Evolutionary Biology

, Volume 40, Issue 2, pp 228–245 | Cite as

The Early Stages of Speciation in Amazonian Forest Frogs: Phenotypic Conservatism Despite Strong Genetic Structure

  • Igor L. Kaefer
  • Bruno M. Tsuji-Nishikido
  • Edvaldo P. Mota
  • Izeni P. Farias
  • Albertina P. Lima
Research Article


Phylogeographic perspectives incorporating multiple classes of characters, especially those relating to sexual signals, are promising for the elucidation of recent evolutionary mechanisms driving speciation. Here, forest frogs were used as a model system to access distinct stages in the process of evolutionary differentiation. We studied 280 individuals assigned to three species: Allobates paleovarzensis, A. nidicola and A. masniger. Samples were collected at 20 localities arranged in two study systems, along the middle Amazon and the lower Madeira Rivers, in Central Amazonia. Mantel tests, analyses of molecular variance, and the spatial distribution of haplogroups indicated that the distribution of genetic variability, as inferred from a mitochondrial DNA marker, was determined by a combination of isolation-by-distance effects and the transposition of large Amazonian rivers. These two factors had contrasting relative influences in each of the study systems, which also differed regarding the estimated time of the major cladogenetic events. Pronounced population genetic structure was observed. However, multivariate discriminant function analyses revealed that the phenotypic (morphological and acoustic) divergence was loosely related with genetic differentiation and did not successfully predict assignment of individuals to genetic groups. The observed distribution of genetic variability showed the important role of genetic drift in the diversification of the mitochondrial marker studied. The phenotypic conservatism among populations was surprising in view of the high genetic structuring observed, and indicates a prevailing role of stabilizing selective forces in the process of sexual signal and morphological differentiation.


Genetic drift Integrative phylogeography Isolation by distance Mitochondrial DNA River barrier Sexual signals 



We thank Anelise Montanarin, Francisco C. de Freitas, Irene da S. Melo, Maria A. Carvalho, Moisés da S. Melo and Raimundo N. Amorim for fieldwork assistance; Daniela Leroy e Waleska Gravena for help in laboratory procedures; Adolfo Amézquita, Adrian Garda, Andrew J. Crawford, Camila Ribas, Heike Pröhl, Janet W. Reid, Jeffrey Podos, Luciana K. Erdtmann, Marcelo Gordo, Marcelo Menin, Marina Anciães, Pedro Ivo Simões, Tomas Hrbek, Vanessa Verdade and Walter Hödl for valuable suggestions during this study. We extend thanks to two anonymous reviewers whose observations led to further improvements in the text. We also thank the Brazilian Conselho de Desenvolvimento Científico e Tecnológico for financial support (CNPq-CT Amazônia 553997/2006-8 and 575572/2008-6).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

This study complies with the current Brazilian laws and was allowed by RAN-ICMBio/IBAMA (licences 13777-2, 18516-1, 20065-2, and 21950-1).

