Arganiella Giusti & Pezzoli , 1980 ( Caenogastropoda : Truncatelloidea : Hydrobiidae ) : a widespread genus or several narrow-range endemic genera ?

Most valvatiform genera of the gastropod family Hydrobiidae are narrow-range taxa. One exception is the genus Arganiella, which is comprised of three congeners: the type species A. pescei from the Apennine Peninsula, A. wolfi from the Iberian Peninsula and A. tabanensis from the Balkans. The genus assignment of the latter two species was based on morphological similarities with A. pescei in the shell, operculum, radula and genitalia. Given that the morphology of hydrobiids is sometimes susceptible to convergence, this study re-evaluates the taxonomic status of species of Arganiella by analysing mitochondrial (mtCOI) and nuclear (18S rRNA) sequences of topotypes or near topotypes to infer their phylogenetic position. Our phylogenetic analyses depicted Arganiella as a non-monophyletic group within Hydrobiidae, and sequence divergence among the three species ranged from 14.5 to 16.7% for mtCOI and 2.0 to 3.8% for 18S. We also re-examined the extent of morphological variation among species of Arganiella and found a few differences among them and other valvatiform genera. Consequently, we propose two new genera for A. wolfi and A. tabanensis. Our results conflict with the classification of valvatiform hydrobiid species solely based on traditional phenotypical methods and suggest further taxonomic evaluation within a molecular framework.

The assignment of A. wolfi and A. tabanensis to Arganiella was based on morphological similarity with the type species in shell, penis and female distal genital features (Arconada & Ramos 2007a, 2007bBoeters et al. 2014). However, such characters are susceptible to convergent evolution in valvatiform hydrobiid genera ) and, therefore, the systematics of the known species of Arganiella needs to be re-evaluated through molecular analyses. To date, DNA sequence data are available only for A. wolfi, and phylogenetic relationships inferred from a multilocus dataset of valvatiform hydrobiid genera from the Iberian Peninsula resolved A. wolfi as the sister taxon of the Iberian genus Iberhoratia (Delicado et al. 2019. Sequence data from the other two species of Arganiella are needed to confidently assess the taxonomic composition of this wide-ranging genus. As might be expected in taxa with poor dispersal capabilities, we hypothesize that phylogenetic analyses would depict species of Arganiella as three unrelated, narrow-range lineages rather than as a widely distributed monophyletic group. This result would challenge the genus assignment of these valvatiform hydrobiids, which was based on morphology, and limit the known geographic distribution of Arganiella to the Apennine Peninsula. To test these assumptions, we analyse new mitochondrial and nuclear DNA sequence data from the type species A. pescei and from A. tabanensis with previously published molecular datasets of valvatiform hydrobiids that also include sequences of A. wolfi (Delicado et al. 2019) in order to infer the phylogenetic relationships of the species of Arganiella and to quantify the degree of divergence among them. We also provide previously missing descriptions of the radular features of A. tabanensis for a morphological comparison. Finally, the taxonomic status of species of Arganiella is discussed in light of the evidence from the phylogenetic analyses and morphological comparisons of these taxa with other valvatiform hydrobiid genera recorded from the same Mediterranean regions.

