Environmental gradients are excellent systems to explore how species and biotic communities respond to gradual changes in abiotic factors. Environmental gradients include latitudinal, elevational or aridity gradients. Elevational gradients are considered natural experiments for testing ecological and evolutionary responses of organisms to variation in geophysical factors. Along with elevational gradients, atmospheric pressure, temperature and clear-sky turbidity decrease, and species exhibit a diverse range of physiological and morphological responses [1].

Biotic communities usually exhibit changes along elevational gradients. Plant species richness and diversity usually decline with elevation due to the decreasing importance of herbs, vines and shrubs [2].

Elevation profoundly affects floral traits, forcing plants to adapt to extreme conditions. Altitudinal gradients constitute a robust framework to analyze the variation of functional traits in plants, since they represent consistent climatic contrasts. As the elevation increases there is a decrease in mean temperature and atmospheric pressure and the growing seasons are shorter; these changes in climatic conditions directly affect the morphology, phenology and physiology of species [1][3]. These patterns make it possible to distinguish the effects of altitude from local site or habitat influences, which are usually more linked to edaphic or microclimatic conditions [4]. Empirical evidence shows that seedling phenology and plasticity in temperature adaptation are predictably adjusted along altitudinal gradients, reinforcing the idea that functional traits respond mainly to large-scale climatic variables [5]. At higher altitudes, flowers tend to be smaller in overall size, but with specific morphological and chromatic adaptations to survive and attract scarce pollinators [6].

For several plant species, elevational changes in flower morphology have been documented as result of changes in abiotic and biotic factors, like temperature and plant-animal interactions [7]. For instance, Devoto et al. [8] found that along elevation gradients, pollinators changed from hymenopters to dipters and from specialist to generalist [9]. When pollinator composition changes along elevation, pollinator services are also modified and flowers respond to these changes [10].

Plants respond to elevational gradients through morphological or physiological changes that allow them to persist in a particular habitat, maximizing their fitness [3][11]. For reproductive structures, several studies have documented positive responses in flower longevity, flowering time and floral traits while other traits like total number of flowers decline with elevation. In Phacelia secunda [12], Aritolochia chilensis [13], Campanula sp. [14][15], Oxalis compacta [16], and Rosmarinus officinalis [17], flower longevity, flowering time and flower size increase with elevation. In contrast, the total number of flowers of Stylidium armeria [18] and Campanula rotundifolia [15] decreases with elevation. Most of these studies have been carried out in iteroparous plant species with periodic reproduction while very few have been conducted on semelparous species that invest in large floral displays in one final reproductive event [19][20]. This is the case of rosette plants with semelparous reproduction, which exhibit a reproductive strategy often associated with bat pollination that often produces large inflorescences and flowers that secrete large quantities of nectar and attract a large pollination guild [19].

The genus Agave consists of semelparous rosette species endemic to the American continent, and it is represented by approximately 166 species [21]. México is the center of diversity, where nearly 150 species are distributed in its territory, representing around 90% of the genus [22]. Many species produce flowers that open at night and nectar secretion is primarily nocturnal [23]-[27]. Very few species of Agave have been studied among environmental gradients. For instance, Silvano-Montellano and Eguiarte [28] studied how flower traits and pollinators change along a latitudinal gradient in northern Mexico. They found that flowers were longer and showed smaller diameters in southern sites and shorter and greater diameters in northern sites. However, no previous study has explored how flower traits vary along elevation in the genus Agave. One of the members of this genus is Agave gentryi, with a distribution in the Sierra Madre Oriental in México, from 1800 to 3200 meters above sea level. A.gentry represents an excellent system to study how flowers respond to spatial changes along an elevational gradient. In this study, we describe patterns of variation of flower traits in three populations inhabiting an elevation gradient. Particularly, we measure nectar volume, sugar concentration, corolla diameter and lengths of the stamens and pistils.

Agave gentryi has an anthesis period that lasts from 4 to 6 days, with protandrous flowers, where stamens grow from 50.5 to 81.2 mm and pistils grow from 44.9 to 70.2 mm. Nectar secretion takes place during both the male and female phases and during the night and the day. The accumulated volume of nectar per flower varies from 1.16 to 2.22 mL (Table 1).

Table 1.Mean (±sd) of morphometric and nectar traits along the elevational gradient.

Discriminant analysis of floral traits between sites showed significant differences along the elevation gradient (Wilks’ λ = 0.009, p < 0.001). The first root managed to explain 95.4 % of the data, with the variable length of the stamen being the ones that contribute the most to these differences and the second root diameter explains the 4.4 % of the differences with corolla diameter being the ones that contribute the most to these differences (Table 2). The length of the stamen tends to decrease with altitude, while the diameter of the corolla increases.

Table 2.Results from the discriminant function analysis.

