Rivaling the world's smallest reptiles: discovery of miniaturized and microendemic new species of leaf chameleons (Brookesia) from northern Madagascar

Abstract

Background: One clade of Malagasy leaf chameleons, the Brookesia minima group, is known to contain species that rank among the smallest amniotes in the world. We report on a previously unrecognized radiation of these miniaturized lizards comprising four new species described herein.

Methodology/principal findings: The newly discovered species appear to be restricted to single, mostly karstic, localities in extreme northern Madagascar: Brookesia confidens sp. n. from Ankarana, B. desperata sp. n. from Forêt d'Ambre, B. micra sp. n. from the islet Nosy Hara, and B. tristis sp. n. from Montagne des Français. Molecular phylogenetic analyses based on one mitochondrial and two nuclear genes of all nominal species in the B. minima group congruently support that the four new species, together with B. tuberculata from Montagne d'Ambre in northern Madagascar, form a strongly supported clade. This suggests that these species have diversified in geographical proximity in this small area. All species of the B. minima group, including the four newly described ones, are characterized by very deep genetic divergences of 18-32% in the ND2 gene and >6% in the 16S rRNA gene. Despite superficial similarities among all species of this group, their status as separate evolutionary lineages is also supported by moderate to strong differences in external morphology, and by clear differences in hemipenis structure.

Conclusion/significance: The newly discovered dwarf chameleon species represent striking cases of miniaturization and microendemism and suggest the possibility of a range size-body size relationship in Malagasy reptiles. The newly described Brookesia micra reaches a maximum snout-vent length in males of 16 mm, and its total length in both sexes is less than 30 mm, ranking it among the smallest amniote vertebrates in the world. With a distribution limited to a very small islet, this species may represent an extreme case of island dwarfism.

Figure 1. Map of northern Madagascar showing distribution of species of the Brookesia minima group.

Type localities in bold, B. dentata, B. exarmata, B. karchei, B. peyrierasi, and B. ramanantsoai not included, because their ranges are located further south; see inset maps in Fig. 2. Orange (dry forest) and green (rainforest) show remaining primary vegetation in 2003–2006, modified from the Madagascar Vegetation Mapping Project (http://www.vegmad.org).

Figure 2. Phylogenetic relationships among species of the Brookesia minima group based on mitochondrial DNA sequences.

Phylogram (50% majority rule consensus tree) from a Bayesian inference analysis of the 568 bp DNA sequence alignment of the mitochondrial ND2 gene showing deep divergences among and low differences within species of the B. minima group. Species of the northern clade (B. tuberculata plus the four new species described herein) are marked in colour. The tree was rooted with Brookesia nasus, and including B. brygooi and B. superciliaris as hierarchical outgroups (not shown). A, B and C refer to major clades as discussed in the text. Inset maps show known distribution ranges (typically single localities) of all species. Asterisks denote posterior probabilities of 1.0.

Figure 3. Phylogenetic relationships among species of the

Phylograms (50% majority rule consensus trees) from Bayesian inference analyses of DNA sequence alignments of (a) the nuclear genes CMOS (846 bp) and (b) RAG1 (1522 bp). (c) Phylogram from BI analysis of concatenated DNA sequences of the nuclear genes CMOS (846 bp) and RAG1 (1522 bp), and the mitochondrial genes ND2 (568 bp) and 16S (580 bp). (d) Brookesia portion of the Bayesian chronogram derived from the BEAST analyses of all four concatenated genes (see Supporting Information S1 for full chronogram with all outgroups). Posterior probabilities are indicated above branches, and bars represent 95% HPDs for mean date estimates. Units on scale are millions of years. Species of the northern clade (B. tuberculata plus the four new species described herein) are marked in colour. The trees were rooted with Brookesia nasus, and including B. brygooi and B. superciliaris as hierarchical outgroups (not shown). A, B and C refer to major clades as discussed in the text. Asterisks denote posterior probabilities of 1.0.

Figure 4. Morphometric differentiation among species of the Brookesia minima group.

The two left graphs are scatterplots of the first two factors of Principal Component Analyses of (a) male and (b) female specimens of the Brookesia minima group based on measurements in Tables 1 and 2, Supporting Information S1 and . Note that Factor 1 is mostly influenced by the size of specimens (see Table 5). The graphs on the right (c–d) are univariate scatterplots of selected measurements and morphometric ratios of male specimens of species in the Brookesia minima group.

Figure 5. Morphometric differentiation among sexes in species of the Brookesia minima group.

Scatterplot of tail length vs. snout-vent length in male and female specimens of the Brookesia minima group, showing larger body sizes in females and a weak trend towards relatively longer tails in males. Based on measurements in Tables 1 and 2, Supporting Information S1 and .

Figure 6. Hemipenes of species in the Brookesia minima group.

The photos show for each species, a general view of the organs and a close-up. For B. desperata, the inset picture shows a non-turgid everted hemipenis where the two apex projections are very prominent. Note that also several other of the shown preparations are not fully turgid, especially in B. ramanantsoai and B. micra. In two other species (B. confidens and B. tristis) the shown hemipenes might not be fully everted.

Figure 7. Dorsal, lateral and ventral views of preserved male holotypes of newly described species.

Scale bar equals 5 mm.

Figure 8. Brookesia micra sp. n. from Nosy Hara, northern Madagascar.

(A) adult male on black background, showing orange tail colouration; (B) juvenile on finger tip; (C) juvenile on head of a match; (D) habitat along a small creek on western flank of Nosy Hara, where part of the type series was collected.

Figure 9. Life history and morphology of Brookesia desperata.

(A) Female (displaying stress colouration) with two recently laid eggs. (B) Figure showing well-developed pelvic spine (1) and lateral spines on tail (2).

Figure 10. Adult specimens of newly described species in life.

(A) male and (B) female of Brookesia tristis from Montagne des Français; (C) male and (D) female of Brookesia confidens from Ankarana; (E) male and (F) female of Brookesia micra from Nosy Hara; (G) male and (H) female Brookesia desperata from Forêt d'Ambre.