Material and methods
We assessed the taxonomic status of the three recognized species of Arganiella using DNA sequence and morphological information from these and other valvatiform genera occurring in the Apennine, Balkan and Iberian peninsulas. One individual of A. pescei was used for the DNA assessment. This specimen was collected from Susanna Springs, in the region of the type locality (i.e., central-eastern Apennine Peninsula), and deposited in the collection of the University of Giessen Systematics and Biodiversity (UGSB) (Diehl et al. 2018) in Germany (UGSB 10365). Following an exhaustive morphological examination, Bodon et al. (2001) had assigned the hydrobiid populations living in these springs to A. pescei. Morphological characters from A. pescei were scored using the original description by Giusti & Pezzoli (1980) and the re-description by Bodon et al. (2001). For the Iberian A. wolfi, we used the morphological description by Arconada & Ramos (2007a) and Boeters & Glöer (2007), and the sequences of a topotype used by Delicado et al. (2019) for the molecular analyses. The shell morphology and anatomy of the Balkan A. tabanensis are as illustrated by Boeters et al. (2014). Additional data on radular and opercular features of this species, as well as partial sequences of the studied DNA markers, were also collected for the present study. We collected ca 50 topotypes from the A. tabanensis type locality (Taban Spring, Montenegro;42.52795° N, 19.21921° E) in 2015 and preserved them in 80% ethanol in the field. Shells and opercula were photographed using a Keyence VHX 2000 3D Digital Microscope. Six adults were dissected, and their buccal mass extracted, under an Olympus SZX12 stereo microscope. Radulae were extracted, cleaned and prepared as described by Delicado et al. (2016) for imaging on a field emission scanning electron microscope (FESEM) DSM982 Gemini (Carl Zeiss GmbH, Germany). The collected specimens were then deposited in the UGSB collection (UGSB 18847).
We isolated total DNA from one individual per species (for A. pescei and A. tabanensis) following the CTAB protocol performed by Wilke et al. (2006). Fragments 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 primers for metazoan 18S (Holland et al. 1991). Amplification conditions for both gene fragments were as previously described by Delicado et al. (2012). The annealing temperature used was 52°C. Samples were sequenced in an ABI 3730 XL sequencer (Life Technologies, Carlsbad, CA, USA) using a BigDye Terminator Kit ver. 3.1 (Life Technologies). The new sequences were deposited in GenBank (Table 1).
We assessed the taxonomy of the three species of Arganiella by analysing the COI and 18S sequences of these species along with those of other European valvatiform and (closely related) non-valvatiform genera available from GenBank (Table 1). Forward and reverse sequences were aligned and edited in Sequencher ver. 5.4.6 (Gene Codes, Ann Arbor, MI). MEGA ver. 7.0.14 (Kumar et al. 2016) was used to assemble the gene-partition datasets and to calculate genetic distances (uncorrected p-distances). The COI dataset was manually aligned also using MEGA. The rRNA 18S fragment was aligned using MAFFT ver. 7.402 (Katoh & Standley 2013), with default settings for gap penalties. According to the corrected Akaike's information criterion (AICc; Akaike 1974;Sugiura 1978;Hurvich & Tsai 1989), jModelTest ver. 2.1.7 (Darriba et al. 2012) selected TrN (Tamura & Nei 1993) + I (invariable sites) + G (rate variation among sites) and TrNef (Tamura-Nei model with equal base frequencies; Tamura & Nei 1993) + I + G models of nucleotide evolution for the COI and 18S datasets, respectively. We used DAMBE ver. 7 (Xia 2018) and the proportion of invariant sites (Pinv = 0.47) obtained in jModelTest to conduct the saturation test (Xia et al. 2003;Xia & Lemey 2009) on the COI partition. The observed saturation (I ss = 0.39) was significantly lower than the critical values (I ss.c = 0.71; p < 0.001), suggesting little saturation in our COI dataset.
Phylogenetic analyses based on maximum likelihood (ML) methods were conducted using the RAxML BlackBox web-server [https://raxml-ng.vital-it.ch/#/; Kozlov et al. 2019] with 10 random starting trees and the optimal substitution models for each gene partition selected in jModelTest. Bayesian inference (BI) analyses were run with mixed substitution models in MrBayes ver. 3.2.6 (Ronquist et al. 2012) for 5 million generations with a sampling frequency of 1000. After verifying convergence of the BI analysis (standard deviation of split frequencies < 0.01), the first 10% of generations were discarded as burn-in. Branch robustness was evaluated by rapid bootstrapping (BS) (Stamatakis et al. 2008) with an automatic cut-off for ML and by Bayesian posterior probability (BPP) for BI. Inferred topologies and branch supports were visualized in FigTree ver. 1.4.3 (Rambaut 2010).

