Teeth morphology and dental sexual dimorphism of three species of the sandskate genus Psammobatis Günther, 1870 from the Brazilian coast (Rajiformes, Arhynchobatidae)

Sets of jaws of the sandskates Psammobatis extenta (n = 10), P. lentiginosa (only adult specimens, n = 8) and P. rutrum (only adult specimens, n = 10) were examined by scanning electron microscopy. Female teeth of the three species and those of the immature P. extenta did not show differences, all having monocuspid crushing teeth, with a small, unpronounced or absent cusp. Monognathic heterodonty was observed in adult specimens in both sexes of the three species analyzed. Immature P. extenta and females of the three species differed from the adult males in having a crushing as opposed to a clutching dentition, implying gynandric heterodonty. Teeth of males of P. extenta have pointed, well-pronounced, rounded cusps, whereas males of P. rutrum and P. lentiginosa have elliptical and similar cusps. Immature specimens of P. extenta have small cusps. Teeth of P. lentiginosa have a longitudinal sulcus on their labial face, a character herein hypothesized as a derived condition.

Studies of species of Psammobatis are mostly limited to reproductive biology (e.g., CHIARAMONTE, 2002;MARTINS;ODDONE, 2017), feeding habits (e.g., MUTO et al., 2001;COUSSEAU, 2004;PEREZ, 2005) and geographical distribution (e.g., MENNI;STEHMANN, 2000;GOMES et al., 2010). Concerning sexual dimorphism, the unpublished study of Paragó (2001) analyzed P. extenta and P. rutrum and sex-related shape of the anterior margin of the disc; Barbini and Lucifora (2012) recorded the gynandric heterodonty in Psammobatis bergi and P. extenta, and Braccini and Chiaramonte (2002) sexrelated differences in measurement variables and in tooth morphology in both sexes of P. extenta to the reproductive habit of the species. Herman et al. (1995), in their extensive batoid study, observed the dentition of P. rudis as a gradient monognathic heterodonty and pointed out its sexual heterodonty as seen in adults only. The same authors affirm that ontogenetic heterodonty is present only in males by a low tooth cusp, which grows to large size in maturing specimens.
In this study, three species of the genus Psammobatis (P. extenta, P. lentiginosa and P. rutrum) were investigated to determine if they show sexual heterodonty and ontogenetic variation.

Material and Methods
Specimens examined belong to the following institutions: Instituto de Ciências Biológicas da Universidade Federal de Rio Grande (FURG), caught between August 2013 and August 2014, by commercial fishing off the coast of Rio Grande do Sul State, between latitudes 34°28'S and 31°29'S, at depths from 40 to 142 Teeth of the sandskate rays Psammobatis m; and Universidade do Estado do Rio de Janeiro, Rio de Janeiro (UERJ), captured off the coast of Cabo Frio, Rio de Janeiro State, between latitudes 22°40'S and 22°41'S at depths of 50 to 55 m. Species identification and the measurements follow Paragó (2001) and Gomes et al. (2010). In the text and tables, the measurements are in cm. The specimens from FURG were received with their ontogenetic stage determined by examining gonads and clasper (MARTINS; ODDONE, 2017). All immature specimens were from UERJ. Twenty-eight sets of jaws (both upper and lower) were prepared and examined by scanning electron microscopy (SEM).
For SEM, the dental plates were cleaned with soft toothbrushes with fine bristles and toothpaste. The samples were subsequently dehydrated in an alcohol series (24 h in 70%, 30 min in 80%, and 30 min in 90%), dried in a 50°C oven, coated with gold ions, and photographed with a Philipps XL 30 SEM of the Central de Microscopia e Microanálise (LabCEMM -PUCRS). This technique, when applied to teeth of P. lentiginosa (males), caused the teeth to crack at the depression on the labial crown face (sulcus). Because of this, the term "sulcus" is maintained in the text even though the images reveal the cracks.
For comparisons and analyses, both upper and lower dental plates were examined for each set of jaws, and three regions from each were highlighted: left lateral, right lateral and symphysis, totaling six regions in each specimen. Each region was compared between males and females of the same species, as well available specimens of each ontogenetic stage (adult/ immature) and between species. The description of the teeth, including size, arrangement, morphology and number, follows Moss (1977) and Rangel et al. (2015), and the shape of the cusp (elliptical or circular) was inferred from the external shape of the tooth. The ratio between the length and width of the symphyseal teeth (an average of fourteen teeth measured in each dental plate) as a function of total length was plotted in graphs to show the relation between tooth development and sexual maturity (see Table 1).
Tooth measurements (Figure 1) follow Gutteridge and Bennett (2014). The dental formula follows Belleggia et al. (2014) and corresponds to the number of rows of teeth in each jaw (minimum and maximum numbers in upper jaw/minimum and maximum numbers in lower jaw).

