﻿Pollen morphology of the genera Hidalgoa and Dahlia (Coreopsideae, Asteraceae): implications for taxonomy

﻿Abstract Hidalgoa and Dahlia are two closely related genera in Asteraceae, tribe Coreopsideae whose limits need to be clarified. Pollen morphology has been useful for delimitation at the genus level in this family. To better define these genera, the morphology of pollen grains was observed and measured using light and scanning electron microscopy. The pollen grains of 25 species of Dahlia and Hidalgoa were acetolyzed and analyzed. Pollen is tricorporate in most of the species studied, although in a few species in Dahlia, grains were found to be hexacolporate. The most outstanding differentiating characters among species of Dahlia and Hidalgoa are colpus length (greater in Hidalgoa) and shape of spines (conical in Hidalgoa). In addition, lalongate ora are larger in Hidalgoa than in Dahlia. A PCA analysis of thirteen pollen characters, identified species of Hidalgoa in a discrete group and Dahliacuspidata as an outlier. These distinctive attributes in pollen morphology support the idea that pollen morphology is useful for delimitation at the generic level in the Dahlia clade. Further evidence from other sources, genetic or anatomical, might contribute to demarcating Dahlia and Hidalgoa, and provide insight into the family’s evolutionary history.


Introduction
of differentiating characters in the taxa of this family are the polar diameter and dimensions of the colpus and endoaperture in Viguiera Kunth (Magenta et al. 2010) and in Xanthium L., the length and number of spines and the number of columellae (Coutinho et al. 2020). Sexine thickness, the type of aperture, and spine dimensions are the differentiating traits for species of Stilpnopappus and Strophopappus (Carrijo et al. 2013), as is pollen surface ornamentation in the complex Phaeostigma of the genus Ajania (Huang et al. 2017). Pollen type and pollen grain shape are taxonomically useful for distinguishing species and genera of the subtribe Lepidaploinae (Marques et al. 2021).
Pollen morphology has not been analyzed in detail for the species of Hidalgoa or Dahlia. Previous palynological research by Wodehouse (1929) on four Dahlia species identified the presence of six apertures (hexacolporate) in pollen grains. This attribute constitutes a notable exception within the family. Further studies examined and de- scribed the meiotic stages of the pollen mother cell in Dahlia to determine the development of these apertures (Wodehouse 1930).
The aims of this study are to compile and compare pollen morphology of the species of Hidalgoa and Dahlia to identify informative characters and understand the relationships and limits of these taxa.

Materials and methods
Twenty-five samples of pollen grains were obtained from herbarium specimens deposited in the IBUG (Instituto de Botánica de la Universidad de Guadalajara) and XAL (Instituto de Ecología, A. C.) herbaria. Vouchers of specimens are included in Table 1.
Pollen grains were acetolyzed according to the methodology of Erdtman (1960), and for difficult material in which compounds formed thin coats on the grains that interfered during the scanning process, the suggestions of Fonnegra (1989) were implemented. The grains were immersed in glacial acetic acid for 24 hours before acetolysis and then transferred to the acetolysis mixture for 1 to 6 hours and the temperature of The following pollen measurements were obtained from 25 grains per sample: polar axis, equatorial diameter, exine thickness, colpus length, colpus width, ora width, ora length, spinae length, spine width at base and number of apertures.
To observe the pollen with a scanning electron microscope (SEM), acetolyzed pollen grains were washed in ethanol and later in water. Grains were sputter-coated with gold and observed using a Carl Zeiss EVO-50 scanning electron microscope. The terminology of Halbritter et al. (2018) was used, and for pollen structure the terminology of Erdtman (1969) was followed. Number of spines/100 μm 2 , colpus end, base of spine and pollen surface ornamentation were described for five grains per sample. Final morphological data are presented in Table 2.
A matrix based on thirteen pollen characters was constructed. To estimate the quantitative variation within Dahlia and Hidalgoa, each character was measured and the average for each species was estimated. Qualitative characters were coded with  (Table 3). Graphical representation displayed distribution of thirteen pollen characters.
Results of the PCA indicate that the first two components explain 46.04% of the observed variation (see Table 3 to for the contribution of each variable to Dim 1 and Dim 2). A bidimensional projection of the axes of the two first components is displayed in Fig. 5. The first principal component explains 25.19% of the variation and is associated with colpus length (Cl) and os width (Ow). The second principal component explains Table 3. Palynological characters used in the multivariate analysis of Hidalgoa and Dahlia species. The contribution of every character for Axis 1 and Axis 2 is indicated (see Fig. 4).

