A peer-reviewed version of this preprint was published in PeerJ on 6June 2018.

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Arenas-Rodríguez A, Rubiano Vargas JF, Hoyos JM. 2018. Comparativedescription and ossification patterns of Dendropsophus labialis (Peters, 1863)and Scinax ruber (Laurenti, 1758) (Anura: Hylidae) PeerJ 6:e4525https://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, Colombia2 Facultad de Ciencias, Universidad del Bosque, Bogotá, Colombia

Corresponding Authors: Angélica Arenas Rodríguez, Julio Mario Hoyos

Email address: angelica.arenas@javeriana.edu.co, jmhoyos@javeriana.edu.co

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 in both species. The tectum nasi is a roof structure located anteriorly in the

100 nasal region, and the ethmoidal plate is a floor structure. The tectum nasi is separated

101 from the orbit by a wall, the lamina orbitonasalis (planum antorbitale). Because of these

102 regions remain only weakly chondrified, the lamina orbitonasalis is not observed in the

103 tadpole stages, and the nasal capsules are visible in later stages. The taenia tecti

104 marginalis is evident and clearly differentiated at GS37 in D. labialis and at GS35 in S.

105 ruber. Neither species we observed a parietal fenestra, nor the taenia tecti transversalis

106 was observed directly on the edge of the fontanella (Fig. 1A).

107 Suprarostral cartilage (cs). In both species, the suprarostral cartilage is composed of a

108 discontinuous cartilaginous plate divided into a corpus suprarostral and a pars alaris,

109 while posterolaterally we observed a distal syndesmotic junction between the corpus

110 and the ala (a). The ala has three processes: two rounded anterolateral processes that

111 join syndesmotically with the cornu trabecula (ct), and one process posterolaterally (Fig.

112 1C). The fenestrations were not observed in the suprarostral cartilage, and in the

113 adrostral cartilage near the processus posterodorsalis (processus dorsalis posterior,

114 sensu Bowatte & Meegaskumbura, 2011) and. In D. labialis the corpus suprarostral is

115 curved, while in S. ruber is straighter and wider distally, articulating proximally with the

116 cornu trabecula. The cornu trabecula is approximately 35% of the total length of

117 chondrocranium (lateral view) in both species, and this is shorter and narrower in D.

118 labialis than in S. ruber. The cornu trabeculae articulate (trabecular horn, sensu

119 Cannatella, 1999) anteriorly with the corpus rostrale and laterally with the pars alaris of

120 the suprarostral cartilage.

121 Cartilago Meckeli (cm). The cartilago Meckeli (Meckel's cartilage, sensu Cannatella,

122 1999) has three processes: the retroarticular (short and blunt), the dorsomedial, and the

123 ventromedial. These processes articulate with both the infrarostral cartilage (ci),

124 composed of two syndesmotically joined flat plates (commissura intramandibularis,

125 sensu Cannatella, 1999), and the processus muscularis quadrati (pmq); the shape of

126 the processus dorsomedialis and the processus ventromedialis are the same in both

127 species. The palatoquadrate cartilage and the commissura quadratocranialis (cqc) are

128 joining anteriorly to the base cranii. Laterally, the palatoquadrate cartilage (pq) forms the

129 arcus subocularis. The process muscularis quadrati is joined to the processus

130 antorbitalis (pars plana sensu Parker, 1876; = lamina externa sensu Higgins, 1921; =

131 processus antorbitalis sensu Roček, 1981; = triangular plane sensu Hall & Larsen, 1998

132 = cartilaginous planum triangulare sensu Púgener and Maglia, 2007) and is placed

133 anterolaterally, projecting above the cornu trabecula. The processus hyoquadrati of the

134 palatoquadrate cartilage articulates ventrally with the ceratohyalia (=ceratohyals) of the

135 hyobranchial apparatus (Fig. 1D).

136 Otic capsule. In the tadpoles this structure is longer and higher than wide, occupying

137 about one-fifth of the total length of the skull. The crista parotica exhibits a more

138 pronounced lateral projection in D. labialis than in S. ruber. The crista parotica (=parotic

139 ridge) is laterally developed, forming a small processus posterolateralis (processus

140 lateralis posterior sensu Bowatte & Meegaskumbura, 2011) a small processus

141 anterolateralis (pal) (more developed in D. labialis). The processus anterolateralis

142 projects vertically descending obliquely and overlapping the ventral posterolateral

143 margin of the palatoquadrate cartilage. The otic capsule is perforated by the fenestra

144 ovalis (fo), occupying 20% of the otic capsule.

145 Hyobranchial apparatus. The large ceratohyal has a processus anterioris hyalis

146 (=anterior process sensu Hoyos et al., 2012; = processus anterior sensu Haas, 1999), a

147 processus posterioris hyalis (pph) (= posterior process sensu Hoyos et al., 2012; =

148 processus posterior sensu Haas, 1999) and a processus anterolateralis hyalis (palh).

149 The first two processes are longer than the processus anteriolateralis hyalis, which

150 extends to meet the transverse crease of the processus lateralis hyalis (plh).

