Ontogeny and Reproductive Aspects of Chiasmocleis Lacrimae

Herpetology Notes, volume 12: 1037-1050 (2019) (published online on 29 October 2019)

Ontogeny and reproductive aspects of Chiasmocleis lacrimae (Anura, Microhylidae, Gastrophryninae) in an unspoiled Atlantic Forest area in the state of Rio de Janeiro, Brazil

Laura Acerb Cordioli1, Vitor Sampaio Matias1, and Ana Maria Paulino Telles de Carvalho-e-Silva1,*

Abstract. Herein we provide data on the reproductive biology of a population of Chiasmocleis lacrimae, including a description of its spawning and the tadpole morphology and ontogeny, from Rio das Pedras Reserve, municipality of Mangaratiba, state of Rio de Janeiro, Brazil. Males called around a temporary pond, 60 meters above sea level. The amplexus was axillary. Males and females differed in size, coloration, and in the presence and distribution of dermal spines. The egg clutch formed round-shaped sets that floated on the water surface, with jelly involving approximately 245 eggs. The development from egg to stage 46 took about 30 days. At stage 25, the tadpoles had on average 10.2 mm in total length and continued growing until stage 38, reaching the maximum size of 20.3 mm. At stage 29, the shape of the third and fifth toe could be observed, and at stage 33 the shape of the second and fourth toes became visible. Only at stage 41 nostril openings could be seen. At stage 42 forelimbs came out simultaneously. The froglets reached 6.9 mm on average, about 1/3 of the adult size (17.7 to 22.8 mm).

Keywords. Tadpole, development, oviposition site, morphometric characteristics, nest, froglet

Introduction is classified by Haddad and Prado (2005) as type 1, with eggs and exotrophic tadpoles in lentic water. The wide Species of the family Microhylidae exhibit various array of developmental strategies, egg-laying patterns levels of fossoriality, with some species living on the and morphological variation is distinct in this family leaf litter and others being actively fossorial (Menin (Vera-Candioti, 2006; Haddad et al., 2008; Forlani, et al. 2007). Their reclusive habits make their capture 2010). difficult, limiting our knowledge about this group The genus Chiasmocleis Méhely, 1904 is currently (Caramaschi and Jim, 1983). Morphological and composed of 36 species, distributed from Panama molecular data support the monophyly of this family and Tropical South America, north and east of the (Ford and Cannatella, 1993; Haas, 2003; Frost et Andes. Twenty-seven of these species are recognised al., 2006; Peloso et al., 2014), although poor data on as inhabitants of Brazil and 18 in the Atlantic Forest, microhylid development hampers further interpretations from the states of Alagoas to Santa Catarina (Frost, about their evolutionary history (Fabrezi et al., 2012). 2019). Chiasmocleis lacrimae Peloso, Sturaro, Forlani, Ontogenetic studies in anurans may then help clarify Gaucher, Motta, and Wheeler, 2014 is the valid name the phylogenetic relationships and taxonomic issues for Chiasmocleis carvalhoi Cruz, Caramaschi and of these animal groups (Costa and Carvalho-e-Silva, Izecksohn, 1997, described from the Horto Florestal de 2008). Many species of this family have a generalised Santa Cruz (currently named Floresta Nacional Mário reproductive mode (Duellman and Trueb, 1986), which Xavier), municipality of Seropédica (22.733651°S, 43.709219°W; 40 m a.s.l.), state of Rio de Janeiro. It is distributed in the lowland forests of the Brazilian states of Bahia, Espírito Santo, Rio de Janeiro and São Paulo (Frost, 2019). In spite of this wide distribution, this species is currently listed as “endangered” in the IUCN 1 Laboratório de Biossistemática de Anfíbios, Departamento de Zoologia, Universidade Federal do Estado do Rio de Janeiro Red List (Pimenta and Peixoto, 2004). – UNIRIO, 22290-24, Rio de Janeiro, RJ, Brasil. The reproductive modes of many species of * Corresponding author. E-mail: [email protected] Chiasmocleis are poorly known (Haddad and Hödl, 1038 Laura Acerb Cordioli et al.

1997). Carvalho-e-Silva et al. (2008) reported in a period of heavy rain that caused the formation of reproduction of C. carvalhoi (= C. lacrimae) from the the temporary pond on the second day (Fig. 1A). In municipality of Mangaratiba, state of Rio de Janeiro, the following years, tadpoles were observed during the Brazil. Wogel et al. (2004) described the external reproductive period. morphology of the tadpole of C. lacrimae from the The pond was made up of three parts separated by municipality of Rio das Ostras, state of Rio de Janeiro, large stones, however interconnected, with variations Brazil, without any comparisons with congeneric in depth from 15 to 78 cm. One of the parts had little species. In this study, we describe the egg-clutch, water exposure, being almost entirely covered by rocks tadpole and the ontogeny of C. lacrimae and provide that formed hiding places from which many individuals data on its reproductive biology. of Chiasmocleis lacrimae vocalised. In periods when the temporary pond remained full, i.e., September Materials and Methods 2006, the relative air humidity was on average 80%, air Our study took place at the Rio das Pedras Reserve temperature 25°C, and water temperature 21°C. (22.991389°S, 44.100278°W), municipality of In the laboratory, tadpoles were euthanised with an Mangaratiba, state of Rio de Janeiro, Brazil, from overdose of chloretone 10% and fixed and preserved September 2006 to March 2009. This area is part of the in formaldehyde 5%, and their stages were identified Parque Estadual Cunhambebe and therefore is currently according to Gosner (1960). Adults and froglets were protected by law. Observations were conducted on the euthanised in chloretone 25%, fixed in formaldehyde right bank of the river named Rio Grande, between two 10% and preserved in ethanol 70%. The description of bamboo clumps, 60 m a.s.l. colour on life of adults, froglets and tadpoles followed Reproduction was accompanied in greater detail by six the Naturalist’s Color Guide (Smithe, 1975). people in September 2006, during a four-day observation Tadpoles were measured with a vernier calliper to

