Identification and characterization of microsatellite loci in two socially complex old world tropical babblers (Family Timaliidae)
© Kaiser et al. 2015
Received: 14 September 2015
Accepted: 10 November 2015
Published: 24 November 2015
Although the highest diversity of birds occurs in tropical regions, little is known about the genetic mating systems of most tropical species. We describe microsatellite markers isolated in the chestnut-crested yuhina (Staphida everetti), endemic to the island of Borneo, and the grey-throated babbler (Stachyris nigriceps), widely distributed across Southeast Asia. Both species belong to the avian family Timaliidae and are highly social, putatively cooperatively breeding birds in which helpers attend the nests of members of their social group. We obtained DNA from individuals in social groups breeding in Kinabalu Park, Malaysian Borneo.
We used a shotgun sequencing approach and 454-technology to identify 36 microsatellite loci in the yuhina and 40 in the babbler. We tested 13 primer pairs in yuhinas and 20 in babblers and characterized eight polymorphic loci in 20 unrelated female yuhinas and 21 unrelated female babblers. Polymorphism at the yuhina loci ranged from 3 to 9 alleles, observed heterozygosities from 0.58 to 1.00, and expected heterozygosities from 0.64 to 0.81. Polymorphism at the babbler loci ranged from 3 to 12 alleles, observed heterozygosities from 0.14 to 0.90 and expected heterozygosities from 0.14 to 0.87. One locus in the yuhina deviated significantly from Hardy–Weinberg equilibrium. We detected nonrandom allele associations between two pairs of microsatellite loci in each species.
Microsatellite markers will be used to describe the genetic mating system of these socially complex species and to measure genetic parentage and relatedness within social groups.
KeywordsBorneo Chestnut-crested yuhina Cooperative breeder Grey-throated babbler Old World tropics Parentage Microsatellite Stachyris nigriceps Staphida everetti
Tropical regions support the highest diversity of bird species than any other region worldwide . Yet, we know little about the social behavior and mating systems of tropical birds [2, 3], especially Old World tropical species . The chestnut-crested yuhina (Staphida everetti) is an endemic, resident bird that lives in tropical montane forests on the island of Borneo [4, 5]. The grey-throated babbler (Stachyris nigriceps) is a common resident of the tropical submontane forests of Northeast Indian subcontinent, southern China, Southeast Asia and Sumatra . Both species belong to the Old World avian family Timaliidae, which is compromised of oscine passerine birds generally known as babblers . Babblers show striking diversity in their social behaviors and mating systems . The chestnut-crested yuhina (hereafter, yuhina) is a highly social bird that forages throughout the canopy in large single-species flocks of 10–30 birds  and the grey-throated babbler (hereafter, babbler) forages in small groups of 5–8 individuals during the breeding months [4, 7]. Both species are putatively cooperative breeders in which helpers attend the nests of their social group members (T. E. Martin unpubl. data). We describe the isolation and characterization of eight polymorphic microsatellite loci in each species that we will use to measure genetic parentage and relatedness between breeders and their offspring and helpers and to investigate the social structure, dispersal, and genetic mating system of these species.
We used 454 GS-Junior shotgun sequencing to develop species-specific microsatellite markers. Genomic DNA was extracted from whole blood stored in lysis buffer  with the DNEasy Blood and Tissue DNA Kit (Qiagen, Valencia, CA). Genomic DNA (2 µg) was sheared on the Q800R sonicator (QSonica, Newton, CT) for 2 min into 300–500 bp fragments. The sheared DNA sample was purified with Sera-Mag Speed Beads (2×) (Thermo Fisher Scientific, Waltham, MA) and eluted in 15 μl of ddH20. Purified samples were prepared for 454 sequencing using a shotgun library preparation protocol . DNA fragments were blunt-ended and short adapters  ligated to the 3′ and 5′ ends of each fragment with the NEB Quick Blunting and Quick Ligase Kits (New England Biolabs, Ipswich, MA). One end of each fragment contained a unique sample-specific 8 bp barcode. Fragments with adapters successfully ligated were reamplified using emulsion PCR (emPCR) primers  and libraries were purified with Sera-Mag Speed Beads in PEG solution and size selected by gel extraction from a 1.5 % agarose gel with the MinElute Gel Extraction Kit (Qiagen). Libraries were quantified with the 454 Library Quantification Kit (Kapa). The yuhina single-stranded DNA (ssDNA) library had an average length of 500 bp and the babbler ssDNA library had an average length of 600 bp. We pooled the barcoded ssDNA libraries with one other individually barcoded species and conducted an emPCR at a concentration of 0.6 copies per bead with Lib-L Roche kits and reagents. The emPCR yielded 3 % enriched beads for sequencing a single PicoTiter plate on the 454 Genome Sequencer Junior System (GS-Junior, 454 Life Sciences, a Roche Company, Branford, CT). We used the Roche software shotgun pipeline for quality filtering on the GS-Junior, resulting in a total of 38,619 sequenced fragments for the three species. The 454 datasets were demultiplexed using a MIDconfig.parse file from the sfffile program. The single run yielded 17,645 reads for the yuhina (range 32–580 bp) and 8029 reads (range 32–548 bp) for the babbler, both with an average read length of 350 bp. We filtered the reads (min length = 60, max length = 400, ambiguity max 1 % of N, mean quality score = 15–25) and trimmed the run of low quality sequences (mean quality score: 5′ = 20, 3′ = 20) with PRINSEQ , resulting in 14,492 good reads for the yuhina and 6591 good reads for the babbler.
