Morphological and phenological variation of flower colour morphs in a wild population of Opuntia streptacantha (Cactaceae)

Background and aims – Opuntia s.s. (Cactaceae) is one of the most diverse genera in the subfamily Opuntioideae, with approximately 220 species. The considerable morphological and anatomical diversity among these species has resulted in a remarkable adaptative plasticity, evident in both intra-and interspecific variability. Our study system is Opuntia streptacantha , which has two flower colour morphs: yellow and orange. The objective is to determine if there are morphological differences in the reproductive and vegetative structures between floral morphs. Material and methods – We measured 8 cladode traits (n = 20 cladodes for each floral morph) and 17 flower traits (n = 30 flowers per morph), and reproductive phenology was recorded for both morphs to describe their phenophases (n = 10 individuals per morph). Key results and conclusion – We found that floral colour morphs of O. streptacantha showed significant differences mostly associated with flower traits. Principal component analysis revealed seven components that explained 80% of the total variation, namely total flower length, number of stamens, distance between anther and stigma, number of pollen grains, style length, equatorial diameter of the ovarian chamber, pericarp width, and number of areole lines. Some individuals of O. tomentosa were classified as floral morphs of O. streptacantha , not having a clear separation between the species. The phenology of the floral colour morphs showed a slight lag in their peak flowering and fruiting. Very high flowering synchrony was found for each floral morph and between them. The modifications found in the flowers of O. streptacantha may be associated with a possible hybridization with O. tomentosa favouring the appearance of the two floral morphs.

Prickly pears in Mexico have a wide distribution, but thrive mainly in arid and semi-arid zones, where the greatest species diversity is found.There are two significant centres of diversity: the Chihuahuan Desert zone and the central-western region (which includes the state of Mexico, Guerrero, and Jalisco) (Golubov et al. 2005;Muñoz-Urias et al. 2008).Prickly pears can also be found, although to a lesser extent, in areas such as tropical and subtropical coniferous forests, as well as tropical and subtropical dry broadleaf and moist broadleaf forest (Esparza-Sandoval 2010;Manzanarez-Villasana et al. 2022).
Opuntia s.s.shows a marked morphological variation to the extent that its taxonomy becomes confusing (Bravo-Hollis and Sánchez-Mejorada 1978;Scheinvar 1995;Reyes-Agüero et al. 2005;Muñoz-Urias et al. 2008).Morphological variations include growth habit, stem size and pubescence, spine length, number of areoles, flower shape and colour, weight and chemical composition of the fruit, seed size, among others; and also phenological variations such as the time in which vegetative and reproductive phenophases are observed, to mention just a few (Wallace and Fairbrothers 1986;Pimienta-Barrios and Mauricio-Leguizamo 1989;Pimienta-Barrios 1994;Pimienta-Barrios and Muñoz-Urias 1995;Fordyce 2006;Muñoz-Urias et al. 2008).An example of such variation can be seen in members of the clade Nopalea.This clade, located in North America, presents modification in its morphology (with its androecium and gynoecium excised from the perianth segments) and in the colour of the flowers (mainly pink) (Majure et al. 2012;Majure and Puente 2014) and thus in fitness of Nopalea members.
It is important to carry out taxonomic, ecological, and genetic studies on plant species with different floral colours, as they can contribute to the taxonomic and phylogenetic delimitation of morphotypes.This, in turn, could lead to the recognition of new species, subspecies, or varieties (Narbona et al. 2014).For example, in the case of Primula vulgaris Huds.(Primulaceae), it has been observed that most populations have yellow flowers, while colour polymorphism is present in some populations in eastern regions.This has led to the taxonomic differentiation of the species into several subspecies (Shipunov et al. 2011).
Opuntia streptacantha Lem. is a wild species endemic to Mexico that has two floral morphs: yellow and orange, of which the yellow floral morph was the first to be described (Bravo-Hollis and Sánchez-Mejorada 1978).The objective of this work is to determine if there are morphological differences in the reproductive and vegetative structures, and phenological differences between the flower colour morphs in O. streptacantha.

