A simplified table for staging embryos of the pipid frog Pipa arrabali

Pipa is a Neotropical genus of frogs that dwell in freshwater environments. It includes four species that lack free-swimming larvae (P. aspera, P. arrabali, P. pipa, and P. snethlageae) and three with tadpoles (P. carvalhoi, P. myersi, and P. parva). Developmental tables such as the one proposed by Nieuwkoop and Faber might be useful for Pipa species with tadpoles. However, for the other Pipa species, to determine stages by this table or by any of the tables already prepared for frogs without tadpoles (e.g., Crinia nimbus, Eleutherodactylus coqui, and Oreobates barituensis) is impossible. By using embryonic, juvenile, and subadult specimens, we generated a staging table for P. arrabali, from the moment limb buds were first observed until birth, based on diagnostic features such as snout-vent length; growth, morphology, and reabsorption of the external tail; growth and differentiation of fore and hind limbs; development of intestine and vent tube; position of the angle of the mouth relative to nostrils and eyes; and color of preserved individuals. Based on these observations, we discuss some noteworthy traits (e.g., posture of hands and feet). We also compare the pattern of development of P. arrabali with that of other anuran species (with and without tadpoles).


inTrOduCTiOn
The conventional idea of frog development is based on the reproductive mode of the so-called 'typical' anurans such as Rana, which is characterized by large clutches of small aquatic eggs and aquatic exotrophic larvae.Anurans, however, exhibit an extraordinary diversity in reproductive patterns including, among others, the bypass of a free-living larval stage.Examples of this reproductive mode are found amongst brachycephalids (Pombal Jr et al. 1994), eleutherodactylids (Townsend and Stewart 1985), hemiphractids (Del Pino and Escobar 1981); pipids (Trueb andCannatella 1986, Fernandes et al. 2011), and rhacophorids (Kerney et al. 2007).In the Neotropical pipid frogs of the genus Pipa, small clutches of eggs develop in brood chambers temporarily formed in the skin of female's back (Rabb and Rabb 1960, Trueb and Cannatella 1986, Fernandes et al. 2011).The eggs hatch as tadpoles OLívIA G.S. ARAúJO, CéLIO F.B. HADDAD, HéLIO R. DA SILvA and LOURDES A. PUGENER that complete their development in a pond in P. carvalhoi, P. myersi, and P. parva (Fernandes et al. 2011, Trueb andCannatella 1986).In these species the eggs are relatively small (Garda et al. 2006).On the contrary, eggs are large and yolky in P. aspera, P. arrabali, P. pipa, and P. snethlageae (Trueb and Cannatella 1986, Garda et al. 2006, Wells 2010), and birth occurs after metamorphosis is completed (Rabb and Rabb 1960, Trueb and Cannatella 1986, Trueb et al. 2000).Of these, P. arrabali, commonly known as Small Surinam Toad, Arrabal's Suriname Toad, or Rana de Celdillas Guayanesa, is the only species in the genus that occurs in ephemeral freshwater ponds not only at elevations inferior to 450 m, like all other species, but also at 860 m, in the tropical moist lowland of Guyana, Suriname, eastern venezuela, and northern and central Brazil (Buchacher 1993, Frost 2015).
Several preceding studies have proposed tables of development for staging anuran embryos and larvae (e.g., Taylor and Kollros 1946, Nieuwkoop and Faber 1956, Gosner 1960, Fabrezi et al. 2009), most of which apply to species that have tadpoles.However, these tables are less useful for staging embryos that complete their development as nonfree living larvae because their external morphology deviates markedly from that of aquatic larvae after neurulation.On the other hand, the tables created for staging egg-brooding anurans, e.g., Gastrotheca riobambae (Del Pino and Escobar 1981) or anurans that put terrestrial eggs and lack free-swimming larval stages, e.g., Crinia nimbus (Mitchell and Swain 1996), Eleutherodactylus coqui (Townsend and Stewart 1985) and Oreobates barituensis (Goldberg et al. 2012), are species-specific and cannot be applied to Pipa.Likewise, the simplified external description of a few specimens of P. pipa by Parker (1876) and Ridewood (1897), and the staging of embryos of the same species based on size by Trueb et al. (2000) render comparisons difficult to make.
Herein we have designed a staging table for embryos of Pipa arrabali in their later stages of development-that is, from the moment limb buds are first observable until birth.We also compared the pattern of embryonic development of P. arrabali with that of other anuran species for which data are available.