Figure 1. A) Temporary pond in the Rio das Pedras Reserve, Rio de Janeiro, Brazil. B) A couple of Chiasmocleis lacrimae in axillary amplexus (UNIRIO 2547, female, SVL= 22.67 mm; UNIRIO 2546, male, SVL= 17.44 mm). C) Egg clutch of C. lacrimae in the water. D) Tadpoles of C. lacrimae swimming in the aquarium. Ontogeny and reproductive aspects of Chiasmocleis lacrimae 1039 the nearest 0.01 mm. The development of tadpoles since we did not find adults near the nests the day after was analysed from stages 25 to 46, by determination spawning. of the following morphometric characters (Altig and In adults, the dorsal body in life is dark brown (Color McDiarmid, 1999): internostril distance (IND), distance 121, Vandyke Brown), with a pale longitudinal dorsal between eye and nostril (END), interocular distance midline (Color 92, Horn) or without this line but with (IOD), distance between eye and snout (ESD), eye spots of the same colour on the dorsal line (Color 92, diameter (ED), tail height (TH), body width (BW), Horn) and dark eyes (Color 119, Sepia). The ventral tail length (TAL), body length (BL) and total length body is lighter (Color 124, Buff) with few spots. The (TL). For adults and froglets we used the following lateral body is light brown (Color 23, Raw Umber) with measurements (Duellman, 1970): internostril distance several light spots (Color 92, Horn). (IND), distance between eye and snout (ES), diameter Adults show a variation in colour pattern: some of the eye (DE), interocular distance (IOD), distance individuals of the same population have a dorsal midline, between eye and nostril (EN), head width (HW), head a midline on the dorsal thigh and two lines in the ventral length (HL), humerus length (HUL), forearm length region forming a “V” shape, with a median line which (FAL), hand length (HAL), thigh length (TL), tibia runs through this “V” below the gular region, lighter length (TIL), tarsus length (TAL), foot length (FTL) and than the colour of the dorsum. In other individuals, snout-vent length (SVL). The material was deposited in median dorsal lines and ventral lines are absent, but the collection of the Laboratório de Biossistemática de there are small irregular light spots distributed all over Anfíbios of the Universidade Federal do Estado do Rio the body. de Janeiro – UNIRIO, Rio de Janeiro, Brazil, under the Females are larger than males in snout-vent length, vouchers: 684, 714, 735, 1346, 2601, 2644, 2704, 2843, with mean of 22.78 mm (SD = 0.44, range = 22.15– 2844, 3391, 3392 (tadpoles); 2659 (froglets); 1030, 23.20 mm, n = 5) whereas males measure in mean 1035, 2007, 2557, 2746 (adult males); 1034, 1037, 17.71 mm (SD = 0.75, range = 16.85–18.85 mm, n = 1476, 1655, 2547 (adult females). 5). All other measurements of females were greater than those of males (Table 1). As evidence of sexual Results dimorphism, the gular region of females had irregular Adults were found only during breeding activity, lighter spots (Color 26, Clay), while in males the gular under heavy rain and after it, from dusk to dawn region is darker (Color 121, Vandyke Brown), similar to accompanied by intense calling activity. At night, pairs the colour of the dorsum, without irregular lighter spots were observed in axillary amplexus (Fig. 1B), and the (Color 92, Horn; Fig. 2). Males have dermal spines reproductive success was determined in the following distributed throughout their body dorsally, on the head morning through observation of egg clutches (Fig. 1C). and thighs, on the fingers and toes (Fig. 3), and ventrally Individuals remained active only during the reproductive only on the abdominal region. Females have fewer and season, in a short period, only less than 12 hours. tiny dermal spines scattered in the inguinal region, The pond where we found the tadpoles was formed inner thighs and cloacal region. These tiny spines were under a shady bamboo grove, and the bottom of observed only with the aid of a stereoscopic microscope the pond consisted of sand and leaf litter. The water with a 25x magnification. was still, yellowish, and acid (pH of 4.5). Tadpoles The tadpoles at stage 36 have a round body in dorsal remained swimming in the water column most of the and ventral view, and oval in lateral view, with the time (Fig. 1D), but did not form schools. Tadpoles of greatest width of the body just behind the eyes, and Ololygon angrensis (Lutz, 1973) were found in the the smaller width nearest the tail; the body corresponds same temporary pond. A single egg clutch was observed to 37.85% (SD = 1.4) of its total length. The eyes are after an amplectant pair spawned in the laboratory. The projected outward, directed laterally with the inter- spawning occurred in the water, and eggs were stuck to ocular distance slightly greater than the width of the one another by thick and viscous jelly, floating on the body. The distance between the eye and the snout is water surface as a small rounded cluster, approximately equal to 53% of the body length (as tadpoles develops, 9 cm long and 8 cm wide. We counted 245 eggs, which that proportion decreases); snout truncate in dorsal and were grey and spherical. It was possible to observe ventral view. Oral disc terminal, semicircular with flaps the formation of embryos a few hours after spawning. suspended in front of the mouth and separated by an Apparently, the species does not display parental care inverted U-shaped medial notch; teeth rows, jaw sheaths 1040 Laura Acerb Cordioli et al.

Figure 2. Colour pattern of adults of Chiasmocleis lacrimae. Dorsal views of (A) Female UNIRIO 2547 (SVL = 22.67 mm) and (B) male UNIRIO 2546 (SVL= 17.44 mm). Ventral views of the same (C) female and (D) male.

Figure 3. Dermal spines on the third finger (2.24 mm) of the hand (A) and on the third toe (4.11 mm) of the feet (B) of male of Chiasmocleis lacrimae (details by a stereoscopic microscope with a 25x magnification). Ontogeny and reproductive aspects of Chiasmocleis lacrimae 1041 and papillae absent. The spiracle is single, ventral and days (96 hours); after about 22 days we found tadpoles slightly oriented to the right, with a wide aperture and at stage 41. For stages 41, 42 and 43, changes occurred beyond the anal tube. The anal tube is ventral, medial within 24 hours, and from stages 43 to 46 changes and adhered to the ventral fin. The dorsal fin originates occurred every four to six hours. In the laboratory, on the body. The tail has greater height in the middle of the development from egg to newly metamorphosed its length, decreasing until the end, where it becomes individuals lasted 30 days. flagellate; tail length equivalent to 58.46% (SD = 5.43) Tadpoles at stage 25 are on average 10.20 mm in total of total length. Tadpoles are filter feeders and the opening length and grow until stage 38, when they reach their of the nostrils is absent until stage 41, after which the greatest length of 20.33 mm. The body growth is small nostrils are visible dorsally in front of the eyes and very between stages 25-26, and accentuated between 26-27, close to the snout; the openings are arranged very close almost doubling in total length, and then again small to one another, measuring 13% of the body width. from 27 to 38 stages. At stage 35, tail length reaches its In tadpoles, the dorsal of the body in life has small largest size (14.72 mm; Table 2), after which resorption dark brown dots (Color 121C, Mikado Brown), with a begins and it decreases in length until 6.93 mm at stage dark brown midline (Color 121, Prout’s Brown) from 46 (SVL, Fig. 4A). At stage 42, the tail is still quite the mouth to the end of the body where it expands to evident, as is the caudal membrane and the flagellum the side of the body. The tail muscle is dark brown of (Figs. 5 and 7). the same colour in the midline of the body (Color 121, At stage 25, the tadpole has an interocular distance of Prout’s Brown); the fins are translucent with small 3.48 mm, which increases until stage 38, when it reaches brown dots, but no colour on the edge ventral fin. The 5.92 mm. After this stage, the interocular distance belly is translucent cream colour (Color 92, Horn), with decreases until stage 46, where it reaches 2.60 mm easy visualisation of the intestines. (Tables 2 and 3). The distance between the eye and the After the formation of the embryos, development snout appears larger in stage 35 (4.22 mm), decreasing occurs quickly. Until stage 25, when they hatch, it is to its smallest size of 1.57 mm at stage 46 (Fig. 4B; possible to follow changes in intervals of two to four Tables 2 and 3). With these changes, the characteristics hours between each stage, reaching stage 26 after four of the adult rostrum gradually appear; the eyes become