Study site
This study was carried out in the southern portion of the Chihuahuan desert known as Queretano-Hidalguense semi-desert, in the wilderness area protected by the Regional Botanical Garden of Cadereyta de Montes "Ing.Manuel González de Cosío", Querétaro, Mexico.Its geographic coordinates are 20°41'15.8"N,99°48'17.7"W,with an elevation of 2,046 m a.s.l., the vegetation type is xerophytic crassicaulous scrub.The climate is semi-dry, temperate with summer rains (Köppen climate group BS1 kw (w) modified by García 2004).The average annual temperature ranges between 12 and 19°C and the average annual precipitation is about 550 mm (Chávez-Martínez and Hernández-Magaña 2009).

Study species
Opuntia streptacantha is an arborescent or shrubby plant, up to 4 m tall, its stems (cladodes) are flattened and racket- shaped, and the flowers are yellow or orange, 5-7 cm long.The fruits are 5 cm long and 3 cm wide, globose to obovoid and usually wine-coloured when ripe.The glochids are short, the pulp is red, and the seeds are 3.8-4.5 mm long by 2.6 mm wide (Bravo-Hollis and Sánchez-Mejorada 1978;Arias et al. 2012;Galicia-Pérez et al. 2023).
Opuntia streptacantha is generally found in xerophytic scrublands and with other co-dominant Opuntia species, they form a vegetation type called "nopaleras" (Arias et al. 2012).According to Bravo-Hollis and Sánchez-Mejorada (1978), it is a wild species of remarkable value because of its edible fruits and stems.
Opuntia streptacantha populations contain individuals with two different floral colour morphs: with yellow flowers (YFM) and with orange flowers (OFM) (Fig. 1).The presence of these colour morphs appears to be consistent throughout the distribution range of the species.Historically, the first description of the colour of the flowers of this species was made by Schumann (1899), who mentioned that the flowers were yellow.However, Bravo-Hollis and Sánchez-Mejorada (1978) observed that the flowers vary from yellow to orange within a population.

Cladode and spines morphometry
Twenty mature cladodes (including those with lateral cladodes or reproductive structures such as buds, flowers, or fruits) were measured, comprising two cladodes per individual, from ten reproductive individuals of O. streptacantha for each floral colour morph.Similarly, 20 young cladodes (considering only lateral cladodes as young) were measured, also two per individual.The sampled individuals were checked to ensure that they had fruits, buds, or flowers, or a combination of them, and were approximately 3 meters tall.The parameters used in the work of Muñoz-Urias et al. (2008) were measured: cladode length (cm), cladode width (cm), maximum distance from the apical to the widest part (cm), maximum distance from the basal to the widest part (cm), number of series of areoles, areole size (mm), distances between areoles (mm), and distance between lines (mm) (Fig. 2).To determine differences in the spines of floral morphs, the type, colour, and number of spines on the central areole of three cladodes per individual were determined for each floral morph (n = 10) (López-Borja et al. 2017;Galicia-Pérez et al. 2023).

Flower morphometry
Thirty-three undamaged flowers were collected at the time of maximum flower opening and during peak flowering from different individuals of each flower colour morph of O. streptacantha and fixed in FAA (formaldehyde, alcohol, acetic acid) (Kiernan 2002).Following Martínez-Ramos et al. (2017), 17 morphological characters were measured to the nearest mm: corolla aperture set in FAA, perianth segment length, total flower length, pericarp length, pericarp width, distance between anther and stigma, stigma width, stigma length, style length, longest stamen length, shortest stamen length, equatorial diameter of the ovarian chamber, polar diameter of the ovarian chamber, and we counted the number of stamens, number of ovules, number of lobes, and number of pollen grains in one anther (Fig. 3).
Morphometric data of cladodes and flowers were tested for differences between floral morphs using Generalized Linear Model (GLM) with Poisson distribution for discrete counts and t-tests for continuous variables.For the spines, a paired t-test was carried out.

