Rhinella bella, Menéndez-Guerrero, Lima dos Santos, Salazar-Nicholls, Green & Ron, 2024

Menéndez-Guerrero, Pablo A., Lima, Sueny P., Santos, Salazar-Nicholls, María-José, Green, David M. & Ron, Santiago R., 2024, Cryptic diversity in toads of the Rhinella marina species group (Anura, Bufonidae) with a subjectively beautiful new species from Western Ecuador, Zoological Journal of the Linnean Society 202 (3), pp. 1-26 : 13-24

RHINELLA BELLA SP. N OV.

( Figs 12, 13, 14, 15; Supporting Information, Table S8)

ZooBank: lsid:zoobank.org:act: E67080E4-D35F-40A7-8C9F- 5E5E3FD6DE65

Holotype ( Fig. 12): QCAZ 23305 , adult male, SVL = 87.11 mm, collected in Ecuador, Guayas Province, between Palmas and Balsas (c. 2.003° S, 80.562° W) at 72 m a.s.l., on 18 March 2003 by Santiago Ron.

Paratypes: Juveniles ( Fig. 14): QCAZ 13893 , collected in Cañar Canton, Manta Real Community (c. 2.554° S, 79.364° W) at 1100 m a.s.l., on 21 December 1998 by Ricardo Oliva. QCAZ 28528 , collected in Santo Domingo Canton, near to secondary road , near Tinalandia , jumping between puddles, (c. 0.303° S, 79.041° W) at 694 m a.s.l., on 05 January 2005 by Giovanna Romero. QCAZ 35433 , collected in Pichincha Province, Pedro Vicente Maldonado , 6 km to the Northwest at airport, (c. 0.104° S, 79.103° W) at 544 m a.s.l., on 17 April 2003 by Santiago Ron. QCAZ 40424 , collected in Esmeraldas Province, Rosa Zarate Parish , Laguna del Cube , (c. 0.415° S, 79.650° W) at 207 m a.s.l., on 22 August 2007 by Santiago Ron. QCAZ 45989 and 46001 , collected in Imbabura Province, Cotacachi-Cayapas Reserve buffer zone , near the Aguas Verdes River , (c. 0.331° S, 78.931° W) at 670 m a.s.l., on 01 November 2009 by Diego Almeida. QCAZ 49447 , collected in Manabí Province, Jama Canton , Reserva Lalo Loor Locality , (c. 0.079° S, 80.148° W) at 150 m a.s.l., on 02 September 2010 by Diego Salazar. QCAZ 50701 and 50702 , collected in Manabí Province , 5 km north of Rocafuerte, San Andrés de Rocafuerte Locality , (c. 0.868° S, 80.464° W) at 334 m a.s.l., on 26 February 2011 by Santiago Ron. Adult males ( Fig. 13): QCAZ 12039 , collected in Santo Domingo Province, 5 km north of Rocafuerte, Hotel Saracay Locality , (c. 0.676° S, 76.399° W) at 334 m a.s.l., on 05 April 1998 by Luis Eduardo López. QCAZ 17965 , collected in Pichincha Province , Guayllabamba , under the bridge over the Guayllabamba river , (c. 0.069° S, 78.372° W) at 1972 m a.s.l., on 23 November 2001 by Verónica Sandoya. QCAZ 23324 , collected in Santa Elena Province, Via Palmas-Balsas Locality , (c. 2.031° S, 80.460° W) at 78 m a.s.l., on 23 November 2001 by Santiago Ron. QCAZ 23486 , collected in Guayas Province, Sendero de Cerro Más Vale Locality , in Cerro Blanco stream approximately 200 m a.s.l., (c. 2.399° S, 79.634° W) on 22 March 2003 by Santiago Ron. QCAZ 24579 , collected in Manabí Province , Puerto Rico, Alándaluz Locality , in lagoon facing the north side beach , (c. 1.640° S, 80.830° W) 18 February 2002 by David Cannatella. QCAZ 39353 , collected in Pichincha Province , Tababela , New Quito International Airport , N-2 reservoir, at the edge of a water reservoir , (c. 0.181° S, 78.334° W), on 09 September 2008 by Fernando Ayala. QCAZ 41327 , collected in Guayas Province , Bosque Protector Cerro Blanco Locality , (c. 2.182° S, 80.018 W), at 237 m a.s.l., on 17 March 