MaTerialS and MeTHOdS
The staging table was created based on observations of the external morphology of 78 preserved embryos and juveniles of Pipa arrabali from Serra do Cachimbo, Novo Progresso, Pará State, Brazil.The specimens were fixed in 10% formalin in the field and are deposited in the Coleção Herpetológica da Universidade de Brasília (CHUnB).Four embryos were removed from the anterior, middle, and posterior sections of the dorsum of 17 females.The snout-vent length and head width of each embryo were measured under a stereomicroscope with the aid of a caliper (Appendix).Some specimens from each stage were prepared as cleared-and-doublestained skeletons following the methodology by Taylor and van Dyke (1985).The length of the incubatory period was not determined because brooding females were never collected at the time of amplexus and fertilization, and therefore, the beginning of incubation was unknown.
Development was divided into two periods.The first one included the early stages of development, from the fertilized egg until the stage previous to the emergence of the limb buds.The second period comprised the later stages of development; from the moment limb buds were first observed until birth.The second period (focus of our research) was divided into 11 stages based on easily discernible features that were used as markers.These features met three conditions, they could be detected with minimal ambiguity, were readily identified by examination with a stereomicroscope, and their presence among individuals was invariable.
The anatomical lexicon used herein for external morphology follows Gosner (1960).For the lateral line system we used the terminology proposed by Shelton (1970), who developed a naming system based on Xenopus that is more similar to Pipa than that used for other frogs.Descriptions and illustrations were made with the aid of a stereomicroscope equipped with a camera lucida and digital camera.

reSulTS and diSCuSSiOn
Although embryonic development is a continuum (Fig. 1), for comparative purposes we divided this process into early and late development (Periods I and II, respectively).The division between both periods is demarcated by a distinct event, the emergence of the limb buds.The key diagnostic characters of each stage are summarized in Table I; additional traits are provided in Table II.
During the first few stages of development of the second period, some aspects of the embryos of Pipa arrabali resemble a generalized biphasic anuran prior to the initiation of the process of metamorphosis (approximately between .The features that contribute to this resemblance include an oblong body, the corner of the mouth rostral to the anterior margin of the eye, and a long and muscular tail.Throughout these stages we also found overall similarities in the sequence of morphological changes that affect the initial development of the hind limbs; i.e., emergence of distally rounded hind limbs during Stage II-1 (about Gosner Stages 26-30; Nieuwkoop and Faber Stages 46-52) and development of a paddle-shaped outline of the foot during Stage II-2 (between Gosner Stages 31-33; ~ Nieuwkoop and Faber Stages 53).The aforementioned embryonic morphology is different from that observed in other anurans that lack a free-living larval stage, such as Eleutherodactylus coqui (Townsend and Stewart 1985, Callery et al. 2001, Elinson 2001).In this species some region develop precociously, displaying what in species with tadpoles would be a mid-metamorphic or even a postmetamorphic morphology.
The mouth of the embryo of Pipa arrabali is terminal, transverse, and slit-like (Fig. 2a and  2c), lacking the oral keratinous structures (i.e., jaw sheath and labial teeth) that most tadpoles usually have (vitt and Caldwell 2014).The same mouth morphology observed in the developing P. arrabali is also shared by other species of Pipa (Sokol 1977,  Pugener et al. 2003), both with and without freeliving larvae.Barbels (= tentacles of Nieuwkoop and Faber 1956) at each corner of the mouth, similar to those found in other extant and extinct pipids such as Silurana, Xenopus, and †Llankibatrakus (Sokol 1977, Baéz andPugener 2003), are absent.Barbellae (i.e., the short and slender marginal structures around the mouth of Rhinophrynus, Orton 1943) also are absent.A keratinized eggtooth such as the one present in, for example, Eleutherodactylus (Hardy Jr 1984, Townsend andStewart 1985) is lacking in P. arrabali.2 Hind limb with paddle-shaped outline of foot (Fig. 2g).