Fruit and seed morphometry
We collect two fruits from ten different reproductive individuals per flower colour morph (n = 20).We assessed fruit diameter (mm), fruit length (mm), number of spiral series, and number of seeds (López-Borja et al. 2017).A paired t-test for continuous variables and a GLM with Poisson distribution for discrete variables were used to find differences between morphs.In addition, the external colour of the fruit, colour of the pulp, colour of the glochids using the HTML colour code, and the shape of the fruit were taken based on the classification given by Moreno (1984).
A sample of ten seeds was randomly selected from each fruit (n = 200 seeds per floral morph), photographed, and measured for size with length and width of each seed in mm using Adobe Photoshop CS6.A paired t-test was used to compare between morphs.

Multivariate analysis
Two tests were performed to compare flower, cladode, fruit, and seed characteristics between morphs (Sokal and Sneath 1963;Cuadras 1981).The first test was a principal component analysis (PCA), to reduce the variables to those that would give us the most taxonomic information.The morphological characters of all the previously mentioned measured structures were considered, the analysis was performed in R, with the packages FactoMinerR v.

Reproductive phenology and flowering synchrony
Reproductive phenology (flowering and fruiting) was registered, taking monthly observations (April 2018 to March 2019) for ten individuals of each floral morph.The data were analysed with circular statistics to determine the flowering and fruiting peaks (Morellato et al. 2010), and the Rayleigh uniformity test (Zar 1999;Mendoza 2020) was calculated to identify if the distribution of the phenophases was uniform, and the non-parametric Mardia-Watson-Wheeler test (Batschelet 1981) was performed to determine differences between flowering and fruiting.Analyses were carried out in R with the circular v.0.5-0 package (Agostinelli and Lund 2022).Flowering synchrony between YFM and OFM was calculated with the index of Mahoro (2002), modified by Osada et al. (2003).For the modified version, the relative number of open flowers in each individual at an interspecific level (in this case, between YFM and OFM) is considered, following the formula below: where s i is the degree of synchrony of species A with species B, Y A,j is the ratio of flowers in morph A, and Y B,j is the ratio of flowers in morph B.
Both indexes take values from 0 to 1, where a value close to one represents perfect synchrony and a value close to zero represents asynchrony.

Cladode and spines morphometry
The morphometry of old cladodes differed significantly between YFM and OFM in two variables: cladode length (t = -2.62,p = 0.01) and cladode width (t = -2.23,p = 0.03), with OFM being the largest (Table 1).No significant differences were found for young cladodes.Both flower colour morphs have ovate cladodes and two types of spines, straight and subulate.The spines have a yellow or white colouration and the number of spines per areole in both morphs was similar (mean ± standard error; YFM = 3.63 ± 0.49, OFM = 3.93 ± 0.52, χ 2 = 0.3569, p = 0.55).

Flower morphometry
Flowers were actinomorphic in both morphs of O. streptacantha (Fig. 1), but we found significant differences in most of the characters (Table 2), with higher values for YFM.In contrast, the gynoecium was very similar for both morphs.

Fruit and seed morphometry
Both floral morphs had ovate fruits, a magenta pericarp with wine-coloured pulp, and opaque-golden glochids.Fruit length was the only difference, with YFM having longer fruits (Table 2).Both floral morphs have funiculate seeds, with an oval to amorphous shape and light brown colour.Significant differences in seed size (Table 2) were found between the morphs.

Multivariate analysis
Of the morphological characters, those showing significant differences between floral morphs for PCA and discriminant analysis were considered.PCA shown seven principal components that explain 80% of the total variation.The first component explains 28.37% (total flower length), the second component 17.50% (number of stamens), the third component 11.01%(distance between anther and stigma), the fourth component 7.20% (number of pollen grains and style length), the fifth component 6.58% (equatorial diameter of the ovarian chamber), the sixth component 5.71% (width of the pericarpel), and the seventh component 4.05% (number of areole lines), considering only the first two components explain 45.87% of the total variation (Fig. 4; Supplementary material 1).It is important to emphasize that six of the seven components are flower morphometric variables.Linear discriminant analysis explained 89.28% of the variation in the first two linear discriminant functions.Opuntia cantabrigiensis was completely separated from the other species, O. tomentosa was grouped with YFM, and OFM was almost separated from O. tomentosa but showed a small overlap with YFM (Fig. 5).The analysis was able to correctly assign 92% of the individuals within species.YFM had the fewest correctly classified individuals (Table 3).

