Herpetology Notes, volume 11: 629-639 (2018) (published online on 20 August 2018)

Ontogeny and behavioural aspects of the tadpoles of Megaelosia goeldii (Baumann, 1912) (Amphibia, Anura, Hylodidae)

Dener das Neves da Silva1, Felipe Cesar Barros da Rosa¹ and Ana Maria Paulino Telles de Carvalho-e-Silva¹,*

Abstract. The genus Megaelosia consists of large size, diurnal stream-breeder species. Although the tadpole of M. goeldii had been described, its ontogeny and behaviour were never detailed. Here, we describe the development of the M. goeldii larvae, as well as some behavioural aspects. Tadpoles were collected between 1983 and 2015 in the municipality of Teresópolis, state of Rio de Janeiro, Brazil. Stages were determined based on Gosner’s table. Tadpoles present accentuated growth on stage 25, but a slow growth from stages 26 to 42. Changes in coloration were observed throughout the tadpole development. Larvae were found in streams of running water, in approximately 1.0–1.2 m depth. During the day they hide among stones in the bottom, and at night they are easily spotted in shallower waters, foraging near the surface. Larvae are observed throughout the year, although the smaller ones are mostly found in August and September, indicating that reproduction occurs in the winter. The tadpoles showed emetic behaviour when collected, probably due to the exposure to stress situations or to scare away predators. We analysed 208 specimens (stages 25 to 40), and 71% presented depigmentation in the oral disc; this phenomenon was more frequently observed in larger tadpoles. The oral depigmentation in these tadpoles indicates the presence of the fungus Batrachochytrium dendrobatidis (Bd), one of the main responsible for the worldwide decline of amphibians. The presence of Bd zoosporangia in the oral disc of M. goeldii tadpoles were detected with histological investigation.

Keywords. development, emetic behaviour, natural history, chytridiomycosis

Introduction Alamillo, 2005; Costa et al., 2010; Alcalde et al., 2011; Fabrezi, 2011; Vera Candioti et al., 2011; Mongin and The morphology and biology of tadpoles attract Carvalho-e-SiIva, 2013). the interest of researchers from different areas. For The family Hylodidae Gunther 1858 is currently example, including information about larval traits and composed of the genera Crossodactylus Duméril and habits on systematics studies, not only helps in the Bibron 1841, Hylodes Fitzinger 1826 and Megaelosia identification of several amphibian species but also can Miranda-Ribeiro 1923, forming a well-defined be used to improve our understanding on phylogenetic monophyletic group (Frost et al., 2006; Grant et al., relationships (Hall et al., 1997). In addition, detailed 2006; Pyron and Wiens, 2011), totalizing 46 species descriptions of ontogeny are important tools for (Frost, 2017). Both adult and larval forms are restricted comparing the growth patterns of different amphibian to streams of the Atlantic Rainforest (Pombal et al., taxa (Costa et al., 2010). Recent studies have focused on 2003; Haddad and Prado, 2005; Lingnau et al., 2008; the importance of tadpoles using different approaches, Silva and Benmaman, 2008). i.e. anatomy, morphology, ecology and even phylogenies The genus Megaelosia comprises large-sized and solely based in larval characters (Haas, 2003; Sheil and diurnal species, associated with lotic sites (Giaretta et al., 1993). All seven species in the genus are restricted to a limited area of the Atlantic Rainforest, in the southeastern Brazil: Megaelosia apuana Pombal, Prado and Canedo 2003; Megaelosia bocainensis Giaretta, ¹ Laboratório de Biossistemática de Anfíbios, Departamento Bokermann and Haddad 1993; Megaelosia boticariana de Zoologia, Instituto de Biologia, Centro de Ciências Biológicas e da Saúde, Universidade Federal do Estado do Giaretta and Aguiar 1998; Megaelosia goeldii Rio de Janeiro, 22290-240, Rio de Janeiro, RJ, Brasil. (Baumann 1912); Megaelosia jordanensis (Heyer * Corresponding author. Email: [email protected] 1983); Megaelosia lutzae Izecksohn and Gouvêa 1987 630 Dener das Neves da Silva et al. and Megaelosia massarti (De Witte 1930). Specimens isolated from tadpoles with depigmented mouthparts of Megaelosia are extremely rare in scientific collections across Brazil’s Atlantic forest (Jenkinson et al., 2016). due to their restricted geographical ranges, cryptic Therefore, patterns of mouthparts depigmentation can behaviour (Giaretta et al., 1993; Melo et al., 1995) and be effectively used as a proxy for Bd infections in Brazil low-density populations (Pombal et al., 2003). They are (Carvalho et al., 2017). very agile animals, exhibiting a rapid response of diving There are no studies regarding the ontogeny of any of in lotic waters when perturbed, consequently reducing the species in the genus Megaelosia. The tadpole of M. the chance of being captured (Miranda-Ribeiro, 1923; goeldii was described by Lutz (1930) and redescribed by Izecksohn and Gouvêa, 1985). Nuin (2003), but its ontogeny and behaviour were never The emesis capacity is a behavioural aspect described detailed. In the present study, we describe the larval for vertebrates and are present in mammals (Borison development of M. goeldii, considering its behavioural and Wang, 1953; Andrews et al., 1990), reptiles (Gans, aspects. We also discuss the evidence of the fungus 1952), birds (Matthews, 1949; Saxena et al., 1977), fish Bd on M. goeldii larvae and the possible consequence (Tiersch and Griffith, 1988; Andrews et al., 1998) and for tadpole growth. We expect larger tadpoles to have amphibians (Naitoh et al., 1989; Naitoh and Wassersug, higher Bd prevalence because they have been inhabiting 1994). In general, vomiting is a defensive mechanism the aquatic habitat for longer and therefore might to eliminate toxins when accidentally ingested (Lang et have been more exposed to Bd zoospores than smaller al., 1986). However, Gans (1952) describes a selective tadpoles. emesis of eggshell performed by snakes from Dasypeltis Wagler 1830 genus frequently carried out to avoid Materials and Methods predation. Naitoh et al. (1989) shows that in anurans, the ability to vomit only occurs after metamorphosis, We examined 215 tadpoles of M. goeldii, collected because tadpoles do not have the necessary features to in sporadic events between 1983 and 2015 at the increase their abdominal pressure and vomit. Parque Nacional da Serra dos Órgãos (PARNASO) Chytridiomycosis is an emergent disease caused - “National Park of Serra dos Órgãos” (22°26’54’’S, by the Chytridiomycota fungus, Batrachochytrium 42°59’01’’W) and the adjacent locality of Vale da dendrobatidis (Bd) Longcore, Pessier and Nichols Revolta (22°26’00’’S, 42°56’00’’W), both belonging 1999. Declines and mass extinctions associated with Bd to the municipality of Teresópolis, Rio de Janeiro state, was reported in Australia (Berger et al., 1998), South Brazil. America (Carvalho et al., 2017), Central America We captured tadpoles using sieves and funnel traps. (Lips 1999), North America (Muths et al., 2003) and After capture, individuals were anesthetized with Europe (Bosch et al., 2001). This pathogenic fungus chloretone 5% (from 1983 until 2011 by A.M.P.T. (Bd) has flagellated aquatic zoospores with a potential Carvalho-e-Silva) and lidocaine 2%, (from 2012 until to rapidly spread within water (Longcore et al., 1999), 2015 by A.M.P.T. Carvalho-e-Silva, F.B. Rosa and facilitating tadpoles infection. B. dendrobatidis infects D.N. Silva), fixed and preserved in 5% formalin. The the keratinizing tissue of amphibians, which in tadpoles identification of tadpoles was based on specimens reared is concentrated in the mouthparts (Berger et al., 1998). in aquarium until metamorphosis and on literature (Lutz The depigmentation pattern in tadpoles due to Bd 1930; Nuin 2003). Observation on the behaviour of infection is unequivocal, although depigmentation tadpoles reared in the laboratory also improved and may also be a result of the exposure to environmental corroborated our in situ observations. The depth of the contaminants (Hayes et al., 1997; Rowe et al., 1998) sampling sites was taken with a standard measuring or due to low temperatures (Rachowicz, 2002). B. tape. The analysed material (see Appendix) is stored in dendrobatidis causes patchy depigmentation with the amphibian collections of Universidade Federal do complete loss of keratin in localized areas compared Estado do Rio de Janeiro (UNIRIO) and Universidade with fully keratinized surrounding areas (Fellers et Federal do Rio de Janeiro (UFRJ/ZUFRJ). al., 2001). Recent studies found a huge proportion of Some specimens were fixed with the mouth open in tadpoles with highly depigmented mouthparts attributed order to evaluate the oral morphology, applying the to Bd. The prevalence of infection was estimated at 95% technique of Carvalho-e-Silva and Carvalho-e-Silva in the Atlantic forest torrent frog Hylodes cf. ornatus (1994). Tadpoles were measured with digital caliper and Lithobates catesbeianus (Shaw 1802) (Vieira et ruler (precision 0.1 mm). The measurements and al., 2013). Thus, more than 100 Bd genotypes were nomenclature were adapted from Altig and McDiarmid Ontogeny and behavioural aspects of the tadpoles of Megaelosia goeldii 631