3
Outline of all five toes visible.Knee articulation evident as a slight curvature (Fig. 2g).

34-35 54-55
Corner of the mouth extending slightly beyond the level of the nostril (Fig. 2a).43 63 4 Toes separated from each other; Toe III is the longest (Fig. 2g).

36-37 --
Corner of the mouth extending to the level of the anterior margin of the eye (Fig. 2a).44 64 5 Forelimbs outside gill chamber.42 57 Fingertips with four minute, terminal lobes (Fig. 2f).
Upper and middle lateral lines visible on both sides of the body (Fig. 2d).

6
Melanophores in the midventral region of the body.
Hands covering the eyes and anterior end of the head.

7
Intestine no longer visible through transparency.
Lobes of fingertips slightly apart from each other; melanocytes visible in the space between lobes (Fig. 2g).
Dermal modification of the upper lip observable at the angle of the mouth opening (Fig. 2e).

--
Corner of the mouth extending to the level of the posterior margin of the eye (Fig. 2a).45 65 9 Subarticular tubercles visible (Fig. 2g).
vent tube absent.45 10 Hands and feet covering the anterior region of the head.

11
Dermal fold on the dorso-posterior region of the head partially covering the eyes (Fig. 2a).
Hands and feet no longer covering the head.

TABLE II (continuation)
In Pipa arrabali there is an early backwards migration (Stage II-8) of the corner of the mouth (to the level of, or posterior to, the posterior margin of the eye) when compared with biphasic species.In the latter, the above-mentioned change occurs close to the end of metamorphosis (e.g., about Gosner Stage 44 in Bombina orientalis and Nieuwkoop and Faber Stage 65 in Xenopus laevis).On the contrary, by Stage II-8 (four stages before the end of metamorphosis) the external morphology of the head of an embryonic P. arrabali is substantially different from its initial appearance, and reminiscent of a late metamorphic stage in biphasic anurans.
Pipa arrabali displays a dermal flange on the upper lip at the angle of the mouth opening by Stage II-7.It consists of a narrow labial pocket surrounding the corner of the mouth, and a simple piece of tissue shaped like a teardrop that partially covers over the posterior end of the lower lip (Fig. 2f).This upper lip adornment grows larger by Stage II-8, decreasing in size in the subsequent stage until almost disappearing by the Stage II-10.A dermal modification of the upper lip occurs in the metamorphic stages of all other small species of Pipa, (i.e., P. aspera, P. carvalhoi, P. myersi, and P. parva) and persists in the adults, although proportionally smaller (Trueb and Cannatella 1986).Apparently, the same structure occurs in P. pipa and P. snethlageae, but it is larger and with an irregular edge.All others species of Xenoanura (Olívia G.S. Araújo, personal observation, Trueb and Cannatella 1986) lack adornments on the upper lip, which suggests that such feature may represent a synapomorphy for the genus Pipa.
Four minute terminal lobes arise at the end of the fingers of Pipa arrabali by Stage II-5.These lobes occupy the entire tip surface, creating a quadripartite digital tip morphology (Fig. 2f).In subsequent stages there is an apparent fusion of the lobes into pairs, resulting in a dorsoventrally flattened fingertip.By Stage II-7, growth of the fingers leads to a slight separation of the lobes, with melanocytes clearly visible between them (Fig. 2f).By Stage II-11 the fingertip lobes are larger and exhibit a radial distribution.This peculiar modification of the adult fingertips is a distinctive characteristic of Pipa, with the exact shape and distribution of the lobes varying by species (Trueb and Cannatella 1986).For example, in the adult P. parva and juvenile P. myersi and P. pipa there are four asymmetrically distributed fingertip lobes; two of the lobes are fused in the adult P. myersi, creating a tripartite digital tip morphology.On the other hand, the adults of P. aspera, P. carvalhoi, P. pipa, and P. snethlageae exhibit an arrangement of the fingertip lobes similar to that described for P. arrabali.Unfortunately, further comparisons are hampered by the lack of information on the timing and process of development of these structures in the other Pipa species.
The body of a Stage II-1 embryo of Pipa arrabali lies on a large, wide-bore, coiled intestine filled with yolk; this condition persists until Stage 1b,and 2c).As development proceeds, however, the yolk reserve slowly diminishes and the lumen of the intestine decreases to about half the size observed in previous stages.The initial length of the intestine is about one and a half coils by Stage II-1, it increases to three coils by Stage II-3, and shortens to about one and a quarter coils by Stage II-5.The axis of the coils is perpendicular to the body and the apex points downward.The concomitant development of the rectus abdominis completely conceals the intestine by Stage II-6 (i.e., the intestine becomes immersed within the pleuroperitoneal cavity).It is worth noting that, although the intestine of the embryo of P. arrabali is coiled, it is extremely short when compared to the seven or eight turns of the intestine of P. carvalhoi and P. parva free-living larvae (Sokol 1977).In the latter, the long, narrow-bore, highly coiled intestine implies a greater surface area available for digestion, whereas the shorter and wider intestine of P. arrabali only seems to fulfill a storage function.A wide intestine with a few coils has also been observed in, for example, embryos of Pseudophilautus sarasinorum (Bahir et al. 2005), a species that lacks free-living larvae.Its function in these embryos is unclear, although it could be related to yolk storage and absorption.