Reproductive phenology and flowering synchrony
Reproductive phenology differed between the floral morphs.Flowering for YFM was significantly (r = 0.9566, p = < 0.001) concentrated in four months, from March to June, with peak flowering in April (Fig. 6A).On the other hand, the OFM showed a significant (r = 0.9443, p = < 0.001) flowering season of five months, from February to June, with a single peak in flowering in May (Fig. 6B).The flowering patterns of the floral morphs were significantly different (W = 43.686,p < 0.0001).
Fruiting in YFM was significant (r = 0.9023, p = <0.001)concentrated in a period of five months (June to October), with peak fruiting in July (Fig. 6C).On the other hand, in the OFM, showed a significant fruiting season (r = 0.8749, p = <0.001),spanning seven months, from May Table 3. Classification of the individuals (columns) based on floral morphometrics using the linear discriminant analysis.to November, with peak fruiting in June (Fig. 6D).The fruiting patterns of the floral morphs were significantly different (W = 206.5,p < 0.0001).

DISCUSSION
We found that the greatest morphological difference between floral colour morphs in Opuntia streptacantha is found for flower characteristics, both in the external part of the flower and in the reproductive structures, with the yellow-flowered morph generally being larger than the orange-flowered morph.
Although cladodes and spines are the most striking morphological characteristics in Opuntia (Del Castillo 1999), they showed few differences between YFM and OFM, which may reflect the fact that both floral morphs are subjected to similar environmental stress.In cacti, spines help regulate plant body temperature and also reduce photosynthetically active radiation (Majure et al. 2023).
YFM fruits are longer and have larger seeds compared to OFM; however, there is no difference in the number of seeds in each fruit.Several studies showed that seed size can vary within populations and within plants in the same species (Janzen 1977;Cavers and Steel 1984;Winn and Gross 1993;Sakai and Sakai 1996).For example, in the species Phaseolus lunatus L. (Fabaceae) it was found that in different regions and in the same population, there is a great variation in fruit and seed characters (Vargas et al. 2003).Another factor to consider is the reproductive success of the species, since the type of reproductive system of a flowering plant may condition in some way the production of fruits and seeds, because many depend on the efficiency of pollination (Galetto et al. 2002).
Floral morphometry studies in cacti are few, but it has been reported that there is variation in flower in some cactus species, such as Lophophora diffusa (Croizat) Bravo (Briseño-Sánchez 2019), where white or pink flowers have been reported, Weberbauerocereus weberbaueri (K.Schum.ex Vaupel) Backeb.(Sahley 1996), where it ranges from bright pink-red to white, and Ariocarpus kotschoubeyanus (Lem.)K.Schum.(Martínez-Peralta et al. 2014), where it ranges from white with a darker tepal line to magenta, with intermediate shades.In the case of L. diffusa and A. kotschoubeyanus, the flowers are visited by bees, while W. weberbaueri flowers are visited by bats and hummingbirds.However, no information is available on possible morphological differences associated with flower colour.Studies on the species Collinsia parviflora Douglas ex Lindl.(Scrophulariaceae) showed a positive relationship between flower size and the number of floral visitors (Elle and Carney 2003), but another study with Ipomoea aquatica Forssk.(Convolvulaceae) found that floral visitors similarly visited flowers of all colours (Hassa et al. 2020).
In the linear discriminant analysis, O. tomentosa and O. cantabrigiensis were included, since they showed flower colour similarities with the floral morphs of O. streptacantha: O. cantabrigiensis has yellow flowers and O. tomentosa orange flowers (Galicia-Pérez et al. 2023).The analysis completely separated O. cantabrigiensis from O. tomentosa, YFM, and OFM, but the latter three were morphologically close, giving the possibility that there are some individuals with hybrid phenotypes between these species.Intermediate phenotypes are common in Opuntia s.s., and morphology supports this interclade hybridization (Majure et al. 2012).It is likely that the existence of YFM and OFM in O. streptacantha is due to a gene exchange with O. tomentosa, which has the greatest number of morphological and phenological similarities with YFM.
In general, the flowering peaks of YFM and OFM were unique, this agrees with the information from several studies where it is mentioned that the cacti studied so far have only one flowering peak (unimodal), although there are species that flower throughout the year and with several flowering peaks (Mandujano et al. 2010).The two flower colour morphs of O. streptacantha had their peak flowering in different months, this may be a strategy to ensure the reproductive success of both or may be a differential response to the environment (Fenner 1998;Matías-Palafox et al. 2017).
The floral morphs of O. streptacantha showed very high synchrony indices, either within the same morph or between morphs.In both cases, flowering occurred in a single period (from February to June).Rathcke and Lacey (1985) mention that simultaneous flowering between different species can be advantageous, since the flowering of one species increases the visitation rate of another species.Martínez-Ramos et al. (2024) found for the same study site as O. tomentosa and O. streptacantha (without distinguishing the morphs), that a high rate of interspecific flowering synchrony was present, adding that this could favour gene flow between these species.Matías-Palafox et al. (2017) found that during peak flowering of Astrophytum ornatum (DC.)Britton & Rose (Cactaceae) and Turbinicarpus horripilus (Lem.)V.John & Říha (Cactaceae), which cohabit the same area, both species showed synchronous flowering, bee pollination and shared floral visitors, this could result in interspecific competition or facilitation when there is a shortage of pollinators.
In conclusion, the differences between the floral morphs of Opuntia streptacantha extend beyond flower colour.The structure in which most of the morphological variation is found is the flower, but the differences between floral morphs are not only morphological, but also ecological since they show differences in flowering phenology.Therefore, it is important to determine whether these floral morphs are already differentiated into another taxonomic category, further research (e.g.hand pollination to evaluate whether the flower colour morphs are sexually compatible and observations to determine floral visitors and pollen flow) will help to understand the role of flower colour polymorphism in O. streptacantha.
Two indices were evaluated to determine the flowering synchrony of the colour morphs.The Marquis (1988) index was evaluated, which considers the number of open flowers per census and the proportion that these flowers represent with respect to the total number of flowers, following the formula below: where, S is the degree of synchrony, x t the number of open flowers per census, is the proportion of open flowers to the total number of flowers, and Pt represents the proportion of the censused individuals at flowering during time t.

