Comparative Morphology of Disk Floret Trichomes of Encelia (Asteraceae)

Abstract

Disk florets of 19 Encelia taxa were examined with scanning electron microscopy to characterize disk floret trichome complement, density, and distribution, to interpret their evolution in light of phylogeny and ecology, and to determine the utility of these characters for phylogenetic analysis and species delimitation. Trichome types present include multicellular biseriate glands, biseriate achene hairs (Zwillingshaare), and uniseriates. It is notable that the two trichome types present on leaves which have proven useful in phylogenetic hypotheses concerning interspeci- fic relationships in the genus are absent or rare on florets. Exten- sive intra-taxon and intra-population variability in trichome complement precludes the use of these characters in phylogenetic analysis using cladistic parsimony, but examination of trichome density using multivariate statistical techniques agrees with existing species boundaries, hypotheses of hybrid origin of two species, and the existence of two major clades in the genus. Introduction

Encelia consists of 15 species of xerophytic shrubs native to arid and coastal regions of western North and South America. All members are interfertile, obligate outcrossers, and two taxa are believed to be of hybrid origin. Pheno- typic and molecular evidence supports the existence of two clades in the genus, with relationships within the clades weakly supported or unresolved. Objectives

Trichomes have proven useful in supporting species boundaries and phylogenetic hypotheses of both intergeneric and interspeci- fic relationships within Encelia, and other closely related genera. In their study of Enceliopsis capitulum morphology, Sanders and Clark (1987) provided evidence based on trichomes supporting the monophyly of the genus, species boundaries, and the place- ment of Encelia nutans within Encelia. Nishida and Clark (1988) cite trichome evidence supporting the monophyly of Geraea. In Encelia, Clark (1986) demonstrated that leaf trichomes support of the hypothesis of two clades within the genus. The purpose of this research is to characterize disk floret trichome complement, distribution, and density in Encelia, to determine the utility of these characters in supporting existing species boundaries, and in phylogenetic analysis, and to reach conclusions pertaining to the evolution of these characters. Materials and Methods

Disk florets from 19 of the Encelia taxa were selected from preserved capitula collected from plants growing in the wild and in cultivation. Florets were sampled from as many different capitula, individuals, and populations as possible. They were then dehydrated in an ethanol series, critical point dried in a CO2 critical point dryer, and coated with gold particles in a sputter coater at 22 mA for around 500 s. They were then mounted on aluminum stubs with colloidal silver conductive paint and examined and photographed in a scanning electron microscope. Principal components analysis (PCA) and multivariate analysis of variance (MANOVA) were carried out on the data set using the SAS System for Windows, release 6.12. Results

Anther

Corolla lobe Throat

Trichomes occur mainly on the corolla tube, the abaxial side of the corolla lobes, and along the furrows of the abaxial anther tips. The throat is Tube ordinarily glabrous. The commonest trichome types are unicellular-based uniseriate hairs (above) and biseriate hairs with glandular tips (“glands”, right). Glandular hairs vary greatly in their size and the length of their stalks. Glands of the corolla tube are generally longer and thinner than those of the corolla lobes and anthers. Biseriate pointed hairs (Zwil- lingshaare, below left), which cover the ovary, extend onto the tube of some taxa.

