Morphological characterization and taxonomic key of tadpoles (Amphibia: Anura) from the northern region of the Atlantic Forest

Although anuran tadpoles are widely distributed and abundant in tropical aquatic habitats, there is a lack of taxonomic keys for the Atlantic Forest. Herein, we developed a dichotomous key for identifying the tadpoles for all species with known larval phase and already recorded in the Atlantic Forest north of the São Francisco River. We analyzed discrete characteristics of 1,042 tadpoles encompassing 63 species of 28 genera from 32 localities. The userfriendly key includes illustration and pictures, and it is a significant step towards improving our knowledge of tadpoles of the Atlantic Forest.


Introduction
The majority of the more than 7,000 currently known species of anurans (AmphibiaWeb 2020, Frost 2020) have a larval phase . Tadpoles occur in a great variety of aquatic habitats, including ponds, streams, phytotelmata, and shallow films of water in splash zones of rivers and waterfalls . Tadpoles experience a variety of selective pressures in aquatic habitats and thus exhibit extensive ecological and morpho-functional variation (Duellman & Trueb 1994, Rolents et al. 2011, Sherrat et al. 2017, 2018. Although the number of studies on Neotropical tadpoles is growing, there are still several knowledge gaps regarding their natural history, habitat use, microhabitat occupation, feeding behavior and ecology (Provete et al. 2012, Rossa-Feres et al. 2015. Brazil harbors the richest amphibian fauna worldwide (AmphibiaWeb 2020), yet tadpoles have been described for only about 60% of its species (Provete et al. 2012). Furthermore, this number is underestimated because many new species have been described without the larval phase being reported (Rossa-Feres et al. 2015). This gap in tadpole descriptions is further compounded by the few identification keys available for Neotropical tadpoles (e.g., Lips & Savage 1996, Rossa-Feres & Nomura 2006, Schulze et al. 2015, which are restricted to specific regions. For the Atlantic Forest, home to a remarkable diversity of anurans (more than 625 species) with high endemism (78%) (Rossa-Feres et al. 2017), identification keys are only available for the Southern and Southeastern regions (Rossa-Feres & Nomura 2006, Machado & Maltchink 2007, Pimenta et al. 2014, Fatorelli et al. 2017, Pezzuti et al. 2019. The limited number of taxonomic keys hampers the correct identification of anuran larvae, thus hindering studies on ecology, systematics and conservation , Andrade et al. 2007).
The Atlantic Forest north of the São Francisco River is also known as the Pernambuco biogeographical sub-region (Ribeiro et al. 2009). This sub-region is characterized by dense ombrophilous vegetation, most of it replaced by monocultures and pastures (Assis 2000, Studer et al. 2015, resulting in a vastly fragmented landscape with most
All examined specimens are housed in the following herpetological collections:  Grande (LHUFCG). Specimens were identified up to species level based on one of the following methods: (i) DNA barcoding approach (fragment of mitochondrial 16S rRNA) gene, (ii) observation of metamorphosed specimens in captivity, (iii) developing from eggs obtained from identified mating pairs, (iv) larvae from the same lot used in the original description of the species, or (v) direct comparison with the original description. Species nomenclature followed Frost (2020).
Because morphological features vary along tadpole's ontogenetic development , Grosjean 2005, Laufer et al. 2013, we evaluated 21 morphological characteristics in tadpoles at development stages 30-40 (Gosner 1960), and 51-58 (Niewkoop & Faber 1956) for Pipa larvae. Nevertheless, if these stages were not available, adjacent younger and older tadpoles were examined. Morphological characterization and terminology followed : body shape in dorsal and lateral views; snout shape in dorsal and lateral views; eye position, nostrils aperture configuration; distance of nostrils from the tip of the snout and eyes; presence and position of the oral disc, distribution of marginal papillae; labial tooth row formula (LTRF); spiracle number and position; vent tube position; dorsal fin origin; dorsal and ventral fins height and contour; relative length of tail; tail tip shape; and presence of flagellum (only for those who have it). All observations were made using a Coleman © NSZ 405 stereomicroscope.
To define body morphogeometric states to standardize nomenclature, at least three taxonomically unrelated species were used. The body contour around those corresponding shapes was blended for a better view of shape variations in one same state seeking to eliminate the effect of rotation and scaling. The resulting shapes are shown in Figure 2.