Table 1. Morphometric measurements (mm) of Chiasmocleis lacrimae (full names of the variables in the text). The values are average (x̅), standardTable 1. deviation Morphometric (SD), measurementsminimum (min) (mm) and of maximum Chiasmocleis (max). lacrimae (full names of the variables in the text). The values are average (x̅ ), standard deviation (SD), minimum (min) and maximum (max).

Froglets (n=5) Females (n=5) Males (n=5) Characters x̅ SD min max x̅ SD min max x̅ SD min max IND 0.65 0.08 0.55 0.80 1.47 0.07 1.40 1.50 1.18 0.16 1.05 1.45 ES 1.57 0.15 1.30 1.75 2.64 0.23 2.40 3.00 2.34 0.09 2.20 2.40 DE 0.69 0.03 0.65 0.75 1.58 0.16 1.40 1.80 1.48 0.08 1.40 1.60 IOD 2.60 0.05 2.55 2.60 4.91 0.11 4.80 5.10 4.41 0.19 4.25 4.70 EN 1.10 0.08 1.00 1.20 3.56 0.24 3.30 3.90 3.00 0.17 2.75 3.15 HW 3.10 0.12 2.80 3.10 8.11 0.33 7.90 8.70 6.23 0.54 5.60 7.10 HL 3.27 0.09 3.10 3.40 8.19 0.07 8.10 8.30 6.73 0.43 6.15 7.10 HUL 1.20 0.20 1.00 1.20 4.22 0.33 3.70 4.55 3.62 0.39 3.25 4.15 HAL 1.55 0.11 1.40 1.70 4.89 0.26 4.50 5.20 3.95 0.44 3.30 4.40 FAL 1.30 0.17 1.10 1.60 4.44 0.27 4.10 4.70 3.57 0.24 3.25 3.90 TL 2.67 0.28 2.35 2.70 8.43 0.48 8.00 9.20 6.78 0.45 6.15 7.30 TIL 2.57 0.23 2.25 2.80 8.27 0.24 7.85 8.45 6.92 0.35 6.30 7.15 TAL 1.60 0.28 1.25 1.80 5.31 0.32 4.80 5.70 4.52 0.26 4.30 4.95 FTL 2.57 0.13 2.40 2.60 9.23 0.27 8.90 9.65 7.65 0.42 7.20 8.00 SVL 6.93 0.25 6.50 7.20 22.78 0.44 22.15 23.20 17.72 0.75 16.85 18.85

Table 2. Morphometric measurements (mm) of 59 Chiasmocleis lacrimae tadpoles in Gosner’s (1960) stages 25 to 36. Values are the mean ± standard deviation, minimum and maximum (in parentheses). TL (total length), TAL (tail length), BL (body length), TH (tail height), ED (eye diameter).

Stage N TL TAL BL TH ED 10.20±0.60 7.82±0.83 3.89±0.55 2.17±0.36 0.43±0.06 25 5 (9.50–11.00) (6.90–8.80) (3.10–4.55) (1.80–2.75) (0.35–0.50) 9.56±1.58 6.62±0.81 3.60±0.54 2.22±0.53 0.59±0.20 26 5 (7.00–10.85) (5.30–7.50) (2.65–4.00) (1.60–2.90) (0.35–0.90) 16.77±1.2 12.14±1.13 6.17±0.70 3.46±0.50 0.96±0.17 27 5 (15.00–17.90) (10.40–13.30) (5.10–6.80) (2.60–3.80) (0.70–1.15) 17.35±0.58 11.77±0.91 6.49±0.17 3.64±0.47 1.01±0.07 28 5 (16.70–18.15) (10.50–12.70) (6.20–6.65) (3.00–4.20) (0.90–1.10) 17.32±0.59 12.44±0.61 6.57±0.41 4.20±0.45 0.92±0.08 29 5 (16.05–17.90) (11.70–13.00) (6.05–6.90) (3.70–4.65) (0.85–1.05) 18.02±0.43 13.06±0.48 6.76±0.11 4.07±0.39 0.87±0.10 30 5 (17.50–18.60) (12.40–13.70) (6.60–6.85) (3.50–4.50) (0.80–1.00) 17.62±1.05 12.59±1.13 6.99±0.48 4.10±0.57 0.94±0.16 31 5 (16.10–18.80) (11.25–14.3) (6.35–7.60) (3.20–4.80) (0.80–1.20) 17.65±1.73 12.45±1.85 7.21±0.43 4.02±0.56 0.82±0.10 32 5 (15.05–19.90) (9.75–14.10) (6.75–7.90) (3.35–4.60) (0.65–0.90) 18.40±0.90 13.37±0.83 7.31±0.30 4.27±0.44 0.92±0.12 33 5 (17.15–19.55) (12.1–14.10) (7.00–7.70) (3.90–4.80) (0.80–1.10) 18.34±1.71 13.20±1.79 6.72±0.74 3.81±0.33 1.03±0.08 34 5 (16.40–20.30) (11.30–16.10) (6.15–7.95) (3.45–4.32) (0.90–1.10) 19.45±1.60 14.72±2.19 7.14±0.88 4.14±1.18 0.98±0.08 35 5 (17.15–21.50) (12.00–17.80) (5.75–7.90) (2.50–5.20) (0.90–1.10) 18.82±1.52 13.42±1.98 6.86±0.76 3.86±0.35 1.01±0.08 36 4 (16.75–20.30) (11.30–16.10) (6.15–7.95) (3.45–4.32) (0.90–1.10)

1042 Laura Acerb Cordioli et al.

Figure 4. Average values of morphometric measurements of tadpoles of Chiasmocleis lacrimae from stages 25 to 45. (A) Measurements are total length (TL), body length (BL) and tail length (TAL); (B) Measurements are eye diameter (ED), distance between eye and snout (ESD), distance between eye and nostril (END) and internostril distance (IND).

