High throughput sequencing enables discovery of microsatellites from the puff-throated bulbul (Alophoixus pallidus) and assessment of genetic diversity in Khao Yai National Park, Thailand

Biochemical Systematics and Ecology 55 (2014) 176e183

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High throughput sequencing enables discovery of microsatellites from the puff-throated bulbul (Alophoixus pallidus) and assessment of genetic diversity in Khao Yai National Park, Thailand Robert B. Page a, *, Wangworn Sankamethawee b, Andrew J. Pierce c, Ken A. Sterling d, David H. Reed e, y, Brice P. Noonan d, Tommaso Savini c, George A. Gale c a

Department of Biology, College of St. Benedict & St. John’s University, Collegeville, MN 56321, USA Department of Environmental Science, Faculty of Science, Khon Kaen University, 40002, Thailand c Conservation Ecology Program, School of Bioresources & Technology, King Mongkut’s University of Technology Thonburi, 49 Soi Tientalay 25, Bangkhuntien-Chaitalay Rd., Thakham, Bangkhuntien, Bangkok 10150, Thailand d Department of Biology, University of Mississippi, Box 1848, University, MS 38677-1848, USA e Department of Biology, University of Louisville, Louisville, KY 40292, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 December 2013 Accepted 29 March 2014 Available online

Bulbuls (family Pycnonotidae) are a diverse family of songbirds that carry out a number of ecologically important functions associated with seed dispersal. Since, 2003, a puffthroated bulbul (Alophoixus pallidus) population in the Mo-Singto Long-term Biodiversity Research Plot in Khao Yai National Park, Thailand has served as a model system for examining how bulbul behavior, movement, and demographics affect Southeast Asian forests. In this study, we used 454 pyrosequencing to discover microsatellites from A. pallidus that will enable the long-term mark-recapture work conducted at Mo-Singto to be complemented by molecular ecology and population genetic studies. In addition, we conducted fragment analysis to examine the level of genetic diversity exhibited by the MoSingto population. In total, we identified 103 DNA fragments containing microsatellite repeats and 66 fragments with sufficient flanking sequences to allow for primer design. Upon screening 26 loci via PCR-based genotyping assays, we identified nine polymorphic loci and used eight of these to examine genetic diversity in the Mo-Singto population. The results of these analyses suggest that the Mo-Singto population is moderately diverse (mean number of effective alleles across eight loci ¼ 3.36, standard deviation ¼ 1.78), is more-or-less in HardyeWeinberg equilibrium, and has not recently been subject to severe population reduction. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Alophoixus pallidus Genetic diversity Mirosatellites Next-generation sequencing Southeast Asia

* Corresponding author. Tel.: þ1 320 363 3174; fax: þ1 320 363 3202. E-mail addresses: [email protected], [email protected] (R.B. Page), [email protected] (W. Sankamethawee), [email protected] (A.J. Pierce), [email protected] (K.A. Sterling), [email protected] (B.P. Noonan), [email protected] (T. Savini), [email protected] (G.A. Gale). y Deceased. http://dx.doi.org/10.1016/j.bse.2014.03.032 0305-1978/Ó 2014 Elsevier Ltd. All rights reserved.