Results
The size range of specimens examined is shown in Table 1. In all species, there was the occurrence of monognathic and gynandric heterodonty and absence of dignathic heterodonty.

Immature specimens
Immature individuals smaller than 20.0 TL (Figure 2A and 2B), both males and females, had monocuspid teeth of the crushing type, with a small cusp (symphyseal teeth) or lacking a cusp (lateral teeth), and a smooth lozenge-shaped crown with rounded margins. Lateral teeth had a greater distance in between ( Figure  2C), whereas for symphyseal teeth this space was almost nonexistent. The length/width ratio of the symphyseal teeth varied from 0.9 to 1.1 in immature specimens, as seen in Figure 3A. This figure shows that the symphyseal cusps of immature and female specimens are smaller than those of adult males. Dental formula was: P. extenta, immature specimens, 16-20/17-20 (n = 3).

Psammobatis extenta
Monocuspid teeth of the clutching type, with pointed and well-pronounced cusps, especially those of the symphyseal region, which had a perpendicular orientation and were smooth and rounded ( Figure  5A). Crown was smooth and lozenge-shaped, and with rounded margins. Monognathic heterodonty was observed in all adult male specimens. In males larger than 30 TL, the lateral teeth had cusps that varied from short to well-developed, even though most teeth were morphologically similar. Specimens smaller than 28 TL had fewer teeth with developed cusps in comparison with larger males, as their lateral regions had only poorly developed cusps ( Figure 5B). The length/width ratio of symphyseal teeth varied from 1.7 to 1.9 ( Figure 3A). Dental formula was 19-23/20-23 (n = 3).

Psammobatis lentiginosa
Monocuspid teeth were of the clutching type, with pointed and well-pronounced cusps, especially on symphysis, with a perpendicular orientation, a longitudinal sulcus on the apical face, and elliptical shape ( Figure 5C). Crown was smooth, lozenge-shaped, and had rounded margins ( Figure 5D). Monognathic heterodonty was observed in all adult males. In specimens greater than 48 TL, the jaws had morphologically similar teeth, with lateral teeth having short to developed cusps. Specimens smaller than 47 TL have fewer teeth with developed cusps in comparison with larger males, as their lateral regions had only poorly developed cusps ( Figure 5E). The length/width ratio of symphyseal teeth ranged from 1.6 to 1.7 ( Figure 3B). Dental formula was 21-22/21-23 (n = 6).

Psammobatis rutrum
Monocuspid teeth were of the clutching type, with pointed and well pronounced cusps, especially on symphysis, with a perpendicular orientation, smooth and elliptical ( Figure 5F). Crown was smooth, lozenge-shaped and had rounded margins. Monognathic heterodonty was observed in all adult males. Specimens smaller than 28 TL had fewer teeth with developed cusps in comparison with larger males, since their lateral regions had only poorly developed cusps ( Figure  5G). In specimens greater than 30 TL, all teeth had developed cusps; the largest cusps always occurred in the symphyseal region. The length/width ratio of symphyseal teeth ranged from 1.7 and 2.0 ( Figure 3C). Dental formula was 25-27/25-27 (n = 8).

