Natural fruit set of some allogamous and autogamous
Abstract
The study aimed at evaluating the mating system of Vanilla mexicana (Orchidaceae) in natural populations in the island of Guadeloupe. A total of 132 V. mexicana samples were collected from 12 sites in Guadeloupe (Basse‐Terre). Five other samples coming from Martinique and Mexico completed our analyses. Reproductive biology experiments excluding pollinators with bagged flowers revealed 53.9% fruit set, a value identical to the natural fruit set measured in the populations. These results suggested that V. mexicana, unlike most Vanilla species, was reproducing by self‐pollination and autogamy. Due to lack of specific DNA markers for V. mexicana, microsatellite markers, previously developed in other Vanilla species, were used for the genetic analyses. Only 6 out of the 33 markers tested were transferable and polymorphic in V. mexicana. A panel of 51 V. mexicana samples genotyped with 3 polymorphic loci was finally retained for Guadeloupe population genetic analyses. A heterozygote deficiency was detected, and the selfing rate was estimated to 74%. These results confirmed the reproductive biology results as self‐pollination and autogamy were the most likely explanation for this deficit. Results were compared to those from allogamous wild Vanilla species and discussed in the light of suggested existence of a pollinator for V. mexicana in other areas (Mexico).
Keywords
- autogamy
- genetic diversity
- Guadeloupe
- microsatellites
- Vanilla mexicana
1. Introduction
Knowledge and management of agricultural genetic resources (AGR) and of their wild relative species [referred to as Crop Wild Relatives (CWR)] are of major importance to ensure the preservation of natural resources, development of sustainable agriculture and food security in a global climate change context. The extremely low genetic diversity in the cultivated vanilla species
Geographically,
The vast majority of
In
Section | Taxonomic group | Species | Natural fruit set (%) | Mating system | |
---|---|---|---|---|---|
0.0 |
Allo | ||||
18.2 |
Allo | ||||
17.9 |
Allo | ||||
14.5 |
Allo | ||||
6.4 |
Allo | ||||
0.8d | Allo | ||||
26.3 |
Allo | ||||
15.0 |
Allo | ||||
0.9 |
Allo | ||||
6.6 |
Allo | ||||
0.1–1.0 |
Allo | ||||
1.1 |
Allo | ||||
42.5 |
Auto | ||||
76.0 |
Auto | ||||
78.0 |
Auto | ||||
53.0 |
Auto | ||||
15.0l | Allo |
In Africa (subgenus
The use of codominant neutral genetic markers such as microsatellites to perform genetic analyses on natural populations [33, 34] is also a method of choice to estimate mating system parameters such as inbreeding rate [35–38]. As no specific markers were available for
2. V. mexicana mating system in Guadeloupe
2.1. Material and methods
2.1.1. Study species
To precisely record morphological descriptors of the studied species, characters were measured to the nearest 0.01 mm using a digital caliper
2.1.2. Study site
Sampling was performed in 2013 by the Association Guadeloupéenne d’Orchidophilie (AGO) mandated by the National Park of Guadeloupe (PNG). According to the inventory of
2.1.3. Plant sampling
Leaves were sampled from 132 accessions of
GPS coordinates of each accession were recorded. Populations were named according to the locality (site) where they were collected (Figure 4). For the genetic analyses, two other
2.1.4. Reproductive biology experiments and fruit set measurements
Flowering rates and season were estimated from June 2014 to June 2015 by surveying on average 96 plants each month in four sites [Habituée (40 plants in mean surveyed per month), Mazeau (22), Moreau (21), and Desbordes (13)]. Plants were checked for the presence of flowers. The lifespan per flower was estimated on 11 flowers from one plant (Desbordes) by measuring the time‐laps between flower opening and its wilting.
From June to July 2014, fruit sets were precisely measured from 16 inflorescences (86 flowers in total) on two accessible Mazeau population plants, which were located at about 2 km distance from each other. Eight inflorescences were covered before flower opening by an insect‐proof bag to exclude insect visits, while the other eight inflorescences (control) were not bagged. Inflorescences being always at the canopy (10–20 m high), access to flowers had to be performed using a 2 × 8‐m‐high ladder.
Fruit set was estimated as the ratio of the number of fruits developed at 30 days by the number of flowers at day 0. The natural fruit set (unbagged lowers) was then compared to the spontaneous fruit set observed in bagged flowers using a Student’s test with the software
Natural fruit set was also assessed globally from June 2015 to June 2016 on 103 inflorescences from 32 plants in four different sites (9 from Habituée, 4 from Desbordes, 8 from Mazeau, and 11 from Moreau), by counting maturing fruits visible using Leica 10 × 40 binoculars. The fruit set was measured as the ratio of the mean number of fruits per inflorescence by the mean number of flowers produced by inflorescence (as determined from the previous Mazeau experiment).