151 The basihyal plate is oval and extends proximally to the copula anterior (= copula I

152 sensu Maglia and Púgener, 1998; Sheil and Alamillo, 2005; = basibranchial I sensu

153 Duellman & Trueb, 1986; = basihyale sensu Haas, 1997 and sensu Haas, 1995) in D.

154 labialis, but in S. ruber is absent). The basibranchial plate is semi-oval, placed between

155 the two hypobranchial plates (planum hypobranchiale, phb) (= planum hypobranchiale

156 sensu Haas, 1999; = plate hyoid sensu Maglia & Púgener, 1998; = hyobranchial plate

157 sensu Sheil and Alamillo, 2005); the branchial bridge is present in both species, being

158 wider in S. ruber than in D. labialis. The junction between each ceratobranchial (cb I-IV)

159 and the planum hypobranchiale is syndesmotic. The ceratobranchialia is united by the

160 commissura terminalis (ct) bearing three spicules (sp) (Fig. 1D).

161 Appendicular skeleton

162 Shoulder girdle. The pectoral girdle is arciferal in both species. The earliest

163 ossification of clavicle (c), coracoid (co) and scapula appears at GS36 (Fig. 3A). The

164 clavicle and the cleithrum are distinct, and the epicoracoids cartilage (ps) is prominent

165 between the clavicle and the coracoid. The epicoracoids are not mineralized in

166 tadpoles. The omosternum (o) in D. labialis is elongated and the sternum has two

167 projections; the omosternum and the sternum (st) are oval in S. ruber. The clavicle is

168 ossified in D. labialis (GS36) but is not in S. ruber, being articulated with the coracoid.

169 The sternun are formed by the epicoracoids and the sternum element (mesosternum)

170 joining the medial junction of the epicoracoids (Fig. 2B).

171 Pelvic girdle. The primordium of the ilium (il) appears at GS34 and is fully developed at

172 GS41 in both species. The ilium is ossified during development, articulating anteriorly

173 with the ventral surface of the lateral margin of the sacral diapophyses at GS42. The

174 iliac crest appears dorsally prominent; the primordia of the pubis (pu) and the ischium

175 (is) appear at GS36, synchondrotically fused at GS38. The sacral diapophysis is wider

176 in D. labialis that in S. ruber. The pubis is totally fused at GS40. The pelvic girdle is

177 completely ossified with the halves fused at the midline, extending anterodorsally

178 forming an angle of 55º with the head of the femur at GS45 (Fig. 3B).

179 Forelimb and hindlimb. The first cartilaginous elements of the forelimbs (radius, ulna

180 and humerus) appear at GS32, and those of the hindlimbs at GS33 (femur, tibia and

181 fibula). The tibia and fibula are fused at D. labialis (GS41) and S. ruber (GS38). We

182 observed ossification of the radius and ulna in D. labialis (GS41) and S. ruber (post

183 metamorphic); these elements are fused in both species. Primordia of the four carpal

184 and five tarsal elements appear at GS33 and complete development at GS41. The

185 phalangeal carpal formula is 3-3-4-4 and the phalangeal tarsal formula is 3-3-4-5-4 in

186 both species. Metacarpals (mc) are curved and phalanges are cylindrical, having a

187 conical shape at the tip of the terminal phalanges. Digits IV (manus and pes) and V

188 (pes) begin to ossify at GS42 only in D. labialis, although all phalanges are ossified at

189 GS45 both species (Fig. 3). The carpalia remain cartilaginous in the specimens and

190 stages examined, and the distal tarsals remain cartilaginous in S. ruber. The relative

191 size of carpal elements (mc) is 3 < 4 < 2 < 1< prehalux (ph) and the tarsal elements (mt)

192 is 4 < 5 < 3 < 2 < 1 < prepollex (pr). Sesamoids are absent from GS25 to GS45. In the

193 figure 3A showed elements as central (ce), fibulare (fi), radiale (rd), tibiale (ti), ulnare

194 and intermedium (ul).

195 Axial skeleton

196 The vertebral column is composed of eight procoelous presacral vertebrae, the sacrum,

197 and the urostyle. The notochord length diminishes as the tadpoles grow, reaching a total

198 resorption at GS44 in both species (Fig. 4). We found that the axial skeleton was more

199 chondrified in D. labialis than in S. ruber. The first postcranial skeletal elements to

200 develop in both species were the nine pairs of semicircular cartilaginous primordia of

201 neural arches, included eight presacral vertebrae, the sacrum, the urostyle (u) and the

202 hypochord (hy). The sacral diapophyseal (sd) primordia are cylindrical. The last

203 postsacral vertebra (first coccygeal or vertebra X sensu Haas 1999) and the second

204 coccygeal vertebra ossified only in D. labialis at GS45. Simultaneously, with the

205 absorption of the notochord, the coccygeal elements fuse and form the urostyle. The

206 urostyle has a bicondylar articulation with the sacral vertebra and the condyles are

207 widely separated (Haas 1999) in both species (Fig. 4).

208 The atlas (a) is concave at its point of articulation with the convex occipital condyles

209 at the base of the skull. Semicircular vertebral centrum begins to develop as procoelous

210 (sensu Jolie 1962) vertebra from GS31 in D. labialis and at GS32 in S. ruber, increasing

211 the thickness of both the neural arches and the transverse process. The neural arches

212 appear cartilaginous at GS33 in both species; these are complete at GS34 in D. labialis

213 and at GS38 in S. ruber. The arches are fused dorsally at the midline at GS38 in S.