We used 454 sequence data to identify microsatellites and design PCR primer pairs. We screened for perfect and imperfect (>85 %) microsatellites (repeats of di-, tri- and tetranucleotides) with minimum repeat lengths of 20, 24, and 28 bp, respectively, with the Phobos plugin  in Geneious . We selected unique repeat motifs with adequate length of flanking sequences (~25 bp) to design primer pairs (~18 bp) for amplifying these repeats. We used BLAST  to identify and remove microsatellite sequences that matched known bacteria over the length of the read. We aligned all reads with microsatellites to remove exact duplicates with Geneious. This resulted in 36 unique microsatellites in the yuhina and 40 in the babbler for primer design. We designed five primer pairs for each microsatellite locus with the Primer3 plugin  and chose the best pair of primers for each microsatellite based on a combination of least dimer (pair, self, and/or hairpin) and matching melting temperatures between primers with a size <300 bp.
Thirteen yuhina primer pairs and 20 babbler primer pairs were tested for amplification, optimized, and screened for polymorphism using DNA from unrelated females each from different social groups (yuhinas = 20 females, babblers = 21 females) sampled in Kinabalu Park, Malaysian Borneo. We amplified 1 μL of genomic DNA from each individual at each locus in a 10 μL PCR containing 4.15 μL dH2O, 1 μL 10× PCR buffer, 1.0 μL 25 mM MgCl2 (2.5 mM final concentration), 1.0 μL 10 mM deoxyribonucleotide triphosphates, 0.4 μL 10 μM forward and pigtail reverse primers, 1.0 μL of 2.5× bovine serum albumin, and 0.05 μL 5.0 U μL−1 AmpliTaq Gold DNA polymerase [Applied Biosystems (ABI), Carlsbad, CA]. We initially used touchdown cycling conditions decreasing by 0.5 °C for each cycle (55–65 °C for yuhinas and 50–60 °C for babblers) with unlabeled forward primers to test for amplification and to optimize the annealing temperatures (TA) for each primer pair. After determining the optimal range of TA, we added a 5′ fluorescent label (6-FAM, 5-HEX; Eurofins MWG Operon; NED, ABI) to the forward primer and a six base-pair ‘pigtail’ (GTTTCT) to the 5′ end of the reverse primer (babbler primers only) to promote adenylation of the 3′ end of the forward strand to improve genotyping accuracy . We ran PCRs with fluorescently labeled forward primers on a DYAD thermal cycler (MJ Research) under the following conditions for yuhina primers: initial denaturing at 94 °C for 8 min, followed by eight cycles of 94 °C for 30 s, primer-specific upper TA for 30 s and decreasing by 0.5 °C for each cycle, 72 °C for 1 min, followed by 35 cycles of 94 °C for 30 s, primer-specific lower TA for 30 s, and 72 °C for 1 min, then a final extension at 72 °C for 30 min. We ran PCRs under the following conditions for babbler primers: initial denaturing at 94 °C for 8 min, followed by 45 cycles of 92 °C for 30 s, primer-specific TA for 40 s, 72 °C for 40 s and a final extension at 72 °C for 7 min. The labeled PCR products were analyzed on an ABI PRISM 3130 Genetic Analyzer (ABI) and allele sizes were scored with the GeneScan 500 ROX size standard (ABI) in Genemapper v.4.1 (ABI). All primers amplified and eight of these primers were polymorphic in each species.
Characteristics of eight microsatellite loci developed and optimized from the chestnut-crested yuhina, Staphida everetti
Primer sequence (5′–3′)
Size range (bp)
GenBank accession no.