2008 by Gustavo Pazmiño. QCAZ 47444 and 47445 , collected in Loja Province, San Bernabé Locality , (c. 4.113° S, 79.304 W), at 1665 m a.s.l., on 03 March 2010 by Elicio Tapia. Adult females ( Fig. 13): QCAZ 50698 , collected in Manabi Province , Puerto Cayo Locality , (c. 1.344° S, 80.732 W), at 29 m a.s.l., on 25 February 2011 by Santiago Ron. QCAZ 50703 , collected in San Andrés de Rocafuerte, 5 km north of Rocafuerte, (c. -0.86 822, -80.46 363) at 334 m a.s.l. by Santiago Ron. QCAZ 47192 , collected on the shore of the Vance river in Esmeraldas Province , (c. 0.3269719, -79.47 686) by Melina Samaniego. QCAZ 33775 , 33778-79 , 33782-83 , collected in Alluriquín, Santo Domingo de los Tsáchilas Province , (c. -0.320 309, -78.99 347) by Elicio Tapia. QCAZ 26345 , collected 2.7 km north of Oña in Azuay Province, (c. -3.454 261, -79.16 227) at 2386 m a.s.l. by Ítalo Tapia. QCAZ 26320 , collected in Oña, Azuay Province , (c. -3.46 123, -79.16 253) by Luis Coloma. QCAZ 17966 -69, collected under the bridge over the Guayllabamba river in Guayllabamba , Pichincha Province , (c. -0.068925, -78.37 195) at 1972 m a.s.l. by Verónica Sandoya. QCAZ 1992 -93, collected in Santo Domingo de los Colorados , Pichincha Province , on the road to Quevedo , (c. -0.256 950, -79.16 612) by Santiago Ron. QCAZ 1994 -97, 1999 , collected in Tonsupa , Esmeraldas Province , (c. 0.8 852 820, -79.80 499) by Santiago Ron. QCAZ 2001 , collected in Guayaquil suburbio , Guayas Province , (c. -2.257 000, -79.87 800) by Santiago Ron. QCAZ 2258 -59, 2263 -64, collected in Tonsupa , Esmeraldas Province , (c. 0.8 852 820, -79.80 499) by Santiago Ron. QCAZ 2265 , collected in Santo Domingo de los Colorados , Pichincha Province , (c. -0.256 950, -79.16 612) by Santiago Ron. QCAZ 2267 , collected in the suburbs of Guayaquil , Guayas Province , (c. -4.2396, -79.2196) by Santiago Ron. QCAZ 10497 , collected in Chalcápac , Valle de Yunguilla , Vía Girón Pasaje , Azuay Province, (c. -3.233 333, -79.2) at 1550 m a.s.l. by Santiago Ron.

Suggested common names

Cane toad (English) or beautiful cane toad (English); sapo bello de la caña (Spanish).

Assignment of new species

We assign the new species to the genus Rhinella based on the phylogeny. We assign the new species to the R. marina species group (sensu Pereyra et al. 2021) based on the phylogeny and the synapomorphy ‘sacral diapophyses with the anterior edge angled posteriorly to the midline axis of the vertebral column’. The other synapomorphy of this group listed by Pereyra et al. (2021; i.e. ‘jagged or scalloped articulation between the medial ramus of pterygoid and parasphenoid alae’) could not be examined. Rhinella bella also shares the following characters with other members of the R. marina group, which, when used in combination, led to differentiate the species from other Rhinella groups (eight out of 12 characters listed by Pereyra et al. (2021) were examined): preorbital crest well developed, supraorbital crest well developed, pretympanic crest well developed, nasal and frontoparietal articulate along the entire margin, inguinal fat bodies, parotid gland approximately ellipsoid, longer than wide or triangular and bulky, tarsal fold, and extensor digitorum longus with an insertion on the metatarsophalangeal joint of digiti IV (Supporting Information, Table S7).