The identification key was elaborated in a taxonomically inclusive way, with terminals at family, genus, species group (following the most current phylogenetic proposals, see below) and species. Exception to two pairs of species: Phyllodytes brevirostris Peixoto & Cruz, 1988 and P. edelmoi Peixoto, Caramaschi & Freire, 2003; and the pairs Physalaemus albifrons (Spix, 1824) and P. cuvieri Fitzinger, 1826, which were reciprocally grouped together in the same terminal because no morphological variations that diagnosed these tadpoles were identified. Characters grouping monophyletic taxa were preferred over those grouping ecomorphotypes. People with different degrees of knowledge about tadpole morphology tested the key.
The characterization of genera and species groups followed the following sequence: (i) list of the species that occur in the region and the locality from where the described tadpoles were obtained, (ii) list of the examined specimens, with the following data: acronym and catalog number in scientific collections, number of individuals analyzed, range of developmental stages, and locality from where the tadpoles were obtained, (iii) morphological characterization of specimens examined, (iv) notes: comparison with tadpoles described from other localities, if morphological differences were detected.
Characteristics: Body ovoid in dorsal view, globular-depressed in lateral view. Snout oval in dorsal and lateral views. Nostrils reniform, closer to snout than to eyes. Eyes dorsal. Oral disc anteroventral, emarginate laterally. Marginal papillae arranged lateroventrally, with a wide dorsal gap. LTRF 2(2)/3. Upper jaw sheath with medial reentrance. Spiracle sinistral. Vent tube medial. Dorsal fin originating at tail-body junction, dorsal and ventral fins parallel to longitudinal axis of tail. Tail length about 70% of total length, tail tip acute.
Notes: The tadpoles of A. olfersioides described by Verdade & Rodrigues (2007) from Tijuca, state of Rio de Janeiro, Brazil, differ from those studied here by having the LTRF 2(1)/3, nostrils circular and dorsal fin originating on the body.
Characteristics: Body elliptical in dorsal view, globular-depressed in lateral view. Snout oval in dorsal and lateral views. Nostrils elliptical, closer to eyes than to tip of snout. Eyes dorsal. Oral disc ventral, with two emarginations on posterior margin. Marginal papillae arranged lateroventrally, with a short dorsal gap. LTRF 3(1,3)/5(1). Spiracle sinistral. Vent tube dextral. Dorsal fin originating at tail, dorsal and ventral fins parallel to longitudinal axis of tail. Tail length about 70% of total length, tail tip acute.
Boana pulchella species group -Body elliptical in dorsal view, globular-depressed in lateral view. Snout oval in dorsal and lateral views. Nostrils reniform, equally distant from eyes and tip of snout. Eyes dorsal. Oral disc ventral, two emarginations on posterior margin. Marginal papillae arranged lateroventrally, with a wide dorsal gap. LTRF 2(1,2)/4(1). Spiracle sinistral. Vent tube dextral. Dorsal fin originating at tail-body junction. Dorsal and ventral fins parallel to longitudinal axis of tail. Tail length approximately 60% of total length, tail tip acute.
Boana punctata species group -Body elliptical in dorsal view, globular-depressed in lateral view. Snout rounded in dorsal view, oval in lateral view. Nostrils reniform, equally distant from eyes and tip of snout. Eyes dorsal. Oral disc anteroventral, two emarginations on posterior margin. Marginal papillae arranged lateroventrally, with a wide dorsal gap. LTRF 2(1,2)/3(1). Spiracle sinistral. Vent tube dextral. Dorsal fin originating at tail-body junction, dorsal and ventral fins parallel to longitudinal axis of tail. Tail length approximately 60% of total length, tail tip acute.
Boana semilineata species group -Body oval in dorsal view, globular-depressed in lateral view. Snout oval in dorsal and lateral views. Nostrils reniform, equally distant from eyes and tip of snout. Eyes dorsal. Oral disc anteroventral, two emarginations on posterior margin. Marginal papillae arranged lateroventrally, with a wide dorsal gap. LTRF 2(2)/4(1). Spiracle sinistral. Vent tube dextral. Dorsal fin originating at tail-body junction, dorsal and ventral fins arched. Tail length approximately 60% of total length, tail tip acute.
