Herpetology Notes, volume 13: 931-935 (2020) (published online on 16 November 2020) Notes on the reproductive biology of Phyllomedusa bicolor (Anura, Phyllomedusidae) in the Amazon Forest of Northern Brazil Yuri Breno Silva e Silva1, Wirley Almeida-Santos2, Andréa Soares Araújo1, and Carlos Eduardo Costa-Campos1,* The Neotropical phyllomedusid genus Phyllomedusa P. rohdei (Wogel et al., 2005, 2006), and P. trinitatis (Wagler, 1830) currently contains 16 species (Frost, (Downie et al., 2013). 2020) and belongs to the Family Phyllomedusidae. Although two studies report reproductive biology of Most species are arboreal and usually walk slowly on P. bicolor from western (Venâncio and Melo-Sampaio, branches and leaves, rarely leaping (Caramaschi and 2010) and central Amazon (Neckel-Oliveira and Cruz, 2002). Phyllomedusa bicolor (Boddaert, 1772) Wachlevski, 2004), there is not much information on it is one of the largest Amazonian tree frogs found in the reproductive biology of this species for the eastern the forests of Brazil, Guianas, Venezuela, Colombia, Amazon. In this sense, the present study we aimed to Peru, and Bolivia (Frost, 2020). The reproductive describe the reproductive biology of the species, with mode (number 24, sensu Haddad and Prado, 2005) is emphasis on the amplexus and oviposition from eastern characterised by the oviposition in suspended leaves Brazilian Amazon, north of the Amazon River. in lentic water bodies and development of exotrophic The amplexus and oviposition were observed at the tadpoles. Parque Natural Municipal do Cancão (0.9026°N, Several species of the family Phyllomedusidae have 52.0050°W), Municipality of Serra do Navio, Amapá aspects of the reproduction described: Callimedusa State, Brazil. The study area covers 370.26 hectares of tomopterna (Neckel-Oliveira and Wachlevski, 2004; primary forest, including terra-firme rainforests, open Najar and Ferrante, 2018); Phyllomedusa bahiana areas and treefall gaps (Drummond et al. 2008; Silva (Santos-Silva et al., 2012), P. bicolor (Neckel-Oliveira e Silva and Costa-Campos, 2018). The local climate and Wachlevski, 2004; Venâncio and Melo-Sampaio, according to Köppen’s classification is Equatorial (Am) 2010), P. burmeisteri (Abrunhosa and Wogel, 2004; characterised by a rainy season from March to July, Srbek-Araujo et al., 2017), P. tarsius (Neckel-Oliveira with annual rainfall of 2,850 mm (Alvares et al., 2013). and Wachlevski, 2004); Pithecopus ayeaye (Oliveira, The mean rainfall was obtained from the Núcleo de 2017), P. azureus (Rodrigues et al., 2007; Filadelfo Hidrometeorologia e Energias Renováveis – Instituto et al., 2013; Dias et al., 2014; Oliveira et al., 2014), de Pesquisas Científicas e Tecnológicas do Estado do P. hypochondrialis (Matos et al., 2000), P. iheringii Amapá. (Dias et al., 2017), P. nordestinus (Faraulo et al., 2019), Samples were collected monthly from August to October 2013 (177 mm) and May to July 2014 (468 mm), always with three observers for four consecutive nights per month in the field, through active searches in breeding sites (Heyer et al., 1994) in the forested areas and temporary ponds in River Amapari trail (0.9008°N, 1 Departamento de Ciências Biológicas e da Saúde, 52.0135°W) in the Parque Natural Municipal do Cancão. Universidade Federal do Amapá, Rodovia Juscelino Clutches were collected, preserved in formaldehyde Kubitschek, km 02, Jardim Marco Zero, Macapá, Amapá 10% and housed in the Herpetological Collection of the 68903-419, Brazil. 2 Secretaria Municipal de Turismo de Serra do Navio, Rua A1 Universidade Federal do Amapá. 533A, Serra do Navio, Amapá 68948-000, Brazil. Phyllomedusa bicolor presents a characteristic typical * Corresponding author. E-mail: [email protected] of species of prolonged breeding, with reproduction 932 Yuri Breno Silva e Silva et al. for more than three consecutive months, asynchronic The clutches were located along a large temporary arrival of females at the breeding sites, and with males pond (maximum width: 15 m; maximum length: 50 m; defending territories (sensu Wells, 1977). The months maximum depth: 0.5 m), located near the Amapari River. with greatest reproductive activity were May to July Eggs clutches were enveloped by three (n = 4) and two 2014, coinciding with the highest recorded rainfall. In leaves (n = 4) from the angiosperm families Bignoniaceae these months heavy rainfall increases the hydroperiod of (n = 5), Cyclanthaceae (n = 1) and Heliconiaceae (n = 2) ponds, forming a suitable environment for development and the arrangement nests varied in length and closure of tadpoles. On the other hand, breeding activity of P. the leaf: partially, totally or open (Fig. 4). Tadpoles bicolor can be also explained by reproductive mode were observed at different development stages in lentic (arboreal eggs, and eggs hatching into exotrophic waters of temporary ponds. tadpoles that drop in lentic water, see Haddad and The spawning of P. bicolor consists of a gelatinous Prado, 2005) and eggs and embryos enveloped in leaves mass with a mean of 751 eggs per clutch, with a above the water protection against desiccation (Altig minimum of 241 and a maximum of 1,722 eggs (SD ± and McDiarmid, 2007). 