Definition ( Figs 12, 13, 15)

Rhinella bella is characterized by: (1) average SVL in adult females 102.16 mm (SD = 13.75, N = 29), adult males 86.51 mm (SD = 13.95, N = 31; see Supporting Information, Table S8); (2) short snout, subacuminated in dorsal view, truncated to rounded in profile; (3) bony knob at angle of jaws absent; (4) low and thick cephalic crests with borders with keratinized spicules, continuous with supraorbital, supratympanic, canthal, rostral, and preorbital crests; reduced infraorbital crests, and preorbital crests absent; (5) heel not reaching posterior margin of eye when hindlimbs depressed; (6) vertebral apophyses absent; (7) dorsal skin with medium and round tubercles usually with a keratinized spicule at the tip, with or without large scattered warts, most are pointed; (8) a thin middorsal line from snout to groin is present; (9) absence of dorsolateral fringe; (10) tympanic membrane and tympanic annulus distinct; (11) parotoid glands relatively big, elongate posteriorly, relatively prominent in dorsal view, with visible small pores, and with a keratinized spicule at the posterior tip, width of parotoid glands varies from 9.17 to 14.27% of SVL; height of parotoid glands can vary from 15.91 to 22.44% of SVL; (12) upper eyelid with keratinized warts; (13) palms without rudimentary basal webbing between fingers, lateral fringes absent; small rounded knobs at tips of digits; finger lengths 3> 4> 2> 1; (14) inner and outer metatarsal tubercles conical, protruding distally, outer usually similar size of inner tubercles; subarticular tubercles present; supernumerary tubercles with keratinized spicule at the tip, distributed linearly on toes on plantar surface; (15) forelimbs and forearms robust with low and rounded warts keratinized; supernumerary tubercles rounded with different sizes, distributed irregularly on palm; palmar tubercle larger than thenar tubercle, both are oval; (16) foot with basal webbing between toes, lateral fringes present; small rounded knobs at tips of digits; toe lengths 4> 5> 3> 2> 1; (16) nuptial pads present.

Diagnosis

Rhinella bella is morphologically similar to R. horribilis and R. marina . However, R. bella differs from populations of R. horribilis by having a smaller body size, along with a proportionally larger femur, wider head, and larger tibia ( Fig. 8; Supporting Information, Tables S3 , S8). Differences among species were also evident in the skull shape (see results of morphometric analyses). Acevedo et al. (2016) reported cranial morphology differences between populations of the east ( R. marina ) and west ( R. horribilis ) side of the Venezuelan Andes, mainly around the pre-maxillar, nasal bones, and occipital region. We also found skull differences between populations on the eastern ( R. marina ) vs. western ( R. bella ) sides of the Ecuadorian Andes, mainly concerning the pre-maxillar and nasal bones. However, the non-monophyletic status of the eastern and Amazon Basin populations ( Vallinoto et al. 2010; this paper) precluded us from making further comparisons with the findings of Acevedo et al. (2016). The new species is distinguishable from R. horribilis by a depressed area on the quadratojugal-squamosal contact region, and the elongation of frontoparietal bones at their most distant region ( Fig. 10; Table 5 ).

Rhinella bella differs in environmental envelope from R. horribilis and R. marina ( Fig. 11; Table 6 ; for details see climatic analysis section from Material and methods and Results) and is separated by high genetic distances from both species as well ( Fig. 3; Table 1 ; see molecular phylogenetic analysis from Material and methods and Results).