Figure 1. Mean values of total length (TL), body length (BL) Figure 2. Mean values of body height (BH) and tail height and tail length (TAL) of Megaelosia goeldii tadpoles in the (TH) of Megaelosia goeldii tadpoles in the Gosner stages 25 Gosner stages 25 to 46. to 46.

(1999): total length (TL), body length (BL), tail length individually observed with a stereoscopic microscope (TAL), body height (BH), tail height (TH), body width following Lambertini et al. (2013). Individuals with jaw (BW), interocular distance (IOD), nostril diameter (ND), sheaths or tooth rows fully pigmented was classified as eye diameter (ED), eye to snout distance (ESD), nostril Bd-negative. We considered Bd-positive specimens if to snout distance (NSD), internarial distance (IND) they exhibited patterns of full or partial depigmentation and oral disc width (OW). Stages were determined of buccal apparatus compatible with depigmentation according to Gosner’s (1960) table. Because of the due to chytridiomycosis (Rachowicz and Vredenburg, great variation of size observed in larvae at the stage 2004; Vieira et al., 2013). We did not considered 25, we chose to categorize its size into three classes for individuals with slight depigmentation in mouthparts comparative purposes: 25(a) (28–54 mm), 25(b) (60–94 or tiny losses due to manipulation process. For Bd mm), and 25(c) (97–126 mm). Schematic drawings were zoosporangia detection we performed histological made with a camera lucida coupled to a stereoscopic techniques in two tadpoles classified as Bd-positive. We microscope (Nikon SMZ800), at different stages of sectioned each specimen’s buccal apparatus, fractioned development. For the description of ontogeny, tadpoles tissue using a scalpel, placed tissue fragments on a glass at the stages 25(a) (n = 20), 25(b) (n = 10), 25(c) (n = slide with a drop of distilled water, and screened for Bd 10), 26 (n = 2), 34 (n = 2), 35 (n = 2), 36 (n = 4), 37 (n zoosporangia using a microscope at 400x magnification = 5), 38 (n = 4), 39 (n = 1), 40 (n = 1), 42 (n = 4), 45 for 30 min according to Vieira et al. (2013). (n = 1), 46 (n = 2), were analysed. For the coloration To evaluate a possible correlation between tadpole description of live specimens, we used the colour guide growth and occurrence of depigmented mouthparts, of Smithe (1975). ten groups were formed with variations of 10–10 mm For Bd detection in the tadpoles, the buccal apparatus (Vieira et al. 2013). A test of linear regression (R²) of 208 tadpoles in the developmental stages 25 to 40 was was further applied aiming to relate the cited variables.

Figure 3. Mean values of body width (BW), interocular Figure 4. Mean values of eye diameter (ED), eye to snout distance (IOD), internarial distance (IND) and oral disc width distance (ESD), nostril to snout distance (NSD) and nostril (OW) of Megaelosia goeldii tadpoles in Gosner stages 25 to diameter (ND) of Megaelosia goeldii tadpoles in Gosner 46. stages 25 to 46. 632 Dener das Neves da Silva et al.