Molecular analyses
The data matrix constructed of COI (658 bp) and 18S (492 bp) sequences yielded an alignment with a total length of 1150 bp. Our ML and BI analyses generated similar tree topologies and branch supports (Fig. 1).
In both inferences, Arganiella did not form a monophyletic group; instead, both A. wolfi and A. tabanensis were distantly related to the type species A. pescei. Also in both analyses, A. wolfi resolved as the sister taxon to the genus Iberhoratia from the Iberian Peninsula (BS = 100%, BPP = 1.00), and A. tabanensis, to the genus Kerkia Radoman, 1978 from the Balkan Peninsula and adjacent islands (BS = 91%, BPP = 0.95). The phylogenetic position of A. pescei was not well resolved in either of the phylogenetic analyses. Sequence divergence among the three species of Arganiella ranged from 14.5 to 16.7% for COI and 2.0 to 3.8% for 18S. Divergence among genera ranged from 9.1 to 22.5% for COI and 0 to 4.5% for 18S.
Overall, our results show that DNA sequence divergence values among the three species of Arganiella fall within the range of inter-generic variation and that both A. wolfi and A. tabanensis not only have a sister group relationship with a genus from their respective Mediterranean peninsulas ( Fig. 1) but also

Revised diagnosis
Shell trochiform; whorls 3.5-4.0; aperture complete, rounded; outer lip narrow, straight in lateral profile; umbilicus wide. Operculum corneous, yellowish, thin, pliable, oval to rounded, paucispiral with a central nucleus, without peg. Two pairs of basal cusps on each central radular tooth. Ctenidium well developed, with approximately 14 gill filaments. Osphradium positioned opposite to approximately the middle of the ctenidium. Stomach without gastric caecum; rectum forms a gentle U-shape in the mantle cavity. Bursa copulatrix medium-sized, pyriform, pedunculated and protruding beyond the posterior edge of the albumen gland; bursal duct shorter than bursal length; unpigmented renal oviduct that makes a complete loop over the pallial gland; one elongated or pyriform seminal receptacle arising from the renal oviduct, just above the insertion point with the bursal duct. Prostate gland bean-shaped, about twice as long as wide. Penis small and simple, gradually tapering. Nervous system unpigmented.

Etymology
The genus is named after Villa Aretiana, the Roman name of the town Aracena, which gives its name to the mountain range where the genus was found (i.e., Sierra de Aracena); gender feminine.

Remarks
Aretiana Delicado & Ramos gen. nov. can be distinguished from Arganiella as the former has a taller shell, a more oval operculum, pigmentation on the body and eyespots, fewer gill filaments, a narrower penis, a larger and pyriform bursa copulatrix located beyond the posterior edge of the albumen gland and fewer cusps on the lateral radular teeth (for comparison, see Giusti & Pezzoli 1980;Boeters & Glöer 2007;Arconada & Ramos 2007a). The new genus differs from the closely related genus Iberhoratia by its taller shell with a narrower umbilicus, the absence of lobes on the inner edge of the penis and of a proximal seminal receptacle (SR2) and presence of two pairs of basal cusps on each central radular tooth (see

Revised diagnosis
Shell valvatiform; whorls ca 3; aperture complete, from rounded to ellipsoidal; outer lip narrow, straight in lateral profile; umbilicus wide. Operculum corneous, yellowish, thin, pliable, rounded, paucispiral with a central nucleus, without peg. Two pairs of basal cusps on each central radular tooth. Ctenidium well developed, with 10-11 gill filaments. Animal unpigmented. Osphradium positioned opposite approximately to the middle of the ctenidium. Stomach without gastric caecum; rectum forms a gentle V-shape in the mantle cavity. Bursa copulatrix small, globular, pedunculated and positioned beyond the posterior edge of the albumen gland; bursal duct longer than bursal length; unpigmented renal oviduct; one pyriform seminal receptacle arising at the insertion point with the bursal duct loop. Penis small and simple, gradually tapering.

Etymology
The new genus is named after Doclea, the name of the Roman city located on the site of modern Podgorica on whose municipal territory a new genus was found; gender feminine.

