Haumania J. Léonard is a plant genus of the family Marantaceae endemic to central tropical Africa (Dhetchuvi, 1996). The genus comprises three species of climbers confined to the understory, gaps, and forest edges of lowland tropical rainforest. Haumania danckelmaniana (J. Braun & K. Schum.) Milne-Redh. is distributed from Cameroon and Equatorial Guinea to Gabon and northern Republic of the Congo. Haumania liebrechtsiana (De Wild. & T. Durand) J. Léonard can be found from Gabon to the Republic of the Congo and the Democratic Republic of the Congo. Haumania leonardiana G. Evrard & Bamps is distributed in northern Democratic Republic of the Congo. With their parapatric distribution pattern across phytogeographic domains in central Africa, species of the genus Haumania are of great interest in the field of phylogeography to extend our knowledge of the patterns and processes of speciation and intraspecific diversification in this tropical region. In this study, we isolated polymorphic microsatellite loci from H. danckelmaniana and showed that they can be transferred to the closely related species H. liebrechtsiana, allowing for further phytogeographic investigations in tropical central Africa.
METHODS AND RESULTS
Microsatellite primers were isolated in H. danckelmaniana at Genoscreen (Lille, France) using the 454 GS-FLX Titanium platform (454 Life Sciences, a Roche Company, Branford, Connecticut, USA) through a procedure that combines multiplex microsatellite enrichment and pyrosequencing (Malausa et al., 2011; Micheneau et al., 2011). Total DNA was extracted from silica gel–dried leaves using the NucleoSpin Plant kit (Macherey-Nagel, Düren, Germany). A mixture of ca. 5 µg of DNA of three species to simultaneously recover microsatellite primers in a single next-generation sequencing run (H. danckelmaniana, Hypselodelphys poggeana (K. Schum.) Milne-Redh., Marantochloa incertifolia Dhetchuvi, including multiple samples from the same locality per species; Appendix 1) was sent to Genoscreen, and was used to isolate microsatellite loci using a 1/32nd GS-FLX plate of the Roche Sequencer (454 Life Sciences, a Roche Company), following the protocol of Malausa et al. (2011).
We used Primer3 (Rozen and Skaletsky, 1999) implemented in the QDD bioinformatics pipeline (Meglécz et al., 2010) with the criteria described in Malausa et al. (2011) to automatically design 5657 primer pairs targeting 1285 microsatellite loci. From these, we selected 80 primer pairs representing the longest di- and trinucleotide repeats (≥8 repeats), and having more than 10 flanking nucleotides between microsatellite motifs and designed primers. The 80 designed primer pairs were first tested individually to verify amplification in H. danckelmaniana, with the following PCR conditions: 1 µL buffer (10×), 0.4 µL MgCl2 (25 mM), 0.3 µL dNTPs (10 mM each), 0.2 µL of each primer (0.01 mM), 0.05 µL Taq polymerase (TopTaq DNA Polymerase, 5 U/µL; QIAGEN, Venlo, The Netherlands), 1 µL of template DNA (of ca. 10–50 ng/µL, specimen ACL0711), and H2O to make a final volume of 10 µL. Amplifications were performed as follows: 94°C (4 min), followed by 40 cycles of 94°C (30 s), 56°C (45 s), 72°C (1 min), and a final extension at 72°C for 10 min. Twenty markers generated products as indicated on a 1% agarose gel stained with SYBR Safe (Invitrogen, Merelbeke, Belgium).
For these 20 markers, unambiguous amplification and levels of polymorphism were tested on up to seven individuals chosen across the entire species' distribution range (Appendix 1) using a modified protocol of Schuelke (2000), which incorporates three primers (M13-like protocol) (for further details see Micheneau et al., 2011). Ten markers presented unambiguous amplification products within the expected size range, and eight of these were polymorphic (for primers see Table 1). These latter 10 loci also amplified in the closely related species H. liebrechtsiana using the same protocol as described for H. danckelmaniana.