2.1.5. DNA extraction
DNA was extracted from each accession from 0.020 to 0.025 g of dehydrated leaf material. Tissues were grinded using a
2.1.6. Microsatellite analyses
Fourteen microsatellite markers isolated from
2.1.7. Genetic analyses
An extended dataset comprising all studied accessions from Guadeloupe, Martinique, and Mexico (137 individuals) for the 6 microsatellite loci was used to calculate the total number of alleles for each locus (Na), the number of private alleles per population (Np) using the
Then, accessions from Martinique and Mexico were excluded from the dataset to calculate for each locus the observed heterozygosity (HO), expected heterozygosity under Hardy‐Weinberg (HW) equilibrium (HE) and fixation index (FIS) as in [44], using the online version of
Linkage disequilibrium between loci was tested using a probability test in
2.2. Results
2.2.1. Reproductive biology
Morphological character measurements from reproductive and vegetative organs (Table 2) fitted the botanical description of
Organ | Length | Width | Thickness | Diameter |
---|---|---|---|---|
Sepal | 44.5 (±6.3) | 12.5 (±1.8) | ||
Petal | 44.4 (±5.4) | 10.9 (±1.9) | ||
Labellum | 25.8 (±2.0) | 11.2 (±0.9) | 11.2 (±0.5) | |
Column | 23.5 (±1.5) | 2.4 (±0.3) | 2.2 (±0.4) | |
Ovary | 40.6 (±10.1) | 2.6 (±0.4) | 2.5 (±0.4) | |
Fruit | 160 (±18.7) | 10.2 (±0.3) | ||
Stem | 96.2 (±25.2)IL | 4.9 (±1,2) | ||
Leaf | 183.4 (±30.4) | 48.5 (±8,9)LW 82.1 (±21,1)LMW |
Results from the reproductive experiments (bagged and unbagged inflorescences) performed on 86 flowers from the Mazeau site are shown in Table 3. The mean number of flower per inflorescence in
Day 0 | Fruit set at day 30 (%) | |||
---|---|---|---|---|
Control | Bagged | Control | Bagged | |
Individual | Nb_fl | Nb_fl | ||
Mazeau 16 | 6 | 6 | 50.0 | 50.0 |
6 | 5 | 83.3 | 80.0 | |
6 | 5 | 66.7 | 60.0 | |
6 | 6 | 50.0 | 50.0 | |
Mazeau 4 | 6 | 3 | 33.3 | 100.0 |
5 | 6 | 80.0 | 50.0 | |
6 | 4 | 16.7 | 25.0 | |
4 | 6 | 50.0 | 16.7 | |
Total | 45 | 41 | ||
Mean ± SE | 5.63 ± 0.7 | 5.13 ± 1.05 | 53.7 ± 21.1 | 53.9 ± 25.3 |
0.30 (NS) |
2.2.2. Genetic analyses
A total of 23 alleles were revealed for the 6 loci in the analyses on the complete dataset (Table 4), with a mean of 3.67 allele per locus, of which nine were private: four alleles to Mexico (with frequencies >0.1), and one in each of the Guadeloupe populations (with N ≥ 5) of Desbordes, Habituée, Léon, Moreau, and Sofaia (with frequencies >0.01). The six loci were polymorphic at the regional scale (Guadeloupe, Martinique, Mexico), and only four were polymorphic in Guadeloupe. Eighteen alleles were revealed in Guadeloupe (Table 4), with a mean of 3 alleles per locus.
Locus | HU03 | HU04 | HU06 | HU07 | HU09 | RO05 |
---|---|---|---|---|---|---|
Na (Guad) | 4(4) | 3(1) | 4(4) | 3(3) | 6(5) | 3(1) |
Size (bp) | 119–127 | 150–161 | 252–260 | 165–171 | 109–203 | 178–180 |
Pol_Reg | Yes | Yes | – | Yes | Yes | Yes |
Pol_Guad | Yes | No | Yes | Yes | Yes | No |
N (Guad) | 113(111) | 42(40) | 43(43) | 57(55) | 126(125) | 48(47) |
Guadeloupe | ||||||
NullMC | 0.00 | – | 0.19 | 0.34 | 0.14 | – |
NullIIM | 0.01 | – | 0.12 | 0.02 | 0.02 | – |
HE | 0.333 | 0.000 | 0.515 | 0.525 | 0.503 | 0.000 |
HO | 0.342 | 0.000 | 0.227 | 0.000 | 0.296 | 0.000 |
FIS | ‐0.026 | – | 0.559 | 1.000 | 0.412 | – |
HW | NS | – | *** | *** | *** | – |
Except for HU03, all other 3 polymorphic loci (HU06, HU07, and HU09) deviated significantly from HW expectations due to strong heterozygote deficits in Guadeloupe. The remaining two monomorphic loci (HU04, RO05) were also homozygous in Guadeloupe (Table 4), but not in Mexico (data not shown).
