Systematic relevance of pollen morphology in tribe Hylocereeae (Cactaceae)

Abstract Hylocereeae is one of the nine tribes in the subfamily Cactoideae (Cactaceae), for which the limits and recognition of genera have been controversial. Essentially, this group comprises epiphytic and hemiepiphytic genera with stems modified as climbing structures. The aim of this paper is to examine pollen attributes in representative species of genera of Hylocereeae, focusing on Selenicereus whose current circumscription comprises Hylocereus and three Weberocereus species, to find whether significant potentially apomorphic and/or autapomorphic systematic characters can be discovered. Utilizing SEM and light microscopy, 25 pollen characters were observed and measured. Tribe Hylocereeae is stenopalynous, with pollen grains isopolar and radially symmetrical monads, mostly tricolpate, except in Kimnachia, Pseudoripsalis and Weberocereus, whose pollen grains are pantocolpate. Seven attributes (five qualitative and two continuous) exhibited useful variation and were coded. The character of brevicolpate pollen grains was shared by Kimnachia ramulosa and Pseudorhipsalis amazonica. Convex quadrangular outline in the polar view was shared by Weberocereus tunilla and S. glaber. The absence of spinules on the exine was shared by S. minutiflorus and S. stenopterus. The largest pollen grain, found in Selenicereus megalanthus, might be correlated with polyploidy. Selenicereus is the taxon with the highest variation in pollen attributes, including species with an exine with or without spinules and variable polar area index and shape (subprolate or oblate-spheroidal).


Introduction
Hylocereeae is one of the nine tribes in subfamily Cactoideae (Cactaceae), in which the limits and recognition of genera have been controversial (Britton and Rose 1920;Buxbaum 1958; Barthlott and Hunt 1993;Bauer 2003;Korotkova et al 2017). With the exception of Acanthocereus, this group comprises epiphytic and hemiepiphytic genera with approximately 82 species with stems modified as climbing structures (Barthlott and Hunt 1993). It includes ornamentals in genera such as Selenicereus, Epiphyllum and Disocactus, whose flowers are night-blooming and known as queen of the night. It also includes Hylocereus with several species producing edible fruits known as dragon fruit, pitaya or pitahaya, which are cultivated in tropical areas around the world (Anderson 2001). Species in the tribe share characters such as angled stems and branches, with ribs, furrows or rarely smooth, aerial roots and usually spiny or hairy ovary areoles ( Fig. 1) (Bauer 2003;Hunt et al. 2006).
The aim of this paper is to examine pollen attributes in representative species of genera of tribe Hylocereeae focusing on the current concept of Selenicereus that includes Hylocereus and three species of Weberocereus to find whether potentially apomorphic and/or autapomorphic character states can be discovered. Traditionally, pollen has provided valuable and significant characters in plant taxonomy (Larson and Skvarla 1962;Nowicke and Skvarla 1979;Ferguson 1985), and in particular for Cactaceae, pollen characters continue to be useful taxonomically (e.g. Anderson and Skillman 1984;Rose and Barthlott 1994;Halbritter et al. 1997;Aguilar-García et al. 2012;Gonzaga et al. 2019;Mouga et al. 2019). Furthermore, pollen attributes have been used as a tool to clarify the taxonomy of diverse and complex angiosperm groups such as Poaceae (Dórea et al. 2017), and of difficult genera such as Rosa or Psidium (Wrońska-Pilarek and Jagodziński 2011; Tuler et al. 2017). Likewise, pollen morphology has been useful in systematic determinations at generic level in large families such as Asteraceae (Zhao et al. 2000), Liliaceae (Du et al. 2014), Ericaceae (Wrońska-Pilarek et al. 2018), and Bignoniaceae (Burelo-Ramos et al. 2009). Leuenberger (1976) compiled the most complete description of pollen morphology of 600 cactus species, and found that aperture ratio was one of the most variable and useful characters at different taxonomic levels, from subfamily to genus. In addition, Kurtz (1963) in his study of pollen in Cactaceae -which included several genera in Hylocereeae -identified relevant variation in pollen size in Hylocereus and Selenicereus, differences in the number of furrows in a number of genera such as Weberocereus, and useful variation in pollen sculpture in the length of spinules and perforation of the exine in several genera. Likewise, the identification of species in Cactaceae using pollen in countries such as Brazil, Peru and Argentina found useful characters such as variation in size, shape, and exine thickness to determine taxa at tribe and genus levels (Santos and Watanabe 1996;Santos et al. 1997;Garralla and Cuadrado 2007;Cuadrado and Garralla 2009;Lattar and Cuadrado 2010;De la Cruz et al. 2013;Miesen et al. 2015;Cancelli et al. 2017).
The study of the pollen of tribe Hylocereeae is part of our current project on the evolution and systematics of the Hylocereus clade. We aim to incorporate pollen characters with morphological and molecular data to better understand the limits and phylogenetic position of this group, along with phylogenetic relationships of its species, the evolution of chemical and fruit characters. In this paper, the pollen attributes of 27 representative taxa of the genera comprising tribe Hylocereeae, with a main focus in the current concept of Selenicereus (including Hylocereus and three Weberocereus species), are examined to determine whether character states are shared or are exclusive in these taxa.