Figure 5. Tadpoles of Chiasmocleis lacrimae in lateral view. Nostrils became visible from stage 41. From stages 42 to 45 forelimbs and hind limbs developed with tail reduction. Scale bar = 1 mm. Ontogeny and reproductive aspects of Chiasmocleis lacrimae 1043 closer together and the nostrils come closer to the snout as they develop (Figs. 4B and 5). At stage 41, the opening of the nostrils becomes evident (Figs. 5Table and 1.7) Morphometric and the distance measurements between (mm) them of isChiasmocleis lacrimae (full names of the variables in the text). The values are average (x̅ ), standard deviation (SD), minimum (min) and maximum (max). 0.5 mm, increasing gradually until reaching 0.65 mm at stage 46. The greater distance betweenFroglets (n=5) the eye and the Females (n=5) Males (n=5) nostril occurs inCharacters stage 41 (2.42 x̅ mm) SD and min decreases max until x̅ SD min max x̅ SD min max stage 46 (1.1 mm),IND i.e., the 0.65 eyes 0.08 become 0.55 closer 0.80 to 1.47 the 0.07 1.40 1.50 1.18 0.16 1.05 1.45 nostril during developmentES 1.57 (Fig. 0.154B; Table 1.30 3). 1.75 2.64 0.23 2.40 3.00 2.34 0.09 2.20 2.40 At stage 26, hind limbs begin to appear. Both forelimbs DE 0.69 0.03 0.65 0.75 1.58 0.16 1.40 1.80 1.48 0.08 1.40 1.60 become apparent at stage 42. At this same stage, the IOD 2.60 0.05 2.55 2.60 4.91 0.11 4.80 5.10 4.41 0.19 4.25 4.70 ventral spiracle disappears and the anal tube begins to EN 1.10 0.08 1.00 1.20 3.56 0.24 3.30 3.90 3.00 0.17 2.75 3.15 suffer changes, forming the opening of the cloaca (Fig. HW 3.10 0.12 2.80 3.10 8.11 0.33 7.90 8.70 6.23 0.54 5.60 7.10 5 and 7). At stage 29, HLthe shape of 3.27the third 0.09 and 3.10 fifth 3.40 toe can 8.19 be 0.07 8.10 8.30 6.73 0.43 6.15 7.10 observed, and HULat stage 33 1.20it is already 0.20 1.00 possible 1.20 to 4.22 see 0.33 3.70 4.55 3.62 0.39 3.25 4.15 the shape of theHAL second and 1.55 fourth 0.11 toe 1.40 also 1.70(Fig. 5 4.89 and 0.26 4.50 5.20 3.95 0.44 3.30 4.40 6). The mouthFAL begins to change 1.30 0.17 at stage 1.10 42, 1.60 but 4.44does 0.27 4.10 4.70 3.57 0.24 3.25 3.90 not project furtherTL than the 2.67 nostril; 0.28 only2.35 at 2.70 stage 8.43 43 0.48Figure 8.00 6. Development 9.20 6.78 of 0.45 the hind 6.15 limbs 7.30 of tadpoles of the changes inTIL the mouth 2.57become 0.23 evident 2.25 and 2.80 project 8.27 0.24Chiasmocleis 7.85 8.45 lacrimae 6.92 from 0.35 stages 6.30 29 to 7.1533. Scale bar = 1 beyond the lineTAL of the nostril. 1.60 At 0.28 stage 1.25 44, 1.80the mouth 5.31 0.32mm. 4.80 5.70 4.52 0.26 4.30 4.95 FTL 2.57 0.13 2.40 2.60 9.23 0.27 8.90 9.65 7.65 0.42 7.20 8.00 SVL 6.93 0.25 6.50 7.20 22.78 0.44 22.15 23.20 17.72 0.75 16.85 18.85

Table 2. Morphometric measurements (mm) of 59 Chiasmocleis lacrimae tadpoles in Gosner’s (1960) stages 25 to 36. Values are the mean ± standardTable deviation, 2. Morphometric minimum measurements and maximum (mm) (in of parentheses).59 Chiasmocleis TL lacrimae (total length), tadpoles TAL in Gosner’s (tail length), (1960) BL (body length), TH (tail height),stages ED (eye 25 todiameter). 36. Values are the mean ± standard deviation, minimum and maximum (in parentheses). TL (total length), TAL (tail length), BL (body length), TH (tail height), ED (eye diameter).

1044 Laura Acerb Cordioli et al.

Table 3. Morphometric measurements (mm) of 42 Chiasmocleis lacrimae tadpoles in Gosner’s (1960) stages 37 to 45. Values are the mean ± standardTable deviation, 3. Morphometric minimum measurements and maximum (mm) (in of parentheses). 42 Chiasmocleis TL lacrimae(total length), tadpoles TAL in (tailGosner’s length), (1960) BL (body length), TH (tail height), EDstages (eye 37 diameter). to 45. Values are the mean ± standard deviation, minimum and maximum (in parentheses). TL (total length), TAL (tail length), BL (body length), TH (tail height), ED (eye diameter).