Advertisement calls of R. bella are significantly shorter, with fewer notes and a shorter rise time than those of R. horribilis and R. marina ( Fig. 4; Table 2 ). The number of notes per call of R. bella (midrange = 23; range 6–40) is also lower than those reported in other species of the R. marina group [e.g. R. arenarum (midrange = 84, range 61–107; Straneck et al. 1993); R. marina (midrange = 176.5, range 18–335; present work); R. poeppigii (midrange = 27.5, range 10–45; De la Riva et al. 1996); and R. diptycha (midrange = 36.5, range 33–40; Köhler et al. 1997)]. Furthermore, R. bella produce calls with higher dominant frequency than R. horribilis and R. marina ( Fig. 6; Tables 2 , 3 ).

Description of holotype

Adult male: robust body; head wider (HW = 36.15 mm) than long (HL = 30.49 mm). Head shape in dorsal view subtriangular with short, squared snout and straight lateral view, tip of the snout not surpassing anterior margin of maxilla in dorsal and ventral views. Numerous rounded tubercles with several keratinized spicules in dorsum. Scattered granulation on top of the head. Presence of low cephalic crests with keratinized borders, continuous with supraorbital, supratympanic, canthal rostral and preorbital crests; reduced infraorbital crests, and preorbital crests absent. Tympanic membrane and tympanic annulus distinct; bony knob at angle of jaws absent; corner of mouth angular; vertebral apophyses absent; parotoid glands relatively big, elongate posteriorly, relatively prominent in dorsal view, with small pores visible, and tubercles keratinized; upper eyelid with keratinized warts. Forelimbs and forearms robust with low and rounded keratinized warts. A longitudinal mid-dorsal cream thin raphe is present from the snout to near the cloacal region. Hand without basal webbing between fingers, lateral fringes absent; small, rounded knobs at tips of digits; finger lengths 3> 4> 2> 1; subarticular tubercles low and rounded; supernumerary tubercles rounded, distributed irregularly on palm; palmar tubercle larger than thenar tubercle, both are oval; nuptial pads on the dorsolateral surface of fingers I and II. Foot longer than tibia (TL/FL = 0.948); foot with basal webbing between toes, lateral fringes present; small, rounded knobs at tips of digits; toe lengths 4> 5> 3> 2> 1; inner and outer metatarsal tubercles conical, protruding distally, outer similar size of inner tubercles (~ 4.18 mm); subarticular tubercles present; supernumerary keratinized tubercles rounded, distributed linearly on toes on plantar surface.

Colour of holotype in preservative

Dorsum light grey, rounded tubercles on dorsum between light cream and dark brown with dark brown spicules, also in dorsal surfaces of tights, shanks, and forelimbs which are lighter than dorsum; few dark grey marks arranged irregularly on dorsum. Nuptial pads dark brown. Ventral surfaces grey becoming yellowish-cream with irregular dark brown marks medially; cream colour with black edges slightly posterior to gular region. Fingertips and subarticular tubercles yellowish-cream. Dark brown knobs at tips of digits; tympanum dark grey ( Fig. 12).

Variation of coloration in preserved specimens

Colour variation in preserved specimens is shown in Figures 12 and 13. Background dorsal coloration varies from light grey (e.g. QCAZ 23305, 50698, 50701) to dark brown (e.g. QCAZ 41327, 24579, 39353), with irregular black and yellowish marks (e.g. QCAZ 40806, 50702, 50698). A thin middorsal line from snout to groin is present, except in QCAZ 47445. Ventral surfaces have a yellowish-cream to dark brown background; and several with irregular grey marks arranged in diverse patterns (e.g. QCAZ 12039, 17965 28528), absent in QCAZ 41327, 47445, 47444.

Variation of coloration in live individuals

Colour variation in live individuals is shown in Figure 15.