Pollen preparation
The acetolysis method proposed by Erdtman (1960) was used for processing the pollen grains for observation. For difficult material such as collapsed grains or delicate pollen, the suggestions of Fonnegra (1989) were implemented. Pollen grains were mounted in jelly and sealed. For observing pollen with scanning electron microscope (SEM), the material was dried at critical point and sputter coated in palladium gold (Boyde and Wood 1969). SEM observations were made and electromicrographs taken with a Jeol JSM-5600LV scanning electron microscope.

Morphometric pollen characters
Eleven morphological continuous pollen characters of the studied species were measured, including 1) equatorial diameter in polar view, 2) apocolpium (area delimited by lines connecting the apices of the colpi at the pole of the pollen grain), 3) mesocolpium (area delimited by lines between the apices of adjacent colpi), 4) polar axis in equatorial view, and 5) equatorial diameter in equatorial view. They were measured for a maximum of 25 pollen grains from at least three preparations of a single specimen for each species, with 800× optical zoom under a Carl Zeiss Fomi III Optical Microscope, equipped with a Cannon Power Shot G9 digital camera. Additionally, under 1250× optical zoom, 10 pollen grains were observed to measure 6) exine thickness (Fig. 2). For further analysis, SEM electromicrographs on 10 pollen grains for each species with 6000× magnification SEM analysis was performed on acetolyzed and non-acetolyzed pollen material. With SEM, the following exine characters were measured: 7) spinule length, 8) spinule base, and 9) perforation diameter. In addition, the following ratios were estimated: 10) PAI (Polar Area Index) PAI= Apocolpium/ Equatorial diameter in polar view and 11) PE (Shape class) PE=Polar axis/Equatorial diameter in equatorial view. Character measurements from optical microscopy were obtained with the software Axio Vision ver. 4.7.2, and characters from SEM were acquired using ImageJ 1.45 software (Abramoff et al. 2004) (Fig. 2). To identify character states, every continuous character was coded following Almeida and Bisby (1984), ordered in boxplot diagrams by median values, and examined for dips or gaps. Gaps based on the first and third quartiles are codified as discontinuities and the corresponding character states are assigned.

Exploratory multivariate analyses
A principal component analysis (PCA) was performed using the packages Factoextra and FactoMine in R (R Development Core Team 2018) to reduce the dimensionality of phenotypic variation and summarize the variables that are correlated. PCA was carried out to identify the characters that explained the greatest proportion of the variability and to identify pollen grains occupying different spaces.

Results
The taxa studied in tribe Hylocereeae are stenopalynous, i.e. there is slight variation in pollen grains. They are isopolar and radially symmetrical monads, mostly tricolpate, with the exception of Kimnachia, Pseudoripsalis and Weberocereus, whose pollen grains are pantocolpate, with 12 to 15 colpi.
In the following paragraphs detailed descriptions of the pollen grains are provided.