Remarks
Docleiana Delicado & Pešić gen. nov. can be distinguished from Arganiella as the former has a more ellipsoidal shell aperture, a narrower base of the penis, a smaller bursa copulatrix that is positioned beyond the posterior edge of the albumen gland, a larger seminal receptacle and more cusps on the lateral radular teeth (Fig. 2; for comparison, see Giusti & Pezzoli 1980;Boeters et al. 2014). The new genus differs from the closely related genus Kerkia by its smaller shell, absence of lobes on the inner edge of the penis, a smaller bursa copulatrix, a more globular seminal receptacle and V-shaped rectum (see Radoman 1978;Bodon et al. 2001). Mean COI sequence divergence for Docleiana Delicado & Pešić gen. nov. was 14.5% with Arganiella and 12.3% with Kerkia.

Discussion
Previous taxonomic studies of valvatiform hydrobiids have relied on morphological similarities to classify species, a practice that can be difficult for this group because of their small size and simplified morphologies. Considering this, more recent systematic revisions have incorporated molecular  (Bole, 1961), later transferred to the genus Bracenica Radoman, 1973by Hofman et al. (2020; Horatia hadei Gittenberger, 1982transferred to Daphniola Radoman, 1973by Falniowski & Szarowska (2011b; and Neohoratia azarum Boeters & Rolán, 1988, later recognized as Islamia azarum (Boeters & Rolán, 1988) by Arconada & Ramos (2006) and currently combined as Deganta azarum by Arconada & Ramos in Delicado et al. (2019). In the case of Arganiella, A. wolfi and A. tabanensis, two endemic species from the Iberian and Balkan peninsulas, respectively, were classified within this genus on the basis of shell and genital similarities with the Apennine species A. pescei (Arconada & Ramos 2007a, 2007bBoeters et al. 2014). Our survey of DNA sequence divergence within Arganiella, although based on a limited number of samples and gene fragments, showed the substantial divergence of these two species from the type species A. pescei (> 14.5% for COI). This degree of DNA sequence differentiation is comparable to those inferred between other recognized valvatiform genera (uncorrected p-distances 9.1-22.5% for COI). Moreover, Arganiella was not recovered as a monophyletic group by our phylogenetic inferences (Fig. 1).
However, incorrect systematic conclusions can be drawn from molecular phylogenies when the specimens have previously been misidentified (e.g., Radomaniola/Horatia in Szarowska & Falniowski, 2014). Detailed morphological examinations of the studied material are therefore needed for a more reliable systematic interpretation. Our comparative morphological study (Table 2) indicated close similarities among the, until now, considered species of Arganiella, especially in those characters related to the penis, ctenidium and radula and, to a lesser degree, the shell, pigmentation and distal genitalia of females. Some character states found in species of Arganiella, such as a simple penis without lobes, the presence of a single distal seminal receptacle and two cusps at both sides of the basis of the central radular tooth, are rarely present in other valvatiform genera Radea et al. 2016) and can, thus, lead to genus misidentification. However, differences in other characters can be observed among the three species. The most dissimilar species is A. wolfi: it has a larger and more trochiform shell, body pigmentation and a larger bursa copulatrix (Arconada & Ramos 2007a;Boeters & Glöer 2007). Arganiella tabanensis is more similar to A. pescei than A. wolfi, especially in shell shape and body pigmentation (Boeters et al. 2014). However, it differs from A. pescei in shell size, the shape of the bursa copulatrix and the bend type of the rectum. On the basis of this DNA and morphologic evidence, we assign the species A. wolfi and A. tabanensis to two distinct genera.
been sequenced, resemble Docleiana Delicado & Pešić gen. nov. in penis morphology but differ in shell size and radular and female genital features; they also lack a ctenidium ( Table 2).
The systematic findings suggesting erroneous assignment of the three geographically disjunct species to the same genus due to morphological similarities conflict with the use of traditional taxonomy to classify valvatiform hydrobiid taxa and highlight the need to integrate morphological and molecular data for more robust taxonomic assessments.