Ranitomeya amazonica ( Schulte, 1999 )

Klein, Benjamin, Regnet, Ruth Anastasia, Krings, Markus & Rödder, Dennis, 2020, Larval development and morphology of six Neotropical poison-dart frogs of the genus Ranitomeya (Anura: Dendrobatidae) based on captive-raised specimens, Bonn zoological Bulletin 69 (2), pp. 191-223 : 195-202

Ranitomeya amazonica ( Schulte, 1999)

Breeding behavior in captivity. The breeding pairs, among the five specimens of R. amazonica , placed the clutches of four to six anthracite eggs in the bromeliad phytotelm. While those egg depositions had no clear frequency, later depositions in a water filled film container occurred every two to five days. Thereby, the clutches were placed directly underneath the water surface of the vertically orientated container, which was placed on the ground next to a large stone. Moreover, at one day a single tadpole at stage 25 was found at the ground of the container, beneath a newly produced clutch.

Larval morphology. The description of the tadpole is based on one specimen at stage 41 ( ZFMK 97374 ). Further voucher specimens are ZFMK 97357 , 97362 , 97366 , 97370–97373 . According to McDiarmid & Altig (1999), R. amazonica tadpoles belong to the exotrophic, lentic, benthic, arboreal larval type. All measurements that were used to calculate the following proportions and its comparison with the other species of this study, can be found in Appendix III .

Dorsal view: Body shape is oval and moderately elongated (MBW/BL= 0.75). The snout is short and moderately pointed (RED/BL= 0.23, BWN/BWE = 0.56), nares are small and elliptical, positioned dorsally and orientated laterally. Nares are situated closer to snout than to eyes (RND/RED = 0.43). Eyes are large (ED/ BL = 0.12), positioned dorsally and orientated laterally. Internarial distance is smaller than interorbital distance (IND/IOD = 0.52). Single, sinistral spiracle is not visible in dorsal view.

Lateral view: Body is depressed (MBH/MBW = 0.59), snout is rounded. The spiracle is positioned below the longitudinal axis, at the second half of the body (RSD/ BL = 0.64), the inner wall is free from the body, opening is round and the spiracle tube is short. The maximum body height is situated between the eyes and the tail. The tail is long and narrowly rounded (TAL/BL= 1.95, TAL/ TL= 0.66). The musculature is well developed (TMH/ MTH = 0.58, TMW/MBW = 0.33). The “V”-shaped myo- septa are visible along the whole length of the tail, particularly at the first half. Upper fin originates posterior to the tail-body junction and the margin of the lower fin. Upper fin is slightly higher than the lower fin. Ventral tube is dextral, emergence from abdomen sagittal, opening is rounded. Hindlimbs are fully developed. Oral ap- paratus is visible in lateral view.

Oral apparatus: Oral disc is elliptical, positioned ven- trally and covers nearly one third of the body width (ODW/MBW = 0.27), emarginated. Marginal, ensiform, rounded and transparent papillae are present at the posterior side, with a moderate medial gap, and absent at the anterior side, except the most lateral part (seven papillae). Submarginal papillae are absent. Anterior labium contains two tooth rows of the same width (A1, A2), large medial gap in second anterior tooth row (A2-GAP). Posterior labium contains three tooth rows (P1, P2, P3), moderate medial gap in first tooth row (P1-GAP). Black jaw sheaths, both with serrations. The upper jaw sheath is wider than the lower jaw sheath. The labial tooth row formula is 2(2)/3(1) ( Fig. 3D ). Characteristic traits and the correlated proportions do not change during the development stages 26 to 41 ( Table 1).

Coloration of a living tadpole of R. amazonica (ZFMK 97374). The dorsum is black to grey, with a yellowish green median stripe and two dorsolateral stripes of the same color, which run parallel to the longitudinal axis, and two lateral stripes ( Fig. 3A 1 , A 2 ). The two dorsolateral stripes originate at one point posterior to the nares, become separated and run next to the eyes to the base of the tail, with a moderate gap on eye level. The median one lies in between the two others, starts at eye level and ends prior to the tail-body junction. The lateral stripes are situated differently. One of the lateral stripes is situated at the first half of the body below the longitudinal axis, while the other one is located above the longitudinal axis at the second half of the body. The hindlimbs are dark bluish with large black spots. The tail shows a brownish coloration and is covered with dark and bright spots, the second half is brighter than the first half. Fins are trans- parent and spotted with beige dots. The density of dots wanes till the tip.

