Character variation during development
Mouthparts (Figs. 5A-B): Papillae: Tadpoles at the earliest stages examined (stages 26 and 27) have three papillae on the lateral corner of each labium. The upper labium does not possess other papillae, while the lower labium already possesses two rows of marginal papillae. Tadpoles at stage 28 possess three to five papillae, with a dominant frequency of three. At stage 29, the tadpoles have four to seven papillae on the lateral corner of each labium, most commonly five, and six for the next stages. Submarginal papillae on the lower labium increase in number and size until stage 30, whereas the marginal papillae continue to increase proportionally to growth until stage 36. At this late stage, the largest ones are five times larger than the submarginal papillae.
Fig. 5. Oral disk of the tadpole of Rana nigrovittata. (A) at stage 26 (MNHN 1998.7119); (B) at stage 29 (MNHN 1998.7179). Scale bar = 1 mm.
Keratodont formula and keratodonts: At stages 26 to 28 keratodonts are poorly developed, especially in rows A2, P1, and P3. These early keratodonts are light-brown or yellow-orange and many are not yet formed. The two upper rows of keratodonts (A1 and A2) are of equal length; the same is observed for the two internal rows of the lower lip (P1 and P2) which are longer that the third one (P3). From stage 29 on, keratodonts are fully developed, and darkbrown. From stage 32 on, the keratodont rows have their definitive relative proportions and a density of about 80 keratodonts per mm2.
At stage 26, five of the eight studied specimens have a keratodont formula of 1:1+1/1+1:2, one specimen has 1/1+1:1, and two have 1/1+1:2. By stage 27, all specimens possess all ridges, and malformations are found occasionally, such as divisions in normally unbroken rows of keratodonts. Jaw sheath coloration: At the first stages (stages 26 to 28), the jaw sheaths are entirely white except for the serrations, which are black and edged by a fine brown halo. From then until the end of the larval period, the coloration extends during development, linked to the keratinization of sheaths, but does not pass the upper half of the jaw sheath. The basal part stays white. The coloration of the upper beak is more extensive than that of the lower beak. Tadpoles in advanced stages may still show light coloration. In one clutch, most of the tadpoles have the medial third of upper beak poked, sometimes without serration. This abnormality become less marked at the stages 32-33, but the missing serrations never reappear.
Glands: The pair of glands on the flanks appears early, at stage 28. At stage 30, the glands between the eyes and the ventral glands appear which at this stage of development have a more or less central position. From stage 31 onwards, all pairs of glands are present.
Coloration: From stage 28 onwards, coloration of the body is identical to that of a tadpole in stage 36. However, the tail is still less pigmented, though the pattern is already noticeable. From stage 30 onwards, the coloration of the tadpole is identical to that of a stage 36 tadpole.
Internal buccal features: This description is based on two tadpoles at stage 26 (MNHN 1998.7103 and MNHN 1998.7129), one tadpole at stage 29 (MNHN 1998.5948) and one tadpole at stage 31 (MNHN 1998.7380).
Buccal floor: Prelingual area papillae and pustules are present from stage 26. The tongue anlage is poorly delimited at stage 26, although lingual papillae are already present; at stages 29 and 31 the tongue anlage is elongate, whereas it is round and better defined at stage 36. At stage 26, the buccal floor arena papillae are already present, although less developed than at stage 36; at stage 29 the size of the BFA papillae approaches that in stage 36. Less than 10 pustules are present within the arena at stage 26, while at stage 29 the number of pustules is identical to that observed at stage 36. Buccal pockets visible at stage 25. Only a few prepocket pustules are present at stage 26, while their definitive number is fixed by stage 29. Only the three lateralmost pairs of projections are present on the ventral velum at stage 26; their definitive number is reached at stage 31.
Buccal roof: The tadpole at stage 26 already has a well-developed prenarial ridge, whereas the number and location of pustules within the prelingual arena seem to vary between individuals, independently of stage. The narial valves and their ornamentations are fully developed from stage 26 onwards. The postnarial papillae are well developed and already pustulose at stage 26, but they attain their typical form between stages 29 and 31. At least four small pustules are present in the postnarial arena at stage 26, growing until stage 36 when they reach their full size. The medial ridge is present but it has not reached its maximum size and lacks most of its pustulations on margin at stage 26; from stage 29 onwards it is similar to that of stage 36. The buccal roof arena is defined by one or two small buccal roof arena papillae at stage 26 and by two or three fully developed buccal roof papillae by stage 29. The arena is short, less extended backwards than in oldest stages, growing regularly, continuing to increase after stage 31, interior with 30-40 pustules at stage 26, 50 pustules at stage 29 and about 60 at stage 31. The posterolateral ridges are well developed from stage 26. The glandular zone is present from early stages onwards. An individual at stage 29 already possesses all features of a stage 36 tadpole, but is merely less developed.
Morphometric variation: When plotting measurements as a function of the developmental stage (Figs. 6A-C), all the graphs show an increasingly inflected curve, except for the gap of the first keratodont row of the lower lip (LKR1I), which shows the opposite trend (Fig. 6C). Size increase in all variables is therefore fast in early stages (until stage 32) and slows down in late stages. The different developmental tadpole stages (Gosner, 1960) do not have an identical duration (Gollmann, 1991). The early stages extend over longer periods of time, thus the tadpole grows significantly during this period. On the contrary, later stages based on hindlimb development take place rapidly; the growth of tadpole within each stage is therefore reduced and a lower variation in the values of morphometric parameters is observed. In the case of the gap of the first keratodont row of the lower lip, the values decrease during the development until stage 30 and then stabilize, which confirms that the keratodont rows grow in the early stages and become stabilized at about stage 30. Nevertheless, the correlation between stages and all parameters is significant (Table 2).
Fig. 6. Correlation between three morphometric parameters and stages. (A) snout-vent length (SVL); (B) lower tail fin (LF); (C) length of the medial gap between portions of first tooth row of lower lip (LTR1I)
Plotting the measurements as a function of tadpole total size (TL), the growth in each parameter is proportional to the total growth of the tadpole (Figs. 7AC). Morphometric parameters were better correlated to total length than to stages (Table 2). The range of extreme values of the coefficient of variation can be great within a parameter (e.g. 9.51-57.43 in V-mHT). Overall, the characters are highly variable in stage 26 and most stable in stage 38. The mean coefficient of variation for each character is given in Table 1. Only a few parameters are very variable (thus unreliable) in the constancy of their values (mean value of their coefficient of variation higher than 10), viz. V-mHT, UF, LF, RN, UKR2I and LKR1I. All other characters have a mean coefficient of variation lower than 10. Among these characters, 10 have a mean coefficient of variation lower than 8 (SS, SU, SVL, VT, TL, ED, PP, NN, NP, ODW) and six have a mean coefficient of variation of 8-10 (BH, BW, HT, DPG, LKR2 and UKR2).
Fig. 7. Correlation between three morphometric parameters and total length (TL). (A) snout-vent length (SVL); (B) lower tail fin (LF); (C) length of the medial gap between portions of first tooth row of lower lip (LTR1I).