Weberocereus Britton & Rose
Pollen. pantocolpate, radially symmetrical, isopolar with convex-cuadrangular contour in polar view. Shape: prolate-spheroidal (P/E=1.07 Qualitatitative pollen characters. Of the fourteen characters examined, five were identified as variable: Amb (the outline of a pollen grain seen in polar view), colpi number, aperture type (colpate or brevicolpate pollen), marginate exine (an area of the exine around an ectocolpous that is differentiated from the remainder of the exine by difference in thickness and the presence of spinules). The other qualitative characters were not variable (Table 1, Suppl. material 1: Table S1). Pollen grains of the studied Table 1. Qualitative and quantitative morphological characters of pollen for the representative species in the tribe Hylocereeae. Spinule length and perforation diameter are coded based on the simple gap method by Almeida and Bisby (1984). PE ratio =Polar axis/Equatorial Diameter; PAI ratio =Apocolpium/Equatorial diameter in polar view (Polar Area Index); Amb: Outline in polar view. Spinule length (0 = 0 μm, 1 = 0.33-0.78 μm, 2 = 1.27-1.86 μm); Perforation diameter (0 = 0.11-0.18 μm, 1 = 0.23-0.72 μm). P=present, A=Absent.  (Figs 3 and 4). Weberocereus, Pseudorhipsalis, and Kimnachia differ from the rest of the tribe by having pollen grains with a small aperture, brevicolpate, with differences in the number of colpi as well. Aporocactus, Acanthocereus, Disocactus, Epiphyllum, and Selenicereus (including Hylocereus) have tricolpate pollen. In addition, two autoapomorphic characters were identified: convex-quadrangular outline in Weberocereus, marginate exine in Pseudorhipsalis (Figs 3, 4). Continuous pollen characters. The size of pollen grains for the 27 taxa examined varies from 55.47 to 154.42 μm in polar axis, and this is large according to Erdtman (1952). Boxplot diagrams ordered by median values found gaps for spinule length and perforation diameter based on the first and third quartiles (Fig. 5). These were coded based on the simple gap method by Almeida and Bisby (1984). Spinule length differentiates Kimnachia from Pseudorhipsalis. For Selenicereus (Hylocereus clade), spinule length was found to be polymorphic because it includes representatives lacking spinules and representatives with spinules of larger dimensions than those of Kimnachia and Pseudorhipsalis (Table 1).

Species
Multivariate analyses. The PCA graph displays projections of pollen characters in a multidimensional space in which the first two components explained 62.4% of the observed variance. PC 1 explains 44% of the variance and is associated with size (equatorial diameter, polar axis, and spinule dimensions), while PC 2, which explains 18.4% of the variance, is associated with proportions (PAI and PE ratios) (Fig. 6A). The length of the arrows in Figure 6A indicates adequate sampling for all characters, except for exine length (EL) and perforation diameter (PD). The size of pollen grains in the polar and in equatorial views had a positive correlation and, similarly, there is  a negative correlation between the PE and PAI ratios, as expected in spherical forms. There is a negative correlation between exine length (EL) and the other measures. Figure 5B displays the species studied along the principal components in relation to pollen characters. The association of species is defined by the size of the pollen grain and its shape from prolate to oblate spheroidal). A core association of species is formed by representative species from all genera, including pollen mainly subprolate to prolatespheroidal. The only species with no representatives in the core group and that appear as outliers are Pseudorhipsalis amazonica and Kimnachia ramulosa, with prolate to oblate pantocolpate pollen. Remarkably, Selenicereus (Hylocereus) megalanthus, along with S. setaceus are two of the species with prolate-spheroidal pollen grains with the largest polar axis; S. minutiflorus and K. ramulosa have the smallest pollen grains (smallest polar axis). Selenicereus minutiflorus is the representative of Selenicereus (Hylocereus) with the unique characters of suboblate pollen and an exine lacking spinules (Fig. 3).

