Vast underestimation of Madagascar’s biodiversity evidenced by an integrative amphibian inventory David R. Vieitesa,1, Katharina C. Wollenbergb, Franco Andreonec,Jo¨ rn Ko¨ hlerd, Frank Glawe, and Miguel Vencesb aMuseo Nacional de Ciencias Naturales, Consejo Superior de Investigaciones Científicas, c/ Jose´Gutierrez Abascal 2, 28006 Madrid, Spain; bZoological Institute, Technical University of Braunschweig, Spielmannstrasse 8, 38106 Braunschweig, Germany; cMuseo Regionale di Scienze Naturali, Via Giolitti 36, 10123 Turin, Italy; dHessisches Landesmuseum Darmstadt, Friedensplatz 1, 64283 Darmstadt, Germany; and eZoologische Staatssammlung Mu¨nchen, Mu¨nchhausenstrasse 21, 81247 Munich, Germany Edited by David B. Wake, University of California, Berkeley, CA, and approved March 27, 2009 (received for review October 26, 2008) Amphibians are in decline worldwide. However, their patterns of Among terrestrial vertebrates, amphibians are characterized by a diversity, especially in the tropics, are not well understood, mainly rapid rate of species discovery (8, 9), with an overall increase in the because of incomplete information on taxonomy and distribution. number of amphibian species globally of 19.4% during the last We assess morphological, bioacoustic, and genetic variation of decade, reaching 6,449 currently recognized species (10). An im- Madagascar’s amphibians, one of the first near-complete taxon portant acceleration in the rate of new discoveries, mainly from samplings from a biodiversity hotspot. Based on DNA sequences of tropical areas, is obvious from many recent studies (11–16). These 2,850 specimens sampled from over 170 localities, our analyses discoveries are not the result of taxonomic inflation (9, 14, 17), but reveal an extreme proportion of amphibian diversity, projecting an correspond to real divergent species (18, 19). Although high almost 2-fold increase in species numbers from the currently numbers of undescribed amphibians have been estimated to exist in described 244 species to a minimum of 373 and up to 465. This poorly studied tropical regions (11, 15), these results remain un- diversity is widespread geographically and across most major verified for complete, highly diverse amphibian faunas. In parallel, phylogenetic lineages except in a few previously well-studied an increase of threatened amphibian species has been reported genera, and is not restricted to morphologically cryptic clades. We worldwide (8, 20, 21). Amphibians are of high conservation con- classify the genealogical lineages in confirmed and unconfirmed cern, with 43% of species being globally threatened (20), most of EVOLUTION candidate species or deeply divergent conspecific lineages based them in tropical regions with high amphibian diversity. on concordance of genetic divergences with other characters. This Madagascar is one of the top priority global hotspots for integrative approach may be widely applicable to improve esti- biodiversity conservation (1), affected by a high rate of habitat mates of organismal diversity. Our results suggest that in Mada- destruction (22). Its fauna and flora evolved largely in isolation gascar the spatial pattern of amphibian richness and endemism (23), and many taxa are characterized by a high degree of must be revisited, and current habitat destruction may be affecting microendemism within Madagascar (24–27). The native amphib- more species than previously thought, in amphibians as well as in ian fauna is constituted by 5 endemic evolutionary lineages of other animal groups. This case study suggests that worldwide frogs with 100% species-level endemism, 2 of which (the man- tropical amphibian diversity is probably underestimated at an tellids and the cophyline and scaphiophrynine microhylids) are unprecedented level and stresses the need for integrated taxo- very species-rich. Large-scale taxonomic inventories conducted nomic surveys as a basis for prioritizing conservation efforts within since 1991 have led to an increase from 133 to 244 described biodiversity hotspots. species, largely due to the exploration of new areas and the application of more efficient techniques. About 46 species were identified during the 1990s mainly based on bioacoustics; the biodiversity estimates ͉ new candidate species ͉ phylogeny ͉ application of combined methods, including molecular genetics, DNA barcoding was crucial in the discovery of 51 new species since 2000 as well as in the resurrection of species formerly considered to represent he current biodiversity crisis demands the study of broad- synonyms. Tscale spatial variation in species richness and endemism to Undescribed diversity may have an important impact on under- identify areas that merit special conservation attention. Global standing the spatial patterns of endemic radiations on the island, efforts to minimize biodiversity loss have led to prioritizing but objective estimates of species numbers are not available so far. biodiversity hotspots (1) which are defined as areas with high We report a comprehensive assessment of morphological, bio- concentrations of endemic species and that are undergoing acoustic, and genetic variation of the anuran fauna of Madagascar. exceptional habitat loss (2–4). A second step is the efficient Our goals are (i) to provide a reliable estimate of the proportion of implementation of conservation measures at a local scale which yet-undescribed amphibian species from Madagascar and their requires an understanding of spatial patterns of richness and phylogenetic and geographic distribution, (ii) to discuss the impact endemism within these hotspots (5). Assessments of such re- of our findings for global estimates of amphibian diversity, and (iii) gional priority areas are often hampered by incomplete distri- to propose a novel terminology to be better able to assess the butional and taxonomic information. The operational units used increasing number of identified but taxonomically undescribed to assess conservation priority areas are described species, candidate species of animals. whereas estimates of undiscovered and undescribed species are usually ignored. Recently, many potential new species have been Author contributions: D.R.V. and M.V. designed research; D.R.V., K.C.W., F.A., J.K., F.G., and identified by DNA barcoding, but a taxonomic validation of M.V. performed research; D.R.V., K.C.W., J.K., and M.V. analyzed data; and D.R.V. and M.V. these species will outdate the short time span left for efficient wrote the paper. biodiversity conservation prioritization, and so far it is not clear The authors declare no conflict of interest. how this undescribed diversity can nonetheless be considered. This article is a PNAS Direct Submission. This undescribed diversity also bears relevance for understand- Data deposition: The sequences reported in this paper have been deposited in the Genbank ing the tree of life: The completeness of taxon sampling is one database (accession nos. FJ559069–FJ559372 and FJ217329–FJ217345) of the major prerequisites for reliable phylogenetic analysis, 1To whom correspondence should be addressed. E-mail: [email protected]. reconstruction of character evolution, and inference of macro- This article contains supporting information online at www.pnas.org/cgi/content/full/ evolutionary processes (6, 7). 0810821106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810821106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 30, 2021 Fig. 1. Phylogenetic, geographic, and historical pat- terns of undescribed amphibian diversity in Madagas- car. (A) Bayesian phylogenetic tree of 236 (out of 244) described species and 258 deeply divergent genealog- ical lineages of Malagasy frogs (among them 129 CCS and 92 UCS and 37 DCL with Ͼ3% 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 Appen- dix). 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 remain- ing primary vegetation (evergreen forests, blue; non- evergreen forests, gray). Current protected area net- work 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 num- bers of described species and CCS, UCS, and DCL in major clades of Malagasy frogs. Results frogs, indicating that they are not the result of taxonomic inflation From our integrative analyses of morphological, bioacoustic, and (e.g., elevating subspecies to specific status). genetic data, we identified many more species of Malagasy frogs New discoveries are also geographically ubiquitous. They occur than there are names available. The number of described species throughout Madagascar, both in poorly explored and in better- of Malagasy frogs has slowly increased during the 20th century, studied areas. Described species are known from 451 sites and reaching 100 described species around 1940, and 133 at the time 87.3% are found in protected areas, whereas CCS and UCS are of the last monographic account in 1991 (28) (Fig. 1). The known from 168 sites and only 66.4% are