Introduction

The tropical Pacific region constitutes one of the world’s 25 designated hotspots of biodiversity (Myers et al. 2000), but at the same time its terrestrial avifauna is threatened in many ways. Population sizes are often small due to limited availability of suitable land surface, human land-use changes are continuing to decrease the original habitat (e.g., Buchanan et al. 2008), and introduced diseases (e.g., Jarvi et al. 2001), predators (e.g., Fritts and Rodda 1998), and non-native competing species greatly impact naïve bird populations (e.g., Steadman 2006). Contrary to traditional views that the islands of the tropical Pacific serve as a sink of biodiversity generated on the mainland, it was recently shown that diversification likely occurs in situ, and lineages that arise in the Pacific may recolonize the Australasian mainland (Filardi and Moyle 2005). Thus, the tropical Pacific constitutes a diverse and threatened region where biodiversity is likely being generated, yet it is relatively understudied.

One of the most widespread bird genera in the Pacific is constituted by the Myzomela honeyeaters (Pratt et al. 1987), a monophyletic group within the Meliphagidae (Driskell and Christidis 2004). The genus is distributed from the eastern Indonesian islands and Australia to the Pacific islands of the Commonwealth of the Northern Mariana Islands (CNMI) and American Samoa. Despite the fact that the Pacific species in this genus are still comparatively common, population trends are often negative. For example, a recent study reported a 72% decline of the Micronesian honeyeater (M. rubrata) population on Rota, CNMI (Amar et al. 2008). Whereas populations from many islands are often treated as a single species, it is plausible that the Myzomela honeyeaters of the Pacific constitute multiple recently diverged taxa, and should be treated as such for conservation purposes.

Here we describe the isolation and characterization of 15 novel tri- and tetranucleotide microsatellite loci from M. cardinalis and M. (cardinalis) rubrata populations on six islands (n = 104) in the northern tropical Pacific: Sarigan, Saipan, Rota (CNMI), Yap, Chuuk, and Pohnpei (Federated States of Micronesia). Blood samples (50–100 μl) were taken from captured birds and stored in lysis buffer. No blood samples were available from the population on Sarigan, in which case we used feather samples for DNA extractions. DNA was extracted using the Qiagen QIAamp DNA Mini Kit using the manufacturer’s protocol for either blood or tissue. DNA extractions from blood samples were used to create an enriched library at Genetic Identification Services, Inc. (Chatsworth, California). Details on the enrichment procedure can be found in Jones et al. (2000). In brief, the extracted DNA was incompletely digested using blunt-end restriction enzymes BsrB1, EcoRV, HaeII, PvuII, RsaI, ScaI, and StuI. HindIII adaptors were attached to fragments of 300-750 bp long. Fragments containing target repeat motifs were captured using 5′-biotinylated probes. Capture molecules with the following repeat motifs were used: AAC, ATG, CATC, and TAGA. Captured fragments were subsequently amplified using a primer complementary to the adaptor, digested with HindIII and ligated into the HindIII site of pUC19. Plasmids were electroporated into E. coli DH5α, and recombinant clones were selected for sequencing on an ABI 377 sequencer using ABI Prism Taq dye terminator cycle sequencing methodology.

Primers were originally designed for 28 loci using WebSat (Martins et al. 2009). Of these, 15 loci amplified correctly and were polymorphic in 104 individuals. Fragments were amplified by means of the M13-hybrid primer process. In this process, a 16-bp fragment is added to the 5′ end of the forward primer for binding of the dye-labeled M13-hybrid primer (Boutin-Ganach et al. 2001; Schuelke 2000). Primer mixes were prepared as follows: 2 μl reverse primer (100 μM); 4 μl forward primer (2.5 μM); 4 μl 6FAM dye-labeled M13-hybrid primer (2.5 μM); 90 μl RNAse/DNAse-free water. Amplification was carried out in 10 μl reactions containing 5 μl Qiagen Multiplex Mastermix, 0.5 μl BSA (10 mg/ml), 1 μl primer mix, 2 μl RNAse/DNAse-free water, and 1.5 μl template DNA (30–100 ng in total). The following cycling conditions were used: an initial step of 95°C for 15 min; 25 cycles of: 30 s at 94°C, 90 s at 59°C, 60 s at 72°C; 20 cycles of: 30 s at 94°C, 90 s at 53°C, 60 s at 72°C; and 30 min at 60°C. PCR products were run on an ABI 3730 capillary sequencer, and allele sizes were scored manually using GS 500-LIZ size standard in GeneMapper v3.7 genotyping software (ABI).

Tests for deviations from Hardy–Weinberg equilibrium (HWE) for each locus and each population, and calculations of observed (H O) and expected heterozygosity (H E) and fixation indices (F is) were carried out in GenAlEx v6 (Peakall and Smouse 2006). Tests for linkage disequilibrium (LD) were performed in Genepop 4.0 (Rousset 2008). After Bonferroni correction for multiple comparisons, no consistent departures from HWE were detected across populations (P > 0.05). One of the primer combinations (Mr 28) amplified two separate regions, which were highly significantly linked (Mr 28A and Mr 28B; P < 0.001). Among the remaining loci, LD was only suggested in the population from Saipan for Mr 40 and Mr 47. The number of alleles ranged from 3 to 23 (Table 1). Observed and expected heterozygosities ranged from 0.067 to 0.913 and from 0.065 to 0.918, respectively. Finally, F is ranged between −0.143 and 0.634.

Table 1 Microsatellite loci developed for Myzomela honeyeaters
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Cross-amplification of primers in other species was evaluated in 18 different Myzomela species and subspecies. DNA was extracted from toe pads collected from museum material using the Qiagen QIAamp DNA Mini Kit according to the manufacturer’s protocol for tissue. Most loci amplified well (Table 2), however, because only a single individual per species was available, polymorphism could not be tested. The microsatellites developed here could be useful in population genetic studies of Myzomela honeyeaters, and in the conservation of members of this genus in the tropical Pacific.

Table 2 Cross-amplification of developed microsatellite loci in members of the genus Myzomela
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