A peer-reviewed version of this preprint was published in PeerJ on 6 June 2018. View the peer-reviewed version (peerj.com/articles/4525), which is the preferred citable publication unless you specifically need to cite this preprint. Arenas-Rodríguez A, Rubiano Vargas JF, Hoyos JM. 2018. Comparative description and ossification patterns of Dendropsophus labialis (Peters, 1863) and Scinax ruber (Laurenti, 1758) (Anura: Hylidae) PeerJ 6:e4525 https://doi.org/10.7717/peerj.4525 Comparative description and ossification patterns of Dendropsophus labialis (Peters, 1863) and Scinax ruber (Laurenti, 1758)(Anura: Hylidae) Angélica Arenas Rodríguez Corresp., 1 , Juan Francisco Rubiano 2 , Julio Mario Hoyos Corresp. 1 1 Facultad de Ciencias, UNESIS (Unidad de Ecología y Sistemática), Pontifica Universidad Javeriana, Bogotá, Colombia 2 Facultad de Ciencias, Universidad del Bosque, Bogotá, Colombia Corresponding Authors: Angélica Arenas Rodríguez, Julio Mario Hoyos Email address: [email protected], [email protected] Although comparative studies of anuran ontogeny have provided new data on heterochrony in the life cycles of frogs, most of them have not included Colombian species. Using different staining techniques, we describe the cranial and poscranial elements development in two hylid species, Scinax ruber and Dendropsophus labialis, providing new data for more comprehensive ontogenetic studies in Neotropical frogs. We examined specimens from Gosner stages 25 to 45. We found differences in the infrarostral and suprarostral cartilages, optic foramen, planum ethmoidale, and the gill apparatus. In the ossification sequence, one of the first elements to ossify were the transverse process of spinal column and atlas in both species, and the parasphenoid in the skull. New descriptions of skeletal development and ossification sequences of larval stages of these two species, especially data concerning the postcranium, contribute with useful information for analysis of sequential heterochrony, because although the hylids are widely known, there are few works (15 of 700 species) about ossification sequence that include the whole skeleton. PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3368v1 | CC BY 4.0 Open Access | rec: 24 Oct 2017, publ: 24 Oct 2017 1 Comparative description and ossification patterns of Dendropsophus labialis 2 (Peters, 1863) and Scinax ruber (Laurenti, 1758)(Anura: Hylidae) 3 Angélica Arenas Rodríguez1, Juan Francisco Rubiano2, Julio Mario Hoyos1 4 1Facultad de Ciencias, UNESIS (Unidad de Ecología y Sistemática), Pontifica Universidad Javeriana, 5 Bogotá, Colombia 6 2Facultad de Ciencias, Universidad del Bosque, Bogotá, Colombia 7 Short Title: Skeletal development of two hylids 8 9 Abstract Although comparative studies of anuran ontogeny have provided new 10 data on heterochrony in the life cycles of frogs, most of them have not included 11 Colombian species. Using different staining techniques, we describe the cranial and 12 poscranial elements development in two hylid species, Scinax ruber and 13 Dendropsophus labialis, providing new data for more comprehensive ontogenetic 14 studies in Neotropical frogs. We examined specimens from Gosner stages 25 to 45. We 15 found differences in the infrarostral and suprarostral cartilages, optic foramen, planum 16 ethmoidale, and the gill apparatus. In the ossification sequence, one of the first 17 elements to ossify were the transverse process of spinal column and atlas in both 18 species, and the parasphenoid in the skull. New descriptions of skeletal development 19 and ossification sequences of larval stages of these two species, especially data 20 concerning the postcranium, contribute with useful information for analysis of sequential 21 heterochrony, because although the hylids are widely known, there are few works (15 of 22 700 species) about ossification sequence that include the whole skeleton. 23 Keywords Ossification sequences, rank, skeletal development, tadpoles 24 25 Introduction 26 Systematicians have been assessing anuran relationships from the 1960´s to the 27 present day, using both molecular and morphological methods. However, studies of 28 morphological characters have widely privileged adults over tadpoles (Alcalde et al. 29 2011). Traditionally, studies of frog larvae have considered external morphological 30 characters, while most skeletal characters (bones and cartilages) have been often 31 neglected, and when skeletal features are considered, studies have been concentrated 32 on describing the chondrocranium more than the postcranium (Orton 1953; Starrett 33 1973; Wassersug 1980; Wassersug and Heyer 1988; Haas 2003). 