Dendropsophus leucophyllatus species group -Body ovoid, elongated in dorsal view, triangular-depressed in lateral view. Snout oval in dorsal and lateral views. Nostrils circular, anteriorly positioned, much closer to tip of snout than to eyes. Eyes laterally positioned. Oral disc anterior, not emarginate. Marginal papillae arranged ventrolaterally, with one dorsal gap and one pair of narrow ventrolateral gaps. LTRF 0/1. Spiracle sinistral. Vent tube dextral. Dorsal fin originating at tail-body junction, dorsal and ventral fins slightly arched. Tail length about 60% of total length, tail tip acute.
Dendropsophus marmoratus species group -Body ellipticalelongated in dorsal view, triangular-depressed in lateral view. Snout rounded in dorsal view, oval in lateral view. Nostrils circular, anteriorly positioned, much closer to tip of snout than to eyes. Eyes laterally positioned. Oral disc anterior, not emarginate. Marginal papillae arranged ventrolaterally, with one dorsal gap and one pair of narrow ventrolateral gaps. LTRF 0/1. Spiracle sinistral. Vent tube dextral. Dorsal fin originating at body, dorsal and ventral fins slightly arched. Tail length about 60% of total length, tail tip acute.
Dendropsophus microcephalus species group -Body ovoid, elongated in dorsal view, triangular-depressed in lateral view. Snout oval in dorsal and lateral views. Nostrils circular, anteriorly positioned, much closer to tip of snout than to eyes. Eyes laterally positioned. Oral disc anterior, not emarginate, modified in a protractile and conic-shaped tube. Marginal papillae absent. LTRF 0/0. Spiracle sinistral. Vent tube dextral. Dorsal fin originating at body or tail-body junction, dorsal fin slightly arched, ventral fin parallel to longitudinal axis of tail. Tail length about 60-70% of total length, tail tip acute, with flagellum.
Dendropsophus minutus species group -Body elliptical-elongated in dorsal view, triangular in lateral view. Snout rounded in dorsal view and truncate in lateral view. Nostrils circular, anteriorly positioned, much closer to tip of snout than to eyes. Eyes laterally positioned. Oral disc anterior, not emarginate. Marginal papillae arranged ventrolaterally, with a wide dorsal gap. LTRF 1/2. Spiracle sinistral. Vent tube dextral. Dorsal fin originating at body, dorsal and ventral fins arched. Tail length about 60% of total length, tail tip acute, with flagellum.
Notes: Tadpoles of D. minutus vary in LTRF, with the specimens analyzed here having 1/2, while the specimens analyzed by Rossa-Feres & Nomura (2006)  Characteristics: Body elliptical in dorsal view, globular-depressed in lateral view. Snout rounded in dorsal and lateral views. Nostrils circular, closer to eyes than to tip of snout or equally distant. Eyes dorsal. Oral disc ventral, not emarginated. Marginal papillae arranged Characteristics: Body ovoid or elliptical in dorsal view, globulardepressed in lateral view. Snout rounded in dorsal view, sloped in lateral view. Nostrils circular, closer to tip of snout than to eyes. Eyes dorsal. Oral disc ventral, not emarginate. Marginal papillae arranged ventrolaterally, with a wide dorsal gap, absent in P. gyrinaethes. LTRF 2(2)/4[5] or 1(1)/5, the latter only for P. gyrinaethes. Spiracle sinistral. Vent tube dextral. Dorsal fin originating at the end of body or at tailbody junction, dorsal fin arched or parallel to longitudinal axis of tail and ventral fins parallel to longitudinal axis of tail. Tail length 65% of total length, tail tip rounded.