214 ruber and at GS38 in D. labialis. Transverse processes are the first elements to ossify in

215 both species (Tables 1 and 2). Both postzygapophyses and prezygapophyses are

216 conspicuous in presacral vertebrae II, III, and IV in both species. Sesamoids are absent

217 from GS25 to GS45.

218 Ossification sequence

219 The earliest stage of the two species was the GS25. The ossification timing in D.

220 labialis begins at GS34 and in S. ruber at GS35 (Fig. 1A). Ossification in D. labialis

221 begins with the atlas and the transverse processes, whereas in S. ruber it begins with

222 the parasphenoid, the I-VII transverse processes and neural arches I-III. The

223 ossification sequence was constructed with the first appereance of bone. We observed

224 that the metamorphic climax (MC) begins at GS41 in D. labialis, and in S. ruber at

225 GS39-40. We identified seven ranks (I–VII) in D. labialis and five ranks (I–V) in S. ruber

226 (Tables 1 and 2). The GS in which we found ossified elements (or bones) in D. labialis

227 were from GS35 to GS45, with 46 elements, and in S. ruber from GS36 to GS43, with

228 26 elements. S. ruber showed intraspecific variation in nine stages (Table 3). The MC in

229 D. labialis was at GS45 with 14 ossified elements, and in S. ruber at GS39-40 with

230 seven ossified elements. Of these, the structures in common were the femur, tibia,

231 fibula, humerus, ilium and radioulna (rad). The two species present both differences and

232 similarities with respect to the ossification of the chondrocranium and postcranium.

233

234 Discussion

235 Notwithstanding the fact that Colombia is the richest country in the planet concerning

236 the number of hylid species, there are very few studies about developmental ossification

237 sequences. The species of the family Hylidae show variation in the taxonomic

238 arrangements, as it was showed by the variety of phylogenetic hypotheses based on

239 molecular, chromosomal, and morphological data from both larvae and adults

240 (Faivovich 2002; Faivovich et al. 2005; Wiens et al. 2010; Pyron and Wiens 2011).

241 There are 15 genera of hylids in Colombia, containing 125 species (Frost 2017), from

242 which 35 belong to Dendropsophus and 17 to Scinax.

243 Cranial morphology has been poorly studied in hylid tadpoles, being known only in

244 Acris crepitans (Maglia et al., 2007), Hyla raniceps, Scinax granulatus and Scinax

245 squalirostris (Alcalde and Rosset, 2003), Scinax species: S. acuminatus, S. uruguayus,

246 S. aff. pinima, S. aromothyella, and S. berthae (Alcalde et al., 2011; Faivovich, 2002),

247 Gastrotheca riobambae (Haas, 1996), Hypsiboas pulchellus (Hoyos et al., 2012),

248 Phyllomedusa vaillanti (Sheil and Alamillo, 2005), and Hyla orientalis (Yıldırım and

249 Kaya, 2014).

250 Chondrocranial morphology and hyobranchial apparatus development is similar

251 between both species. We found differences between the two species in the shape of

252 the suprarostral (i), size and width of infrarostral cartilages (ii), length of processus

253 articularis (iii), thickness of palatoquadrate (iv), size of optic foramen (opf) (v), the

254 presence of operculum (vi) and processus posterolateralis of the auditive capsule (vii),

255 thickness of the processus muscularis quadrati (viii), the presence of quadratoethmoid

256 and pseudopterygoid (ix), the attachment of the ascending process to the braincase (x),

257 thickness of the planum ethmoidale (xi), the development of the branchial apparatus

258 (xii), the presence of copula I (xiii), and the type of junction between ceratobranchialia

259 and planum hypobranchiale (xiv) (Fig. 1 and 2). These differences likely represent

260 species-specific differences among the taxa examined. There are no admandibular

261 cartilages attached to the anteroventral margin of the cartilago Meckeli as described by

262 Hoyos et al. (2012) in Hypsiboas pulchellus.

263 The processus ethmoidalis of the quadrate in S. ruber is wide, and it is not clearly

264 distinct from the processus articularis, while in D. labialis the processes are easily

265 distinguishable, similar to that described by Alcalde and Rosset (2003), who also found

266 the same feature in Hyla raniceps comparing with the Scinax species group (S.

267 squalirostris and S. granulatus, Scinax ruber group). The palatoquadrate is similar

268 between species but the processus ascendens of the palatoquadrate in D. labialis is

269 wider than in S. ruber, while the distal side of the cornu trabeculae extends posteriorly

270 toward the otic capsule and the anterior region of the palatoquadrate is distinctively

271 broader in S. ruber than in D. labialis. In S. ruber the dorsomedial process is wider than

272 the ventromedial process in D. labialis. The lateral development of the crista parotica is

273 more prominent in S. ruber than in D. labialis. It is possible that some outstanding

274 anatomical structures of the otic capsule (oc) allow to perceive vocalizations of their

275 own species in adult stages, but experiments must be conducted to check the

276 relationship of anatomical structures with hearing physiological functions (Ruggero and

277 Temchin 2002; Boistel et al. 2013).

278 The chondrification of skull in S. ruber was faint when it is viewed laterally, and foramina

279 are not clearly visualized. By contrast, in D. labialis, more blue coloration was observed,