Character
Axis 1

Discussion
The pollen grains of the 25 species of Dahlia and Hidalgoa we studied share the pollen type common to tribe Coreopsideae: more or less spheroidal, round in both views, tricolporate, ora lalongate, tectum microperforate, echinate, spines irregularly distributed, conical to long-pointed and smooth or distended bases with perforations (Blackmore et al 2009). The pollen of the species studied is quite homogeneous, with little variation in size and shape. Pollen grains size ranges from 25.3 to 39.4 μm in Dahlia and 25.7 to 34 μm in Hidalgoa, and the ratio of polar axis and equatorial diameter is 0.90-1.04 (Dahlia: 0.90-1.04, Hidalgoa: 1.02-1.04). The largest grains were observed in D. neglecta and the smallest in D. australis and H. pentamera. Based on the classification proposed by Erdtman (1969), the pollen of both genera corresponds to mediumsized grains (25-50 μm), like those described by Tellería (2017) for tribe Coreopsideae. Pollen grains in Dahlia and Hidalgoa are radially symmetrical, isopolar, and mostly spheroidal, similar to those described in Coreopsis (Tadesse et al. 1995).
Apertures are the most variable attribute between Dahlia and Hidalgoa, but not within the genera. Pollen in Hidalgoa is always tricolporate, while in pollen grains of Dahlia, the aperture varies from tricolporate to hexacolporate. Hexacolporate species observed here were: D. cuspidata, D. dissecta, D. imperialis, D. merckii, D. parvibracteata, D. rudis and D. sorensenii. Wodehouse (1930) recognized the same pattern in D. brevis, D. coccinea, D. pinnata and D. imperialis. However, Wodehouse (1930) described these six apertures as uniform in all pollen grains; the observations were made on species belonging to the San Francisco Dahlia Society, plants that are probably of hybrid origin. The specimens collected from the herbariums for this study do not display characters of hybrid origin. Furthermore, hexacolporate grains have been indeed reported in Old World Vernonieae and Adenanthemum (Blackmore et al 2009). Other members of Coreopsideae such as Bidens also vary in the number of colpi from 3-4 (Tadesse et al. 1995) or polypentoporate (Younis et al. 2020).
The os and colpus displayed more variation in Dahlia than in Hidalgoa. In Hidalgoa the os is lalongate, the widest is up to 13 μm width, and the longest colpus is approximately 17 μm, with apices always acute. These traits of Hidalgoa are similar to those observed in Bidens (Tadesse et al. 1995). In Dahlia the ora are either lalongate or lolongate. The os is slightly wider than larger and apices are obtuse, rarely acute. The  widest and largest colpus was observed in D. cuspidata (5.89 μm and 9.2 μm respectively) and D. scapigera (ca. 7 μm), and the smallest in D. australis (2.9 μm). Variation in the apertures like those of Dahlia has not been reported in other genera of the tribe Coreopsideae (Blackmore et al. 2009).
Spines are variable between Dahlia and Hidalgoa. In Hidalgoa they are conical and smaller (4.08 to 6.77 μm) while in Dahlia they are deltate and larger (4.29 to 12.26 μm), with exception of D. linearis (smaller). Spines in Dahlia varied more in shape and size. They are commonly triangular or deltate with a broadened base (distended base), as described by Tellería (2017) for tribe Coreopsideae. Sometimes spines emerge abruptly from the exine surface, e.g. D. campanulata, D. imperialis, D. parvibracteata, D. scapigera. These spines are similar to those described in Coreopsis (Tadesse et al. 1995). The transition between the microperforate basal portion of the spine and the unperforated apical portion is abrupt in almost all species, except in D. cordifolia, D. linearis and D. sorensenii. Exine thickness did not vary among Hidalgoa and Dahlia species. The thinnest was observed in D. cuspidata and D. sorensenii (1.6 μm) and the thickest in D. neglecta (4.2 μm).
Multivariate analyses did not reveal a clear clustering among species of Dahlia according to the sections proposed by Sørensen (1969), based mainly on life form and in the phylogeny of Saar et al. (2003). Nevertheless, D. cuspidata and the Hidalgoa species are significantly different from the other Dahlia species included in this study. Hidalgoa species have long colpi and wide ora, and D. cuspidata has long ora. Dahlia cuspidata possesses unusual morphological characters such as large involucral outer bracts and cuspidate leaf shape. Thus, further research might decide the position of this species.
The most recent phylogeny that included Dahlia and Hidalgoa (Sánchez-Chávez et al. 2019) identified Hidalgoa within the Dahlia clade. However, both genera are morphologically complex (Sørensen 1969;Turner 2010), and contrasting characters such as life form, number and arrangement of fertile and sterile flowers have been used to separate these two groups. A further phylogeny, including all species, may help us to better understand pollen evolution in the genus.

Conclusions
The palynological descriptions for Hidalgoa presented here are the first, and despite the similarities in its floral morphology to that of Dahlia, its pollen is remarkably different, mostly in colpus length and shape of their spines. Hidalgoa has pollen grains with large colpi and small, conical spines. In addition, the length of the lalongate ora differ. Hexacolporate grains with a distended base, were found in a number of Dahlia species but have not been identified in Hidalgoa. Likewise, morphological characters such as pistillate fertile ray florets, cypselae with two apical lateral cusps and twisting petioles in Hidalgoa contrast with the ray florets, which can be fertile, pistillate or sterile, cypselae with pappus absent or present with 2(5) small teeth or of two weak filiform, caducous bristles of Dahlia; characters that have been utilized to tell these two genera apart. The results obtained in this palynological study support the idea that pollen morphology is useful for delimitation at the generic level in the Dahlia clade. These differentiating attributes in pollen morphology in the species of Dahlia and Hidalgoa indicate that they should be recognized as separate genera. However, as indicated above, additional anatomical and molecular characters are needed to make the taxonomic decision and help us understand evolution in the genera, and their relationship to other genera in Coreopsideae.