Systematic evaluation of cryptic freshwater snails from central Chile, including the enigmatic Littoridina santiagensis (Gastropoda, Truncatelloidea) Author Collado, Gonzalo A. Author Aguayo, Karina P. Author Cazzaniga, Néstor J. Author Gutiérrez Gregoric, Diego E. Author Lucía, Micaela De Author Haase, Martin text European Journal of Taxonomy 2019 2019-05-14 524 1 15 journal article 26826 10.5852/ejt.2019.524 20c6f816-bec3-4494-bd4c-78327477c5bd 2824346 6A434946-471E-4927-8235-A793F249D560 Potamolithus santiagensis ( Biese, 1944 ) comb. nov. Figs 1–5 Littoridina santiagensis Biese, 1944 : 187 –188, fig. 21 (Estero Dehesa, Cerro Manquehue, Barnechea, east to Santiago , Chile , type locality). Littoridina santiagensis– Stuardo, 1961 : 17 . — Valdovinos, 1999 : 128 , 2006: 90. — Sielfeld, 2001 : 3 . Heleobia santiagensis ( Biese, 1944 ) . Hershler &Thompson, 1992 : 55 . — Collado et al. , 2011 : 51 , 54–56, fig. 2R–S. Type material Lectotype CHILE • Santiago ; 200611 , MNHNCL ( Collado et al. 2011 ). Description SHELL. Ovate-conic, dark brown, relatively thick, with about 5.5 whorls ( Fig. 3 A–D). Shell length about 3.0 mm ( Table 1 ). Periostracum brown. Protoconch smooth ( Fig. 3E ), with about 1.3 whorls and 400 μm length (± 10 μm of standard deviation, n = 9). Aperture ovate, slightly angled adapically, lip thickened. Umbilicus imperforate or absent. Operculum ovate, thin, multispiral, light brown-transparent, with eccentric nucleus ( Fig. 3 F–G); attachment scar occupying almost half of the internal surface ( Fig. 3G ). Mantle black with a conspicuous gray band on anterior margin, head black, somewhat depigmented in center ( Fig. 3 H–I). Foot black. FEMALES. With nuchal node, white lips, tentacles grayish or black ( Fig. 3 H–I). Some specimens with a white band at base of tentacles where eyes are located. Males were not observed. RADULA. Taenioglossate (formula 3-1-3), with two marginal teeth and a lateral tooth placed on each side of the central tooth ( Fig. 3 J–K). Central tooth trapezoidal, dorsally concave, ca. 20 μm wide; basal tongue U-shaped; with 5–6 lateral cusps on each side of median cusp and 3–4 pairs of basal cusps, first basal cusps arise from tooth face and are larger than those on cutting edge. Median cusp of central tooth Fig. 2. Shells of truncatelloidean freshwater snails observed in the present study. A–B . Slender morphotype from El Yeso Spring (A) and Lo Carreño (B) assigned to Potamopyrgus antipodarum (Gray, 1843) . C–E . Thicker morphotype from El Yeso Spring (C), Lo Carreño (D) and El Colorado (E) assigned to Potamolithus santiagensis (Biese, 1944) comb. nov. F . Thicker morphotype from Viña Casas del Maule assigned to Potamolithus sp. Scale bar = 1 mm. well-developed and pointed. Lateral tooth with 11 cusps and median cusp well-developed and pointed. Inner marginal teeth with about 30 cusps ( Fig. 3K ). Fig. 3. Potamolithus santiagensis (Biese, 1944) comb. nov. , Yeso Spring, Chile. A . Shell imaged using SEM. B–D . Shell of the same specimen photographed under a stereo microscope (frontal, dorsal, lateral views). E . Protoconch. F–G . Opercula of two specimens (outer, inner sides, respectively). H . Head of a female. I . Head of another female having a nuchal node. J . Anterior-central section of radular ribbon. K . Central teeth. Abbreviations: f = foot; h = head; l = lip; lt = left tentacle; nn = nuchal node; rt = right tentacle. Scale bars: A–D = 1.0 mm; E = 250 μm; F–G = 500 μm; H–I = 0.5 mm; J = 50 μm; K = 10 μm. Ecology Potamolithus santiagensis comb. nov. is a herbivorous-detritivorous species that inhabits small water bodies like springs and small streams. Distribution Yeso Spring in Cajón del Maipo, Región Metropolitana , Central Chile ( Biese 1947 ; Collado et al . 2011 ; present study). This spring is a small tributary of the Yeso River, which empties in the Maipo River. The species also inhabits small irrigation canals in the Región de O’Higgins (Lo Carreño) and Región del Maule (El Colorado). Snail collections made in 2011, 2014, 2015 and 2017 at the type locality Estero Dehesa have not provided specimens of the species. This ecosystem has been invaded by P. antipodarum ( Collado 2014 ) and Physa sp. (unpublished data). In Yeso Spring, P. santiagensis comb. nov. coexists with P. antipodarum and snails of the genus Chilina Gray, 1828 . Morphometric analysis The mean values of the shell variables obtained for the freshwater snails inhabiting central Chile are shown in Table 1 . Although the Kruskal-Wallis tests considering the six variables analyzed were statistically significant, only 23 of 60 pairwise post-hoc comparisons among native Potamolithus populations and P. antipodarum were significant, providing evidence of the difficulties in distinguishing these snails ( Table 2 ). However, in the PCA the Potamolithus populations were well separated from P. antipodarum Fig. 4. PCA of Potamolithus populations and Potamopyrgus antipodarum (Gray, 1843) collected from central Chile. Table 1. Average shell dimensions (± standard deviation) of six linear variables used in the comparative study of Potamolithus populations and Potamopyrgus antipodarum (Gray, 1843) from central Chile.