Table 1. Measurements (mm) of tadpoles of Megaelosia goeldii. Means ± SD are followed by the range in parentheses. TL = total length, BL = body length, TAL = tail length, BW = body width, BH = body height, TH = maximum tail height, IOD = interorbital

Tabledistance. 1:

Stage N TL BL TAL BW BH TH IOD 42.4 ± 6.2 16.3 ± 3.5 26 ± 5.1 7.6 ± 2 6.5 ± 1.4 9.1 ± 1.6 5.2 ± 1.2 25 (a) 20 (28.2–53.4) (11.1–22.8) (12.9–34.1) (3.7–11) (4.3–9.4) (7.1–12.3) (3.4–7.3) 81.2 ± 11.6 34.2 ± 9.1 45.9 ± 4.1 20.2 ± 6.6 14.8 ± 4.1 18.6 ± 2.6 8.9 ± 0.7 25 (b) 10 (60.9–93.1) (15.8–40) (40.8–53.2) (10.2–33.5) (9.7–24.3) (14–21.8) (8–10.1) 111.7 ± 11.2 46.6 ± 3.8 65.1 ± 8.3 25.3 ± 4.2 20.2 ± 2.8 25.2 ± 3.7 9.5 ± 0.7 25 (c) 10 (97.4–126) (41.2–52.6) (54.6–77.6) (19.2–31.6) (16.9–25.8) (21.3–31) (8.3–10.7) 117.1 ± 23.9 47.3 ± 8.8 69.7 ± 15.3 29.5 ± 14.2 21.8 ± 8.3 26.9 ± 6.3 13.2 ± 2 26 2 (100.2–134) (41–53.5) (58.9–80.5) (19.4–39.5) (15.9–27.7) (22.4–31.3) (11.8–14.6) 116.7 ± 0.7 49.6 ± 4.7 67.2 ± 4 23 ± 4.1 19 ± 4 28.2 ± 1.8 13 ± 1.6 34 2 (116.2–117.2) (46.2–52.9) (64.3–70) (20.1–25.9) (16.2–21.8) (26.9–29.5) (11.9–14.1) 117.1 ± 3 49.1 ± 0.6 68 ± 3.7 26.2 ± 7.6 21.1 ± 5.3 30.8 ± 7.5 14.2 ± 0.8 35 2 (114.9–119.2) (48.6–49.5) (65.4–70.6) (20.8–31.6) (17.3–24.8) (25.5–36.1) (13.6–14.8) 115.5 ± 3.5 46.8 ± 2.8 69.6 ± 3.2 24.4 ± 3.2 19.8 ± 1.9 27 ± 5.1 15.2 ± 2.3 36 5 (111.6–119.1) (42.5–49.7) (65.3–72.6) (21.6–29.5) (17.5–22) (21.3–33.6) (12.7–17.7) 116.7 ± 13 50.8 ± 4 71.3 ± 18 26.3 ± 5.1 22.5 ± 4.1 27 ± 5.1 14.3 ± 1.6 37 5 (97–128.9) (45.7–56.4) (47.1–96.7) (20.3–33.4) (18–28.4) (21.3–33.6) (12.1–16.7) 124.4 ± 1.7 59.2 ± 12.3 72.6 ± 4.4 26.4 ± 3.9 21.2 ± 4.1 27.3 ± 2.9 13.6 ± 0.3 38 4 (122.1–126) (50.9–77.2) (68–78.5) (22.5–30) (16.8–25.9) (24.1–30) (13.2–13.9)

39 1 107.3 43.2 64.1 26.8 20.5 25.7 14

40 1 118.2 45.5 72.7 27.3 22.7 27.1 13.9

121.1 ± 10.9 49.4 ± 1.1 71.7 ± 11.7 20.7 ± 3.8 15.7 ± 1.6 18.8 ± 1.1 11.3 ± 1 42 4 (109.4–138) (47.8–50.7) (59.2–90.4) (16.2–25.9) (13.2–17.6) (17–19.7) (10–12.7)

45 1 79.1 44.4 34.7 19 17.6 12.9 11.6

52.1 ± 1.5 52.1 ± 1.5 18.9 ± 1.5 13.3 ± 1.75 10.1 ± 0.05 46 2 0 0 (50.6–53.7) (50.6–53.7) (17.4–20.4) (11.6–15.1) (10.1–10.2)

Statistical analyses were implemented in R version considerable modifications, only minor increase of BH 3.3.2 (R Development Core Team, 2016); we used R and TH is observed along the development (Fig. 2). The package Rcmdr. maximum BH is in the stage 40, with = 22.7 mm, whereas TH attains 30.8 mm in the stage 35. Results From stage 40 on, there is an abrupt reduction in TH until metamorphosis (Fig. 2, Fig. 5C – D). Tadpoles of M. goeldii have a great size variation in The greatest variation of BW, OW, IOD and IND occur the stage 25. In the stage 25(a), they present 42.4 in the stage 25 and posteriorly there is no substantial mm of TL and attain 111.7 mm in the stage 25(c) modification (Fig. 3). After stage 40, the BW narrows (Fig. 1, Table 1). From stage 26 on, they show little if compared to earlier stages (Fig. 3). In contrast, OW increase in size until reaching stage 42 (Fig. 1). In the reaches its maximum values in the final stages of larval stage 38, tadpoles acquire their largest TL ( 124.4 development. mm) and BL ( 59.2 mm) (Fig.1, Table 1). The tail The eyes gradually move away from the snout grows until stage 42, and partially regresses in the stage beginning in the stage 25 until stage 34, when they are 45 (Fig. 1, Fig. 5D), being completely absorbed in the displaced toward the more central region of the body, stage 46 due to metamorphosis. reaching a maximum distance of = 13.9 mm (Fig. 4, The greatest range in BH and TH also occur at stage Table 1). In the late stages of development (40–46), the 25, respectively with 6.5 mm and = 9.1 mm eyes are again close to the snout due to changes in the in the stage 25(a), 20.2 mm and 25.2 mm larval body. A similar pattern of gradual displacement in the stage 25(c) (Fig.2, Table 1). After these initial is noticed with the nostrils, which are more distant from Ontogeny and behavioural aspects of the tadpoles of Megaelosia goeldii 633