Etymology

‘ Bella ’, in Latin meaning ‘beautiful’, in contradiction to ‘ horribilis ’, the name of its sister species. We think that ‘ horribilis ’ is an undeserved name that stems from anachronistic views of nature common in the 19th century. Judgements of the beauty of organisms are highly subjective, something that we highlight with the name of the new species. Additionally, the precise geographic transition between R. bella and R. horribilis is still unknown, just as is the border between beauty and ugliness.

Vocalization

Based on 17 males recorded at six localities from western Ecuador, the advertisement call of R. bella is a short trill with a mean duration of 1.51 s (range 0.94– 2.56 s), a mean call rise time of 0.94 s (range 0.65– 1.79 s), and a mean dominant frequency of 803.05 Hz (range 668.00–961.83 Hz). The call consists of 10–34 notes, with 2–5 pulses per note. The mean pulse rate is 91.31 pulses per second (range 66.95–113.08 pulses per second), and the mean call repetition rate is 8.57 calls per minute (range 5–14 calls per minute; Table 3 ).

Distribution, ecology, and conservation status

This species is restricted to the western side of the Andes, from sea level up to 2900 m a.s.l. Among Ecuadorian amphibians, it has one of the widest elevation ranges and occurs in six out of ten natural regions: Chocó Tropical Rainforest, Coastal Deciduous Forest, Coastal Dry Scrubland, Inter-Andean Scrubland, Western Montane Forest, and Western Piedmont Forest (regions as defined by Ron et al. 2022). It is most frequently found in artificial open areas including agricultural fields, grasslands, and urban and suburban habitats. It occurs infrequently in undisturbed forests. Reproduction takes place in ponds, rivers, and swamps. Reproductive activity (calling males or amplectant pairs) have been observed mainly in April, June, and July. As in other species of the R. marina species group, eggs are deposited in strings in water. Tadpoles are aquatic and have a high resistance to heat, up to 42.5 °C ( Pintanel et al. 2022). The complete distribution of R. bella remains unknown, mainly due to lack of distributional information in the Colombian Chocó Bioregion. Ecuadorian localities in northern Ecuador are close to the border with Colombia. Nearby Colombian records of ‘ R. horribilis ’ in iNaturalist (e.g. observation numbers 36168677 in Llorente, San Andrés de Tumaco and 102430734 in Tumaco) morphologically resemble R. bella and we consider them as unconfirmed records of R. bella in Colombia. Moreover, the limited seasonal temperature variation of the Colombian Chocó Bioregion matches the climatic envelope of R. bella , further suggesting its occurrence in southern Colombia (see results of climatic envelopes; Fig. 11; Table 6 ). This species also occurs in northwestern Peru, south to 6°S of latitude (reported as ‘ R. horribilis ’ by Armijos-Ojeda et al. 2021).

The new species occurs in the following protected areas: Reserva Ecológica Manglares Churute, Reserva Ecológica Los Ilinizas, Parque Nacional Machalilla, Reserva Ecológica Mache Chindul, Reserva Tesoro Escondido, Bosque Protector Mindo-Nambillo, Centro Científico Río Palenque, Bosque Petrificado de Puyango, Bosque Integral Otonga, Estación Biológica Bilsa ( Páez-Rosales and Ron 2022 as ‘ R. horribilis ’). The new species may be abundant in artificial open areas. Its tolerance of habitat disturbance, large distribution range along a wide elevation gradient, and occurrence in diverse natural regions (six different regions in Ecuador, one of the highest among Ecuadorian anurans) allow us to recommend its assignment to the Least Concern category in the Red List (based on the IUCN guidelines).

