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. 2020 Feb 21;15(2):e0229324.
doi: 10.1371/journal.pone.0229324. eCollection 2020.

One Step Beyond a Broad Molecular Phylogenetic Analysis: Species Delimitation of Adenomera Marmorata Steindachner, 1867 (Anura: Leptodactylidae)

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One Step Beyond a Broad Molecular Phylogenetic Analysis: Species Delimitation of Adenomera Marmorata Steindachner, 1867 (Anura: Leptodactylidae)

Carla S Cassini et al. PLoS One. .
Free PMC article


Taxonomists always have had intense discussions about how species should be delimited and recently many studies have used integrative approaches by combining molecular, morphological, and bioacoustic data. Although these studies are paramount for understanding species diversity, few of them actually formalize species delimitations to the final step of nomenclatural acts. Historically, the Neotropical frog genus Adenomera has been considered as a difficult taxonomic group because it comprises many morphologically similar species exhibiting high levels of intraspecific polymorphism. A recent work using molecular data shed light on the phylogenetic relationships within the genus and identified several lineages that may correspond to undescribed species but did not delimit species boundaries. In the Atlantic Forest, a clade formed by A. marmorata and two putative species (Adenomera sp. J and Adenomera sp. K) were identified. In this paper, we combine morphological, acoustic, and molecular data in order to evaluate species limits within this Atlantic Forest Adenomera clade. We provide a redescription of A. marmorata and restrict its type locality to the Tijuca Massif, in the Municipality of Rio de Janeiro, Brazil. Our results do not support A. marmorata and the two candidate species as diagnosable distinct species. Therefore A. marmorata corresponds to a species with pronounced morphological and acoustic variation in the genus and a complex phylogeographic structure.

Conflict of interest statement

The authors have declared that no competing interests exist.


Fig 1
Fig 1. Bayesian inference and genetic samples.
50% majority rule consensus tree from Bayesian inference analysis of concatenated nuclear (POMC and RAG) and mitochondrial (COI, CYTB, and 16S) genes, and map containing sampled localities for molecular analyses. Numbers above branches indicate Bayesian posterior probabilities (pp) and numbers below branches indicate non-parametrical bootstrap support. Asterisks indicate clades fully supported (1.0 [pp] or 1000 [bootstrap]). Colors of the clades match colors of the localities.
Fig 2
Fig 2. Statistical parsimony networks of phased nuclear loci for CCS Ma, CCS J and CCS K.
(A) POMC; (B) fragment RAG1; (C) map containing RAG1 sampled localities for CCSs Ma, J and K. Haplotypes are shown as circles proportional in size to haplotype frequency.
Fig 3
Fig 3. Variation of Snout-Vent Length among CCS Ma, CCS J, CCS K and other Adenomera species.
Male (left) and female (right) specimens.
Fig 4
Fig 4. Morphological variation regarding the presence or absence of tibial tubercles.
(A) CFBH 34402, CCS Ma, Rio de Janeiro, RJ; (B) CFBH 36131, CCS Ma, Nazaré Paulista, SP; (C) CFBH 17130, CCS J, Alcatrazes Island, SP; (D) CFBH 23923, CCS J, Santos, SP; (E) CFBH 36001, CCS K, Ubatuba, SP; (F) CFBH 444653, CCS K, Itatiaia, RJ.
Fig 5
Fig 5. Advertisement call of the candidate species CCS Ma, CCS J and CCS K.
Spectrogram (above) and oscillogram (bellow) of one call of (A) CCS Ma (clade Ma1), Tijuca Massif, Rio de Janeiro, RJ; (B) CCS Ma (clade Ma2), Nazaré Paulista, SP; (C) CCS K (clade K1), Itatiaia, RJ; (D) CCS K (clade K2), São Luís do Paraitinga, SP; (E) CCS J (clade J1), Mogi das Cruzes, SP; (F) CCS (clade J2), Santo André, SP.
Fig 6
Fig 6. Principal Component Analysis (PCA) of call characters of CCS MA, CCS J and CCS K.
Fig 7
Fig 7. Topotype of Adenomera marmorata, CFBH 34403.
(A) Dorsal, (B) Ventral, and (C) lateral views. Scale bar = 5 mm.
Fig 8
Fig 8. Holotype of Adenomera marmorata, NHMW 16453.
Photographs by Dr. Heinz Grillitsch.
Fig 9
Fig 9. Traversed area by the “Novara Reise” [Novara Expedition] crew (August, 1857).
In red, route taken for the city of Rio de Janeiro forested areas recognition, through the Tijuca Massif (type locality of Adenomera marmorata). In blue, route taken via rail and road transports to the municipality of Petrópolis. Blue dots indicate islands visited during an excursion through Guanabara Bay (image modified from INDE–Infraestrutura Nacional de Dados Espaciais, available at:

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Grant support

This study was part of C. S. C.’s PhD degree and postdoctoral research and P. P. G. T.'s Master’s degree. C. S. C. thanks FAPESP (grant #2010/51606-8) for doctorate scholarship and CNPq for current postdoctoral research funding. P. P. G. T. thanks for Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for MSc fellowship and FAPESP (grant #2019/04076-8) for current funding. São Paulo Research Foundation (FAPESP) and Programa de Pós-Graduação em Zoologia da UFMG gave field financing. T. R. C. has received scholarship from FAPESP (#2012/15763-7, #2015/13404-8, and #2017/08489-0). This work has benefited from an “Investissement d’Avenir” grant managed by Agence Nationale de la Recherche (CEBA, ref. ANR-10-LABX-25-01). C. F. B. H. thanks FAPESP (grant #2013/50741-7) and CNPq for financial support. P. C. A. G. thanks Fapemig and CNPq for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.