The test for genotypic disequilibrium for each pair of locus revealed no significant linkage between loci (p > 0.05). No large‐allele dropout was detected.
Possible null alleles were detected with
The analyses per population on the selected complete dataset of 51 individuals for 3 loci (HU03, HU07, and HU09) revealed that the three studied populations with N > 5 individuals (Mazeau, Moreau, and Sofaia) deviated significantly from HW expectations due to a heterozygote deficit (Table 5). Deviation from HW expectations was also significant at the scale of Guadeloupe (Table 5). Selfing rate was estimated as 79% in Mazeau and 74% in Guadeloupe as a whole (Table 5). Global diversity HE was 0.44 (Table 5). FST value across all populations was calculated as 0.157 using
Population | N | Na | Ap | HE | HO | FIS | S | HW |
---|---|---|---|---|---|---|---|---|
Mazeau | 14 | 6 | 0 | 0.342 | 0.119 | 0.652 | 0.79 | ** |
Moreau | 13 | 7 | 0 | 0.350 | 0.205 | 0.415 | 0.59 | ** |
Sofaia | 7 | 6 | 0 | 0.389 | 0.143 | 0.633 | 0.78 | ** |
Guadeloupe | 51 | 9 | 2 | 0.438 | 0.183 | 0.582 | 0.74 | ** |
2.3. Discussion
Autogamy and self‐pollination (53.9% fruit set in bagged inflorescences) explained the totality of the observed natural fructifications (53.7%) for the species
It is noteworthy that it was suspected that
Autogamy is therefore found either in subgenus
Population genetic parameters indicated a significant deviation from HW equilibrium and/or a homozygote excess for five loci out of six tested (not for HU03) in Guadeloupe vanilla population. Deviation from HW equilibrium was also detected in all the populations with more than five individuals studied, including Mazeau in which reproductive biology experiments were conducted. On the contrary, populations from allogamous species
Deviation from HW equilibrium and homozygote excess could be due not only to homozygosity but also to null alleles, commonly encountered with microsatellite markers. This possibility was therefore also tested.
In autogamous species, only plant seeds ensure efficient gene dispersion whereas pollen also contributes in allogamous species [55, 56]. This has important consequences on the genetic diversity organisation, with autogamous species populations being more strongly differentiated, but less variable than populations from allogamous species [55, 56]. A metadata analysis [55] confirmed that annual or autogamous plants, or with gravity‐dispersed fruits, allocate genetic variability among populations rather than within, with therefore high FST (0.34–0.42) and low HE (0.41–0.47). On the contrary, long‐lived or allogamous taxa, or with wind or ingested dispersed seeds, are more variable within populations than between and show low FST (0.13–0.22) and high HE (0.61–0.68). The calculated FST value in
Intra‐population diversity (HE) value in
It was suggested that
From the set of 14 microsatellites developed from the
3. Conclusion
Our preliminary results obtained with the set of 6 heterologous microsatellite primers allowed the confirmation of the reproductive biology results and showed that
Acknowledgments
This work was funded under the VaBiome project by ANR # 11‐EBIM‐005‐06 to Parc National de Guadeloupe, ANR # 11‐EBIM‐005‐01 and Réunion Regional Council # DGADD/PE/20120590 to UMR 53 PVBMT Réunion, as part of the EU Era‐Net NetBiome call for projects. The authors thank Alain Ferchal (PNG, Parc National de Guadeloupe) for drawing the map in Figure 4; Alain Rousteau (UAG, University of Antilles Guyane) for the help in habitat names translation; Céline Lesponne (PNG) for support to Chloé Goulié mapping work during her Master 2 thesis; Monique Citadelle and Marie‐France Barre (AGO) for their participation in field work; Danièle Roques (Cirad Guadeloupe) for her participation in flowering monitoring; Philippe Feldmann (Cirad), Thierry Guillon and Pascal Segrétier (PNG), and Claudine and Pierre Guezennec for precious information given on
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