Vast underestimation of Madagascar's biodiversity evidenced by an integrative amphibian inventory

David R. Vieites; Katharina C. Wollenberg; Franco Andreone; Jörn Köhler; Frank Glaw; Miguel Vences

doi:10.1073/pnas.0810821106

New Research In

Edited by David B. Wake, University of California, Berkeley, CA, and approved March 27, 2009 (received for review October 26, 2008)

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Fig. 1.
Phylogenetic, geographic, and historical patterns of undescribed amphibian diversity in Madagascar. (A) Bayesian phylogenetic tree of 236 (out of 244) described species and 258 deeply divergent genealogical lineages of Malagasy frogs (among them 129 CCS and 92 UCS and 37 DCL with >3% genetic divergence to nearest described neighbor) based on a fragment of the mitochondrial 16S rRNA gene. Circles represent CCS (brown), UCS (orange), and DCL (light orange). Inset photos show some of these forms (see SI Appendix). He, Heterixalus (Hyperoliidae); Dy, Dyscophus; Sc, Scaphiophryninae; Co, Cophylinae (Microhylidae); Sp, Spinomantis; Md, Mantidactylus; Mt, Mantella; Gu, Guibemantis; Ge, Gephyromantis; Bo, Boophis; Bl, Blommersia; Ag, Aglyptodactylus (Mantellidae). (B and C) Maps of collecting localities of described species (B) and of CCS and UCS (C) of Malagasy frogs, and remaining primary vegetation (evergreen forests, blue; nonevergreen forests, gray). Current protected area network is shown in red. (D) Cumulative number of species currently considered as valid per 10-year intervals (2001–2008 for the current decade), and the CCS, UCS, and DCL identified in the present paper. (E) Total numbers of described species and CCS, UCS, and DCL in major clades of Malagasy frogs.
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Fig. 2.
Examples of combined phylogeographic, bioacoustic, and morphological evidence used to classify divergent mitochondrial lineages as CCS or DCL. (A) Boophis majori and B. aff. majori are sympatric CCS; despite their only low genetic divergence, they have distinct and constant differences in tadpole morphology and qualitative differences in advertisement calls, without signal of genetic admixture. (B and C) B. luteus (B) and Guibemantis liber (C) consist of deeply divergent genealogical lineages classified as DCL, because the call differences only affect quantitative parameters such as note-repetition rate in B. luteus, and there are no morphological or ecological differences between the populations. Furthermore, genetic admixture of the lineages was detected in G. liber at one locality.

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Table 1.

Definitions of proposed categories of candidate species

Category	General definition	Definition used in Malagasy frogs
Unconfirmed Candidate Species (UCS)	Default category for deep genealogical lineages of unknown status. The genetic differentiation must be above a threshold value typical for comparisons among closely related species in the group of animals under study. Data deficient for morphology, ecology, and distribution.	Uncorrected pairwise genetic divergences in 16S rRNA gene >3% to all other described species. No data on morphology and bioacoustics due to unavailability of voucher specimens or immature state of vouchers.
Confirmed Candidate Species (CCS)	Specimens or populations characterized by a detectable genetic differentiation to all described species, not necessarily above any threshold, but in concordance with at least one of the following criteria: a distinct differentiation in a character that mediates a premating reproductive barrier, a diagnostic morphological difference in a character that in the respective group of animals is known to be of low intraspecific variability and of high value to discriminate species, sympatric occurrence without admixture, and with at least one phenotypic character state difference, even if subtle, strictly correlated to the genealogy inferred from a neutral molecular marker.	Uncorrected pairwise genetic divergences in 16S rRNA gene to all other described species in most cases >3%, sometimes only 1–2%. Concordance of this molecular differentiation with one of the following: a qualitative difference in advertisement calls, diagnostic difference in at least one morphological character known to be generally species-specific in Malagasy frogs: e.g., presence and extent of dermal spines, ridges and tubercles, extension of terminal finger tips, morphology of femoral glands, relative tympanum size, iris color, or tadpole mouthparts.
Deep Conspecific Lineage (DCL)	Deep genealogical lineages above a threshold value typical for comparisons among closely related species in the group of animals under study. One or several of the following must apply: morphological and chromatic data reveal no differences to topotypic populations of a described species, or only subtle differences in characters that are known to show intraspecific variability, or only quantitative differences (in characters such as body size), if characters relevant for premating isolation are known, then these show no or only quantitative differences to topotypic populations of described species, phylogeographic studies are available and show indications for genetic admixture with other genealogical lineages within a species.	Uncorrected pairwise genetic divergences in 16S rRNA gene > 3% to all other described species in combination with one or several of the following: no morphological differences, or differences only in characters known to show intraspecific variability: e.g., size, relative hindlimb length, body color and pattern, no difference in advertisement calls, or only differences in variables known to show intraspecific variability, co-occurrence with other deeply divergent haplotypes within the same populations, not concordant with any distinct or subtle morphological difference among individuals.