280 may be due to the abundant chondrification of these parts or to the early developmental

281 stages of the larvae in this region, which allowed differentiation of craniopalatine carotid

282 foramina, contrary to what was found by Alcalde and Blotto (2006). D. labialis exhibited

283 more ossified elements with stronger chondrification and less intraspecific variation,

284 while S. ruber showed more intraspecific variation and less overall chondrification. This

285 variation suggests that may be caused by the intrinsic factor that determines the timing

286 of development or because extrinsic factors may affect the osteogenesis (Vera and

287 Ponsa, 2014).

288 Haas (1996) observed that the ceratohyalia II-IV are fused in Scinax ruber and

289 Megophrys montana nasuta (former Megophrys montana), separating them from other

290 species; this character has to be taken into account when taxonomic studies based on

291 morphology are carried on. This character was found in S. ruber but not in D. labialis.

292 The ceratohyal in D. labialis presents a process on the articular condyle that is not

293 present in S. ruber. Alcalde and Rosset (2003) found this process in both S. granulatus

294 and S. squalirostris. Spicules I-III on the posterior margin of the hypobranchial plate are

295 present in D. labialis and S. ruber, but the spicule IV is not. The copula II cp (II) is

296 present in both species, but the copula I ca (I) is absent in S. ruber and it is present in

297 D. labialis, just as in S. squalirostris, but in S. granulatus, Hyla raniceps (Alcalde and

298 Rosset 2003) and H. microcephala (Vera and Haas 2004) is absent. Although the

299 presence of copula I is extremely variable in hylids, and is shared by all non-hylid

300 macrophages (Vera and Haas 2004), a relation of this structure with the ecological

301 function that it can perform, for example in relation to the type of food, has not been

302 found.

303 The urostyle in D. labialis and S. ruber presents a bicondylar articulation with the

304 sacral vertebra with the condyles are widely separated. The shoulder girdle of both

305 tadpoles presents differences in the shape of omosternum and sternum at GS 45. D.

306 labialis and S. ruber present suprascapular processes in tadpoles and adults similar to

307 those in other species of the family Hylidae: Hypsiboas lanciformis (De Sá 1988),

308 Hypsiboas pulchellus (Hoyos et al. 2012), Pseudacris crucifer, Acris blanchardi (Havens

309 2010) and A. crepitans (Maglia et al. 2007).

310 The variation of larval characters has been evaluated in the genera Scinax and

311 Dendropsophus in several phylogenetic studies (Fabrezi and Vera 1997; Haas 1996;

312 1999; 2003; Alcalde and Rosset 2003; Vera 2007). In our study, the skeleton shows

313 significant differences between the species Scinax ruber and Dendropsophus labialis,

314 beginning with fact that elements ossified in S. ruber exhibit more intraspecific variability

315 than in D. labialis (see Table 3).

316 Regarding the ossification sequence, the first bones ossified in the cranium were the

317 exoccipital, the frontoparietal and the parasphenoid at GS 36. Haas (1999) found that in

318 S. ruber, and in other species of Hyla (some species named after as Dendropsophus),

319 this occurred at GS37. The ossification of the vertebrae begins from centra of the

320 presacral vertebrae and continuing ventrally along the notochord, forming osseous rings

321 around the notochord in both species similar to those that Haas (1999) described for

322 other species of Hylidae. Haas (1999) also recorded the time of the first ossification

323 timing of the transverse process of the presacral vertebrae II- III; we observed these

324 elements beginning at GS36 in both species. The ossification of the neural arches I to

325 IX was in D. labilis (GS37) whereas in S. ruber (GS36) was I to III; the ossification of the

326 arcs in these species is completed in the GS37. Interestingly, the first elements to ossify

327 were those from the postcranium: the transverse process of the vertebral column and

328 the atlas in D. labialis (Figure 4). The ossification process of the vertebra in the two

329 species started ventrally from the centrum of each vertebra, and followed dorsally to

330 close the vertebrae in advanced stages, as Haas (2003) recorded for Litoria nannotis.

331 The differences between the ossification sequences of these two species are also

332 seen with regard to the ossification ranks and number of ossified bones; in particular, D.

333 labialis presented the highest number of ranks and elements that began the process of

334 ossification. With respect to the postcranium, the number of elements ossified appears

335 earlier in D. labialis than in S. ruber. The occurrence of events in the development of

336 each species is the relative timing of each event in the ossification sequence, allowing

337 us to compare the relative timing of developmental events across different taxa. For this

338 reason we did not take the developmental GS and we take the order of begining of the

339 ossification of each element, because these are based on the appearance of external

340 morphological structures that are the same in both species studied. Moreover, Nunn

341 and Smith (1998:86) considered “that ontogeny may be ordered by age, size, or stage,

342 none of these measures are useful for comparing ontogeny across significantly

343 divergent taxa”.

344 Analysis of the ossification sequences with different methodologies, for example

345 Parsimov, could be found that the heterochronic cranium elements, or ossification

346 timing appearing at different times, were the parasphenoid and prootic when we

347 compared S. ruber with H. pulchellus (Hoyos et al. 2012), but in Dendropsophus labialis

348 and Pseudis platensis were the frontoparietal, dentary and maxilla (Fabrezi and

349 Goldberg 2009). Ossification sequences of D. labialis and S. ruber showed that the

350 ossification timing of the parasphenoid was at the first rank. Meanwhile, the timing of

351 ossification of the exoccipital, frontoparietal and prootic was at the second rank in both

352 species.