Notes: Morphologically, P. gyrinaethes differs from all congeners by having a bell-shaped body in dorsal view, wider posteriorly and with lateral reentrances in the midbody; snout flat with a deep indentation at the anterior margin in dorsal view. Peixoto et al. (2003) reported specimens of P. edelmoi with LTRF 2(2)/5[6], while the specimens analyzed in the present study had LTRF 2(2)/5. Scinax Wagler, 1830 Species occurring in the region. Scinax rostratus species group (Faivovich et al. 2005) -Scinax auratus (Wied-Neuwied, 1821). Although this species was included in the Scinax ruber clade, it was not currently associated with any species group by Faivovich et al. (2005). Here this species was treated as related to S. rostratus group by overall morphological resemblance of their tadpoles, and for sharing some diagnostic larval characteristics with this group  (1975) Characteristics: Scinax rostratus species group -Body elliptical in dorsal view, triangular-depressed in lateral view. Snout rounded in dorsal view, oval in lateral view. Nostrils circular, slightly closer to eyes than to tip of snout. Eyes lateral. Oral disc anteroventral, two emarginations on lower margin, labial arm present. Marginal papillae arranged ventrolaterally, with a wide dorsal gap, absent at the labial arm. LTRF 2(2)/3(1), with the P3 located at end of labial arm. Spiracle sinistral. Vent tube dextral. Dorsal fin originating at posterior third of body, dorsal fin and ventral fins slightly arched. Tail length about 65% of total length, tail tip acute.
Scinax ruber clade -Body elliptical in dorsal view, triangular or triangular-depressed in lateral view. Snout rounded in dorsal view, oval or truncate in lateral view. Nostrils oval, closer to tip of snout than to eyes. Eyes lateral. Oral disc anteroventral, two emarginations on lower margin. Marginal papillae arranged ventrolaterally, with a wide dorsal gap. LTRF 2(2)/3 [exception in S. pachycrus: 2(2)/3(1)]. Spiracle sinistral. Vent tube dextral. Dorsal fin originating at the middle or end of body, dorsal and ventral fins arched. Tail length about 70% of total length, tail tip acute.
Specimens examined. Sphaenorhynchus prasinus: MZUESC (these tadpoles were obtained in a recent sampling, right on with the closing of Universities during COVID-19 pandemic, preventing us from getting voucher numbers for these specimens, n =10, stages 33-38), municipality of Ilhéus, state of Bahia, Brazil, located at south of the São Francisco River. They were included because although there are records of this species for the northern Atlantic Forest (Almeida et al. 2016), there are no tadpole specimens.
Characteristics: Body ovoid in dorsal view, triangular-depressed in lateral view. Snout rounded in dorsal view, sloped in lateral view. Nostrils reniform, closer to tip of snout than to eyes. Eyes lateral. Oral disc anteroventral, not emarginate. Marginal papillae arranged ventrolaterally, with a wide dorsal gap, few large marginal papillae (highlighted papillae) on the anterolateral and posterolateral margins (large papillae about twice the size of the small papillae and alternating among them). LTRF 2(2)/3(1). Spiracle sinistral. Vent tube medial. Dorsal fin originating at tail-body junction, dorsal fin parallel to longitudinal axis of tail and ventral fin slightly arched. Tail length about 60% of total length, tail tip acute.
Specimens examined. Trachycephalus mesophaeus: MUFAL 15457 (n = 6, 31-34), municipality of Porto Seguro, state of Bahia, Brazil, located at south of the São Francisco River. They were included because although there are records of this species for the northern Atlantic Forest (Almeida et al. 2016), there are no tadpole specimens.
Specimens examined. Adenomera sp.: LHUFCG 0110 (n = 1, stage 36; Figure 7F), northeastern Brazil. The populations occurring in the Atlantic Forest domain are probably an undescribed taxon and there are no tadpole specimens available, so they were not included in the key. However, due to the phylogenetically conserved morphology of these endotrophic tadpoles, we present here a general characterization for the genus based on specimens obtained in the Cerrado biome.
Characteristics: Body ovoid in dorsal view, oval-depressed in lateral view. Snout rounded in dorsal view, oval in lateral view. Nostrils circular, located closer to tip of snout than to eyes. Eyes dorsal. Oral disc ventral, not emarginate. Marginal papillae with broad dorsal gap. LTRF 0/0. No spiracle. Vent tube medial. Dorsal fin originating at tail-body junction, dorsal fin and ventral fin parallel to longitudinal axis of tail. Tail length approximately 65% of total length, tail tip acute.
Leptodactylus Characteristics: Leptodactylus fuscus species group -Body elliptical or ovoid in dorsal view, globular-depressed in lateral view. Snout oval or rounded in dorsal view, oval in lateral view. Nostrils circular, generally located closer to tip of snout than to eyes. Eyes dorsal. Oral disc anteroventral, not emarginate. Marginal papillae arranged ventrolaterally, with a wide dorsal gap, anterior margin of oral disc totally fused with body wall. LTRF 2(2)/3(1). Spiracle sinistral. Vent tube medial. Dorsal fin originating at tail-body junction, dorsal and ventral fins slightly arched. Tail length approximately 60% of total length, tail tip acute.