Discussion
Pollen grains of the representative species of the genera of tribe Hylocereeae studied here share the pollen type common to Caryophyllales: tricolpate to pantocolpate with the exine spinulose and perforate (Nowicke and Skvarla 1979). Furthermore, in particular for Cactaceae, tricolpate pollen has been described in all tribes of subfamilies Pereskioideae and Cactoideae (Lehuenberger 1976;Kurtz 1948Kurtz , 1963 and is the most common pollen type in eudicots (Erdtman 1952;Furness and Rudall 2004).
From the 25 pollen characters analyzed, only seven attributes (five qualitative and two continuous) exhibited useful taxonomic variation. Four genera in Hylocereeae: Epiphyllum, Acanthocereus, Disocactus, Selenicereus (comprising Hylocereus and three species of Weberocereus), and Aporocactus have pollen grains with essentially similar morphology. That being said, Disocactus and Epiphyllum form part of the Phyllocactoid clade while Selenicereus and Weberocereus form part of the Hylocereoid clade in the molecular phylogeny constructed by Korotkova et al. (2017).
Despite the fact that the majority of taxa studied here share many pollen attributes, certain characters were common to limited groups of species. By way of example, Kimnachia ramulosa and Pseudorhipsalis amazonica are the only two species included in our study that share the attributes of brevicolpate pollen grains with small apertures. Kimnachia is a recently described genus whose sole species was previously included in Pseudorhipsalis (Korotkova et al. 2017). In addition, Kimnachia ramulosa and Pseudorhipsalis amazonica also share the character of pollen grains with 12-15 colpi with Weberocereus. Furthermore, Selenicereus glaber and Weberocereus tunilla share the character of convex quadrangular contour in polar view (Amb); S. glaber was previously included in Weberocereus (Barthlott and Hunt 1993).
Two species in Selenicereus (S. minutiflorus and S. stenopterus) stand out for lacking spinules in the exine in tribe Hylocereeae. They were retrieved in the Hylocereus clade in the plastid phylogeny of Korotkova et al. (2017) and transferred with all Hylocereus spp. to Selenicereus. They have remarkable morphology with miniature plants bearing pinkish flowers in contrast to the rest of the species in the current concept of Selenicereus whose flowers are white. Bauer (2003) transferred these two species from Selenicereus to Hylocereus and classified them in the Salmdyckia group of Hylocereus. Previously, Britton and Rose (1920) included these taxa (S. minutiflorus and S. stenopterus) in Mediocactus, a genus with intermediate morphological characteristics between Selenicereus and Hylocereus, with spines on the pericarpel.
The Salmdyckia group, including S. ocamponis, S. setaceus and S. megalanthus, possesses the largest pollen grains in the genus. Of these three species, Selenicereus megalanthus had the largest pollen grains, with a pollen grain size that could be correlated with polyploidy, a process that can produce large to very large pollen grains (Muller 1979). Chromosome counts for this species indicate that it is tetraploid (Tel-Zur et al. 2004;Tel-Zur et al. 2011).
Furthermore, multivariate analyses corroborated the results of discrete and qualitative characters, displaying species such as Selenicereus megalanthus, S. stenopterus, S. multiflorus and Kimnachia ramulosa as outliers in the multidimensional space. Variation in these analyses was found to be mainly associated with size (equatorial diameter, polar axis, and spinule dimensions).
Of the genera in Hylocereeae, Selenicereus in its current concept including the species previously considered in Hylocereus and three species formerly classified in Weberocereus, is the taxon with the greatest variation in pollen grains. For instance, it includes species with and without spinules in the exine, variable shape (subprolate to oblate-spheroidal), and polar area index is either small, medium or large. Moreover, the generic limits of Hylocereus and Selenicereus have changed over time Cruz et al. 2016;Korotkova et al. 2017). Pollen size, the absence of spinules and the morphological characters in species such as S. minutiflorus and S. stenopterus suggest they might belong to a genus other than Hylocereus or Selenicereus.
Pollen research that concentrates on finding crucial taxonomical characters in Cactaceae has been scarce. Nevertheless, current studies in other plant groups have demonstrated their utility and that of other data sources (e.g. Kriebel et al. 2017;Niu et al. 2018;Siniscalchi et al. 2017;Wrońska-Pilarek et al. 2018). The most complete study on the palynology of the entire Cactaceae by Leuenberger (1976b) was published in a series of dissertations and is difficult to acquire. Probably the delicate pollen in Cactaceae which is difficult to process is one of the causes of the limited number of studies on pollen.
The pollen attributes identified here and that are shared by a number of species belonging to different genera that have recently been segregated or grouped together, suggests that additional evidence should be gathered and new phylogenetic analyses performed to clarify boundaries. Circumscription of the genera in tribe Hylocereeae has only been carried out based on a set of molecular or morphological characters. Our project on the Hylocereus clade will include the palynological characters determined here, along with other sources of attributes such as their morphological, ecological and molecular traits.