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Phylogeny and Diversification of Pomatorhinus ruficollis using Mitochondrial and Nuclear Genes

Background Information

While there has been much interest in Asian avifauna, the diversity of species present today is still unknown despite its profound implications for preserving the integrity of long-standing ecosystems. This research focuses on the bird family Timaliidae (commonly known as babblers) as they are a key component in this region and can help us understand the biogeography of southeast Asia. Babblers can be studied to assess general diversification patterns however, species-limits in many taxa within this family still need to be resolved. Previous studies have been far from systematic and the Tamiliidae family has been considered a ‘dustbin’ by taxonomists and systematists. To understand the phylogeny of one babbler species, Pomatorhinus ruficollis, both mitochondrial and nuclear genes were studied. Since mitochondrial genes evolve at a faster rate than nuclear genes, we will test whether the signal from both these sources are in agreement as to the diversification patterns within this species.


  • Establishing phylogenetic relationships of P. ruficollis birds collected from various locations
  • Determining if there is genetic and geographic structure across populations of P. ruficollis
  • Comparing the influence of mitochondrial versus nuclear genes

Materials & Methods

  • Specimen Collection:  Expeditions from University of Kansas collected a total of 48 tissue samples from the following seven sites in China:
    • Shiwandashan National Nature Preserve (SNNP)
    • Diding Headwater Nature Preserve (DHNP)
    • Dawei Shan National Park (DSNP)
    • Dongan Shun Huang Shan National Park (DSHSNP)
    • Dashahe Nature Preserve (DNP)
    • Kuan Kuo Shui Nature Reserve (KKSNR)
    • Libo, 8 km N 
  • Extraction of DNA from samples:  DNA was extracted from 46 tissue samples using the Qiagen DNeasy Tissues Kit.  For the other two tissue samples, DNA was extracted using the OMEGA SQ Tissue DNA Kit. 
  • DNA Amplification: The complete mitochondrial gene ND3 (351 bp) and the nuclear TGFb2 intron 5 locus (~500-600bp) were amplified using standard PCR procedures. Each sample underwent a 25 μL reaction containing: Promega GoTaq Green Master Mix NonHotStart 2X, 10μM solution of forward and reverse primers, nuclease-free water, and 2μL of DNA.  The primers used to amplified ND3 were H11151 and L10755 and the primers used to amplify TGF were TGFb2ex5 and TGFb2ex6.
  • Sequencing of genes:  PCR products were purified using the Agencourt AMPure XP protocol.  Clean products were placed in cycle sequencing with 5 μM solution of forward and reverse primers, ABI BigDye 3.1, and a dilution buffer of Tris-MgCl2 solution.  Sequencing reactions were cleaned using ethanol and EDTA precipitation.  
  • Data Analysis: Generated sequences were edited using Geneious Pro 5.0.4.  Both forward and reverse strands were used to ensure the quality  of the generated sequences.  The sequences were aligned using MUSCLE and the trees were generated using the maximum likelihood method in GARLI 1.0 and viewed in FigTree v1.3.1.


Fig 1. Map of locations in which the P. ruficollis specimens were collected. Each shape represents a different location. The abbreviations correspond with the site locations listed in Materials & Methods.

Fig. 2.  Maximum likelihood tree of  P. ruficollis specimens using the mitochondrial ND3 gene.  Xiphirhynchus superciliaris was used as an outgroup.  54 taxa were included in the analysis.

Fig. 3. Maximum likelihood tree of  P. ruficollis specimens using the nuclear TGF gene.  Xiphirhynchus superciliaris was used as an outgroup.  38 taxa were used in the analysis.

Fig. 4. Maximum likelihood tree of P. ruficollis specimens combining the ND3 gene and the TGF gene. Xiphirhynchus superciliaris was used as an outgroup. 34 taxa were included in the analysis.


  • Of the 351 characters, the ND3 gene had 41 parsimony-informative characters and of the 530 characters, the TGF gene had 7 parsimony-informative characters. Therefore, it can be reasonably inferred that the mitochondrial genes have more signal at this level of phylogenetic analysis. 
  • Both the ND3 tree and the combined tree show that populations on either side of the Pearl River (Ji Xiang) are distinct. Figure 3 shows that the TGF does not display a pattern such that specimens from the same locality have similar genetic traits likely due to a slower rate of evolution in this nuclear gene. Despite this, there is evidence for some genetic structure as there are two divergent clades and further analysis is needed to assess this phenomenon. 
  • The trees show that while the specimens studied are related to other species within the same family, they are clearly distinct species themselves. This corroborates with Reddy & Moyle’s (in prep) results that these Chinese populations consist of at least two distinct evolutionary lineages.

Future Directions

The data collected at this point is the beginning for determining the phylogenetic relationships of the Chinese streak-breasted scimitar babblers. At present, one mitochondrial gene (ND3) and one nuclear gene (TGF) have been sequenced and analyzed.  The mitochondrial genes ND2 & Cytb and the nuclear genes FIB5 & MUSK will be examined next.


Specimens: University of Kansas Biodiversity Institute for tissue samples; Field Museum of Natural History for access to the DNA sequencer
Support: Sushma Reddy for guidance; Rachel Baker and John Juranek for comments and assistance
Funding:  National Science Foundation DEB-0962078 and  REU supplement DEB-1026960; Loyola University Chicago Biology Summer Research Fellowship


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Sarah Sharief,
Aug 12, 2010, 9:17 AM