Stage N TL TAL BL TH ED 18.49±1.00 13.39±1.36 6.96±0.35 3.77±0.39 0.99±0.09 37 5 (17.50–19.60) (11.20–14.90) (6.50–7.45) (3.20–4.10) (0.90–1.10) 20.33±0.64 14.42±0.70 7.21±0.19 4.16±0.27 0.98±0.08 38 5 (19.60–21.20) (13.50–15.30) (7.00–7.50) (3.80–4.50) (0.90–1.10) 18.83±1.46 13.26±1.14 7.02±0.28 3.92±0.40 1.01±0.10 39 5 (17.00–20.40) (11.50–14.60) (6.65–7.25) (3.30–4.30) (0.85–1.10) 19.12±1.10 13.44±0.82 7.21±0.15 4.00±0.27 0.93±0.04 40 5 (17.80–20.60) (12.50–14.60) (7.00–7.40) (3.60–4.30) (0.90–1.00) 18.75±1.20 13.51±1.32 6.84±0.26 3.31±0.41 0.95±0.05 41 5 (16.85–20.10) (11.20–14.35) (6.40–7.10) (2.80–3.90) (0.90–1.00) 15.33±2.55 8.71±3.59 7.30±0.37 2.60±1.04 0.92±0.06 42 5 (11.20–17.40) (4.50–12.3) (6.90–7.90) (1.30–3.90) (0.85–1.00) 11.90±1.31 6.50±0.97 6.84±0.52 159±0.16 0.95±0.23 43 5 (10.00–13.40) (4.90–7.20) (6.20–7.60) (1.35–1.80) (0.65–1.20) 8.77±0.40 3.50±0.44 6.33±0.32 1.40±0.26 0.87±0.06 44 3 (8.40–9.20) (3.00–3.80) (6.10–6.70) (1.20–1.70) (0.80–0.90) 7.10±0.69 1.51±0.37 6.46±0.11 1.08±0.21 0.63±0.10 45 4 (6.55–8.10) (1.00–1.80) (6.30–6.55) (0.90–1.30) (0.50–0.70)

Table 4.

Stage N ESD IOD BW 2.02±0.19 3.48±0.40 3.29±0.35 25 5 (1.70–2.20) (3.00–3.90) (2.90–3.75) 2.00±0.37 3.21±0.66 3.10±0.55 26 5 (1.40–2.30) (2.25–3.80) (2.35–3.65) 3.25±0.29 5.45±0.47 5.06±0.51 27 5 (3.00–3.75) (4.90–6.05) (4.60–5.90) 3.23±0.46 5.30±0.16 5.22±0.20 28 5 (2.50–3.75) (5.10–5.55) (5.00–5.50) 3.36±0.17 5.41±0.35 5.25±0.29 29 5 (3.10–3.50) (4.85–5.70) (4.80–5.50) 3.98±0.97 5.34±0.88 5.32±0.16 30 5 (3.40–5.70) (3.80–5.90) (5.10–5.50) 3.49±0.17 5.64±0.18 5.32±0.27 31 5 (3.30–3.70) (5.45–5.90) (5.10–5.70) 3.67±0.21 5.37±0.42 5.47±0.35 32 5 (3.35–3.90) (4.85–5.85) (5.10–6.00) 3.78±0.29 5.46±0.83 5.49±0.22 33 5 (3.35–4.10) (4.00–6.00) (5.20–5.70) 3.77±0.26 5.43±0.44 5.28±0.78 34 5 (3.40–4.10) (5.05–6.10) (4.30–6.10) 4.22±1.04 5.42±0.80 5.37±0.63 35 5 (3.30–6.00) (4.10–6.20) (6.00–4.40) 3.73±0.28 5.48±0.48 5.52±0.64 36 4 (3.40–4.10) (5.05–6.10) (4.75–6.10)