353 Cranial and postcranial characters described (Table 4) show that among these Hylid

354 species, the number of ranks that includes elements of the skull and postcranium vary

355 from one to five. The ranks increase when postcranial elements are included,

356 demonstrating the importance of not excluding them. Weisbecker and Mitgutsch (2010),

357 Harrington et al. (2013), and Sheil et al. (2014) used these data for reconstructing

358 phylogenetic trees of amphibians with ossification sequences of cranial and postcranial

359 elements of Leptodactylidae, Ranidae and Bufonidae.

360 Some biological implications of the heterochronies, which are well known in

361 amphibians (Alberch 1985; Reilly et al. 1991), are to observe changes of structures in

362 shape and size, and the evolution of relative rates of growth of developmental events

363 among taxa (Raff 1996; Smith 2001; 2002; 2003). Some scholars recognize these

364 developmental events as modules with evolutionary implications, which can reveal

365 interactions or restrictions on development with individual events (Wagner 1996).

366 The intraspecific variation of the ossified elements could be linked with the genes

367 involved in these variations (Raff 1996). We should note that it is possible that the

368 difference in cartilage formation between the two species is due to paracrine factors

369 induced in cells that express mesodermal transcription factors involved in the activation

370 of gene specific to cartilage (Gilbert 2000; Kozhemyakina et al. 2015); the present study

371 did not account for these factors.

372 The detection of more intraspecific variability in Scinax ruber than in Dendropsophus

373 labialis could be due to the great intra-generic diversity within the clade Scinax ruber,

374 this issue has been poorly studied, then it could be studied in greater detail when

375 comparing the adults of these species (Fig. 5). Although amphibians are organisms that

376 have been extensively studied (Canatella and Trueb 1988; de Sá and Hillis 1990; Haas

377 2001; Baez and Pugener 2003; Roelants and Bossuyt 2005; Faivovich et al. 2005, Frost

378 et al. 2006; Pyron and Wiens 2011; Duellman et al. 2016) would solve taxonomic and

379 morphology voids.

380 Conclusions

381 The contribution of the ontogenetic data provided here is supported by the knowledge

382 that affords opportunity to understand the interactions between ontogeny and phylogeny

383 in morphological and ecological diversity of frog genera. Likewise, ossification

384 sequences data for the skeletal elements, combined with evolutionary hypotheses, may

385 shed light about patterns of development to support future phylogenetic hypotheses as

386 Larson (2003) claimed: “the variation in chondrocranial morphology in larval anurans

387 can be phylogenetically informative, even among closely related taxa”.

388 Acknowledgements

389 We thank to the Pontificia Universidad Javeriana and the Paläontologisches Institut

390 und Museum, Universität Zürich, for partial funding of this work. We want to thank

391 Timothy Sosa for assistance with the English translation.

392 The authors declare that they have no conflicts of interest of any kind.

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582 Figure legends

583 Fig. 1 Larval chondrocranium of Dendropsophus labialis (GS 34 - MUJ 9250) and

584 Scinax ruber (GS 34 - MUJ 6178). A. Dorsal view, B. Ventral view, C. Lateral view, D.

585 Ventral views of hyobranchial apparatus in Dendropsophus labialis (GS36 - MUJ 9250)

586 and Scinax ruber (GS36 - MUJ 3727). Scale 1 mm.

587 Chondrocranium: a, alae suprarostralis; ci, cartilago infrarostralis; cm, cartilago Meckeli;

588 cqc, commissura quadratocranialis; cs, suprarostral cartilage; ct, cornu trabeculae; fo,

589 fenestra ovalis; jf, jugular foramen; pal, processus anterolateralis; pmq, processus

590 muscularis quadrati; pof, prootic foramen; pq, palatoquadrate; oc, otic capsule; of,

591 oculomotor foramen; opf, optic foramen, ts, tectum sinoticum.

592 Hyobranchial apparatus: ca (I), copula anterioris; cb I–IV; ceratobranchialis I–IV; cp (II),

593 copula posterioris; ct; commisura terminalis; pah, processus anterioris hyalis; palh,

594 processus anteriolateralis hyalis; phb, planum hypobranchiale; plh, processus lateralis

595 hyalis; pph, processus posterioris hyalis; pr, pars reuniens; sp, spicula.

596 Blue: cartilage, light blue: fontanella.

597 Fig. 2 Appendicular skeleton of Dendropsophus labialis (GS45 – MUJ497) and Scinax

598 ruber (GS45 – MUJ6018). A. Scapula, B. Pectoral girdle, C. Ventral view pelvic girdle.

599 Scale 1 mm.

600 c, clavicle; co, coracoid; e, epicoracoid; ps, processus suprascapularis; o, omosternum;

601 st, sternum; il, ilium; is, ischium; pu, pubis

602 Red, ossified; blue, condrificated.

603 Fig. 3 Dorsal view of manus and pes of Dendropsophus labialis (GS45 – MUJ497) and

604 Scinax ruber (GS45 – MUJ6018). A. Manus, B. Pes. Scale 1 mm.

605 ce, centrale; fi, fibulare; mc, metacarpal, mt, metatarsus; ph, prehallux; pr, prepollex; rd;

606 radiale; rad, radioulna; ul, ulnare and intermedium; ti, tibiale.