D I SC USSI O N

Our analyses reveal that populations from western Ecuador represent an independent lineage from all other known species of the R. marina group. We base this conclusion on the congruence observed between genetic, bioacoustic, morphological, and climate envelope information. Western Ecuadorian populations are highly different genetically from other species of the group. This mtDNA divergence is much higher than that observed between uncontroversial species pairs of the same group like R. arenarum and R. icterica or R. diptycha and R. poeppigii . Differences in advertisement calls are additional, clear indications that those populations represent a candidate species within the R. marina group. We provide conclusive evidence, based on an integrative approach combining different sources of information, that there are two, independent lineages of R. marina group toads west of the Andes in northern South America and Central America. One, R. horribilis , ranges north through Central America as far as southern Texas, whereas the other, R. bella , occurs in Ecuador and probably as far north as southwestern Colombia and as far south as extreme northern Peru.

Past studies have shown two genetically distinct species within R. marina from the east and west sides of the Venezuelan Andes ( Slade and Moritz 1998), including the recent work of Acevedo et al. (2016), who suggested that the R. horribilis name should be revalidated for the western Andes and Central American populations. Additionally, Mulcahy et al. (2006), Pramuk (2006), and Vallinoto et al. (2010) had previously found important levels of genetic divergence between Central American and Ecuadorian samples; however, incomplete sampling (only very few were included from western Ecuador) and the lack of bioacoustic, morphological (but see Pramuk 2006), and ecological information, limited their conclusions. We provide novel evidence showing an independent evolutionary lineage consistent with a separate species within the R. marina species group, confirming that the western Ecuadorian populations ( R. bella ) are a new species distinct from the populations of Central America ( R. horribilis ), and from the populations from the Amazon Basin ( R. marina ).

In contrast to the cryptic morphological diversity notable throughout the genus Rhinella ( Graybeal 1997, Pramuk 2006, Acevedo et al. 2016), including the R. marina species group, our morphometric analyses (including the geometric morphometric-based analysis) suggest that R. bella (western Ecuadorian populations) is overtly distinguishable from R. horribilis (Central American populations) and from R. marina (Amazon Basin populations) as currently understood. Isolated from all other R. marina group toads except for R. horribilis by the Andes, R. bella appears to be among the most geographically isolated and morphologically distinctive of this group of species.

We report significant acoustic differences between R. horribilis , R. marina , and R. bella (see Figs 4–6) despite the generally conservative structure of advertisement calls among species of the R. marina group ( Maciel et al. 2007). The distinctly lower number of notes per call in R. bella relative to other species within the R. marina group is significant. Similarly, the higher dominant frequency of R. bella calls in comparison with calls of R. horribilis and R. marina is remarkable as this call trait is among the few not strongly dependent on operational temperature ( Fig. 6), and has been proven to be useful to assess species limits ( Köhler et al. 2017). This acoustic distinctiveness of R. bella among R. marina group species, and especially from R. horribilis , suggests that calls may have diverged at faster rates compared to morphology in this group, as has also been shown in other anuran species groups (e.g. Padial et al. 2008, Angulo and Icochea 2010, Funk et al. 2012). These patterns could be due to sexual selection promoting rapid divergence in both male traits and female preferences, or strong selection on species recognition ( Panhuis et al. 2001). This may be particularly relevant to the calls of R. bella vs. R. horribilis in that vocal character displacement, where these two species meet (probably in southern Colombia), could promote mating isolation. Alternatively, mate-recognition traits as calls may diverge faster than morphology due to a strong stabilizing selection on morphological traits ( Funk et al. 2012), which may be imposed by several ecological factors ( Bickford et al. 2007). Previous studies have not fully evaluated call differences within the R. marina species group, although acoustic information seems to be particularly useful for the delimitation of cryptic species ( Funk et al. 2012).

The comparison of climatic envelopes among studied populations provided important complementary evidence for species separation (see Fig. 11). Toad populations occurring in western Ecuador ( R. bella ) have a climatic envelope with lesser temperature seasonality than populations of R. horribilis occurring further north in South America and Central America. Temperature seasonality can be a major climatic factor limiting dispersal of amphibians ( Wiens et al. 2006), and Neotropical anurans demonstrate climatic specializations along temperature seasonality axes ( Graham et al. 2004). We would not expect notable and substantial morphological divergence between R. bella and R. horribilis if speciation occurred mainly via geographic isolation related to climatic components unless climate imposed selection on morphology. However, the relative importance of climatic factors in population divergence and speciation of these lineages deserves further investigation.