Figure 7. Tadpoles of Chiasmocleis lacrimae at stages 39 (nostril absent; TL = 17.00 mm), 41 (nostril present; TL = 16.85 mm) and 42 (nostril present; TL = 11.20 mm in dorsal view). Stages 43 (TL = 10.00 mm), 44 (TL = 8.40 mm) in lateral view. Froglet amidst leaves at stage 46 in dorsal view (SVL= 6.50 mm). Ontogeny and reproductive aspects of Chiasmocleis lacrimae 1045 does not exceed the height of the eye-line, and only in 28, Olive – Brown) with several light brown spots (Color stage 45 the mouth surpasses the eye-line, although it 24, Buff). The eyelids have a light brown spot (Color will only be fully formed at stage 46 (Fig. 5 and 7). 24, Buff) that joins the front of the face to the snout. All The froglets (stage 46) were not sexually dimorphic, limbs are lighter than their dorsal surface, brown (Color because there are no spines on the fingers or in the anal 25, Fawn) with light brown colour scores (Color 24, region. The gular region shows the same coloration of Buff). The region above the cloaca shows a dark colour Table 3. Morphometric measurements (mm) of 42 Chiasmocleis lacrimae tadpoles in Gosner’s (1960) adultstages males. 37 to 45. They Values have are twothe mean lines ± standardthat form deviation, a “V” minimumbelow and(Color maximum 119, (in Sepia) parentheses). forming TL a “V”. The ventral region of the(total gular length), region. TAL (tailAfter length), the absorption BL (body length), of the TH tail (tail above height), EDthe (eye body diameter). is cream colour (Color 54, Cream) with the side the anal region, the newly metamorphosed individuals of the belly slightly darker (Color 124, Buff). The gular Stage N TL TAL BL TH ED also present a dark brown patch in a “V” shape. The region shows a light colour (Color 124, Buff), which 18.49±1.00 13.39±1.36 6.96±0.35 3.77±0.39 0.99±0.09 dorsal37 and 5 ventral skins are smooth. (17.50–19.60) (11.20–14.90) (6.50–7.45) becomes(3.20–4.10) a little(0.90–1.10) darker near the mouth (Color 26, Clay). The newly metamorphosed individuals, like adults, Just below the gular region, there are two clear lines, 20.33±0.64 14.42±0.70 7.21±0.19 4.16±0.27 0.98±0.08 38 5 find shelter in (19.60–21.20)the leaf litter. They(13.50–15.30) are small, with(7.00–7.50) snout- which(3.80–4.50) form a “V”,(0.90–1.10) with brown pigmentation (Color 33, vent length of 6.9318.83±1.46 mm, and are13.26±1.14 brown, resembling7.02±0.28 the Cinnamon–Brown)3.92±0.40 1.01±0.10 that accompanies the lighter line. 39 5 color of the leaf(17.00–20.40) litter, thus hampering(11.50–14.60) their visualisation(6.65–7.25) (3.30–4.30) (0.85–1.10) 19.12±1.10 13.44±0.82 7.21±0.15 4.00±0.27 0.93±0.04 in 40their natural 5 environment (Fig. 7, Table 1). Discussion The dorsal surface(17.80–20.60) of the froglets(12.50–14.60) is dark brown(7.00–7.40) (Color (3.60–4.30) (0.90–1.00) 18.75±1.20 13.51±1.32 6.84±0.26 3.31±0.41Wells (1977) 0.95±0.05 considered two temporal patterns of 41 5 (16.85–20.10) (11.20–14.35) (6.40–7.10) reproductive(2.80–3.90) behaviour(0.90–1.00) in anurans: explosive and the 15.33±2.55 8.71±3.59 7.30±0.37 2.60±1.04 0.92±0.06 42 5 prolonged. Explosive reproduction extends for a few (11.20–17.40) (4.50–12.3) (6.90–7.90) (1.30–3.90) (0.85–1.00) Table 4. Morphometric measurements (mm) of 59 days, while prolonged for several weeks. Adults of 11.90±1.31 6.50±0.97 6.84±0.52 159±0.16 0.95±0.23 43 5 Chiasmocleis lacrimae(10.00–13.40) tadpoles in(4.90–7.20) Gosner’s (1960)(6.20–7.60) stages 25 Chiasmocleis(1.35–1.80) (0.65–1.20) lacrimae were only observed during to 36. Values are the mean ± standard deviation, minimum and the breeding season confirming their explosive 8.77±0.40 3.50±0.44 6.33±0.32 1.40±0.26 0.87±0.06 44 3 maximum (in parentheses).(8.40–9.20) ESD (3.00–3.80)(distance between (6.10–6.70) eye and breeding(1.20–1.70) strategy.(0.80–0.90) This behaviour is common within snout), IOD (interocular7.10±0.69 distance), 1.51±0.37 BW (body width).6.46±0.11 From Microhylidae,1.08±0.21 0.63±0.10 such as Chiasmocleis ventrimaculata 45 4 stages 25 to 36 the(6.55–8.10) nostrils are not(1.00–1.80) visible, so we suppressed(6.30–6.55) (Andersson,(0.90–1.30) 1945),(0.50–0.70) Ctenophryne geayi Mocquard, measurements END (distance between eye and nostril) and 1904, Elachistocleis bicolor (Guérin-Méneville, 1838) IND (internostril distance) in this table. Table 4. and E. erythrogaster Kwet and Di- Bernardo, 1998 (Gallardo, 1987; Rodrigues and Duellman, 1994; Kwet Stage N ESD IOD BW and Di-Bernardo, 1998; Rodrigues et al., 2008). 2.02±0.19 3.48±0.40 3.29±0.35 25 5 The coloration of the adult may vary with the presence (1.70–2.20) (3.00–3.90) (2.90–3.75) or absence of one longitudinal dorsal line, the presence 2.00±0.37 3.21±0.66 3.10±0.55 26 5 (1.40–2.30) (2.25–3.80) (2.35–3.65) of numerous spots distributed throughout the body and 3.25±0.29 5.45±0.47 5.06±0.51 a “V” shape on the ventral region. This characteristic 27 5 (3.00–3.75) (4.90–6.05) (4.60–5.90) has likely no taxonomic value but merely represents a 3.23±0.46 5.30±0.16 5.22±0.20 variation within populations (Peloso et al., 2014). 28 5 (2.50–3.75) (5.10–5.55) (5.00–5.50) All measurements were larger in adult females than 3.36±0.17 5.41±0.35 5.25±0.29 29 5 males, as well as C. carvalhoi (=lacrimae) by Cruz et (3.10–3.50) (4.85–5.70) (4.80–5.50) al. (1997) and similar results were found for E. bicolor, 3.98±0.97 5.34±0.88 5.32±0.16 30 5 (3.40–5.70) (3.80–5.90) (5.10–5.50) E. ovalis and E. erythrogaster (Kwet and Di-Bernardo, 3.49±0.17 5.64±0.18 5.32±0.27 1998; Rodrigues et al. 2008). However, the average 31 5 (3.30–3.70) (5.45–5.90) (5.10–5.70) body sizes of females and males found here (22.15 mm 3.67±0.21 5.37±0.42 5.47±0.35 for females and 17.71 mm for males), were larger than 32 5 (3.35–3.90) (4.85–5.85) (5.10–6.00) those described by Cruz et al. (1997) (20.20 mm for 3.78±0.29 5.46±0.83 5.49±0.22 33 5 females and 11.30 mm for males) in a population from (3.35–4.10) (4.00–6.00) (5.20–5.70) Seropédica city, Rio de Janeiro. 3.77±0.26 5.43±0.44 5.28±0.78 34 5 (3.40–4.10) (5.05–6.10) (4.30–6.10) Tiny dermal spines were observed as characteristics 4.22±1.04 5.42±0.80 5.37±0.63 of sexual dimorphism (e.g., Nelson, 1973; Zweifel and 35 5 (3.30–6.00) (4.10–6.20) (6.00–4.40) Myers, 1989; Cruz et al., 1997; Lehr and Trueb, 2007), 3.73±0.28 5.48±0.48 5.52±0.64 corroborating Peloso et al. (2014), who found the same 36 4 (3.40–4.10) (5.05–6.10) (4.75–6.10) pattern in 14 species of this genus. Besides these, tiny

1046 Laura Acerb Cordioli et al.

Table 5. Morphometric measurements (mm) of 42 Chiasmocleis lacrimae tadpoles in Gosner’s (1960) Table 5. Morphometricstages 37 measurements to 45. Values are(mm) the of mean 42 Chiasmocleis ± standard deviation, lacrimae minimum tadpoles and in Gosner’s maximum (1960) (in parentheses). stages 37 to ESD 45. Values are the mean ± standard(distance deviation, between minimum eye and snout),and maximum END (distance (in parentheses). between eye ESD and (distance nostril), IODbetween (interocular eye and distance), snout), END (distance between eye andIND nostril), (internostril IOD (interocular distance), BW distance), (body width). IND (internostrilFrom stages distance),37 to 40 the BW nostrils (body are width). not visible, From so stages the 37 to 40 the nostrils are not visible,measurements so the measurementsEND and IND onlyEND appear and IND from only stage appear 41. from stage 41.

