrafia Szarowska & Falniowski, 2011(see Szarowska & Falniowski 2011) and the American subfamily Nymphophilinae (see Hershler et al. 2003;Hershler & Liu 2009;Hershler et al. 2011, among others).However, in the absence of a well supported relationship, this feature could be a synapomorphy of these clades or a result of convergent evolution.More comprehensive molecular studies will help resolve the phylogenetic relationship of this newly discovered hydrobiid clade.This type of information is indeed essential to unveil the first evolutionary events that gave rise to the major clades of the family Hydrobiidae.

Fig. 2 .
Fig. 2. Maximum likelihood inference based on COI dataset.Bootstrap values and BPP's are given when ≥ 70% and 0.90, respectively.Black bars on the right indicate subfamily assignments.Scale bar: expected change per site.

Fig. 4 .
Fig. 4. Radula of Bullaregia tunisiensis gen.et sp.nov.from a spring in Djebba, Tunisia. A. Rows of teeth of the radula.B. Detail of central teeth.C. Detail of lateral and marginal teeth.

Table 1 .
Taxa included in the molecular analyses with their corresponding locality names, GenBank accession numbers and original references.