Table 1 (continue): Measurements (mm) of tadpoles of Megaelosia goeldii. Means ± SD are followed by the range in parentheses. IND = internarial distance, OW = oral disc width, NSD = nostril to snout distance, ESD = eye to snout distance, ND = nostril diameter, ED = eye diameter 7 8 Stage N IND OW NSD ESD ND ED 9

4.1 ± 0.9 3.7 ± 0.8 2.7 ± 1.1 5.3 ± 1.4 0,7 ± 0,3 1.5±0.4 25 (a) 20 (3–6) (2.6–5.5) (0.7–4.3) (3.7–8.3) (0.2–1.2) (0.5–2.2) 9 ± 1.7 8.4 ± 2.1 6.0 ± 1.4 7.0 ± 2.1 1.3 ± 0.3 2.6 ± 0.8 25 (b) 10 (6.5–11.6) (4.65–11.1) (4.25–9.1) (7.2–13.5) (0.9–1.8) (0.7–3.4) 11 ± 1.2 11.8 ± 1.3 7.0 ± 0.6 10.1 ± 1.5 1.7 ± 0.2 3.3 ± 0.4 25 (c) 10 (8.9–12.9) (9.7–13.8) (6.1–7.9) (8.9–13.9) (1.3–2.0) (2.2–3.7) 9.4 ± 2.5 11.7 ± 1.5 7.3 ± 0.6 13.2 ± 3.2 1.7 ± 0.2 3,4 ± 0,7 26 2 (7.6–11.2) (10.6–12.7) (6.8–7.7) (11–15.5) (1.5–1.8) (2.9–3.9) 9.6 ± 0.8 11.5 ± 1.8 7.9 ± 3.3 13.9 ± 2.6 2.0 ± 0.0 4.1 ± 0.2 34 2 (9–10.2) (10.2–12.7) (5.5–10.2) (12.0–15.7) (2.0–2.0) (3.9–4.2) 10.6 ± 0.5 13.8 ± 1.3 5.6 ± 3.0 11.8 ± 0.1 1.9 ± 0.1 3.4 ± 0.9 35 2 (10.2–10.9) (12.9–14.7) (3.4–7.7) (11.7–11.8) (1.8–2.0) (2.7–4.0) 11 ± 2 11.8 ± 1.5 8.2 ± 1.6 13.3 ± 3.8 1.7 ± 0.6 3.4 ± 0.3 36 5 (9–13.1) (9.6–13.1) (6.2–10.4) (9.7–18.1) (0.9–2.4) (3.1–3.8) 10.6 ± 0.7 13.3 ± 1.6 6.2 ± 1.8 11.5 ± 0.9 2.0 ± 0.3 3.6 ± 0.4 37 5 (9.6–11.2) (11.3–15.5) (3.5–7.9) (10.8–12.9) (1.5–2.2) (3.2–4.3) 9.8 ± 0.1 12.2 ± 0.4 5.2 ± 1.3 11.6 ± 1.0 1.8 ± 0.1 3.5 ± 0.2 38 4 (9.7–9.9) (11.7–12.6) (4.2–7.1) (10.3–12.5) (2.4–3.0) (3.3–3.7)

39 1 10.2 11.4 5.5 13.9 1.7 3.2

40 1 9.7 12.8 2.8 9.2 2.0 4.1

8.3 ± 0.5 10.7 ± 0.7 5.1 ± 1.8 10.4 ± 1.8 2.5 ± 2.6 4.6 ± 0.6 42 4 (7.8–9) (9.9–11.8) (2.2–7.0) (7.6–12.6) (0.9–6.9) (4.2–5.7)

45 1 6.6 14.8 3.9 7.8 1.0 4.9

6.25 ± 0.15 16.1 ± 0.15 3.95 ± 0.25 8.35 ± 0.75 0.3 ± 0.1 5.4 ± 0.2 46 2 (6.1–6.4) (16–16.3) (3.7–4.2) (7.6–9.1) (0.2–0.4) (5.2–5.6)

the snout in the intermediate stages (34 to 39); at the end the spiracle opening is light beige (Colour Buff, 24) of development (46) they again become closer to the contrasting with the dark brown present in the spiracle. snout (Fig.4). These changes are related to the complete From the 215 individuals analysed, 72.5% belonged metamorphosis (Fig. 5). to the stage 25. Most specimens found at this stage, The ED increases continuously during development in addition to the notable size and colour variations, (Fig. 4). During the notorious reduction of the BW (Fig. suggest that the larvae remain a long time period in this 3), the position of eyes is more lateral, which probably stage and may stay for more than a year as tadpole. amplifies the peripheral vision of the tadpoles at the The tadpoles of M. goeldii were found in lotic water end of the metamorphosis. The ND increases during streams (Figure 6). During the day, they hide in crevices development, but in the stages 45 and 46 it returns formed by rocks at the bottom of the streams, usually at to reduce, and nostrils resemble in size with those of 1.0–1.2 m depth. In several occasions, tadpoles were tadpoles of initial stage 25 (a). seen at night in shallower sites, scraping the substrate Coloration of live tadpoles varied between the stages probably in search of food. The greatest activity of larvae of development. In stage 25(a) the colour is uniformly at night is possibly related to negative phototaxis, which dark brown (Colour Vandycke Brown, 121) and after was observed in situ and at laboratory. With exposure this stage tadpoles become lighter (Colour Verona, to artificial lightning, individuals remained under rocks 223B) with dark (Colour Raw Umber, 223) and light in the aquarium, leaving this refuge in the absence of spots (Colour Fawn, 25). The lateral line pores are light. Tadpoles were observed in all months of the year well marked with beige (Colour Buff, 24) and golden due to its extended larval period, but those of smaller highlights in the body and in the tail. The colour around size and earlier stages are mainly found in August and 634 Dener das Neves da Silva et al.