Figure 2 shows the centroids of the six groups. We can see that there are no differences between day and night except for the case of the PPF site (p = 0.03), the growth of the corolla and the length of the stamens were greater during the night than during the day at this site.

Figure 2.Chart showing centroids of the six groups: square: pinyon pine forest (low elevation site); triangle: oak-pine forest (middle elevation site); diamond: pine forest (high elevation site). Open figures correspond to diurnal and closed figures correspond to night values.

Studies of elevational gradients have contributed to our understanding of how organisms respond to gradual changes in abiotic factors [1]. Along altitudinal gradients, biotic interactions can also be important for generating local adaptation [34]. The magnitude of the changes observed in altitude, species diversity, functional diversity, and network structure, when they occur simultaneously, indicates that environmental factors influence the functional and phylogenetic diversity of plants and pollinators, thus impacting the configuration of the ecological network [35].

In this study we have shown that A.gentryi exhibits significant differences in flower traits along an elevational gradient in the Sierra Madre Oriental in México. In this gradient, stamen length and nectar volume decrease, and corolla diameter increases with elevation (Figure 3).

Other studies have documented similar patterns. For instance, Malo and Baonza [36] detected that in Cytisus scoparius, flower size increases with elevation. Similarly, in Campanula rotundifolia, Maad et al. [15] recorded that corolla diameter increases with elevation. For the case of the genus Agave, previous studies have documented significant differences in flower traits along latitudinal but not along elevational gradients. In A.lechuguilla, corolla diameter increases but flower size decreases with latitude [28]. Lebron-Liriano et al. [37] mention that a part of the morphological diversity observed in Agave individuals within the Sierra de Bahoruco National Park is determined by the influence of environmental factors. Abiotic conditions show great variation along altitude gradients; factors such as temperature, solar radiation, humidity and the availability of nutrients in the soil depend mostly on altitude and have direct effects on plant activity [38].

Figure 3.Changes in the morphology of Agave gentry flowers (a) along an elevation gradient and (b) during the day and night periods.

The observed changes in flower morphology in the studied gradient also might be associated with changes in pollinator composition and abundance. Therefore, the observed changes in the characteristics of the flowers in the elevation gradient could be due to the changes in the available pollinators [39]. For instance, inTrollius ranunculoides, flower diameter changes with altitude due to changes in pollinator composition, with flies in high elevation sites and bees in lower elevation sites [40]. In Cytisus scoparius, flower size increases with elevation because of changes in the size of available pollinators [36]. In temperate systems, this pollinator-led selection has been shown to generate variations in floral characteristics along altitudinal gradients. Populations of different plant species can develop specific adaptations to their local pollinators, leading to divergences in floral traits. These traits are subject to natural selection processes mediated by pollinators, aimed at optimizing the reproductive success of plants [41].

In our case, we hypothesize that changes in pollinator composition may be the underlying cause of the observed changes in flower traits. Bats may be the major pollinators in low elevation sites while bumblebees, bees and hummingbirds might be the major pollinators at high elevation sites. This hypothesis requires an evaluation of the composition and abundance of pollinators in our study sites.

Significant changes in corolla diameter and stamen length between day/night periods were detected just for the pinyon pine forest at the low elevation site. In this site, during the flowering season maximum temperatures reach 26˚C while minimum temperatures reach 1˚C, values that are on average 2˚C - 3˚C greater than in the other sites [42]. Greater nocturnal elongation of flowers may be associated with heat stress during the day in the lower elevation site. This elongation pattern may be a strategy to reduce transpiration and water loss during the day. Other studies have documented that Agave flowers tend to secrete nectar and anther dehiscence mainly during the night [26]. In A.gentryi, however, no significant differences were detected for nectar secretion between day and night. This species has a wide latitudinal distribution along the Sierra Madre Oriental from Queretaro to Coahuila and might exhibit a similar pattern of variation of flower traits as in our elevational gradient, as reported to A.lechuguilla [28].

Our study shows clear differences in flower traits along an elevational gradient. Further studies are needed on the pollinators, fruit and seed set, clonal growth and sugar composition in nectar along the elevational gradient to have a better understanding of the environmental factors that influence reproduction in Agave gentryi. Future studies should consider an evaluation of fruit and seed sets and the relative importance of sexual and asexual reproduction in this Agave. Finally, we would like to suggest that this species should be studied along a latitudinal gradient to explore how flowers respond to both types of gradients.

We thank Arturo Mora Olivo, Clara Tinoco Ojanguren and Erick Ruben Rodriguez Ruiz for the review critical comments and Adán De Leon Contreras, Oscar López Hernández and José F. Martinez for lab and field assistance. This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACYT), as well as Proyect UAT10-NAT 0101 of Dirección de Estudios de Posgrado e Investigación de la Universidad Autónoma de Tamaulipas.

*Variation on floral traits of A.gentryi along an elevational gradient.

^#Corresponding author.