We also found that R. marina from the Amazon Basin remains non-monophyletic, as was previously found by Vallinoto et al. (2010) and Rivera et al. (2022). Samples of R. marina are nested within only one well supported clade (Clade E in Fig. 2) as the sample MJH 3678 of ‘ R. marina ’ from Clade D is probably misidentified and most likely corresponds to R. poeppigii . Clade E is composed of ‘ R. poeppigii ’ from Bolivia, R. diptycha and R. marina from the eastern side of the Ecuadorian Andes, and R. marina from the Amazonian Basin. The sample MNCN/ ADN6044 of R. poeppig ii, however, is likely misidentified and corresponds to either R. marina or R. diptycha . Furthermore, corroborating Vallinoto et al. (2010) and Ron et al. (2015), our results placed R. crucifer as sister to the R. marina group according to mtDNA sequence information. Although R. crucifer is not formally considered to be in the R. marina species group ( Pramuk 2006), morphological similarities and a close cytogenetic and phylogenetic relationship between it and members of the R. marina group have been noted previously ( Blair 1972, Cei 1972, Duellman and Schulte 1992, Baldissera et al. 1999, Pauly et al. 2004, Pramuk 2006).

The discrepancy between phylogenetic relationships and the taxonomic classification of species within the R. marina species group could be influenced by several factors. First, the lack of an integrative taxonomy (i.e. species delimitation based on multiple sources of evidence; Padial et al. 2010) that provides a more effective solution to refine the systematics of the R. marina species group. Second, DNA sequences of some lineages (e.g. some populations previously known as ‘ R. horribilis ’) available for only a small number of samples and/or with limited representation of the geographic distribution ( Maciel et al. 2010, Vallinoto et al. 2010). Third, the evident lack of prominent diagnostic morphological characters for some lineages in this group can result in the misidentification of specimens. For instance, Venegas and Ron (2014) reported on 10 specimens of R. poeppigii deposited at Museo de Zoología , Pontificia Universidad Católica del Ecuador (QCAZ) that had previously been misidentified as R. marina . Finally , genetic introgression resulting from interspecific hybridization, which is known to occur among numerous closely related species of bufonid toads in sympatry (e.g. Masta et al. 2002, Smith and Green 2004), may confound phylogenetic reconstruction by obscuring topology and divergence estimates. This could be the case for some populations of R. marina and R. diptycha in the Amazon Basin ( Sequeira et al. 2011) and will likely continue to present challenges for delineating species boundaries within the entire R. marina group east of the Andes ( Rivera et al. 2022). West of the Andes, however, the genetic differentiation between R. bella and R. horribilis is clearly demonstrable, although the nature and extent of any putative contact zone between the two species, possibly in southwest Colombia and/or extreme northwest Ecuador, remains unknown.

Cane toads are economically important and invasive outside of their native range, having been introduced on many Caribbean and Pacific islands ( Easteal 1981, 1985, Lever 2001, Shine 2010, 2018), where they are often now considered pests. The impact of such a large, voracious, prolific, and toxic amphibian on the ecology of northern Australia, for example, has been profound ( Shine 2018). Most of the evidence and historical record indicates that the cane toads currently in the Pacific originated from native populations in Venezuela, via Puerto Rico, Cuba, and Florida ( Slade and Moritz 1998). Considering the extent of cryptic taxonomic diversity of R. marina species toads, however, and the probability that new species such as R. bella remain to be discovered within the complex, it is possible that the cane toads infesting Australia and many Pacific Islands may not actually be R. marina . If this is the case, some aspects of our current knowledge on global biological invasions of these species (e.g. Tingley et al. 2014) would deserve reconsideration.