This document summarizes a study that analyzed genetic variation in the Pygmy Antwren (Myrmotherula brachyura) across its range in Amazonia. Tissue samples were collected from five countries and sequenced to analyze the mitochondrial ND2 gene. Four well-supported clades were identified that corresponded to geographic regions divided by the Amazon River and its major tributaries. Genetic divergence across rivers was low and uniform for M. brachyura compared to related understory species, suggesting it responds less to river barriers. Vocal variation also divides M. brachyura into three groups matching the genetic clades, providing evidence that it may comprise multiple biological species.

1. Patterns of genetic differentiation in Myrmotherula brachyura (Pygmy Antwren)
Adam Martin1,2 and R. Terry Chesser2,3
1Department of Fisheries and Wildlife, Oregon State University, Corvallis, OR; 2Division of Birds, Department of Vertebrate Zoology, National Museum of Natural
History, Washington DC, USA; 3USGS Patuxent Wildlife Research Center, National Museum of Natural History, Washington DC, USA
Methods
• Tissue samples of M. brachyura from localities in Brazil, Guyana,
Ecuador, Peru, and Bolivia (Fig. 1) were obtained from five museum
collections (see Acknowledgments).
• Two samples of the sister species M. obscura were used as the
outgroup.
• Genomic DNA was extracted from all samples using QIAGEN extraction
kits.
• The mitochondrial gene NADH Dehydrogenase 2 (ND2) was amplified
using standard PCR protocols.
• Samples were sequenced using an ABI PRISM 3130 Genetic Analyzer,
and Sequencher 4.10.1 was used to align and edit the sequences.
• Maximum Parsimony (MP) and Maximum Likelihood (ML) analyses
were conducted using PAUP* 4.0b10 and RAxML, respectively.
• Support for phylogenies was assessed using 1000 parsimony bootstrap
replicates and 100 likelihood bootstrap replicates.
Discussion
• Maximum genetic divergence within canopy species M. brachyura (2%)
was much lower than that within understory species M. menetriesii (5%)
and M. longipennis (6%) (Chesser, unpubl. data).
• Divergence across individual rivers in M. brachyura was lower than that
of M. menetriesii and M. longipennis, except for the Rio Negro.
• The variability in divergence across rivers was much greater in M.
menetriesii and M. longipennis than in M. brachyura.
• Preliminary analyses of vocalizations of M. brachyura (M. Isler, unpubl.
data) identified three distinct groups corresponding to the NE, SE, and
SC+W genetic clades.
• Vocal and genetic data suggest that M. brachyura is likely comprised of
at least 3 biological species and of at least 4 phylogenetic species.
Acknowledgments
We thank:
• The Smithsonian Natural History Research Experiences Program, Cristián Samper, Elizabeth
Cottrell, Eugene Hunt, and Virginia Power.
• Robert Fleischer, Jesus Maldonado, and Nancy Rotzel of the Center for Conservation and
Evolutionary Genetics.
• The following institutions for contributing tissue samples to this project: Louisiana State
University Museum of Natural Science, Baton Rouge; Museu Paraense Emilio Goeldi,
Belém, Brazil; National Museum of Natural History, Washington, DC; Field Museum of
Natural History, Chicago; Academy of Natural Sciences, Philadelphia.
• Mort Isler for contributing the base map and unpublished data on vocalizations.
• Yesha Shrestha and Tammy Wilbert for laboratory assistance.
Literature cited
• Burney, C. W., and R. T. Brumfield. 2009. Ecology predicts levels of genetic differentiation in
Neotropical birds. Amer. Nat. 174: 358-368.
• Snethlage, E. 1913. Über die verbreitung der vogelarten in unteramazonien. J. Ornithol. 61: 469-
539.
• Wallace, A.R. 1852. On the monkeys of the Amazon. Proc. Zool. Soc. London 20: 107-110.
Introduction
• Distributions of many Amazonian species and subspecies are delimited
by the Amazon and its major tributaries, including the rios Negro,
Madeira, and Tapajós (Wallace 1852, Snethlage 1913).
• The monotypic species Myrmotherula brachyura is a suboscine
passerine bird found in canopy and sub-canopy of lowland forest
throughout the Amazon Basin.
• Canopy species are thought to respond to riverine barriers to a lesser
extent than understory species (Burney and Brumfield 2009),
suggesting that M. brachyura may show little genetic structure across
Amazonia.
• However, vocalizations of M. brachyura are known to be
geographically variable (M. Isler, unpubl. data) – differences in
vocalizations suggest that distinct evolutionary units may exist
within M. brachyura.
• We studied genetic variation in M. brachyura across its geographic
range to address the following questions:
1. Does M. brachyura show genetic variation across Amazonia?
2. If so, is this variation consistent with major rivers as barriers to
gene flow?
3. Is genetic divergence across rivers lower in M. brachyura than
in co-distributed congeners that inhabit forest understory (e.g.,
M. menetriesii and M. longipennis)?
4. Does genetic variation mirror vocal variation in M. brachyura?
5. Do genetic and vocal data suggest that M. brachyura is
comprised of more than one species?
Figure 2. One of 750 most parsimonious trees. Numbers above branches indicate
bootstrap support (MP/ML). Individuals of M. brachyura are identified by locality
and color coded by geographic region.
Figure 1. Map showing localities of M. brachyura samples. Ranges of genetically
distinct clades are color coded to correspond to colors on the phylogenetic tree.
Table 1. Comparison of genetic divergence across major Amazonian rivers for
species of Myrmotherula antwren that inhabit different forest strata.
Results
• ND2 sequences were obtained for 48 individuals: 46 M. brachyura and 2
M. obscura.
• Individuals of M. brachyura grouped into four well-supported and
geographically coherent clades.
• Phylogenetic relationships among clades were poorly resolved.
• Geographic boundaries between clades coincide with the lower Amazon
and three main tributaries: the Rio Madeira, Rio Negro, and Rio Tapajós.
• Genetic divergence across these rivers was virtually uniform, ranging
from 1.7% to 2.0%.
species stratum Amazon Tapajós Madeira Negro
M. brachyura canopy 2.0% 1.8% 1.7% 1.8%
M. menetriesii/
M. longipennis understory 4.5 - 6% 3 - 5% 2% 1%
obscura
Nazare Braz 1
Nazare Braz 2
Nazare Braz 3
Florida Bol
Mamore Braz
Fe Deus Braz 1
Fe Deus Braz 2
Fe Deus Braz 4
Fe Deus Braz 6
Fe Deus Braz 5
Fe Deus Braz 3
Nazare Braz 4
Juanjui Peru
Ramon Braz
Oran Peru
Zancudo Ec
Vainilla Peru
Pithecia Peru
Japura Braz 1
Japura Braz 2
Papagaio Braz
Manaus Braz 1
Manaus Braz 2
Manaus Braz 3
Manaus Braz 7
Manaus Braz 4
Manaus Braz 5
Manaus Braz 6
Manaus Braz 8
Baramita Guy 1
Baramita Guy 2
Baramita Guy 3
Baramita Guy 4
Baramita Guy 5
Baramita Guy 6
Acarai Guy 1
Acarai Guy 2
Linden Guy
Waruma Guy
Iwokrama Guy 1
Iwokrama Guy 2
Iwokrama Guy 3
Iwokrama Guy 4
Altamira Braz
Aucre Braz 1
Aucre Braz 2
1 change
100/100
97/100
97/99
98/96
–/–
67/–
100/100
South-Central
West
Northeast
Southeast
PhotobyThierryNogaro
Background photo retrieved from http://awillforthewoods.blogspot.com
W SE
SC
NE