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1. Introduction Bulbuls (family Pycnonotidae) are a widespread, diverse family of songbirds that are found throughout much of Africa, the Middle East, and Asia. In many communities, they serve as important seed dispersers (e.g., Fukui, 1995; Graham et al., 1995; Sankamethawee et al., 2011), and they have been associated with a variety of important ecological phenomena including plant persistence and dispersal in anthropogenically fragmented habitats (Weir and Corlett, 2007), range expansions of introduced plant species (Jordaan et al., 2011; Kawakami et al., 2009; Linnebjerg et al., 2009), and local distributions of parasitic plants (Green et al., 2009). Because pycnonotids often play important ecological roles, a thorough understanding of their behavioral and population dynamics has the potential to provide insights into community structure that go beyond those normally offered by autecological studies. In Southeast Asian forests, bulbuls are known to be important seed dispersers (Kitamura et al., 2002; Sankamethawee et al., 2011). However, a more precise understanding of their ecological roles in these communities has been elusive due to limited information on their movement patterns (see Khamcha et al., 2012). Since 2003, the puff-throated bulbul (Alophoixus pallidus), a frugivorous omnivore that is common in the evergreen forests of Southwestern China, Vietnam, Laos, Cambodia, Thailand, and Myanmar, has been the focus of a series of studies in Khao Yai National Park, northeastern Thailand (14 260 N 101 220 E). Amongst other things, these studies have revealed that puff-throated bulbuls are facultative cooperative breeders (Pierce et al., 2007) with female biased dispersal (Sankamethawee et al., 2010) and that movement frequencies and distances in A. pallidus are correlated with seasonal variation in fruit availability (Khamcha et al., 2012). A logical next step in further elucidating the behavioral and population dynamics of the puff-throated bulbul is to use techniques from molecular ecology and population genetics to address questions about gene flow, population structure, and the relationship between genetic relatedness and group structure/cooperative breeding. For each of these endeavors, microsatellites are the genetic marker of choice. However, despite the availability of such resources for some pycnonotids (Bardeleben, 2004; Lokugalappatti et al., 2008; Wu et al., 2011), microsatellite loci have not been isolated from the genus Alophoixus. In order to further develop A. pallidus as a model system for studying pycnonotid behavior and population dynamics in the tropical forests of Southeast Asia, we used Roche 454 pyrosequencing to generate 1518 sequencing reads that varied in length from tens to hundreds of base pairs (bp). We then scanned these fragments for microsatellite repeats and designed and tested PCR-based genotyping assays for a subset of the potentially amplifiable loci (PALs) that we identified via 454 sequencing. Herein, we summarize the results of our 454 sequencing experiment and describe the PALs that we discovered. In addition, we present information on several population genetic parameters that were estimated from a panel of individuals that we used to screen a subset of the loci identified via 454 sequencing. 2. Materials and methods 2.1. Study site, sample collection, and DNA isolation Sampling was conducted at Khao Yai National Park on the 30-ha Mo-Singto Long-term Biodiversity Research Plot (Brockelman et al., 2011). The plot is in a mature, seasonally wet evergreen forest with undulating ridges and valleys at 725e 815 m in elevation. Puff-throated Bulbuls have been mist-netted and banded in the Mo-Singto plot since January 2003 with a unique color combination of two colors and one Royal Forest Department numbered aluminum ring that enables each bird to be uniquely identified. From April 2006eJuly 2008, blood was collected from the tarsus or brachial vein of 178 birds, and used to isolate genomic DNA via application of the JETQUICK Blood DNA Spin Mini Kit (GenoMed, Leesburg, FL, USA) according to the manufacturer’s protocol. In the Mo-Singto plot, Puff-throated Bulbuls live in territorial groups that range in size from a single breeding pair to seven individuals. Social interactions within some (but not all) of these groups are characterized by cooperative breeding, in which one or more adults (i.e., helpers) that are not members of the breeding pair care for nestlings and/or fledglings via delivering food, removing fecal sacs from the nest, and defending the nest from predators. Of the 178 DNA samples that we collected, 88 were derived from individuals that were participants in one of 14 cooperatively breeding groups that are the subject of a forthcoming study on the relationship between genetic relatedness and cooperative breeding. The remaining 90 birds were not assigned to a cooperatively breeding group during the period when samples were collected and are therefore presumed not to be members of any cooperatively breeding group. Because relatedness among individuals within cooperatively breeding groups could complicate the interpretation of population genetic parameter estimates, the data presented herein are based on a subset of the 90 individuals that are presumed not to be cooperative breeders and therefore correspond to a random sample of non-cooperatively breeding members of the Mo-Singto population rather than the Mo-Singto population at large. 2.2. Roche 454 sequencing, microsatellite discovery, primer design, PCR conditions, and fragment analysis To generate a pool of random genomic fragments from which microsatellite markers could be identified, we selected DNA from a single individual and included it in a pool with nine other species that were differentiated by terminal barcodes (Meyer et al., 2007). DNA was prepared using the manufacturer’s 454 FLX shotgun library preparation protocol, which resulted in an