607 I–V, phalanges.

608 Red, ossified; blue, condrificated.

609 Fig. 4 Ventral view of ossification development in vertebral column of Dendropsophus

610 labialis and Scinax ruber at GS 26-45. Scale 1 mm.

611 h, hypochord; a, Atlas; np, neural process; d, diapophysis; sd, sacral diapophysis; u,

612 urostyle

613 Red, ossified; blue, condrificated.

614 Fig. 5 Adult male of Dendropsophus labialis (MUJ 497) A. Ventral view, B. Dorsal view

615 and Scinax ruber (MUJ 9037) C. Ventral view, D. Dorsal view (Photo by Jorge Enrique

616 García Melo jorgegarciabiophoto.com). Scale 5 mm.

617 Red, ossified; blue, condrificated.

618

Table 1(on next page)

Table 1

Ossification sequence of cranial and postcranial elements in Dendropsophus labialis (Peters,

1863)

Rank

Development

stage*

(Number

specimens with

ossification)

Bone

Cranium Postcranium

I 35 (1) Transverse process I–V

II 36 (2) Parasphenoid Transverse process VI–VIII

III 37 (3)Frontoparietal,

exoccipitalNeural arches I–VIII

IV 38 (1) Hypochord

V 41 (3)

Femur, tibiofibula, humerus,

ilium, radioulna, clavicle, pubis,

metatarsal III–V, coracoids

VI 42 (3) Metacarpal IV, urostyle

VII 45 (2)

Mentomeckelian,

premaxilla, maxilla,

angulosplenial,

dentary

Manus IV proximal phalange,

Metacarpal III and V, scapula,

pedal digit IV proximal

phalange, Metacarpal I and II,

metatarsal I, prepollex

VIII 46 (1)

Neopalatine, nasal,

pterygoid, vomer,

septomaxilla,

squamosal

*Development stage sensu Gosner (1960)

Table 2(on next page)

Table 2

Ossification sequence of the cranial and postcranial elements in Scinax ruber (Laurenti,

1758).

1

Bones

Rank

Development

stage*

(Number

specimens with

ossification)

Cranium Postcranium

I 36 (6) Parasphenoid Transverse process I–VII

II 37 (9) Neural arch I–III

III 38 (3)Frontoparietal Transverse process VIII,

neural arch IV–VIII

IV 39–40 (3)

Femur, tibiofibula, humerus,

ilium, radioulna, scapula,

hypochord

V 43 (3) Ischium

VI 46 (1)

Mentomeckelian,

premaxilla,

maxilla,

angulosplenial,

dentary,

neopalatine,

pterygoid, vomer,

septomaxilla,

squamosal

2 *Development stage sensu Gosner (1960)

3

Table 3(on next page)

Table 3

Onset of ossification of cranial and poscranial elements of Dendropsophus labialis and Scinax

ruber

Specie Dendropsophus labialis Scinax ruber

Gosner Stage

(No. specimens)

35

(1)

36

(2)

37

(3)

38

(1)

41

(3)

42

(3)

43

(1)

45

(2)

36

(10

)

37

(11

)

38

(4)

39/4

0 (3)

41

(5)

42

(5)

43

(3)

44

(4)

45

(2)