During metamorphosis, the dorsal coloration of tadpoles changed in regard to the different development stages ( Fig. 4 ). Reaching stage 25 some specimens displayed a few isolated yellowish green spots while the majority showed no coloration. At stage 28 some parts of the medial and dorsolateral stripes were present at the first half of the dorsum. In comparison to the final coloration, those areas were yellowish green instead of yellow and lacked a continuous connection. At stage 36 the color pattern was yellow, the dorsolateral stripes reached the second half of the body and the medial stripe ended close to the posterior margin of the eyes. While the dorsolateral stripes were continuous, the medial stripe was spotted. At stage 41, the dorsolateral stripes reached the tail-body junction and the medial line ended at the second half of the body. Moreover, each flank displayed the initiation of the ventrolateral stripes posterior to the forelimb pouches, which were visible in dorsal view, as well as the typical color pattern of the hindlimbs ( Fig. 3C 1 , C 2 ).

Coloration of a preserved tadpole of R. amazonica (ZFMK 97374). The dorsum is dark gray, with a brownish area at the forelimb pouches. Dorsolateral and median stripes are whitish and run on top or parallel to the longitudinal axis, clearly discernible on the head and the first half of the body. Dorsolateral stripes originate and bifurcate at one point posterior to the nares and run next to the eyes, with a moderate gap on eye level. The median stripe runs in between the eyes, not fusing with the origin of the dorsolateral stripes. The hindlimbs are bluish gray, spotted with dark dots. The tail is brownish; the first half is darker than the second one, which is almost transparent. Fins are transparent and spotted with beige dots. Ventral side has a dark grey to brown coloration, except one bright spot at the chin, posterior to the oral disc.

Larval staging. During their embryonic development, all four to six eggs of the same clutch develop at the same pace, except the reduction of the gills. While the majority of the eggs swam separately beneath the water surface, two in each clutch stayed as a pair ( Fig. 5B ). Eggs up to stage 10 were not pigmented ( Fig. 5A ). At stage 10, when the dorsal lip was visible, the pigmentation start- ed and the eggs became brownish. After three days, the neural plate was discernible ( Fig. 5C ). Reaching stage 18, a whitish yolk sack was present at the ventral side of the embryo and body parts were slightly differentiat- ed ( Fig. 5D ). At stage 19, the embryo slowly assumed a larval shape. The head and tail region were visible and the larva had a dun coloration with beige spots. While the gill buds, the opening of the mouth and the ventral tube emerged, the eyes were absent. At stage 20, the gills and the correlated circulation were present while the whole body was elongated ( Fig. 5E , Table 2). Upper and lower tail fins were slightly visible and the myosepta were present. Between stages 21 and 22, the tail and the gills were even more elongated, the overall body size increased and the pigmentation of all structures was denser. Tail fins were transparent, the tail was pointed. At stage 22, eyes were visible, nares were discernible and the decrease of the yolk sack was initiated ( Fig. 5F ). During the transition from stage 23 to 24, the sinistral gills were present while the dextral gills were completely reduced ( Fig. 5G ). The yolk sack was almost fully atrophied and the oral apparatus was formed. The transition to a free living and mobile tadpole started at stage 25, while the majority of the clutch was no longer enclosed by the jelly layer and the yolk sack was fully reduced. The spiracle was formed on the left, and after eleven to twelve days of development, the hatchlings swam freely within the water column ( Fig. 5H–I ).

Right after hatching, the free-swimming larvae had a surface area of 0.10 ± 0.02 cm ² ( Table 3). Between the stages 25 to 27, while the hindlimb bud was slightly developed, the surface increased by 330%, resulting in an area of 0.33 ± 0.20 cm ². At stage 28, which half of the tadpoles had reached after 49 to 67 days (median = 56 days), they had a mean surface area of 0.74 ± 0.12 cm ² ( Table 3). The hindlimb bud was as long as wide and the dorsal color pattern was slightly visible at the first half of the body. Between stages 29 to 40, where the completion of the hindlimb development took place, the larvae had a mean surface area of 0.98 ± 0.19 cm ². Thus, all toes, the metatarsal tubercles and the subarticular patches were discernible. With an area of 1.17 ± 0.12 cm ², half of all tadpoles reached stage 41 after 69 to 88 days (median= 84 days). Forelimb buds were visible and the hindlimbs showed the typical color pattern of the adult frog ( Fig. 3A 1, B1 ). Furthermore, colored dorsolateral stripes were discernible and the ventral tube as well as the oral apparatus was still present. While the forelimbs grew inside the body, during the transition from stage 41 to 42, the larvae reached their maximum size with a surface area of 1.19 ± 0.12 cm ². After 82 to 94 days (median = 89 days), 50% of all metamorphs had emerged forelimbs, while the surface area decreased to 1.08 ± 0.17 cm ². The resorption of the tail started after 91 to 99 days (median = 96 days), while the tadpoles had a mean surface area of 0.86 ± 0.15 cm ². During the next days, the tail atrophied until the larva completed the metamorphosis, whereby the area of the larva was reduced to 0.82 ± 0.15 cm ². Thus, the transition from a free-swimming larva to a froglet with a remnant of the tail lasted 91 to 99 days (median = 96 days), while some individuals needed less (84 days) and others more time (105 days, Fig. 6 ). An additional and more detailed staging table, based on stereomicroscopic determinations of 17 specimens between stages 25 to 41, can be found in the Table 4.