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average fragment size of w700 bp. Sequencing employed the 454 FLX Titanium sequencing kit and the protocol was run on ¼ 70  75 cm picotiter plate. Primer pairs were designed via batch processing of repeat containing 454 reads via Primer3 (Rozen and Skaletsky, 2000) using the method of Castoe et al. (2010). Oligonucleotide pairs were modified by the addition of an M13(-21) sequence to the 50 end of either the forward or reverse primer of each respective primer pair (Schuelke, 2000). Optimal annealing temperatures were identified for each primer pair via gradient PCR, and genotyping reactions were carried out using the nested PCR approach described by Schuelke (2000) and 6-FAM as a label. All PCRs were 25 ml reactions that contained 2 ml of template (total template amount w 10e100 ng per reaction), 5 ml of 5 buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.8 mM of nonM13(-21)-twinned primer, 0.8 mM of 6-FAM labeled M13(-21) primer, 0.2 mM M13(-21)-twinned primer, and 0.625 units of GoTaq polymerase (Promega). Genotyping reaction conditions were: 2 min at 94  C followed by 25 cycles of (1) 30 s at 94  C, (2) 30 s at a primer pair-specific annealing temperature (Table 1), and (3) 40 s at 72  C, followed by 8 cycles of (1) 30 s at 94  C, (2) 30 s at 53  C, and (3) 40 s at 72  C, followed by a final cleanup step of 30 min at 72  C. The number of cycles for the primer pair-specific series of cycles was occasionally reduced to 20 cycles to ameliorate signal saturation. 6-FAM labeled PCR products were sent to the Arizona State University DNA Lab, where fragment analysis was performed using an ABI 3730. Pherograms were inspected and genotypes were scored manually using Peak Scanner Software, Version 1.0 (Applied Biosystems, Foster City, CA, USA). In total, we screened 26 PALs. Initially, 6-FAM labeled PCR products from 8 to 12 individuals per PAL were subjected to fragment analysis and additional genotyping was carried out for polymorphic loci that were reasonably easy to score (i.e., loci for which there was not excessive stuttering or amplification artifacts). A subset of the samples genotyped at each locus that passed this initial screen were genotyped at least one additional time from independent PCRs to assess the degree of genotyping error associated with these markers. 2.3. Allele binning and statistical analyses We graphically examined the rank-ordered (smallest to largest) fragment size distributions for each locus, so that we could identify discrete breaks in amplicon sizes that could be used to define the size range of each allele (see Guichoux et al., 2011). We then wrote functions in Microsoft Excel to bin the data from each respective locus into discrete classes that were defined by each allele’s empirically determined size range. We used Microsatellite Analyzer (MSA; Dieringer and Schlotterer, 2003) to calculate several summary statistics (e.g., allele frequencies, number of alleles per locus, and expected and observed heterozygosity) and MicroChecker (Van Oosterhout et al., 2004) to examine each locus for evidence of null alleles. We then used GenePop, Version 4 (Rousset, 2008) to test each locus for departures from HardyeWeinberg proportions, to test all pairs of loci for departures from genotypic equilibrium, and to calculate the Weir and Cockerham (1984) estimator of FIS. In addition, we calculated the effective number of alleles for each locus and M ratios for loci with allelic size distributions that were consistent with a stepwise mutation model (Garza and Williamson, 2001). 3. Results 3.1. Roche 454 sequencing results In total, 1518 reads were generated from A. pallidus (Supplemental Data File 1). The distribution of the lengths of these reads is shown in Fig. 1. PAL_Finder (Castoe et al., 2010, 2012) identified a total of 103 fragments containing microsatellite Table 1 Locus names, repeat motif, primer sequences, annealing temperatures, and size ranges for the nine loci that passed initial screening. Locus

Motif

Primer sequences

Annealing temp. (C)

Size range (bp)