Cranium Paraesfenoides 1 1 1 3 3 1 2 3 5 2 3 3 3 3 3 2

Exoccipital 1 1 1 3 3 1 2 3 3 3 4 3 3 2

Frontopariental 1 2 3 1 2 2 2 2 1 3 3 2

Poscranium

Transverse Process

I 1 6 9 3 3 3 4 3 3 2

II 1 1 1 1 3 3 1 2 5 6 3 3 3 4 3 3 2

III 1 1 1 1 3 3 1 2 5 6 3 3 3 4 3 3 2

IV 1 1 1 1 3 3 1 2 5 6 3 3 3 4 3 3 2

V 1 1 1 1 3 3 1 2 3 6 3 3 3 4 3 3 2

VI 1 1 1 1 3 3 1 2 3 6 3 3 3 4 3 2 2

VII 1 1 1 1 3 3 1 2 1 4 2 3 3 3 3 2 2

VIII 1 1 1 3 3 1 2 1 2 2 3 3 2 3 1

IX 1 1 3 1 1 3

Neural arch

I 1 1 1 3 3 1 2 3 3 3 3 4 3 3 2

II 1 1 3 3 1 2 3 3 3 3 3 3 3 2

III 1 2 3 3 3 3 3 3 2

IV 1 3 3 3 3 3 2 2

V 1 2 3 3 3 3 2

VI 1 2 3 3 1 3

VII 1 1 3 2 3

VIII 1 1 3 1 3

IX

Manus

Metacarpal I 1 1

Metacarpal II 1 1

Metacarpal III 1 2

Metacarpal IV 1 1 2

Metacarpal V 1 2

Metatarsal distal

tarsal IV 1 2

Metatarsal distal

tarsal IV 1 1

Prepollex 1 1

Pes

Metatarsal I 1 1

Metatarsal II 1 1 2

Metatarsal III 1 1 2 2

Metatarsal IV 1 1 2 2

Metatarsal V 1 1 2 2

Atlas 1 1 1 1 3 3 1 2 3 3 3 4 3 3 2

Femur 1 2 3 1 2 3 1 3 1

Fibula 1 2 3 1 2

Tibia 1 2 3 1 2

Tibiofibula 1 2 3 1 2 3 1 1 3 1 1

Scapula 1 2 2 2 3 1 3

Suprascapula 1 2 2 2

Clavicle 1 2 2 2 3 1 3 1

Coracoids 1 2 2 2

Humerus 1 2 3 1 2 1 1 3

Ulna 1

Radioulna 1 2 3 1 2 3 3 3

Table 4(on next page)

Table 4

Ossification sequences of different species of the family Hylidae including postcranial

elements.

Specie ElementNo.

ranksOssification sequence References

Hyla orientalis

C 5 ps [ex, po] fp [ns, sm] [an, de, ma, pm, pt, sq, vo]Yıldırım & Kaya,

2014C, P 6

ps [ex, hu, na, po, ve][ct, fe, fr, il, mc, tf, tl, ru][fp, cl, co, mt, pf, ph][ns, sm][an, de, is, ma, pm, pt, sq, pu, vo] qj

Dryophyteschrysoscelis

C 8 ps [ex][fe][fp][sm, pm, po][ma][de, ns, an, sq][vo]Sherman andMaglia, 2014C, P 10

ps [ex, na, cn][fe][sc, cl, co, hu, ra, ul, il, ti, fi, tb,mc, mt][ph, pf][fp][sm, pm, po][ma, is][de, ns, an, sq][vo]

Hyla versicolor

C 6 ps [ex, fp] [ma, pm, po] [an, de, sq] [pa, pt, qj]

Sheil et al., 2014C, P 7

ps [cl, co, fe, fi, fr, hu, il, na, ra, sc, ti, tl, ul] [ex, fp, mc, ph, pf, ve] [ma, pm, po] [an, de, sq] [ns, me] [pa, pt, qj]

Smilisca baudinii C 7fp [ex, ps, sm] [ag, de, ma, pm, sq] [ns, pt, qj] [me, pa, pv][cm, sp] po Trueb, 1966

[ex, fp, ps, sm][ma, pm, sq][ns, pt][pa, qj, vo][cm, et] so, po Gaudin, 1973

Triprion petasatus C 6 fp, ns [an, de, ex, ma, me, pa, pm, ps, pt, qj, sm, sq][cm, pv] sp, po Trueb, 1970

Dendropsophuslabialis

C 3 ps [ex, fp, po] [an, de, ma, me, np, pm, sm, sq, vo]

This studyC, P 8

[ve] [ps] [ex, fp, po] [cl, co, fe, hu, il, mt, ru, tf] [mc, sc][an, de, ma, me, np, pm, sm, sq, vo]

Pseudis platensis C 5 [fp, ps] [ex, po] [ns, pm, sq] ma [de, pt, vo]Fabrezi &

Goldberg, 2009

Scinax ruber

C 3 ps, [fp, po] [an, de, ma, me, np, pc, pm, sm, sq, vo]

This studyC, P 7

ps, ve [ex, fp, po][fe, hu, il, ru, sc, tf, tl] is [an, de, ma, me, pm, pc] [np, sm, sq, vo]

Hypsiboaslanciformis

C 8 fp, ps, ex, po [ pm, sm] [ns, ma][an, de, sq][sp, me, qj, vo, pa, pt]

De Sá, 1988C, P 9

fp [ps, ve] ex [fr, cl, co, ct, fe, hu, mc, mt, po, sc, tf, tl] pf [pm, sm] [ns, ma][an, de, is, sq][ap, me, pa, ph, pt, qj, sp, vo]

Hypsiboas C 2 [ex, fp, ps] [an, de, ma, pm, po, sq] Hoyos et al.,

pulchellus 2012C, P 4[ex, fp, ps, ve][fe, hu, il, ru, sc, tf][cl, co, ct, hy, mc, mt, pf, ph][an, de, ma, pm, po, sq]

Osteopilusseptentrionalis

C

3 fp, sm [ag, cm, de, et, ex, ma, me, ns, pa, pm, po, ps, pt, qj, sq, vo] Trueb, 1966

7 fp, sm [an, de, ex, ma, ns, pm, ps, pt, pv, sq] pa, qj [me, po, sp] cm Trueb, 1970

5 [fp, sm][ag, de, ex, ns, ma, pm, ps, pt, pv, sq] pa, qj [po, sp] Gaudin, 1973

C, P 13ps, ve, fe [hu, il, ra, su, ul] [ct, ex, fp, fr, sc, tf, tl] [cl, co, mt, pf, po] [mc, ph] is [ma, pm, sm] ns [an, vo] [me, pt, qj, qu, sq] sp

Pseudacris triseriata C 4 [ex, fp, pm] [de, ma, ns, pt, qj, sq, vo][m, po][cm, ha, pa, ps, sp]Stokely & List