33.9, n = 3). Mean egg diameter from all clutch means Most of the individuals were observed in the rainy was 2.6 mm (SD ± 0.2, n = 30 eggs), with minimum and season (May, n = 17 individuals, June n = 28, July, n = maximum mean values of 1.9 and 3.5 mm per clutch. 23, Fig. 1). Calling males of P. bicolor were recorded in The clutch size is similar than the data reported by the months of highest rainfall found on the vegetation close to the ground (range 1 – 4 m, n = 12). The peak of vocalisation activity of the species occurred between 20:00 and 23:00 h (Fig. 2). Male territorial behaviour was observed on two different occasions: vocalisation and physical combats to defend its territory (Fig. 3A, B). We observed Dendropsophus counani, D. leucophyllatus, Leptodactylus petersii, L. mystaceus and Chiasmocleis hudsoni calling syntopically in the same pond. We observed pairs in amplexus between 19:00 and 23:00 h (n = 8); the greatest number of the pairs (n = 6) was found at approximately 22:00 h. Pairs in amplexus were found on the ground (n = 1, Fig. 3C) and perched on edge vegetation (n = 7, Fig. 3D), although spawning Figure 1. Number of individuals (males and females) of was not observed. During the amplexus, of the cephalic Phyllomedusa bicolor observed in each month in relation to type, the male remained with the gular region on the accumulated rainfall (mm) at the Parque Natural Municipal do cephalic region of the female. Cancão, Municipality of Serra do Navio, Amapá State, Brazil, Two males of P. bicolor (amplectant male snout- between the April to October 2013 and May to July 2014. vent length [SVL] = 105 mm, intruder male SVL = 74 mm) were observed engaging in amplexus simultaneously with the same female (SVL = 116 mm) in a tree (Celastraceae, Fig. 3E) located at the margin of a temporary pond (Fig. 3F). The amplected male performed leg stretching behaviour for 15 minutes, until the intruder male gave up. The occurrence of multiple amplexus has also been observed in other species of the family Phyllomedusidae: Callimedusa tomopterna (Najar and Ferrante, 2018), Phyllomedusa bahiana (Santos-Silva et al., 2012), P. distincta (Woehl and Woehl, 2000; Prado et al., 2006), P. iheringii (Oliveira et al., 2014), Pithecopus ayeaye (Oliveira, 2017), P. azureus (Dias et al., 2012), P. megacephalus (Oliveira Figure 2. Peak of the call activity of the males of Phyllomedusa et al., 2012) and P. rohdei (Wogel et al., 2005), although bicolor in the Parque Natural Municipal do Cancão, this is the first record for P. bicolor. Municipality of Serra do Navio, Amapá State, Brazil. Notes on the reproductive biology of Phyllomedusa bicolor in Northern Brazil 933 Figure 3. Breeding biology of Phyllomedusa bicolor in Amapá State, Brazil. (A) and (B) Physical combat between to males for a female Phyllomedusa bicolor. (C) Amplexing pair on the ground. (D) Pair in amplexus perched on riparian vegetation. (E) Multiple amplexus of Phyllomedusa bicolor involving two males and one female in a tree (Celastraceae). (F) Temporary pond. Photos by Wirley Almeida-Santos (A, B, E) and Eduardo Campos (C, D, F). Neckel-Oliveira and Wachlevski (2004) and Venâncio This species is composed of deeply structured genetic and Melo-Sampaio (2010), from 895.5 – 1,202 eggs per groups, constituting more than one lineage across the clutch, respectively. Brazilian Amazonia (Mota et al., 2020). In this sense, 934 Yuri Breno Silva e Silva et al. Figure 4. Spawn of Phyllomedusa bicolor and variations in closure nests: (A) and (B) Partially closed nests (Bignoniaceae). (C) Totally enclosed nest (Heliconiaceae). (D) open nest (Celastraceae). Photos by Wirley Almeida-Santos. these data contribute to the knowledge the natural Acknowledgements. We thank Igor Kaefer for reviewing and history and reproductive biology of this species of wide suggestions to this manuscript. I would like to thank the Instituto distribution from different locations, especially for Brasileiro de Meio Ambiente e Recursos Naturais Renováveis for providing the permits for capture and collection of specimens in eastern Brazilian Amazon, north of the Amazon River. the field (Process n. 32651-2). Notes on the reproductive biology of Phyllomedusa bicolor in Northern Brazil 935 References giant monkey frog (Phyllomedusa bicolor: Phyllomedusidae). Amphibia-Reptilia 41: 1–11. Abrunhosa, P., Wogel, H. (2004): Breeding behavior of the leaf- Najar, T., Ferrante, L. (2018): The journey of life of the Tiger- frog Phyllomedusa burmeisteri (Anura: Hylidae). Amphibia- striped Leaf Frog Callimedusa tomopterna (Cope, 1868): Notes Reptilia 25: 125–135. of sexual behaviour, nesting and reproduction in the Brazilian Altig, R., McDiarmid, R.W. (2007): Morphological diversity Amazon. Herpetology Notes 11: 531–538. and evolution of egg and clutch structure in amphibians. Neckel-Oliveira, S., Wachlevski, M.