Stage N ESD END IOD IND BW 3.69±0.31 5.75±0.19 5.47±0.62 37 5 — — (3.25–4.10) (5.55–6.00) (5.05–6.50) 3.63±0.08 5.92±0.28 5.36±023 38 5 — — (3.70–3.50) (5.7–6.4) (5.20–5.70) 3.51±0.14 5.70±0.26 5.31±0.15 39 5 — — (3.40–3.75) (5.35–6.00) (5.20–5.50) 3.57±0.16 5.87±0.41 5.26±0.29 40 5 — — (3.35–3.80) (5.50–6.55) (4.90–5.50) 3.33±0.12 2.42±0.22 5.15±0.15 0.50±0.16 4.85±0.26 41 5 (3.20–3.50) (2.60–2.10) (4.90–5.30) (0.30–0.70) (4.50–5.20) 3.14±1.01 2.00±0.37 4.34±0.60 0.55±0.04 7.30±0.37 42 5 (2.20–4.80) (1.50–2.50) (3.60–5.10) (0.50–0.60) (6.90–7.90) 2.32±0.13 1.53±0.13 3.73±0.32 0.54±0.04 3.48±0.22 43 5 (2.20–2.50) (1.30–1.60) (3.40–4.20) (0.50–0.60) (3.30–3.85) 1.93±0.06 1.50±0.10 3.37±0.06 0.52±0.08 6.33±0.32 44 3 (1.90–2.00) (1.40–1.60) (3.33–3.40) (0.45–0.60) (6.10–6.70) 1.55±0.21 1.09±0.06 3.05±0.60 0.53±0.13 2.85±0.62 45 4 (1.30–1.80) (1.00–1.15) (2.50–3.90) (0.40–0.70) (2.00–3.50)

dermal spines were also found scattered in the inguinal tadpole from the União Biological Reserve (22.431048°S, region and on the inner thighs of females. 44.033814°W), municipality of Rio das Ostras, Rio de The single egg cluth of C. lacrimae formed a floating Janeiro, with oval body in lateral view and rectangular cluster on the water surface with the eggs attached in dorsal and ventral view; this difference may represent to one another by a thick viscous jelly, similar to C. a population difference or an artifice of interpretation. shudikarensis (Lima et al., 2006), C. ventrimaculata Nascimento and Skuk (2006) reported that the tadpole (Schlüter and Salas, 1991) and C. antenori (Walker, of C. alagoana Cruz, Caramaschi, and Freire, 1999 does 1973) (Krugel and Richter, 1995). This contrasts with not present any diagnostic characters that could securely the egg clutch of C. leucosticta, which was described by distinguish of C. lacrimae; they described the tadpole Haddad and Hödl (1997) as another reproductive mode, of C. alagoana as having an oval body in dorsal and consisting of a bubble nest made up of air bubbles ventral view and triangular in lateral view. adhering to mucus between the eggs (Vilela et al., Tadpoles of C. lacrimae differs from other species 2011). In addition, clutches of C. hudsoni are deposited of Chiasmocleis by body shape, rounded in dorsal between roots in the soil, on fallen tree trunks near view (rectangular body in C. mantiqueira Cruz, Feio, ponds, starting the development of tadpoles out of water and Cassini, 2007, C. hudsoni and C. shudikarensis; (Rodrigues et al., 2008). The number of eggs in the globular body in C. leucosticta and C. antenori; single clutch we obtained in the lab (= 245) was greater prismatic in C. albopunctata); body shape, oval in than those of C. albopunctata, C. anatipes, C. antenori, lateral view (globular body in C. leucosticta, triangular C. avilapiresae, C. bassleri, C. capixaba, C. hudsoni, C. body in C. alagoana, rounded body in C. antenori and leucosticta, C. royi, C. schubarti, C. schudikarensis and acuminated body in C. albopunctata); snout shape, C. ventrimaculata (Peloso et al., 2014). truncated in dorsal view (rounded in C. mantiqueira, The tadpole of C. lacrimae, like in most species of C. anatipes, C. antenori, C. hudsoni and C. leucosticta); Microhylidae, is free-swimming and exotrophic. It has snout shape, truncated in lateral view (rounded in C. a rounded body in dorsal and ventral view, and oval in ventrimaculata, C. hudsoni, C. anatipes, C. antenori lateral view. However, Wogel et al. (2004) described the and C. shudikarensis, acuminate in C. mantiqueira Ontogeny andreproductiveaspectsof Table 6. Morphological characteristics of tadpoles of Chiasmocleis species, followed by the authors of each description. Table 6. Morphological characteristics of tadpoles ofChiasmocleis species, followed by the authors of each description.

Species Body Body Snout Snout Anal tube Dorsal fin Dorsal fin Ventral fin Flagellum Oral disc (dorsal view) (lateral view) (dorsal view) (lateral view) inserction C. lacrimae Rounded Oval Truncated Truncated Adhered High ⅓ of the body High Present Semicircular flaps (present study) C. alagoanus Oval Triangular Truncated Truncated Adhered High Body-tail limit High Present Semicircular flaps (Nascimento and Skuk, 2006) C. albopunctata Rounded* Oval* Rounded* Acuminated* — High ⅓ of the body High Present Semicircular flaps (Oliveira-Filho and (Prismatic) (Acuminated) Giaretta, 2006)

C. anatipes Oval* Globular* Rounded Rounded — High Body-tail limit High Present — Chiasmocleis lacrimae (Duellman 1978) C. anteroni Oval* Rounded* Rounded Acuminated* — High Body-tail limit Low Not present — (Krugel and Richter, Globular 1995) C. hudsoni Rectangular Oval Rounded Rounded Adhered Low Body-tail limit Low Not present Larger upper lips (Rodrigues et al., 2008) C. leucosticta Rounded Globular Rounded Inclinated Adhered High Body-tail limit High Not present Semicircular flaps (Langone et al, 2007) C. mantiqueira Rectangular Oval Rounded Acuminated Adhered High Anteriorly to the High Present Semicircular flaps (Santana et al., 2004) body-tail limit C. shudikarensis Oval* Oval Truncated Rounded Adhered Low Body-tail limit High Present Larger upper lips (Menin et al., 2011) Rectangular C. ventrimaculata Rounded* Oval* Rounded Rounded — Low Body-tail limit Low Present* Larger upper lips (Schlüter and Salas, 1991) ⃰ interpretation based on figures of the articles by several authors.