With the eight polymorphic markers, preliminary population genetic analyses were carried out in two populations per species (H. danckelmaniana: Ivindo [Gabon, N = 22, coordinates: 0°11′21.84″S, 12°33′32.04″E] and Pallisco [Cameroon, N = 96, coordinates: 3°28′52.32″N, 13°34′41.88″E]; H. liebrechtsiana: East Mont de Cristal A and B [Gabon, N = 27, coordinates A: 0°35′48.84″N, 11°12′16.92″E and N = 72, coordinates B: 0°33′16.20″N, 11°6′20.16″E]). Seven of these loci were amplified in one multiplex reaction (see Table 1) using the QIAGEN Multiplex kit in a final 15-µL reaction volume. PCRs were carried out as follows: 7.5 µL Multiplex PCR Master Mix, 1.5 µL primer mix (with Q-tailed forward primers at 0.7 µM and reverse primers at 2 µM), 0.15 µL of each fluorescent Q1–Q4 primers (10 µM), 1.5 µL DNA, and 3.9 µL H2O. The multiplex PCR program consisted of 95°C for 15 min; followed by 23–30 cycles each of 94°C (30 s), 57°C (90 s), and 72°C (90 s); followed by 10 cycles each of 94°C (30 s), 53°C (45 s), and 72°C (45 s); and a final extension at 60°C for 30 min. The remaining locus (F80) was amplified in a separate PCR with a Taq DNA polymerase (QIAGEN) under the following conditions: 94°C (4 min); followed by 20 cycles each of 94°C (30 s), 56°C (45 s), and 72°C (1 min); plus 10 cycles each of 94°C (30 s), 53°C (45 s), and 72°C (45 s); and a final extension at 72°C for 10 min. One microliter of each PCR product was directly added to 12 µL HiDi formamide and 0.2 µL GeneScan 500 LIZ Size Standard and run on an ABI 3730 sequencer (Applied Biosystems, Lennik, The Netherlands). Alleles were defined using Peak Scanner software (Applied Biosystems). In a few individuals, some primer pairs did not yield amplification. Expected (H e) and observed heterozygosities (H o) and tests for deviation from Hardy–Weinberg equilibrium (HWE) were estimated in SPAGeDi version 1.3 (Hardy and Vekemans, 2002). Results were adjusted for multiple comparisons using a sequential Bonferroni correction.
Table 1.
Characterization of 10 microsatellite markers isolated from Haumania danckelmaniana.
H e and H o ranged from 0.354 to 0.720 and 0.318 to 0.810 (H. danckelmaniana Ivindo population), from 0.081 to 0.683 and 0.083 to 0.625 (H. danckelmaniana Pallisco population), from 0.073 to 0.866 and 0 to 0.818 (H. liebrechtsiana East Mont de Cristal A population), and from 0.135 to 0.859 and 0.085 to 0.775 (H. liebrechtsiana East Mont de Cristal B population), respectively (Table 2). Two loci showed a deviation from HWE in the Cameroon population of H. danckelmaniana and two and five loci showed the same in H. liebrechtsiana in the populations from Gabon and Cameroon, respectively. To check whether departure from HWE at a given locus might be explained by the presence of null alleles, we used the software INEST (Chybicki and Burczyk, 2009), which jointly estimates inbreeding and null allele frequencies under the individual inbreeding model. Null allele frequency estimates were significantly different from zero at all except two loci that deviated from HWE, indicating the presence of null alleles. In all cases, the estimated frequency of null alleles was <23%.
CONCLUSIONS
The simple sequence repeat markers herein described are the first developed for H. danckelmaniana. These microsatellites are important tools for genetic studies in H. danckelmaniana and may be used to evaluate the genetic variability of the related species H. liebrechtsiana, aiming to elucidate questions regarding genetic diversity, spatial genetic structure, mating system, and gene flow.
Table 2.
Characterization of eight polymorphic microsatellite loci from two populations of Haumania danckelmaniana and H. liebrechtsiana.a