Figure 5. Tadpoles of Megaelosia goeldii in lateral view: A– UNIRIO 987, stage 25; B– ZUFRJ 6357, stage 36; C– UNIRIO 1155, stage 42; D– UNIRIO 1579, stage 45. Scale bars = 10 mm.

Tadpoles performed emetic behaviour after being captured (100% between stages 25 to 40) altering the abdominal musculature, expelling their stomach contents and releasing debris into the water. After this practice, we noticed that the larvae showed a more flaccid abdomen. This emetic behaviour was frequently recorded probably because of stress or as a mechanism to avoid predation. The stomach content expelled by larvae was fixed for future studies comprising the diet of M. goeldii. The occurrence of depigmentation at mouthparts was very frequent in the species (Fig. 7), detected in more than 70% of the examined individuals. There was a positive correlation between size and depigmentation of oral disc (R² = 0.66; P = 0.0042; P<0.05), and this abnormality was mostly seen in large tadpoles (Table 2, Fig. 7).

Discussion

Figure 6. A– Paquequer River in the National Park of Serra The accentuated growth at the stage 25 in Megaelosia dos Órgãos, Teresópolis, Brazil; B– Megaelosia goeldii goeldii was also observed by Giaretta et al. (1993) in tadpole at the bottom of a stream of Paquequer River. tadpoles of M. massarti. A similar elevated growth at the stage 25 was observed on tadpoles of Hylodes asper (Müller 1924) and Hylodes nasus (Lichtenstein 1823); (A.M.P.T. C. S., pers. obs.). Tail height starts to reduce September, indicating that reproduction of this species in stage 40, prior to metamorphosis, whereas its length is occurs during the winter. becomes shorter after stage 42. This indicates a primary Ontogeny and behavioural aspects of the tadpoles of Megaelosia goeldii 635

laboratory and the nocturnal habit of these tadpoles may be a trait to reduce risks of predation as the major dark coloration of larvae would evidence them to visual predators (such as birds, snakes and mammals) during daylight. However, Giaretta et al. (1993) indicated that M. massarti tadpoles were active during the day, suggesting a plausible plasticity for the genus. The emetic behaviour performed by M. goeldii tadpoles is the first and only record for the genus. However, this defensive mechanism might be present in other species of the family, as we also observed such behaviour in Hylodes nasus tadpoles. Naitoh et al. (1989) analysed the effects of some emetic substances in adults and tadpoles of Lithobates catesbeianus (Shaw 1802), Lithobates clamitans (Latreille 1801) and Xenopus laevis (Daudin 1802). Although no treatment caused any emetic behaviour in tadpoles, during the end of metamorphosis some of them threw up just after the first feeding event. The authors highlighted the influence of the sacroiliac complex during the emesis in adults; and suggested that because this structure is not developed in pre-metamorphic tadpoles, they Figure 7. Oral disc of Megaelosia goeldii tadpoles. A– cannot deform the abdomen and increase pressure in Normal condition; B–Abnormal condition (depigmentation); this region, what would result in the elimination of C– Zoosporangia (white circle) of Batrachochytrium the stomach content (Naitoh et al., 1989). On the other dendrobatidis (Bd) in microscope (400X) UNIRIO 2348. Scale bars = 1 mm. hand, M. goeldii tadpoles possess a well-developed abdominal musculature, a feature that may enable this species to proceed with the emetic behaviour. Depigmented mouthparts in tadpoles may be related to several factors: seasonal changes (Rachowicz, absorption of the marginal tissue that constitutes the tail, followed by the regression of the musculature. Such modifications was also reported in tadpoles of Bokermannohyla circumdata (Cope 1870, Mongin and Carvalho-e-Silva, 2013). Giaretta and Aguiar (1998) found young tadpoles of M. boticariana at November (spring) whereas we found smaller M. goeldii tadpoles in August and September (winter). To reproduce in winter might be an advantage for M. goeldii because of the characteristics of the studied area, which presents a dry winter and wet summer, with intense rainfalls, substantially altering the aquatic environment where larvae live. During daylight, tadpoles of M. goeldii hide in sheltered sites of the stream, often in moderately deep pools (1.0–1.2 m depth), showing higher activity at night Figure 8. Linear regression between total length and percentage and in shallower areas. This behaviour corroborates the of specimens of Megaelosia goeldii with depigmented observations of Nuin (2003) for tadpoles of M. goeldii, mouthparts in each class. X-axis mean total length classes. M. apuana (Pombal et al., 2003) and M. boticariana Size classes (in mm): 1 = 25.5–35.5; 2 = 35.6–45.6; 3 = 45.7– (Giaretta and Aguiar, 1998). The observed negative 55.7; 4 = 55.8–65.8; 5 = 65.9–75.9; 6 = 76–86; 7 = 86.1–96.1; phototaxis of M. goeldii larvae in situ and in the 8 = 96.2–106.2; 9 = 106.3–116.3; 10 = 116.4–126.5. 636 Dener das Neves da Silva et al.

Table 2. Occurrence of depigmented mouthparts in Megaelosia goeldii tadpoles according to different total length classes. 1 TABLE 2. Occurrence of depigmented mouthparts in Megaelosia goeldii tadpoles according to different total length classes. 2

Total length classes Total Depigmented mouthparts

(mm) N n %

1: 25.5 – 35.5 6 2 33.3 2: 35.6 – 45.6 65 16 24.6 3: 45.7 – 55.7 19 14 73.6 4: 55.8 – 65.8 8 7 87.5 5: 65.9 – 75.9 8 8 100 6: 76.0 – 86.0 19 19 100 7: 86.1 – 96.1 20 19 95 8: 96.2 – 106.2 25 25 100 9: 106.3 – 116.3 21 21 100