Shell variable	n	SW	AL	AW	BWL	SPL
P. santiagensis comb. nov. El Yeso	26	1.7 (± 0.1)	1.3 (± 0.1)	0.9 (± 0.1)	2.1 (± 0.1)	0.6 (± 0.1)
P. santiagensis comb. nov. El Colorado	17	1.9 (± 0.1)	1.6 (± 0.1)	1.1 (± 0.1)	2.4 (± 0.2)	0.9 (± 0.1)
P. santiagensis comb. nov. Lo Carreño	9	1.7 (± 0.1)	1.3 (± 0.1)	1.2 (± 0.1)	2.1 (± 0.1)	0.8 (± 0.1)
Potamolithus sp. Viña Casas del Maule	6	2.0 (± 0.1)	1.6 (± 0.2)	1.1 (± 0.1)	2.4 (± 0.2)	0.7 (± 0.1)
P. antipodarum El Yeso	30	1.8 (± 0.2)	1.4 (± 0.1)	1.0 (± 0.1)	2.2 (± 0.2)	1.2 (± 0.1)
P. antipodarum Lo Carreño	7	2.6 (± 0.2)	2.1 (± 0.2)	1.4 (± 0.1)	3.2 (± 0.2)	1.7 (± 0.3)

Fig. 5. Bayesian tree based on COI sequences. Numbers at nodes indicate posterior probability values (only given if Ż 0.95). Names in bold refer to new sequences reported in this paper. Numbers following taxa refer to GenBank sequences. in the morphometric space ( Fig. 4 ). In this analysis, the first two components accounted cumulatively for 95.27% of the variance (PC1: 79.78%; PC2: 15.49%). Molecular analysis The COI phylogenetic analysis grouped the original sequences and those downloaded from GenBank into two main clades, Tateidae and Cochliopidae , respectively, both supported by posterior probabilities (p.p.) of 1.00 ( Fig. 5 ). Our original sequences clustered in the first clade, either in the genus Potamolithus or Potamopyrgus , both groups inferred with high node support (0.97 and 1.00 p.p., respectively). The sequences of the slender morphotype from El Yeso Spring and Lo Carreño were identified as P. antipodarum , and those of the thicker one as representatives of Potamolithus ( Fig. 5 ). The snails from El Colorado, Viña Casas del Maule, Puerto Chico, “ Heleobia ” sp. from Uspallata and “ Heleobia ” hatcheri from Aguas Negras in Argentina retrieved from GenBank were also recovered among Potamolithus species. The COI genetic distances among Tateidae species/populations from South America ranged between 0.2% and 17.0% ( Table 3 ). The genetic distances between P. santiagensis comb. nov. and snails from Puerto Chico were estimated at 6.7–7.2%. In the Bayesian tree, they were placed in different subclades. The population from Viña Casas del Maule grouped with “ Heleobia ” sp. from Uspallata (1.00 p.p.); they differed genetically from P. santiagensis comb. nov. by about 5.0%. On the other hand, the sequence divergence between the population from El Colorado and P. santiagensis comb. nov. from Yeso Spring and Lo Carreño was low (0.0–0.6%).