Figure 3 .
Figure 3. Morphometric variables of Opuntia streptacantha flowers.A. Corolla aperture set in FAA.B. Perianth segment length.C. Total flower length.D. Pericarp length.E. Pericarp width.F. Stigma width.G. Stigma length.H. Style length.I. Equatorial diameter of the ovarian chamber.J. Polar diameter of the ovarian chamber.For each morphological character, the unit of measurement was mm.Illustration by Rafael Ríos/CONABIO, ComenTuna et al. (2009), own modification.Scale bar = 5 cm.

Figure 5 .
Figure 5. Linear discriminant analysis using floral morphometrics of three Opuntia species in Cadereyta de Montes, Queretaro, Mexico.

Figure 6 .
Figure 6. Rose diagram representing the months and phenology of floral morphs of Opuntia streptacantha in Cadereyta de Montes, Queretaro, Mexico.A. Flowering for the yellow floral morph.B. Flowering for the orange floral morph.C. Fructification for the yellow floral morph.D. Fructification for the orange floral morph.The blue arrow indicates the accumulation of data for flowering based on the Rayleigh uniformity test.The red arrow indicates the accumulation of data for fruiting based on the Rayleigh uniformity test.

Table 1 .
Mean and standard error (±) of cladode characteristics of both floral morphs of Opuntia streptacantha in Cadereyta de Montes, Querétaro, Mexico, tested with t-tests and generalized linear model with Poisson distribution.Contrasts are marked in bold type and with * (p < 0.05).n = 20 young or old cladodes per floral morph.

Table 2 .
Mean and standard error (±) of floral characteristics of both floral morphs of Opuntia streptacantha in Cadereyta de Montes, Querétaro, Mexico.Contrasts are marked in bold.*: p < 0.05; n.s.non significant.