10: 116.4 – 126.5 17 17 100 Total 208 148 71 3 4

2002), pollution (Bacon et al., 2013), temperature natural history of tadpoles are extremely important for (Rachowicz and Vredenburg, 2004) and diseases, such understanding the population dynamics and to help as the chytridiomycosis (Vieira et al., 2013). Dias and develop practices for species conservation. Considering Carvalho-e-Silva (2012) reported abnormalities in the endemism of the genus in the Atlantic Forest biome mouthparts of Proceratophrys appendiculata (Günther and the restricted distribution of the species within the 1873) tadpoles at PARNASO, which can be often seen environment they occupy, it is evident the urgency of in other tadpoles at streams of this locality (pers. Obs.). further research that explores both the biology of adults Smith et al. (2007) and Vieira et al. (2013) reported a and the larvae of the genus Megaelosia. positive relationship between the loss of keratin in the oral apparatus and the growth of larvae infected with Acknowledgements. The authors thank Dr. Sergio Potsch (UFRJ) and students of the Laboratório de Biossistemática de Anfíbios- Bd. This association is similarly inferred to tadpoles of LABAN (UNIRIO) for their help with field work; MSc. Marcia M. goeldii, as large individuals were most affected by dos Reis Gomes, biologist of Department of Zoology (UFRJ) for oral dekeratinization as we expected. The association access to collection; Dra. Joice Ruggeri (UNICAMP) for your between growth and the prevalence of tadpoles with oral help in identifying Bd in our sample; Dr. Maurício R. Fernandes, depigmentation might be due to the time of exposure biologist of Department of Zoology (UNIRIO) for the critical to the pathogen in the aquatic habitat as larger larvae revision and translate of the manuscript; Rafael L. Macedo for minor revision. We also thank IBAMA/RAN for providing our stay a longer period inside the water (Vieira et al., 2013; collecting license (number 13256-1). Catenazzi et al., 2013). Also, a recent study carried out study in the same locality of the present work showed References a high prevalence of Bd in tadpoles of streams in comparison with those of temporary ponds (Ruggeri et Alcalde, L., Candioti, F.V., Kolenk, F., Borteiro, C., Baldo, D. al. 2017). As the tadpoles of M. goeldii are exclusively (2011): Cranial anatomy of tadpoles of five species of Scinax (Hylidae, Hylinae). Zootaxa 2787:19–36. found in streams, these results explain the high rates of Altig, R., McDiarmid, R.W. (1999). Body plan. Development and oral depigmentation found in this population. Morphology. In: Tadpoles, the biology of anuran larvae, p. 24– Chyitridiomycosis is one of the main responsible for 51. McDiarmid, R.W., Altig, R., Eds., Chicago, The University the worldwide decline of amphibians (Berger et al., of Chicago Press. 1998; Daszak et al., 1999; Stuart et al., 2004; Lips et al., Andrews, P.L.R., Davis, C.J., Bingham, S., Davidson, H.I.M., 2006), and stream-breeding amphibian seem to be the Hawthorn, J., Maskell, L. (1990): The abdominal visceral innervation and the emetic reflex: pathways, pharmacology, most affected species (Kriger and Hero 2007). Therefore and plasticity. Canadian Journal of Physiology and Bd requires much concern, especially considering the Pharmacology 68(2): 325–345. restricted geographical range of Megaelosia species. Andrews, P.L.R., Sims, D., Young, J.Z. (1998): Induction of Finally, studies concerning the development and emesis by the sodium channel activator veratrine in the lesser Ontogeny and behavioural aspects of the tadpoles of Megaelosia goeldii 637