Embryos of Pipa arrabali have a muscular tail that, at full length, slightly exceeds the snout-vent length.In the first stages of Period II it consists of a muscular stem and a low membranous fin (including dorsal and ventral portions), with a round distal tip.The dorsal portion of the fin originates at about the area of the tail-body junction.The tail is both oriented to the left and right, and occasionally even folded under the body in older embryos.By Stage II-5 the tail muscles are weaker, and reabsorption is well under way by Stage II-6.At Stage II-7 the tail appears as a narrow and translucent piece, and by Stage II-11 it is extremely reduced or absent.The reduction of the muscles of the tail is synchronic with an increase in fin vascularization.By Stage 7-II, the muscular portion is almost absent whereas the fin size remains unchanged and the blood vessels are more conspicuous.Furthermore, the increase in fin vascularization is coincident with the reduction in size of the intestinal lumen and the incorporation of the gut inside the pleuroperitoneal cavity.We do not know if there is a correlation between gut size and tail vascularization; if it does indeed exist, perhaps it could indicate a role in nutrient absorption from the wall of the brood chamber once the yolk stored in the embryo´s intestine is depleted.Fernandes et al. (2011) argued in favor of some type of interaction between embryonic and maternal tissues in P. carvalhoi.This suggestion was based on the presence of a large number of blood capillaries in the skin of the embryo, and a thin epidermis lacking a stratum corneum and a heavily vascularized dermis forming the walls of the brood chambers (Greven andRichter 2009, Fernandes et al. 2011).A vascularized tail may also suggest a role in gas exchange.A function of this kind has arguably been proposed for the highly vascularized tail in embryos of Eleutherodactylus coqui (Townsend and Stewart 1985) and Pseudophilautus (Bahir et al. 2005).
Pipa arrabali seems to exhibit a synchronous initial development of fore-and hind limbs.This pattern is similar to that reported for Discolossus pictus (Schlosser 2001), Eleutherodactylus coqui (Townsend and Stewart 1985), and Pseudis platensis (Fabrezi et al. 2009).This synchrony may be more widespread among anurans than usually reported.However, because in most free-swimming tadpoles forelimbs develop hidden inside an opaque gill chamber, their developmental morphology, prior to the external emergence through the skin that covers the gill chamber, do not constitute an informative feature that can be used in the staging process (e.g., Gosner 1960).
At Stage II-6, embryos of Pipa arrabali use their hands to cover the anterior region of the head (Fig. 1d) and by Stage II-10 the feet are also used in a similar way (Fig. 1e and 1f).By Stage II-11, however, neither hands nor feet are found in this position (Fig. 1g).We are not aware of any reports describing this peculiar position of the limbs neither in other pipids, nor in other anurans lacking free-swimming tadpoles such as Eleutherodactylus (Townsend and Stewart 1985, Callery et al. 2001, Elinson 2001), Arenophryne rotunda (Anstis et al. 2007), Myobatrachus gouldii (Anstis et al. 2007), Philautus silus (Kerney et al. 2007), andGastrotheca (Del Pino andEscobar 1981).It is likely, however, that other species of Pipa that are born as froglets may display similar postures; this, in fact, was observed in embryos of P. pipa (Olívia G.S. Araújo, personal observation).We think that this posture may represent a protective reflex.Rabb and Snedigar (1960) reported embryos of P. pipa moving away from light.Likewise, the limb postures described herein may represent a response to the killing process.
This study provides staging criteria for embryos of Pipa arrabali as a basis for future research on this species, as well as on the other three species of Pipa that have no free living tadpoles.In addition, by correlating this table with the staging tables by Gosner (1960) and Nieuwkoop and Faber (1956) we offer a basis for comparison with species that develop as free swimming tadpoles.The availability of this staging table is crucial for understanding the mosaic of morphological and developmental features that characterize Pipidae in general, and Pipa in particular.