Pp5

AC

57

92e96

Pp40

AAAC

54

232e236

Pp47

AAAC

60

243e251

Pp49

AAAC

57

66e78

Pp51

ATCT

54

216e236

Pp63

AGTTT

58

264e303

PpMS3

AC

57

165e187

PpMS4

ATGGG

57

84e124

PpMS5

AAAAG

F: gac att aaa tat ctt tgt gtg agt ggga R: aag aat tct ctg ctg ttg tga gg F: gga act aga atc ttt cca tat ttc tgca R: acc aat ttg aac cgt cct gg F: gaa agg ctg aag ttc tgt tct gc R: ctc agc cat ccc tac aca gca F: aaa aga cag aca acg agg gga R: gga ctt ctt ctt tgg gct gg F: cca cca aac tag aaa tgt atg act gca R: agg ctt cat gac ctc aaa tcc F: agg ctc tct caa gga aag cg R: acc agc ctc agt tcg agt cca F: aat ttg aat tct tca tga gag gtg ga R: gcg ggc tgt gta aat gaa cc F: aga gga aga atg gga gtg gg R: aac tgc atc acc ctt gga gga F: ctg ccc tag aaa ttg ctg cca R: aac tgc tcc ctc agg ttt gc

59

191e336

a

Indicates the primer in a given primer pair whose 50 end was fused with the M13(-21) sequence.

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Fig. 1. The size distribution of the 1518 reads generated by 454 pyrosequencing.

repeats and of these, 66 contained sufficient flanking sequence on either side of the microsatellite sequence to facilitate primer design (i.e., were PALs; Supplemental Table 1). Fig. 2A shows how these PALs are distributed across several repeat motif size classes and Fig. 2B shows the distribution of times that both primer sequences from a primer pair occurred in the correct orientation among all the fragments generated via pyrosequencing (i.e., the number of occurrences for each amplifiable primer pair amongst all reads). Sunflower plots showing the distributions of the number of perfect tandem repeats and the number of total tandemly repeated nucleotides (for ‘compound’ and ‘broken’ microsatellite sequences; Castoe et al., 2010) are shown for PALs with dinucleotide through hexanucleotide repeat motifs in Fig. 3A and Fig. 3B respectively. 3.2. Initial screening of microsatellite loci and quality control Of the 26 primer pairs that we screened (w39.4% of those designed), nine (w34.6%) passed initial screening (Supplemental Table 1). Of the nine genotyping assays that passed preliminary screening, one (PpMS5) was revealed to have an unacceptably high error rate (w8.8%) early during the data generation phase and was therefore dropped. For the remaining eight primer pairs, we either did not detect any genotyping disagreements among replicate reactions (Pp5, Pp47, Pp49, Pp51, Pp63, PpMS3, PpMS4), or the genotyping error rate was well under 5% (Pp40, error rate w 2.5%). Of the eight PALs that we pursued in detail, MicroChecker detected evidence for null alleles at Pp5 and Pp51.

Fig. 2. (A) The number of potentially amplifiable loci (PALs) discovered via 454 pyrosequencing by repeat size. Di ¼ dinucleotide, Tri ¼ trinucleotide, Tet ¼ tetranucleotide, Pen ¼ pentanucleotide, and Hex ¼ hexanucleotide. (B) The distribution of times that the primer pairs associated with the various PALs occurred in the correct (i.e., amplifiable) orientation across all 454 reads.

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Fig. 3. (A) Sunflower plot of the distribution of perfect tandem repeats for dinucleotide-hexanuleotide (i.e., 2e6 on the x-axis) potentially amplifiable loci (PALs). (B) Sunflower plot of the distribution of total tandemly repeated nucleotides for dinucleotide-hexanucleotide PALs.