spotted dogfish Scyliorhinus canicula (Chondrichthyes Biology 38(4): 390–411. Elasmobranchii). Journal of the Marine Biological Association Fellers, G.M., Green, D.E., Longcore, J.E. (2001): Oral of the United Kingdom 78: 1269–1279. chytridiomycosis in the mountain yellow-legged frog (Rana Bacon, J.P., Fort, C.E., Todhunter, B., Mathis, M., Fort, D.J. muscosa). Copeia 2001(4): 945–953. (2013): Effects of multiple chemical, physical, and biological Frost, D.R. (2017). Amphibian species of the World: on Online stressors on the incidence and types of abnormalities observed Reference. Available at: http:// http://research.amnh.org/vz/ in Bermuda’s cane toads (Rhinella marina). Journal of herpetology/amphibia/index.php. Last accessed on 7 November Experimental Zoology Part B: Molecular and Developmental 2017. Evolution 320(4): 218–237. Frost, D.R., Grant, T., Faivovich, J., Bain, R.H., Haas, A., Baumann, F. (1912): Brasilianische Batrachier des Berner Haddad, C.F.,… Wheeler, W.C. (2006): The Amphibian Tree Naturhistorischen Museums nebst Untersuchungen über of Life. Bulletin of the American Museum of Natural History die geographische Verbreitung der Batrachier in Brasilien. (297): 1–370. Zoologische Jahrbücher 33: 87–172. Gans, C. (1952): The functional morphology of the egg-eating Berger, L., Speare, R., Daszak, P., Green, D.E., Cunningham, adaptations in the snake genus Dasypeltis. Zoologica 37: 209– A.A., Goggin, C.L., Slocombe, R., Ragan, M.A., Hyatt, A.D., 244 McDonald, K.R., Hines, H.B., Lips, K.R., Marantelli, G., Giaretta, A.A., Bokermann, W.C., Haddad, C.F. (1993): A review Parkes, H. (1998): Chytridiomycosis causes amphibian of the genus Megaelosia (Anura: Leptodactylidae) with a mortality associated with population declines in the rain forests description of a new species. Journal of Herpetology 276–285. of Australia and Central America. Proceedings of the National Giaretta, A.A., Aguiar Jr, O. (1998): A new species of Megaelosia Academy of Sciences 95(15): 9031–9036. from the Mantiqueira Range, Southeastern Brazil. Journal of Borison, H.L., and Wang, S.C. (1953): Physiology and Herpetology 80–83. pharmacology of vomiting. Pharmacological Reviews 5(2): Grant, T., Frost, D.R., Caldwell, J.P., Gagliardo, R.O.N., 193–230. Haddad, C.F., Kok, P.J., Wheeler, W.C. (2006): Phylogenetic Bosch, J., Martı́nez-Solano, I., Garcı́a-Parı́s, M. (2001): Evidence systematics of dart-poison frogs and their relatives (Amphibia: of a chytrid fungus infection involved in the decline of the Athesphatanura: Dendrobatidae). Bulletin of the American common midwife toad (Alytes obstetricans) in protected areas Museum of Natural History 1–262. of central Spain. Biological Conservation 97(3): 331–337. Gosner, K.L. (1960): A simplified table for staging anuran embryos Carvalho-e-Silva, S.P., Carvalho-e-Silva, A.M.P.T. (1994): and larvae with notes on identification. Herpetologica 16(3): Descrição das larvas de Ololygon albicans e de Ololygon 183–190. trapicheiroi com considerações sobre sua biologia (Amphibia, Haas, A. (2003): Phylogeny of frogs as inferred from primarily Anura, Hylidae). Revista Brasileira de Biologia 54(1): 55–62. larval characters (Amphibia: Anura). Cladistics 19(1): 23–89. Carvalho, T., Becker, C.G., Toledo, L.F. (2017): Historical Haddad, C.F., Prado, C.P. (2005). Reproductive modes in amphibian declines extinctions in Brazil linked to frogs and their unexpected diversity in the Atlantic Forest of chytridiomycosis. Proceedings of the Royal Society B 284: Brazil. BioScience, 55(3): 207–217. 20162254. Hall, J.A. Larsen, J.H., Fitzner, R.E. (1997). Postembryonic Catenazzi, A., Von May, R., Vredenburg, V. T. (2013): High ontogeny of the spadefoot toad, Scaphiopus intermontatus prevalence of infection in tadpoles increases vulnerability (Anura, Pelobatidae): external morphology. Herpetology to fungal pathogen in high-Andean amphibians. Biological Monographs. 11: 124–178. Conservation 159: 413-421. Costa, P.N., Carvalho-e-Silva, A,M.P.T., Flaksman. (2010). Eggs Hayes, T.B., Wu, T.H., Gill, T.N. (1997): DDT-like effects as a cluth and larval development of Phasmahyla cruzi Carvalho- result of corticosterone treatment in an anuran amphibian: e-Silva, Silva and Carvalho-e-Silva, 2019 (Amphibia, Anura, Is DDT a corticoid mimic or a stressor?. Environmental Hylidae). Herpetology Notes 3: 221–228. Toxicology and Chemistry 16(9): 1948–1953. Costa, P.N., Lourenço, A.C.C., Almeida-Santos, P., Sluys M.V. Heyer, W.R., Rand, A.S., Da Cruz, C.A.G., Peixoto, O.L., Nelson, (2010): Tadpole of the hylid frog Bokermannohyla gouveai C.E. (1990): frogs Kriger, K.M.., Hero, J. M. (2007): The (Peixoto and Cruz, 1992). Zootaxa 2418.1: 61–64. chytrid fungus Batrachochytrium dendrobatidis is non-randomly Cruz, C., Ribeiro, U. (2003): Metodologia científica: teoria e distributed across amphibian breeding habitats. Diversity and prática. Rio de Janeiro, Brasil, Axcel Books do Brasil. Distributions 13(6): 781–788. Daszak, P., Berger, L., Cunningham, A.A., Hyatt, A.D., Green, Izecksohn, E., Gouvêa, E. (1985): Nova espécie de Megaelosia D.E., Speare, R. (1999): Emerging infectious diseases and de Itatiaia, Estado do Rio de Janeiro (Amphibia, Anura, amphibian population declines. Emerging Infectious Diseases Leptodactylidae). Arquivos da Universidade Federal Rural do 5(6): 735. Rio de Janeiro 8: 17–22. Dias, P.H., Carvalho-e-Silva, A.M.P.T. (2012): Records of Jenkinson, T.S., Román, B., Lambertini, C., Valencia-Aguilar, A., abnormalities in Proceratophrys appendiculata (Günther, Rodriguez, D., Nunes-de-Almeida, C.H.L., Ruggeri, J., Belasen, 1873) (Anura; Cycloramphidae; Alsodinae) Herpetology Notes A.A., da Silva Leite, D., Zamudio, K.R., Longcore, J.E., 5:197–199. Toledo, L.F., James, T.Y. (2016): Amphibian-killing chytrid in Fabrezi, M. (2011): Heterochrony in growth and development Brazil comprises both locally endemic and globally expanding in anurans from the Chaco of South America. Evolutionary populations. Molecular Ecology 25(13): 2978–2996. 638 Dener das Neves da Silva et al.