34 The family Hylidae is one of the largest families of frogs with a great number of 35 interspecific variations that have helped to clarify specific taxonomic groups. It is 36 predominantly distributed across the Neotropical region (Frost et al. 2006) and 37 comprises 948 species commonly subdivided into three subfamilies: Hylinae, 38 Pelodryadinae and Phyllomedusinae (Faivovich et al. 2005; Wiens et al. 2010; Frost 39 2017). Within the Hylidae, the ossification sequences have been studied for 25 species, 40 of which only six species, Dryophytes chrysoscelis (former Hyla chrysoscelis Sherman 41 and Maglia 2014) Hypsiboas pulchellus (Hoyos et al. 2012), Hypsiboas lanciformis (De 42 Sá 1988), Phyllomedusa vaillanti (Sheil and Alamillo 2005), Pseudacris crucifer and 43 Acris blanchardi (Havens 2010), have included the postcranial skeleton. 44 Taking into account that variations in the ossification sequence, placed in a 45 phylogenetic context, will help to recognize informative characters and evolutionary 46 relationships among species (Weisbecker and Mitgutsch 2010; Harrington et al. 2013), 47 the goal of the present study is to provide information on the larval morphology (cranium 48 and poscranium development) of two species of frogs Andean hylids, Dendropsophus 49 labialis and Scinax ruber. These species were chosen with the aim of giving a 50 contribution to the knowledge on the timing and sequences of ossification, and in order 51 to provide comparative characters for anuran morphology and systematics of these 52 Colombian species. 53 54 Materials and methods 55 The number tadpoles cleared and double-stained (Dingerkus and Uhler 1977) of each 56 species were 146 (32 of D. labilis, and 114 of S. ruber) and one adult male. These 57 speciemens belong to the Museo de Historia Natural “Lorenzo Uribe” at the Universidad 58 Javeriana (MUJ) and the Instituto de Ciencias Naturales at the Universidad Nacional in 59 Bogotá – Colombia (ICN). The larval stages of D. labialis were collected from the 60 Municipio Tenjo, Cundinamarca Departament, 3200 m (MUJ 9250) and adult stages 61 from the Mun. Fomeque, Cundinamarca Dep., 3150 m (MUJ 497). The larval stages of 62 S. ruber were collected from the Mun. Neiva, Huila Dep., 570 m; Mun. Granada, Meta 63 Dep., 470 m (MUJ 3727, MUJ 6178, ICN 46015-46017) and adult stages from the Mun. 64 La Dorada, Caldas Dep., 490 m (MUJ 9037). All of these were staged according to 65 Gosner’s (1960) proposal. 66 Observations and photographs were made with a stereomicroscope (Advanced 67 optical), a camera (Infinity 1 Lumenera Corporation) with white LED light and Image Pro 68 Insight program (version 8.0.3). The drawings were made using a digitizing tablet 69 (Bamboo connect pen) and edited using Adobe Illustrator 5. Anatomical nomenclature 70 for tadpoles is based on (Parker, 1876; Higgins, 1921; Jolie 1962; Roček, 1981; 71 Duellman & Trueb, 1986; Haas, 1995; Haas, 1997; Hall & Larsen, 1998; Maglia and 72 Púgener, 1998; Cannatella, 1999; Haas, 1999; Sheil and Alamillo, 2005; Púgener and 73 Maglia, 2007; Bowatte & Meegaskumbura, 2011; Hoyos et al., 2012); on the other hand, 74 the adult nomenclature is based on Avilán & Hoyos (2006), using the Latin names given 75 by the ICVAN (1973), taking into account that it does not exit a Nomina Anatomica 76 Batrachologica. 77 In the ossification sequences we used the metamorphic climax (MC) sensu Banbury 78 and Maglia (2006). This concept involves major modifications, and fundamental 79 structural changes within Gosner stages, ending in the loss of most of the larval 80 characters. We also used the term "rank" refering to the assignment of a position of the 81 beginning of ossification timing of the elements and in the case that two or more events 82 are simultaneously present (beginning of the ossification at the same time), they are tied 83 in the same rank within the developmental sequence (ossification sequence) (Nunn and 84 Smith 1998). 85 86 Results 87 Individual development of cranial and poscranial elements in Dendropsophus labialis 88 and Scinax ruber are showed in table 1 and 2, each one showing ossification 89 sequences. 90 Chondrocranium 91 The overall width of the chondrocranium in Dendropsophus labialis and Scinax ruber 92 is approximately 80-90% of this total length (Fig 1). The chondrocranium in D. labialis is 93 wider (dorsal view) and lower (lateral view) than S. ruber (Fig 1A, 1B, 1C). The 94 basicranial fenestrae did not differentiated with Alcian Blue in both species. We 95 observed a stronger blue coloration in D. labialis, and the jugular (jf), prootic (pof) and 96 oculomotor (of) foramen were clearly differentiated, whereas in S. ruber we could not 97 these foramina. The cartilaginous area of taenia tecti medialis and tectum sinoticum (ts) 98 both represents a quarter of that basis cranii, extending from the frontoparietal 99 fontanella