Leptodactylus latrans species group -Body ovoid, elongated in dorsal view, globular-depressed in lateral view. Snout oval in dorsal and lateral views. Nostrils circular, generally located closer to tip of eyes than to snout. Eyes dorsal. Oral disc anteroventral, not emarginate. Marginal papillae arranged ventrolaterally, with a wide dorsal gap, anterior margin of oral disc totally fused with body wall. LTRF 2/3. Spiracle sinistral. Vent tube medial. Dorsal fin originating at tail-body junction, dorsal and ventral fins slightly arched. Tail length approximately 60% of total length, tail tip slightly rounded.
Leptodactylus melanonotus species group -Body oval, elongated in dorsal view, globular-depressed in lateral view. Snout oval in dorsal and lateral views. Nostrils circular, located closer to eyes than to snout. Eyes dorsal. Oral disc ventral, not emarginate, anterior margin of oral disc totally fused with body wall. Marginal papillae arranged ventrolaterally, with a wide dorsal gap. LTRF 2/3. Spiracle sinistral. Vent tube medial. Dorsal fin originating at tail-body junction, dorsal and ventral fins slightly arched. Tail length approximately 60% of total length, tail tip acute.
Characteristics: Body ovoid in dorsal view, globular-depressed in lateral view. Snout oval in dorsal and lateral views. Eyes dorsal. Nostrils circular, generally located closer to eyes than to tip of snout. Oral disc ventral, emarginate laterally, anterior margin of oral disc totally fused with body wall. Marginal papillae with wide dorsal gap, narrow ventromedial gap, and two small ventrolateral gaps (not ventromedial and ventrolateral gaps in P. caete). LTRF 2(2)/3(1)[2]. Spiracle sinistral. Vent tube dextral. Dorsal fin originating at tail-body junction, dorsal fin arched and ventral fin parallel to longitudinal axis of tail. Tail length approximately 60% of total length, tail tip acute.
Notes: Pombal & Madureira (1997) described the vent tube of P. caete as medial even though their illustration shows the tube sloping towards the right side, which is characteristic of a dextral opening. The P. cuvieri tadpoles characterized by Rossa-Feres & Nomura (2006) from Nova Itapirema, state of São Paulo, Brazil, differs from those studied here by having a medial vent tube opening. Bokermann (1962)  Characteristics: Body oval, elongated in dorsal view, globulardepressed in lateral view. Snout oval in dorsal view, sloped in lateral view. Nostrils circular or oval, generally located at midway between the eyes and tip of snout. Eyes dorsal. Oral disc anteroventral, emarginate laterally. Marginal papillae arranged ventrolaterally, anterior margin of oral disc totally fused with body wall. Marginal papillae with wide dorsal gap. LTRF 2(2)/3(1). Spiracle sinistral. Vent tube medial. Dorsal fin originating on the body and slightly arched, and ventral fin parallel to longitudinal axis of tail. Tail length approximately 65% of total length, tail tip acute.
Notes: The tadpoles of P. diplolister described by Peixoto (1982) from Mossoró, state of Rio Grande do Norte, Brazil, differ from those studied here by having a sinistral vent tube opening, although the illustration clearly shows a dextral opening.
Pseudopaludicola Miranda-Ribeiro, 1926 Species occurring in the region. Pseudopaludicola mystacalis (Cope, 1887) ( Figure 9A Characteristics: Body elliptical or ovoid in dorsal view, globulardepressed in lateral view. Snout rounded or oval in dorsal view, oval in lateral view. Nostrils circular, located closer to eyes than to tip of snout. Eyes dorsal. Oral disc ventral, emarginate laterally. Marginal papillae with wide dorsal gap and two small ventrolateral gaps, anterior margin of oral disc totally fused with body, LTRF 2(2)/2. Spiracle sinistral. Vent tube dextral. Dorsal fin originating at tail-body junction, dorsal fin arched and ventral fin parallel to longitudinal axis of tail. Tail length approximately 60% of total length, tail tip acute.  Characteristics: Body elliptical in dorsal view, globular-depressed in lateral view. Snout oval in dorsal and lateral views. Nostrils reniform, closer to eyes than to tip of snout. Eyes dorsal. Oral disc anteroventral, emarginate laterally. Marginal papillae arranged ventrolaterally, with a wide dorsal gap. LTRF 2/3(1). Spiracle sinistral. Vent tube dextral. Dorsal fin originating at posterior portion of body, dorsal fin arched and ventral fin parallel to longitudinal axis of tail. Tail length about 60% of total length, tail tip acute.