Discussion
Adults of the species examined showed monognathic heterodonty, mainly in the anterior teeth (symphyseal) when compared to the lateral ones. Females of the three species were differentiated from adult males by having a crushing type of dentition as opposed to a clutching type of dentition in males, thus indicating also a gynandric heterodonty. Specimens of P. extenta also showed ontogenetic heterodonty with the immature males showing monocuspid teeth of the crushing type, with a negligible or lacking cusp; while adult males had monocuspid teeth of the clutching type, with pointed and well-pronounced cusps, especially those of the symphyseal region.
This ontogenetic heterodonty is related to different feeding habits regarding P. extenta. It is in line with Barbini and Lucifora (2012) who mentioned that "small individuals consume amphipods and small crabs, and that large individuals consume cumaceans [small crustaceans] and isopods". Muto et al. (2001) and Braccini and Perez (2005) also analyzed diets and found no differences between adult males and adult females. However, the ontogenetic and seasonal patterns seem in feeding habits correlated with the skate's body size and prey availability, respectively; and these features seem to be of more importance to the species' ecological role.
The development of the cusp of males' teeth of all three species coincides with sexual maturity: between 25 and 26 TL for P. extenta and P. rutrum and between 40 and 42 TL for P. lentiginosa (GOMES et al., 2010;PERIER et al., 2011;MARTINS;ODDONE, 2017). The analysis of the length/width ratio of symphyseal teeth in the adults of the three species examined here ( Figure  3) demonstrated that teeth in males can be almost twice as long as wide. This does not occur in adult females of the three species or in the immature specimens of P. extenta, in which the ratio is close to 1. The variations in tooth shape in males, related to length/width ratio and the respective size range and sex, support the hypothesis that the observed gynandric heterodonty is related to the mating strategies. This same pattern was found for P. bergi and P. extenta (BARBINI; LUCIFORA, 2012). The relationship between sexual maturation and development of teeth in males is also reported for Amblyraja doellojuradoi (Pozzi 1935) (Rajidae) (DELPIANI et al., 2012), Aptychotrema rostrata (Shaw 1794) (Trygonorrhinidae) (GUTTERIDGE; BENNETT, 2014), Atlantoraja cyclophora (Regan 1903) (Arhynchobatidae) (OLIVEIRA; ODDONE, 2012;RANGEL et al., 2015), and Hypanus sabinus (Leuseur 1824) (Dasyatidae) (KAJIURA; TRICAS, 1996;KAJIURA et al., 2000); in all these cases, males exhibit a copulatory behavior of "hugging" females with their jaws. A. doellojuradoi and H. sabinus show differences in diets between males and females, but A. rostrata and A. cyclophora seem to have a narrower niche, as males and females have a significant overlap in their feeding habitats (KAJIURA; TRICAS, 1996;KYNE;BENNETT, 2002;DELPIANI et al., 2013;VIANA;VIANNA, 2014).
Regarding their taxonomy, P. extenta is differentiated from P. rutrum by their dental formula (33-46 tooth rows on upper jaw vs 50-54 and 42-45, respectively) and from P. lentiginosa by the shape of the cusp (circular, rounded shaped vs elliptical). McEachran (1983) reported 36-50 upper tooth rows and used this range to distinguish P. glansdissimilis McEachran (= P. extenta) from P. rutrum, which possesses 43-66. Braccini and Chiaramonte (2002) recorded for P. extenta 34-46 rows of teeth in the upper jaw in both sexes. P. lentiginosa is distinguished from P. rutrum and P. extenta by the presence of a sulcus on the apical face, which is absent in the latter species. These new data may help in the discrimination of these species, since they are morphologically very similar. Comparing the dental formulas of immature and adults of P. extenta, the number of tooth rows appears to increase proportionally to specimen size, as suggested for this species by Braccini and Chiaramonte (2002) and for Mustelus henlei (Gill 1863) by Bellenggia et al. (2014). Unfortunately, immature individuals of P. lentiginosa and P. rutrum were not available for this study, but comparing small adults (approx. 44 TL and Teeth of the sandskate rays Psammobatis 25 TL for P. lentiginosa and P. rutrum, respectively) with large adults (approx. 47 TL and 28 TL for P. lentiginosa and P. rutrum, respectively) the pattern seems to be the same as in P. extenta.
The sulcus on the apical face of the teeth of P. lentiginosa not seen in other Psammobatis or related species so far, is present in a similar condition in Dasyatis hypostigma (RANGEL et al., 2014). Morphological (MCEACHRAN; DUNN, 1998) and molecular  analyses have shown that Dasyatidae is not closely related to Arhynchobatidae, which leads us to assume that the presence of a sulcus is most parsimoniously interpreted as having occurred independently in D. hypostigma and P. lentiginosa.