3.3. Genetic diversity and population genetic estimates Upon applying the multiple testing correction of Holm (1979), Pp51, Pp5, and Pp49 exhibited significant departures from HardyeWeinberg proportions. Similarly upon applying Holm’s (1979) correction, Pp40 and PpMS3 showed evidence of genotypic disequilibrium. Sample sizes, genetic diversity estimates, M ratios, and inbreeding coefficients for each locus are given in Table 2, as are measures of central tendency and dispersion across loci. 4. Discussion 4.1. 454 pyrosequencing In comparison with other studies that have used high throughput sequencing to discover microsatellite loci (e.g., Castoe et al., 2010; Gardner et al., 2011; Hunter and Hart, 2013; Schobel et al., 2013), we discovered a small number of loci from A. pallidus. This result reflects the small number of reads obtained from our ¼ plate run (28,343), and the fact the A. pallidus sample was on the low end of the read distribution obtained across all ten multiplexed samples (Fig. 4A). While it is unclear why such a small number of reads were generated during our ¼ plate run, our results demonstrate that there can be a fairly wide distribution in read number across multiplexed samples (Fig. 4A). Thus, our results suggest that a tighter read number distribution may be realized by resisting the urge to massively multiplex, or by spreading a comparable number of samples (10 in our ¼ plate run) across a larger proportion (e.g., ½ or whole) of a picotiter plate. In addition to these technical considerations, it is well known that there is a paucity of repetitive sequence in avian genomes (Hughes and Piontkivska, 2005; International Chicken Genome Sequencing Consortium, 2004; Warren et al., 2010). Examination of the proportion of reads

Table 2 Number of female (Nfemale) and male (Nmale) birds sampled, genetic diversity estimates, Weir and Cockerham FIS estimators, and M-ratios for the eight loci used to assess genetic diversity. Locus or summary statistic

Nfemale

Nmale

No. of alleles

HE

HO

FIS

Effective no. of alleles

M

Pp5a Pp40b Pp47c Pp49b Pp51c Pp63b PpMS3c PpMS4c Mean St. dev.

28 37 25 28 27 31 37 32 30.6250 4.5020

35 46 32 35 26 35 40 34 35.3750 5.8049

4 4 3 5 6 9 10 9 6.2500 2.7124

0.3064 0.6394 0.4867 0.6147 0.7759 0.6180 0.7751 0.8626 0.6349 0.1781

0.2063 0.7108 0.4912 0.5397 0.3962 0.5606 0.8052 0.8636 0.5717 0.2175

0.3283 0.1126 0.0093 0.1229 0.4917 0.0935 0.0391 0.0012 0.1093 0.2043

1.4367 2.7435 1.9322 2.5631 4.3215 2.5859 4.3484 6.9474 3.3598 1.7778

1.0000 N/A 1.0000 N/A 1.0000 N/A 0.8333 1.0000 0.9666 0.0746

a Only one allele out of sequence with the expectations of a stepwise mutation model. Estimates of M are based on removing the single out of sequence allele. b Allele sizes violate the assumptions of a stepwise mutation model and M was therefore not calculated. c Allele sizes are consistent with a stepwise mutation model. N/A ¼ not applicable.

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Fig. 4. Boxplots showing the distributions of (A) number of reads and (B) proportion of reads containing microsatellites across all ten samples included in our ¼ plate 454 sequencing run. In addition to the A. pallidus sample (denoted by horizontal gray lines), samples from one lizard, one frog, one spider, and six ant species were included in the multiplex.