Proceratophrys Miranda-Ribeiro, 1920 Species occurring in the region. Proceratophrys cristiceps (Müller, 1883) ( Figure 10A Characteristics: Body elliptical-elongated in dorsal view, triangularobtuse in lateral view. Snout oval in dorsal and lateral view. Nostrils circular, located anteriorly, closer to tip of snout than to eyes. Eyes lateral. Oral disc anterior, posterior margin visible in dorsal view, not emarginate. Marginal papillae arranged lateroventrally, with a wide dorsal gap. LTRF 2(2)/3. Spiracle located ventrolaterally. Vent tube dextral. Dorsal fin originating at tail-body junction, dorsal fin parallel to tail musculature and ventral fin arched. Tail length about 60% of total length, tail tip acute with flagellum.

Discussion
The great morphological diversity of tadpoles is related to the large number of species, genera and families of anurans occurring in the Atlantic Forest north of the São Francisco River, as well as to the diversity of habitats and habits of their tadpoles. The tadpoles of the 63 species from the northern region of the Atlantic Forest with known larval phase represent almost all guilds (sensu Rossa-Feres et al. 2004) with diverse feeding habits such as suctorials, raspers, carnivorous, macrophages and endotrophics (Dubeux et al. 2019). Most tadpoles develop in lentic or lotic environments, but five in phytotelmata (Frostius pernambucensis and Phyllodytes spp.), one in association with rocky streams (Corythomantis greeningi) and one probably has endotrophic tadpoles that complete development in a foam nest placed in an underground burrow (Adenomera aff. hylaedactyla). Unfortunately, the feeding habit and microhabitat use for several tadpoles remain unknown (Dubeux et al. 2019).
Tadpole identification remains an enormous challenge in the Neotropics mostly due to the difficulty in recognizing and distinguishing morphological characteristics. Dichotomous keys are important tools for identifying organisms. Although the first identification keys for tadpoles were published more than a century ago (Boulenger 1892, Altig 1970, Lips & Savage 1996, Mijares-Urrutia 1998, they included few characteristics and few illustrations without enough details. Currently, a dozen or so identification keys are available for tadpoles, most of them for small groups of species (Haas et al. 2009, see below) or restricted regions (e.g., Hero 1990, Rossa-Feres & Nomura 2006. The first tadpole key for Brazilian species was developed by Hero (1990) and included 36 species that occur in the municipality of Manaus, state of Amazonas. Despite this beginning, most taxonomic keys available for tadpoles in Brazil are concentrated in the South and Southeastern regions. Rossa-Feres & Nomura (2006) included 22 species in a key for tadpoles of northwest region of São Paulo state. A year later, Machado & Maltchink (2007) published a key included 44 taxa (species and genera) of the state of Rio Grande do Sul. A tadpole key published in a book included 49 species obtained in the municipalities of Conceição do Mato Dentro, Alvorada de Minas and Dom Joaquim, all in state of Minas Gerais (Pimenta et al. 2014). Four years later, an identification key for 11 species from Ilha Grande, state of Rio de Janeiro, was published (Fatorelli et al. 2017). Recently, an interactive identification key was made available online, including 66 species from a region designated Quadrilátero Ferrífero, in the state of Minas Gerais (Pezzuti et al. 2019). Herein, we provide an key for tadpoles in the northern region of the Atlantic Forest, the first for Northeastern Brazil, contributing to fill one of the knowledge gaps identified during the "Tadpoles International Workshop" (Rossa-Feres et al. 2015): "How can we improved taxonomic identification of larval stages?". We expect this key and the morphological characterization of tadpoles presented here will stimulate further studies with Brazilian larval anurans.
Tamí Mott: Contribution to the conception and design of the work, data analysis and interpretation; writing and critical review of the manuscript, adding intellectual content.