Lambertini, C., Rodriguez, D., Brito, F.B., Leite, D.S., Toledo, chytridiomycosis. Herpetological Review 33(4): 263–264. L.F. (2013): Diagnóstico do fungo quitrídio: Batrachochytrium Rachowicz, L.J., Vredenburg, V.T. (2004): Transmission of dendrobatidis. Herpetologia Brasileira 2: 12–17. Batrachochytrium dendrobatidis within and between amphibian Lingnau, R., Canedo, C., Pombal Jr, J.P. (2008): A new species life stages. Diseases of aquatic organisms 61(1-2): 75–83. of Hylodes (Anura: Hylodidae) from the Brazilian Atlantic Rowe, C.L., Kinney, O.M., Congdon, J.D. (1998): Oral deformities forest. Copeia 2008(3): 595–602. in tadpoles of the bullfrog (Rana catesbeiana) caused by Lips, K.R. (1999): Mass mortality and population declines of conditions in a polluted habitat. Copeia 1998(1): 244–246. anurans at an upland site in western Panama. Conservation Ruggeri, J., Toledo, L.F., Carvalho-e-Silva, S.P. (2017): Stream Biology 13(1): 117–125. tadpoles present high prevalence but low infection loads Lips, K.R., Brem, F., Brenes, R., Reeve, J.D., Alford, R.A., of Batrachochytrium dendrobatidis (Chytridiomycota). Voyles, J., ... Collins, J.P. (2006): Emerging infectious disease Hydrobiologia 1–9. and the loss of biodiversity in a Neotropical amphibian Saxena, P.N., Chawla, N., Johri, M.B.L., Iqbal, S. (1977): Nature community. Proceedings of the National Academy of Sciences of receptors involved in apomorphine responses in pigeons. of the United States of America 103(9): 3165–3170. Psychopharmacol 53: 89–95 Longcore, J.E., Pessier, A.P., Nichols, D.K. (1999): Sheil, C.A., Alamillo, H. (2005): Osteology and skeletal Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid development of Phyllomedusa vaillanti (Anura: Hylidae: pathogenic to amphibians. Mycologia 219-227. Phyllomedusinae) and a comparison of this arboreal species with Lutz, A. (1930). Observações sobre os Batrachios brasileiros: a terrestrial member of the genus. Journal of Morphology 265(3): Taxonomia e biologia das Elosiinas. Memórias do Instituto 343–368. Oswaldo Cruz 24: 195–249. Silva, H.R.D., Benmaman, P. (2008): A new species of Hylodes Matthews, L.H. (1949): The origin of stomach oil in the petrels, Fitzinger from Serra da Mantiqueira, State of Minas Gerais, with comparative observations on the avian proventriculus. The Brazil (Anura: Hylodidae). Revista Brasileira de Zoologia Ibis 91: 373–392. 25(1): 89–99. Melo, A.S., Recco-Pimentel, S.M., Giaretta, A.A. (1995). The Smith, F.B. (1975): Naturalist’s Color Guide. New York, USA, karyotype of the stream dwelling frog Megaelosia massarti American Museum of Natural History. (Anura, Leptodactylidae, Hylodinae). Cytologia 60: 49–52. Sonnini, C.S., Latreille, P.A. (1801): “An. X”. Histoire Naturelle Miranda-Ribeiro, A.D. (1923): Elosia, Tsch. e os gêneros des Reptiles, avec Figures dissinées d’après Nature. Second correlatos. Revista do Museu Paulista 13: 813–821. Edition. Deterville, Paris. Mongin, M.M., Carvalho-e-Silva, A.M.P.T. (2013): Descrição da Stuart, S.N., Chanson, J.S., Cox, N.A., Young, B.E., Rodrigues, morfologia oral interna, ontogenia e redescrição do girino de A.S., Fischman, D.L., Tiersch, T.R., Griffith, J.S. (1988): Bokermannohyla circumdata (Cope, 1870) (Amphibia: Anura: Apomorphine-induced vomiting in rainbow trout (Salmo Hylidae). Boletim do Museu Paraense Emílio Goeldi. Ciências gairdneri). Comparative Biochemistery and Physiology 91(A): Naturais 8: 133–152. 721–725. Muths, E., Corn, P.S., Pessier, A.P., Green, D.E. (2003): Evidence Vera Candioti, M.F., Nuñez, J.J., Úbeda, C. (2011): Development for disease-related amphibian decline in Colorado. Biological of the nidicolous tadpoles of Eupsophus emiliopugini (Anura: Conservation 110(3): 357–365. Cycloramphidae) until metamorphosis, with comments on Naitoh, T., Wassersug, R.J., Leslie, R.A. (1989): The physiology, systematic relationships of the species and its endotrophic morphology, and ontogeny of emetic behavior in anuran developmental mode. Acta Zoologica 92(1): 27–45. amphibians. Physiological Zoology 62(3): 819–843. Vieira, C.A., Toledo, L.F., Longcore, J.E., Longcore, J.R. (2013): Naitoh, T., Wassersug, R.J. (1994): Emesis in larval salamanders, Body length of Hylodes cf. ornatus and Lithobates catesbeianus Hynobius nebulosus (Hynobidae). Journal of Herpetology 28: tadpoles, depigmentation of mouthparts, and presence of 245–247. Batrachochytrium dendrobatidis are related. Brazilian Journal Nuin, P.A.S. (2003): Description of the Tadpole of Megaelosia of Biology 73(1): 195–199. goeldii (Leptodactylidae, Hylodinae) with Natural History Waller, R.W. (2004): Status and trends of amphibian declines and Notes. Herpetological Review 34(1): 27–27. extinctions worldwide. Science 306(5702): 1783–1786. Pombal Jr, J.P., Prado, G.M., Canedo, C. (2003): A new species of giant torrent frog, genus Megaelosia, from the Atlantic Rain Forest of Espirito Santo, Brazil (Amphibia: Leptodactylidae). Journal of Herpetology 37(3): 453–466. Pyron, R.A., Wiens, J.J. (2011): A large-scale phylogeny of Amphibia including over 2800 species, and a revised classification of extant frogs, salamanders, and caecilians. Molecular Phylogenetics and Evolution 61(2): 543–583. R Development Core Team (2016): R: A Language and Environment for Statistical Computing, version 3.3.2. R Foundation for Statistical Computing, Vienna, AUS. Rachowicz, L.J. (2002): Mouthpart pigmentation in Rana muscosa tadpoles: seasonal changes without Ontogeny and behavioural aspects of the tadpoles of Megaelosia goeldii 639

Appendix Megaelosia goeldii- Tadpoles (n = 208): BRAZIL: Rio de Janeiro: Teresópolis: Sítio Vale da Revolta, UNIRIO: 190, 590, 599, 600, 783, 837, 886, 929, 985, 987, 1068, 1112, 1217, 1155; Sítio Vale da Revolta, ZUFRJ 570, 1320, 3922, 4305, 5775, 6328, 6357; Rio de Janeiro: Teresópolis: Parque Nacional da Serra dos Órgãos, UNIRIO 598, 784, 785, 1124, 1320, 1450, 1538, 1579, 1745, 1757, 1828, 1829, 1960, 2153, 2154, 2163, 2222, 2223, 2348, 2536, 2654, 2655, 4391, 4474, 4626, 5182, 5183, 5184, 5265, 5269; ZUFRJ 6217, 6086, 5618, 6268, 8134, 9809. Froglet: 5384. Adults: UNIRIO 2224.

Accepted by Werner Conradie