Effect of Manipulating Flowers In Situ on Sexual Reproduction of Cyrtopodium macrobulbon (La llave & Lex.) Romero-González (Orchidaceae) in Tamaulipas, México

1. Introduction

Mexico is home to a remarkable wealth of orchids, which belong to one of the five most diverse vascular plant families; around 1,260 species belonging to 170 genera have been described (Hágsater et al., 2005; Soto et al., 2007). It is one of the most vulnerable families due to destruction of their habitat and to clandestine extraction, the reasons that 181 species are found in some risk category (SEMARNAT, 2010).

The genus Cyrtopodium belongs to the subfamily Epidendroideae, tribe Cymbidieae Pfitzer and subtribe Cyrtopodiinae Benth. (Dressler, 1993; Szlachetko, 1995; Chase et al., 2003; Pridgeon et al., 2009) and comprises approximately 50 species. The greatest diversity is found in the west central region of Brazil, and the rest is distributed from Florida and Mexico to northern Argentina (Barros et al., 2003). The species of the genus are extracted because of their ornamental and ethnopharmacological value (Vieira et al., 2000; Neto & Morais, 2003; Barreto & Parente 2006; Brasileiro et al., 2006; Romero-González et al., 2008). Some species such as C. andersonii R. Br. and C. cardiochilum Lindl. are used to treat colds, tuberculosis, and hemoptysis (Corrêa, 1975), and in Brazil, to treat inflammatory and immunoregulator disorders (Barreiro et al., 2004; Barreto & Parente, 2006). Extracts of the C. paranaense Schltr. pseudobulbs are used to cure lesions in general (Pereira & Felcman, 1998; Boscolo & Valle, 2008; Boscolo et al., 2010).

C. macrobulbon La Llave & Lex. is widely distributed in Mexico. It grows in tropical climates in rocky substrates where it is continuously exposed to pillage and destruction of its natural habitat (Carranza-Álvarez et al., 2015). The pseudobulbs are used as medicine to treat abscesses and as a balm agent in the states of Morelos and Yucatán (Martínez, 1979; Arellano et al., 2003; Monroy-Ortiz et al., 2013; Garay et al., 2018).

Moreover, it has been documented that in Mexico City it is used to treat male urinary infections, and the gum extracted from the pseudobulbs is used as glue in book binding and diverse handcrafts (García-Peña & Peña, 1981).

Floral evolution has been associated with differences in pollination syndromes. In orchids, this is extremely diverse and includes reward and deceit systems (Phillips et al., 2017). Species of the genus Cyrtopodium do not offer rewards to their pollinators, but they attract bees of the Centridini and Euglossini families by deceit (Chase et al., 1992; Van Der Cingel, 2001; Pansarin et al., 2008). Regarding orchid reproductive and evolutionary biology, fruit set and seed production are the most important processes used to determine plant reproductive success. For this reason, the importance of pollinators for this family is reflected in their complex pollination mechanisms. In the reproductive system of orchids self-compatibility prevails (Proctor et al., 1996; Tremblay et al., 2005), and thus, conservation depends on the existence of biotic interactions within the community, for example with mycorrhizal fungi and pollinating organisms. Therefore, the disappearance of one of them would also cause a reduction or loss of natural orchid populations affecting humans to some degree, as in the case of the species of the genus Cyrtopodium. The objective of this study is to determine the mechanism of pollination for reproductive success in a wild population of Cyrtopodium macrobulbon.

2. Materials and Methods

2.1. Study Area

The study was conducted in the ejido Gallos Grandes, Tula, Tamaulipas, Mexico. The study site has elevations from 1,035 to 1,100 m. The climate is predominantly warm steppe; mean annual temperature is 18 °C, with highs of 43 °C and lows of 1 °C. Soil is regosol from sedimentary rock, and vegetation is rosetophyllous scrub (INEGI, 1999; Treviño-Carreón and Valiente-Banuet, 2005).

From March to September 2018, we visited the study site and selected a total of 23 Cyrtopodium macrobulbon La Llave & Lex. reproductive individuals with developed inflorescence.

2.2. Measurement of Cyrtopodium macrobulbon Floral Structures

We measured the C. macrobulbon floral structures and described the morphology of the species. To this end, we collected 10 flowers from 10 individuals and took measurements with a Surtek Vernier.

2.3. Pollination Treatments

We established five pollination treatments during the flowering period: a) control, in which the flowers were not manipulated; b) self-cross, in which the pollinia from one flower was placed in another flower of the same individual and later isolated in organza-type bags; c) automatic pollination, flowers were isolated in organza-type bags without removing the pollinias; d) cross pollination, the pollinias were removed, placed in flowers of a different individual, and isolated in organza-type bags; and e) emasculation, in which the pollinias were removed from the flowers. Each pollination treatment was marked with thin wires of different colors.

2.4. Analysis of Fructification Data by Pollination Treatment

The percentage of fruits produced by each treatment was determined. An analysis of variance and a Tukey comparison of means test at 0.05 were performed for the five pollination treatments. The data were analyzed using SAS 9.0.