Figure 2 -
Figure 2 -Camera lucida drawing of Pipa arrabali embryos.a -Left lateral view of the head of Stage II-2 (CHUnB 35027-D), II-4 (CHUnB 35000-D), II-8 (CHUnB 35056-D), and II-11 (CHUnB 35053-D) showing the differences in the position of the angle of the mouth relative to nostrils and eyes; in II-11 also showing the dermal fold covering partially the eye.b -Lateral view of left forelimb, inside gill chamber, of Stage II-2 (CHUnB 35027-D), showing the elbow joint (in the drawing was omitted the cover skin of the gill chamber, focusing only on the forelimb).c -Fronto-ventral view of Stage: II-2 (CHUnB 35027-D) showing the coiled intestine full of yolk.d -Left lateral view of the trunk, in between arm and leg, of Stage II-5 (CHUnB 35045-D) showing the pores of the upper and middle lateral lines.e -Left lateral view of the mouth of Stage II-7 (CHUnB 35030-D) showing the dermal modification of the upper lip.f -Latero-frontal view of the fingertip morphology of Stages II-5 (CHUnB 35045-D) and II-7 (CHUnB 35030-D).g -ventral view of the right hind limb of Stages II-2 (CHUnB 35027-D), II-3 (CHUnB 35055-D), II-4(CHUnB 35000-D), II-8 (CHUnB 35056-D), and II-9 (CHUnB 35051-D) showing feet development and differentiation.

TaBle ii All discernible features of Pipa arrabali embryos discriminated by stage. When applicable, approximate Gosner (1960) and Nieuwkoop and Faber (1954) stages are indicated for some of the traits. Stage Specimens Head and body
OLívIA G.S. ARAúJO, CéLIO F.B. HADDAD, HéLIO R. DA SILvA and LOURDES A. PUGENER