containing microsatellites from each of the samples included in our ¼ plate run supports the idea that this atypical feature of avian genome structure also contributed to the limited number of loci identified by our sequencing efforts (Fig. 4B). Despite the limited number of loci identified from A. pallidus, some features of our 454 data are similar to the findings of larger-scale studies on avian/reptilian species. For example, the preponderance of tetranucleotide repeats that we observed relative to smaller and larger repeat size motifs (Fig. 2A) has been reported for other bird and snake species (Castoe et al., 2010, 2012). As another example, the proportion of reads containing microsatellite repeats (6.8%) and PALs (4.3%) observed in our study is only slightly higher than those obtained by Castoe et al. (2012) for Gunnison sage-grouse (4.6% and 1.5% respectively) and Clark’s nutcracker (3.6% and 1.4%) via paired end reads from an Illumina Genome Analyzer IIx. Thus, despite their limited scope, the data generated in the current study suggest that the frequency and nature of the microsatellite loci identified from A. pallidus are not particularly unusual for a bird. 4.2. Microsatellite screening via PCR-based genotyping assays Our efforts to produce robust genotyping assays for polymorphic loci by batch processing 454 fragments using PAL_Finder were moderately successful. Slightly more than a third of the loci we examined passed our initial screens, and of these, one (PpMS5) was subsequently dropped because the genotyping assay was not robust. The primary reason for abandoning markers was the presence of only one peak or one repetitive peak pattern in all of the samples that we initially screened (collectively labeled ‘monomorphic’ in Supplemental Table 1). Other reasons for marker abandonment included the failure of loci to amplify upon the addition of an M13(-21) tag to one of the primers in a pair, non-specific amplification across a wide range of annealing temperatures, and the presence of excessive stuttering and PCR artifacts. While little can be done to increase the utility of monomorphic loci, problems due to amplification failure and stuttering may be remedied by assay redesign (Guichoux et al., 2011). Indeed, some of the loci (e.g., Pp48 and Pp55) that we abandoned due to excessive artifacts or poor amplification appeared to be polymorphic and are therefore high priorities for primer redesign. Similarly, PpMS5 was clearly highly polymorphic, and it is likely that primer redesign would make PpMS5 a high utility marker. All PpMS5 genotyping errors involved scoring heterozygotes for various alleles as homozygotes for a particular allele that was shared by all of the pertinent individuals. This finding suggests there may be unequal and allele-specific amplification efficiencies associated with the current assay of PpMS5 due to mutations in one or both priming regions. Collectively, these results indicate that it is likely that some of the loci we abandoned during the current study can be developed into robust markers via assay redesign. 4.3. Genetic diversity and population genetic parameters As suggested by the substantial number of monomorphic and/or nearly monomorphic loci observed during our initial screens, the puff-throated bulbul population in the Mo-Singto plot appears to have a somewhat modest amount of genetic diversity. While five of the eight polymorphic loci that we surveyed in detail had  five alleles, only one locus (PpMS4) had an effective number of alleles > five, and the mean number of effective alleles across loci was 3.36. In addition, of the three loci

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that deviated from HardyeWeinberg expectations (Pp51, Pp5, and Pp49), all exhibited homozygote excess. There was no evidence that sampling bias for philopatric males (Sankamethawee et al., 2010) explains variation in the inbreeding coefficients of different loci, as there was no relationship between FIS and the ratio of males to females sampled at each locus. Nevertheless, there is little evidence for pronounced inbreeding, as neither mean FIS, nor the mean difference between observed and expected heterozygosity statistically differed from zero when assessed by one-sample and paired t-tests respectively. These tests are not sensitive to the non-independence of Pp40 and PpMS3, as the same conclusions were reached when these analyses were repeated treating the average of these two loci as data from a single locus. Finally, there was no evidence that the relatively modest amount of genetic diversity we observed is associated with a recent bottleneck, as the M ratios we estimated are not indicative of a severe, contemporary reduction in population size. Overall, these results are consistent with the view that the Mo-Singto population is stable, moderately diverse, and more-or-less in HardyeWeinberg equilibrium. 5. Conclusion Using the relatively long reads generated via 454 pyrosequencing we were able to leverage a comparatively small amount of data (550,844 bp from 1518 fragments) from A. pallidus to discover well over 50 potentially amplifiable microsatellite loci and dozens of additional loci that could be further developed via simple molecular biology techniques, such as primer walking. In addition, we designed many genotyping primers in batch (66 primer pairs) and screened a substantial proportion of the associated loci/assays (w40%) for polymorphism and robustness. Ultimately, eight polymorphic loci with seemingly robust genotyping assays were identified and used to assess genetic diversity in a population of A. pallidus from Khao Yai National Park, Thailand. These results suggest that this population has a modest amount of genetic diversity and has not been affected by recent bottlenecks. The resources that we have developed will enable us and others to examine the population genetics and molecular ecology of puff-throated bulbuls, and pursue the relationship between cooperative breeding and genetic relatedness in this ecologically important species. Acknowledgments We dedicate this work to our friend and colleague, Dr. David H. Reed, who passed away suddenly in October 2011 due to heart failure. This work was supported in part by funding obtained by David from the Wallace Endowment to the University of Louisville Department of Biology. Appendix A. 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