2.5. Analysis of Seed Weight Data of the Fruits by Pollination Treatment

The seeds of the C. macrobulbon fruits produced by each treatment were weighed in a Sartorius analytical balance. An analysis of variance and a Tukey comparison of means test at 0.05 were performed and the data were analyzed using SAS 9.0.

3. Results

3.1. Morphological Description of Cyrtopodium macrobulbon

Cyrtopodium macrobulbon La Llave & Lex. belongs to the Orchidaceae family. It has a terrestrial growth habit and grows between bare rocks located in open spaces of the low deciduous forest; it is associated with small patches of rosetophyllous scrub affected by the water stress conditions of these environments. Reproductive individuals reach heights of 0.6 to 1.3 m and are found in groups of erect pseudobulbs. The leaves are linear with a pointed apex. The inflorescence is lateral paniculate type and can have more than 100 flowers. The labellum measures 3.0 cm on average, while the column reaches a length of 5.0 mm. The pollinia is 2 mm long on average, while the fruits are capsular with an oblong to ovate shape and can reach up to 10 cm in length.

3.2. Percentage of Fruits Formed, by Treatment

According to the results of the pollination treatments (Table 1), the self-cross treatment produced the highest percentage of fruits, followed by the emasculation treatment, which produced 10% less.

The ANOVA for fruit production by treatment (Table 2) indicated that there are statistically significant differences among treatments (P < 0.05). Given that the value of P of the treatment test is less than 0.05, there is a statistically significant difference between the fruits of one treatment and those of the next, with a confidence level of 95%. Moreover, this statistically significant difference is also present in the source of flower variation.

In the test of comparison of means of the five pollination treatments with grouped standard errors (Table 3), the control treatment shows a statistically significant difference with respect to the emasculation treatment. However, the treatments cross pollination, automatic pollination, and self-cross pollination are statistically similar.

Given that the value of P of the F test is lower than 0.05 (Table 4), there is a statistically significant difference in percentage between treatments, with a confidence level of 95%.

The comparison of means for the seed mass of the fruits by treatment shows that the treatments control, self-cross and emasculation are significantly different from the cross pollination and automatic pollination treatments (Table 5).

3.3. Analysis of Fruit Seed Mass, by Treatment

The analysis of variance for seed mass of the fruits from five pollination treatments (Table 6) indicated that there is a statistically significant difference between treatments. The results indicate that there is a statistically significant difference in C. macrobulbon seed mass, by treatment, given the value of P < 0.05, while the comparison of means for the seed masses from fruits of the five treatments (Table 7) showed that there are differences among treatments.

The test of comparison of means of the seed mass C. macrobulbon of fruits revealed statistically significant differences among the treatment’s cross pollination, self-cross pollination, and automatic pollination.

4. Discussion

Fruit production in orchids can be limited by a variety of conditions, such as absence of pollinators, herbivores, or limited resources in the mother plant (Ackerman & Montalvo, 1990).

The results indicate that Cyrtopodium macrobulbon is an orchid species that does not reward floral visitors, it is self-compatible but is dependent on pollinators due to a low percentage of self-compatibility. It is necessary to have a pollen vector to transport pollen, even for self-pollination, and cross pollination is favored in most of the species (Van der Pijl & Dodson, 1966; Pansarin, 2003; Mickeliunas et al., 2006).

Speciation does not guarantee an adequate rate of fructification in natural conditions since studies on diverse species have shown reproductive success below 10% (Carvalho & Machado, 2002; Martini et al., 2003; Pansarin, 2003; Pansarin et al., 2006; Mickeliunas et al., 2006; Carvalho & Machado, 2006; Pansarin & Amaral, 2008; Pansarin et al., 2008a; Pansarin & Pansarin, 2010; Rech et al., 2010; Storti et al., 2011).

Ackerman (1989) mentions that many fruits may form but can be lost before maturity for several reasons, such as the effect of non-viable pollen, attack by pathogens, mechanisms of post-zygotic incompatibility, and herbivores.

Another reason for low reproductive success in many orchid species is the low frequency of pollinator visits, especially when the species does not offer any type of reward, but has the strategy of floral mimesis, as is the case of Cyrtopodium polyphyllum (Vell.) Pabst ex F. Barros., which is very infrequently visited by different bees that are not efficient pollen transferers (Van der Pijl & Dodson, 1966; Pansarin et al., 2008). This species has adapted, and some type of abiotic pollination occurs (Pansarin et al., 2008b).

Diverse studies have observed that orchids that produce nectar tend to obtain a higher percentage of pollination and production of fruits than those that do not (Ackerman et al., 1994; Neiland & Wilcock, 1998; Smithson & Gigord, 2001).

5. Conclusion

Of the pollination treatments, self-cross pollination produced the highest percentage of fruits (56.70%), followed by the emasculation treatment, which had 10% less. Moreover, the analysis of variance indicated that the difference is statistically significant, while the comparison of means showed that the control treatment is statistically different relative to the emasculation treatment. The analysis of variance for seed weight of the five pollination treatments showed that there are statistically significant differences, and the comparison of means revealed that the largest quantity of seed mass was produced by cross pollination, which is therefore an effective method for reproductive success of Cyrtopodium macrobulbon. The self-cross and automatic pollination treatments show that the species is self-compatible at low rates.