1047 1048 Laura Acerb Cordioli et al. and inclined in C. leucosticta); anal tube adhered to The developmental stages of C. lacrimae tadpoles are the ventral fin (not adhered in C. ventrimaculata); different in some stages from that described by Gosner dorsal fin height, high (low in C. ventrimaculata, C. (1960). Hind limbs at stage 29 already display the shape hudsoni and C. shudikarensis); insertion of the dorsal of the third and fifth toe (Fig. 6), which Gosner (1960) fin, 1/3 posterior of the body (body-tail junction in C. described only at stage 31. At stage 33, it is possible to ventrimaculata, C. antenori, C. anatipes, C. hudsoni, C. observe the formation of the second, third, fourth and shudikarensis, C. alagoana and C. leucosticta); ventral fifth toes (Fig. 6), which Gosner (1960) described only fin height, high (low in C. ventrimaculata, C. antenori at stage 35. and C. hudsoni); end of the tail, flagellum (not present The loss of vent tube in C. lacrimae occurs only in C. ventrimaculata, C. antenori and C. hudsoni). The at stage 42, although in Dermatonotus muelleri it mouth of C. lacrimae has the terminal semicircular disappears already at stage 40 (Fabrezi et al., 2012). flaps suspended in front of the mouth, separated by an Other differences in the development of these inverted, U-shaped medial notch and is different from Microhylidae species are that the nares open (appear) C. ventrimaculata, C. shudikarensis and C. hudsoni by at stage 41 in C. lacrimae and C. hudsoni (Rodrigues the larger upper lips, fleshy and covering the opening et al., 2008), but only at stages 43 in C. shudikarensis (Duellman, 1978; Schlüter and Salas, 1991; Krugeland (Menin et al., 2011), and sooner in D. muelleri (stages Richter, 1995; Wogel et al., 2004; Oliveira-Filho and 38 to 39); the caudal fins and tail musculature begin Giaretta, 2006; Nascimento and Skuk, 2006; Langone to differentiate already at stage 38 in C. lacrimae, but et al. 2007; Rodrigues et al., 2008; Menin et al., 2011; only at stage 44 in D. muelleri (Fabrezi et al., 2012). Santana et al., 2012). Morphological differences between At stage 42, the mouth appears before the line of the the tadpoles of Chiasmocleis species are summarised in nostril and mouth, and at stage 43 it already exceeds Table 6. the line of the nostril, similar to which occurs with the Tadpole of C. lacrimae differs of the colour in life bufonid Incilius valliceps (Wiegmann, 1833), and the described by Wogel et al. (2004) by absence of a pair in ranid Lithobates pipiens (Schreber, 1782), described bracket-shaped marks between the eyes in 101 tadpoles in the table of Gosner (1960). At stage 44, the mouth examined by us. The tadpole differs too by absence of a of C. lacrimae does not extend beyond the line of peculiar thickening on the fins that was also observed in the eye, different from I. valliceps and L. pipiens. In tadpoles of C. alagoana, C. panamensis, C. leucosticta some anurans, the left forelimb appears first due to the and C. shudikarensis (Nascimento and Skuk, 2006; spiracle opening (Duellman and Trueb, 1986), but for C. Vera-Candioti, 2006; Langone et al., 2007; Menin et lacrimae (Microhylidae), which has a ventral spiracle, al 2011), and Dermatonotus muelleri (Boettger, 1885) the forelimbs appear simultaneously. (Lavilla, 1992). The development of the embryos and tadpoles of The total length of tadpole of C. lacrimae at stage 35 C. lacrimae occurred rapidly, as expected for species was greater than that reported by Wogel et al. (2004) that exhibit explosive breeding (Carvalho-e-Silva and (16.60 mm, range =15.5 to 17.3 mm) at the same stage. Carvalho-e-Silva, 1994). It is important to note that It was also greater than the tadpole of C. alagoana body growth during larval development is marked at (average = 17.74 mm, range = 16.00 to 18.50 mm, early stages (26 to 27), almost doubling the total length, stage 35; Nascimento and Skuk, 2006). Some of our and then slowly increases between 27 and 38 stages. measurements had larger averages than described by The newly metamorphosed individuals are about 1/3 Wogel et al. (2004), such as tail length (14.72 vs. 10.40 the size of adults (6.90 mm compared to 22.8 mm for mm), distance between the eye and the nostril (4.22 vs. females and 17.7 mm for males), with the same dark 1.60 mm), inter-ocular distance (5.42 vs. 2.90 mm) and brown coloration. Data on ontogeny and reproductive eye diameter (0.98 vs. 0.84 mm). These differences may biology are important to fill gaps in knowledge about reflect population differences, since they are nearly 220 the phylogeny of animal taxa. The differences in km distant from each other. The snout-vent length of C. morphological characters of the tadpoles and adults lacrimae froglet was also greater than that reported by found in this work, together with more detailed studies Rodrigues et al. (2008) for C. hudsoni (6.93 vs. 4.97 may help clarify taxonomic problems or even indicate mm), probably because the tadpole was greater at the a new taxon. same stage (17.62 vs. 11.47 mm in stage 31; Rodrigues et al., 2008). Ontogeny and reproductive aspects of Chiasmocleis lacrimae 1049

Acknowledgments. The authors thank P.N. Costa, P.H.S. identificación. Agropecuaria, Buenos Aires. Dias, M. Mongin-Aquino, V.G.D. Orrico, F.C.B. Rosa, and Gosner, K.L. (1960): A simplified table for stating anuran embryos P.A. Valadares for their help with the field work and laboratory and larvae with notes on identification. Herpetologica 16: 183– activities; M.R. Fernandes for his assistance on the work 190. revision and U. Caramaschi for his pre-peer review; D.N. Silva Haas, A. (2003): Phylogeny of frogs as inferred from primarily and L.T. Carvalho-e-Silva for his assistance image editing. We larval characters (Amphibia: Anura). Cladistics 19: 23–89. also thank the staff of Reserva Rio das Pedras by collect and Haddad, C.F.B., Hödl, W. (1997): New reproductive mode in providing field support. The first author received scholarships anurans. Bubble nest in Chiasmocleis leucosticta (Microhylidae). from CNPq (Conselho Nacional de Desenvolvimento Científico Copeia 1997: 585–588. e Tecnológico) and FAPERJ (Fundação de Amparo à Pesquisa do Haddad, C.F.B., Prado, C.P.A. (2005): Reproductive modes in Estado do Rio de Janeiro). IBAMA (Instituto Brasileiro do Meio frogs and their unexpected diversity in the Atlantic Forest of Ambiente e dos Recursos Naturais Renováveis) provided our Brazil. BioScience 55: 207–217. capture permit (number 207/06, process 02022.003111/96-14). Haddad, C.F.B., Toledo, L.F., Prado, C.P.A. (2008): Anfíbios da Mata Atlântica. Guia dos anfíbios anuros da Mata Atlântica. References Editora Neotropica, São Paulo. Kwet, A., Di-Bernardo, M. (1998): Elachistocleis erythrogaster, Altig, R., McDiarmid, R.W. (1999): Body plan: Development and a new microhylid species from Rio Grande do Sul, Brazil. morphology. 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Accepted by Renato Nali