Supplementary material

11692_2012_9205_MOESM1_ESM.pdf (23 kb)
Online Resource 1 Advertisement call measurements of individuals of Allobates paleovarzensis in each study locality in Brazilian Amazonia. Values are presented as mean (above) and standard deviation (below). Call traits are: Note rate (NR, in notes/s); Note duration (ND, in s); Internote interval, as the silent interval between two consecutive notes of a call (InI, in s); Call rate (CR, in calls/s); Call duration (CD, in s); Intercall interval, as the silent interval between two consecutive calls (IcI, in s); Maximum (peak) frequency, as the frequency of higher intensity calculated for the entire note by a power spectrum function of Raven Pro 1.3 (MF, in Hz); Lowest frequency (LF, in Hz); Highest frequency (HF, in Hz); Note modulation, as the difference between the highest and the lowest frequencies of the call (NM, in Hz). Sampling locality codes, sample sizes and respective geographic coordinates are given on Table 1. (PDF 23 kb)
11692_2012_9205_MOESM2_ESM.pdf (19 kb)
Online Resource 2 Advertisement call measurements of individuals of Allobates nidicola and A. masniger in each study locality in Brazilian Amazonia. Values are presented as mean (above) and standard deviation (below). Call traits are: Note duration (ND, in s); Interval between notes (IN, in s); Lowest frequency (LF, in Hz); Highest frequency (HF, in Hz); Note modulation, calculated as the difference between the maximum and minimum frequencies (NM, in Hz); and Peak frequency (PF, in Hz). Sampling locality codes, sample sizes and respective geographic coordinates are given on Table 1. (PDF 18 kb)
11692_2012_9205_MOESM3_ESM.pdf (37 kb)
Online Resource 3 Morphometric measurements (in mm) of individuals of Allobates paleovarzensis in each study locality in Brazilian Amazonia. Values are presented as mean (above) and standard deviation (below). Morphometric traits are: Snout-vent length (SVL); Head length from angle of jaws to tip of snout (HL); Head width at level of angle of jaws (HW); Snout length from anterior corner of eye to tip of snout (SL); Eye to nostril distance from anterior corner of eye to centre of nostril (EN); Inter-nostril distance (IN); Eye length from anterior to posterior corner (EL); Inter-orbital distance (IO); Diameter of tympanum (TYM); Forearm length from proximal edge of palmar tubercle to outer edge of flexed elbow (FAL); Upper arm length from trunk insertion to outer edge of flexed elbow (UAL); Hand length from proximal edge of palmar tubercle to tip of fingers I, II, III and IV (HAND1, HAND2, HAND3 and HAND4); Width of disc on finger III (WFD); Tibia length from outer edge of flexed knee to heel (TL); Foot length from proximal edge of outer metatarsal tubercle to tip of toe IV (FL); Femur length (LL); Diameter of palmar tubercle (DPT); Width of tenar tubercle (WTT); Width of disc on toe IV (WTD); Width of finger III (WPF). Sampling locality codes, sample sizes and respective geographic coordinates are given on Table 1. (PDF 37 kb)
11692_2012_9205_MOESM4_ESM.pdf (37 kb)
Online Resource 4 Morphometric measurements (in mm) of individuals of Allobates nidicola and A. masniger in each study locality in Brazilian Amazonia. Values are presented as mean (above) and standard deviation (below). Morphometric traits are described in Online Resource 3. Sampling locality codes, sample sizes and respective geographic coordinates are given on Table 1. (PDF 37 kb)
11692_2012_9205_MOESM5_ESM.pdf (24 kb)
Online Resource 5 Distribution of 16S rDNA haplotypes of Allobates among 20 sampled localities in Brazilian Amazonia. Collection numbers of vouchers (INPA-H) and GenBank accession numbers are provided. Sampled localities are numbered according to Table 1. (PDF 24 kb)
11692_2012_9205_MOESM6_ESM.pdf (335 kb)
Online Resource 6 Mismatch distributions obtained from pairwise nucleotide site differences among 16S rDNA sequences composing the clusters of the (A) paleovarzensis and the (B) nidicolamasniger systems. Clusters were determined via Bayesian Analysis of Population Differentiation. Pairwise differences were not calculated for Cluster 4 of Allobates paleovarzensis due to the small number of samples that compose it. (PDF 334 kb)
11692_2012_9205_MOESM7_ESM.pdf (18 kb)
Online Resource 7 Classification matrix based on the discriminant function analysis, using phenotypic data from males of Allobates paleovazensis pertaining to four genetic clusters. Clusters are coded from C1 to C4, according to Table 4. Body-shape traits were used to discriminate individuals based on morphology. Acoustic measurements were adjusted for temperature and for both temperature and body size through linear regressions. The number and percentage of individuals correctly assigned to each genetic cluster are indicated. (PDF 18 kb)
11692_2012_9205_MOESM8_ESM.pdf (24 kb)
Online Resource 8 Classification matrix based on the discriminant function analysis, using phenotypic data from males of Allobates nidicola and A. masniger pertaining to seven genetic clusters. Clusters are coded from C1 to C7, according to Table 4. Body-shape traits were used to discriminate individuals based on morphology. Acoustic measurements were adjusted for temperature and for both temperature and body size through linear regressions. The number and percentage of individuals correctly assigned to each genetic cluster are indicated. (PDF 24 kb)


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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Igor L. Kaefer
    • 1
  • Bruno M. Tsuji-Nishikido
    • 2
  • Edvaldo P. Mota
    • 2
  • Izeni P. Farias
    • 2
  • Albertina P. Lima
    • 1
  1. 1.Programa de Pós-Graduação em EcologiaInstituto Nacional de Pesquisas da AmazôniaManausBrazil
  2. 2.Instituto de Ciências BiológicasUniversidade Federal do AmazonasManausBrazil

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