Characteristics of eight microsatellite loci in the grey-throated babbler, Stachyris nigriceps
Primer sequence (5′–3′)
Size range (bp)
GenBank accession no.
Availability of supporting data
SK developed grey-throated babbler microsatellite markers and JD and LB developed chestnut-crested yuhina microsatellite markers. SK analyzed the data and drafted the manuscript. RF designed the study and helped to draft the manuscript. All authors read and approved the final manuscript.
We thank T. Martin and the numerous field technicians for collection of Staphida everetti and Stachyris nigriceps blood samples in Borneo. All research activities were performed under protocols approved by the Animal Care and Use Committees of the authors’ institutions and all federal and international permits were in hand when the research was conducted. We are grateful to Sabah Parks and the Sabah Biodiversity Centre in Malaysia for help in facilitating this study. This research was supported by a National Science Foundation Grant awarded to T. Martin, R. C. Fleischer, and E. Martinsen (DEB 1241041).
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Stutchbury BJM, Morton ES. Behavioral ecology of tropical birds. San Diego: Academic; 2001.Google Scholar
- Macedo RH, Karubian J, Webster MS. Extrapair paternity and sexual selection in socially monogamous birds: are tropical birds different? Auk. 2008;125:769–77.View ArticleGoogle Scholar
- Tori WP, Durães R, Ryder TB, Anciães M, Macedo RH, Uy JAC, Parker PG, Smith TB, Stein AC, Webster MS, Blake JG, Loiselle BA. Advances in sexual selection theory: insights from tropical avifauna. Ornitol Neotrop. 2008;19:151–63.Google Scholar
- Myers S. Birds of Borneo: Brunei, Sabah, Sarawak, and Kalimantan. Princeton: University Press; 2009.Google Scholar
- Collar N, Robson C. Chestnut-crested Yuhina (Staphida everetti). In: del Hoyo J, Elliot A, Christie D, editors. Handbook of the birds of the world: Picathartes to tits and chickadees, vol. 12. Barcelona: Lynx Edicions; 2007.Google Scholar
- Moyle RG, Andersen MJ, Oliveros CH, Steinheimer FD, Reddy S. Phylogeny and biogeography of the core babblers (Aves: Timaliidae). Syst Biol. 2012;61:631–51.View ArticlePubMedGoogle Scholar
- Collar N, Robson C. Grey-throated Babbler (Stachyris nigriceps). In: del Hoyo J, Elliot A, Christie D, editors. Handbook of the birds of the world: Picathartes to tits and chickadees, vol. 12. Barcelona: Lynx Edicions; 2007.Google Scholar
- White PS, Densmore LD. Mitochondrial DNA isolation. In: Hoelzel AR, editor. Molecular genetic analysis of populations: a practical approach. New York: University Press; 1992. p. 50–1.Google Scholar
- Hofman CA, Rick TC, Hawkins MTR, Funk WC, Ralls K, Boser CL, Collins PW, Coonan T, King JL, Morrison SA, Newsome SD, Sillett TS, Fleischer RC, Maldonado JE. Mitochondrial genomes suggest rapid evolution of dwarf California channel islands foxes (Urocyon littoralis). PLoS One. 2015;10:e0118240.PubMed CentralView ArticlePubMedGoogle Scholar
- Schmieder R, Edwards R. Quality control and preprocessing of metagenomic datasets. Bioinformatics. 2011;27:863–4.PubMed CentralView ArticlePubMedGoogle Scholar
- Mayer C: Phobos. 3.3.11. 2006–2010. http://www.rub.de/speezoo/cm/cm_phobos.htm.
- Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Mentjies P, Drummond A. Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28:1647–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.View ArticlePubMedGoogle Scholar
- Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG. Primer3—new capabilities and interfaces. Nucleic Acids Res. 2012;40:e115.PubMed CentralView ArticlePubMedGoogle Scholar
- Brownstein MJ, Carpten JD, Smith JR. Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. Biotechniques. 1996;20:1004–10.PubMedGoogle Scholar
- Excoffier L, Laval G, Schneider S. Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evol Bioinform. 2005;1:47–50.Google Scholar
- Rousset F. GENEPOP’007: a complete re-implementation of the GENEPOP software for Windows and Linux. Mol Ecol Resour. 2008;8:103–6.View ArticlePubMedGoogle Scholar
- Van Oosterhout C, Hutchinson WF, Wills D, Shipley P. MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes. 2004;4:535–8.View ArticleGoogle Scholar