The complete development, from the embryogenesis through hatching and larval period to metamorphosis, was observed under constant conditions with a temperature of 24 °C while the annual mean temperature within the natural distribution area of R. amazonica is slightly higher (T Mean = 25.2 °C, T Max = 28.5 °C, T Min = 21.8 °C: Karger et al. 2017a,b; Fig.7 )

Ranitomeya benedicta Brown, Twomey, Pepper & San-

Figures

Fig. 3. Illustrations of the tadpole of Ranitomeya amazonica, stage 41 of Gosner (1960). A1. Dorsal view, photograph. A2. Dorsal view drawing. B1. Ventral view, photograph. B2. Ventral view, drawing. C1. Lateral view, photograph. C2. Lateral view, drawing. D. Drawing of the oral disc. LTRF=2(2)/3(1).

Fig. 1. Known distribution of Ranitomeya amazonica (green), R. benedicta (light blue), R. imitator (red), R. reticulata (yellow), R. sirensis (blue) and R. vanzolinii (orange). Darker shades of gray indicate higher elevations. The inset map displays the distribution of all six compared species, which are shown in the upper right corner of that figure with the corresponding color code.

Fig. 2. Landmarks andmeasurements of a tadpole body and definitions of the oral apparatus.A. Lateral view.B. Dorsal view.C. Oral apparatus. Abbreviations A–B: BL=body length; TAL=tail length; TL=total length; TMH =tail muscle height; MTH=maximum tail height; IND=internarial distance; IOD=interorbital distance; TMW=tail muscle width. The dotted line indicates the accurate progression of the measurement which represents the body length. Abbreviations C: A1=first anterior tooth row;A2=second ante- rior tooth row;A2-GAP=medial gap in second anterior tooth row; AL=anterior (upper) labium; LJ=lower jaw sheath; LP=lateral process of upper jaw sheath; M=mouth; MP=marginal papillae; OD=oral disc; PL=posterior (lower) labium; P1=first posterior tooth row; P2=second posterior tooth row; P3=third posterior tooth row; SM=submarginal papillae; UJ=upper jaw sheath.

Fig. 4. Development of the color pattern of Ranitomeya amazonica. A. Tadpole at stage 25 with isolated colored spots. B. Tadpole at stage 25 without isolated colored spots. C–D. Different densities at Gosner stage 30. E. Tadpole at Gosner stage 36. F. Tadpole at Gosner stage 41, typical color pattern on hindlimbs.

Fig. 5. Embryos and hatchlings of Ranitomeya amazonica. A. Isolated egg at stage 8. B. Egg pair at stage 9–12.C. Embryo at stage 13–14. D. Embryo at stage 18–19. E. Hatchling at stage 20. F. Hatchlings at stage 22. G. Hatchling at stage 23–24. H. Hatchlings at stage 25. I. Free swimming larva at stage 25–26.

Fig. 6. Developmental series of six Ranitomeya species during the transition from a hatchling to a froglet. The upper panel illustrates the increase of surface area over time, whereby the surface area [cm²] is highly correlated with the body mass. Warmer colors indicate a higher sample density; each circle represents one data point. Loess function represented by the red lines, whereby the outer lines display the 95% confidence interval. The lower panel illustrates the temporal occurrence of the following traits: 1=no limb bud discernible; 2=hindlimb bud discernible; 3=forelimb pouches discernible; 4=forelimbs emerged; 5=initiation of tail resorption.

Fig. 7. Climatic characteristics within the geographic ranges of six Ranitomeya species in terms of annual mean temperature, minimum and maximum temperature of the coldest / warmest month. Boxplots show the 95% range, lower and upper hinge enclosing 50% of the samples and the median based on a random sample per 10 km2.