1954

Pseudacris regilla C 6 ps, fp [ex, po] pv [ma, ns, pm, sm, sq] [ag, de, pt][cm, me, pa, qj, sp] Gaudin, 1973

hcris blanchardi

C 9 ps [ex, fp, po] [pm, sm]ma, ns, vo [an, de][me, pt, qj, qu, sq] sp

Havens, 2010C, P 11

ps, ve [ex, fe, fp, po][fi, hu, mt, pf, ra, sc, ul, tf, ti][cl, co, ct, il, mc, ph][is,pm, sm]ma, ns, vo, [an, de][me, pt, qj, qu, sq] sp

Phyllomedusavaillantii

C 4 [ex, po, ps] fp [ma, pm] [an, de, sm, sq]Sheil & Alamillo,

2005C, P 5[na, ve] [cl, co, ex, fe, fi, fr, hu, il, mc, mt, po, ps, pf, ph, ra, sc, ti, tl, ul] fp [is, ma, pm] [an, de, sm, sq]

C, cranium; P, poscranium

ag, angular; an, angulosplenial; ap, plectral apparatus; cl, clavicle; cu, columella; co, coracoid; ct, cleithrum; de, dentary;

et, ethmoid; ex, exoccipital; fe, femur; fi, fibula; fp, frontoparietalis; fr, fibulare; ha, hyoid apparatus; hu, humerus; hy,

hypochord; il, ilium; is, ischium; ma, maxilla; mc, metacarpals; me, mentomeckelian; mt, metatarsals; na, neural arches;

nc, neural center; np, neopalatine; ns, nasal; pa, palatine; pc, coronoid process; pr, presacral vertebrae; pf, phalanges of

feet; ph, phalanges of manus; pm, premaxilla; po, prootic; ps, parasphenoid; pt, pterygoid; pv, pre vomer; qj, quadratojugal;

qu, quadrate; ra, radius; ru, radioulna; sc, scapula; sm, septomaxilla; sp, sphenethmoid; sq, squamosal; su, suprascapula;

ti, tibia; tf, tibiofibula; tl, tibiale; tp, transverse process; ul, ulna; ve, vertebra (including na, nc, pr, tp); vo, vomer.

C = Cranial elements; P = Postcranial elements.

[Rank = absolute time of ossification of various structures simultaneously]

Figure 1

Figure 1

Larval chondrocranium of Dendropsophus labialis (GS 34 - MUJ 9250) and Scinax ruber (GS 34 - MUJ 6178).

A. Dorsal view, B. Ventral view, C. Lateral view, D. Ventral views of hyobranchial apparatus in

Dendropsophus labialis (GS36 - MUJ 9250) and Scinax ruber (GS36 - MUJ 3727).

Scale 1 mm. Chondrocranium: a, alae suprarostralis; ci, cartilago infrarostralis; cm, cartilago Meckeli; cqc,

commissura quadratocranialis; cs, suprarostral cartilage; ct, cornu trabeculae; fo, fenestra ovalis; jf, jugular

foramen; pal, processus anterolateralis; pmq, processus muscularis quadrati; pof, prootic foramen; pq,

palatoquadrate; oc, otic capsule; of, oculomotor foramen; opf, optic foramen, ts, tectum sinoticum.

Hyobranchial apparatus: ca (I), copula anterioris; cb I–IV; ceratobranchialis I–IV; cp (II), copula posterioris; ct;

commisura terminalis; pah, processus anterioris hyalis; palh, processus anteriolateralis hyalis; phb, planum

hypobranchiale; plh, processus lateralis hyalis; pph, processus posterioris hyalis; pr, pars reuniens; sp,

spicula. Blue: cartilage, light blue: fontanella.

Figure 2

Figure 2

Appendicular skeleton of Dendropsophus labialis (GS45 – MUJ497) and Scinax ruber (GS45 –

MUJ6018). A. Scapula, B. Pectoral girdle, C. Ventral view pelvic girdle. Scale 1 mm. c, clavicle;

co, coracoid; e, epicoracoid; ps, processus suprascapularis; o, omosternum; st, sternum; il,

ilium; is, ischium; pu, pubis Red, ossified; blue, condrificated.

Figure 3

Figure 3

Dorsal view of manus and pes of Dendropsophus labialis (GS45 – MUJ497) and Scinax ruber

(GS45 – MUJ6018). A. Manus, B. Pes. Scale 1 mm. ce, centrale; fi, fibulare; mc, metacarpal,

mt, metatarsus; ph, prehallux; pr, prepollex; rd; radiale; rad, radioulna; ul, ulnare and

intermedium; ti, tibiale. I–V, phalanges. Red, ossified; blue, condrificated.

Figure 4

Figure 4

Ventral view of ossification development in vertebral column of Dendropsophus labialis and Scinax ruber at

GS 26-45.

Scale 1 mm. h, hypochord; a, Atlas; np, neural process; d, diapophysis; sd, sacral diapophysis; u, urostyle

Red, ossified; blue, condrificated.

Figure 5

Figure 5

Adult male of Dendropsophus labialis (MUJ 497) A. Ventral view, B. Dorsal view and Scinax

ruber (MUJ 9037) C. Ventral view, D. Dorsal view (Photo by Jorge Enrique García Melo

jorgegarciabiophoto.com). Scale 5 mm. Red, ossified; blue, condrificated.