Moniliform hairs (right), common on the leaves of some species, are ex- ceedingly rare on the flowers. Anther Corolla Corolla Tube Tube glands glands uniseriates Tube glands uniseriates Zwillingshaare Taxon mean s.d. mean s.d. mean s.d. mean s.d. mean s.d. mean s.d. actoni 7.94 5.48 0.60 1.35 50.25 26.13 18.20 19.35 2.75 3.84 0.00 0.00 asperifolia 1.28 1.79 1.30 1.59 10.37 2.91 17.10 11.51 0.00 0.00 0.03 0.18 californica 0.00 0.00 2.93 3.32 7.50 4.05 39.27 29.94 0.13 0.35 0.00 0.00 canescens 0.33 0.79 1.42 2.61 19.36 19.37 12.58 18.17 1.08 2.16 0.03 0.17 conspersa 1.00 1.58 0.91 1.58 0.18 0.40 3.33 4.23 0.33 0.78 0.00 0.00 densifolia 0.00 0.00 4.38 0.92 5.56 1.94 38.00 5.87 0.00 0.00 0.10 0.32 farinosa 0.75 0.97 0.00 0.00 0.05 0.22 52.00 10.56 0.00 0.00 1.10 1.55 frutescens 2.25 1.70 0.97 1.79 25.87 10.84 8.93 6.72 19.87 19.05 0.00 0.00 glandulosa 0.00 0.00 5.43 1.09 31.79 5.04 6.29 4.84 6.71 5.53 0.14 0.53 halimifolia 3.44 1.24 0.00 0.00 6.90 2.56 20.40 9.66 22.50 6.52 0.00 0.00 palmeri 0.00 0.00 0.00 0.00 3.90 1.52 4.20 3.08 8.80 4.71 0.00 0.00 phenicod. 0.14 0.35 0.00 0.00 0.57 1.22 68.19 15.82 0.00 0.00 2.08 2.41 radians 0.00 0.00 0.00 0.00 0.00 0.00 56.00 8.43 0.00 0.00 0.00 0.00 ravenii 1.40 0.97 4.00 2.05 0.00 0.00 42.70 9.98 0.00 0.00 1.30 1.16 resinifera 1.25 1.48 10.10 3.80 20.70 7.65 14.45 10.39 0.30 0.57 31.50 35.34 stenophylla 6.55 2.73 5.09 2.26 25.45 5.22 12.64 4.92 2.27 1.27 0.00 0.00 tenuifolia 0.13 0.35 2.80 4.69 7.30 2.58 5.42 8.02 0.00 0.00 0.00 0.00 ventorum 0.11 0.33 3.44 2.40 9.33 4.00 19.50 4.97 0.00 0.00 0.80 1.03 virginensis 0.05 0.22 0.05 0.22 46.25 22.53 3.80 3.12 13.40 13.73 0.00 0.00 Trichome counts by organ, type, and taxon. 6 5 5 4 4

3 3

2 2

Factor 2 1 Factor 2 1

0 0

-1 -1 -2 -2 -3 -2 -1 0 1 2 3 4 -3 -2 -1 0 1 2 3 4 Factor 1 Factor 1

The first two PCA factors are plotted above; other factors show different patterns but the clusters are the same. On the left, individual species are color-coded; most form discrete, but overlapping, clusters. Coloring on the right shows the two major clades. Again, there is overlap, but the results of MANOVA analysis (Wilks’ Lambda = 0.436; F = 46.633; a < 0.0001) show that the clades are distinct. The two species of putative homoploid hybrid origin each cluster roughly between the parent species.

6 3

E. actoni 4 2 E. californica E. frutescens 2 1 ssp. glandulosa

Factor 3 0 E. virginensis Factor 2 0

-2 -1 E. asperifolia E. frutescens ssp. frutescens -4 -2 -3 -2 -1 0 1 -2 -1 0 1 2 Factor 2 Factor 1 Discussion

Comparisons with Enceliopsis, Geraea and cauline and foliar trichomes in Encelia—The trichome complement of disk florets is restricted in comparison with other organs in the genus. Three types of trichomes found on leaves, pe- duncles, phyllaries, and paleae are absent or rare on the disk florets. These include moniliform trichomes, broad multicellular-based uniseriate trichomes, and curly unicellular- based uniseriate trichomes. It is notable that the two types of trichomes which have proven useful phylogenetically in previous studies (Clark,1986)—the moniliform trichomes and the broad-based uniseriate trichomes—are among those absent. Both of these trichome characters support the hypothesis of two major clades in the genus. Unlike Encelia, the outgroup genera Enceliopsis and Geraea have only uniseriates and biseriate glands on both disk florets and other organs. Phylogenetic utility of disk floret trichomes—Even a cur- sory examination of the trichome data reveals extensive intra-taxon and intra-population variability in trichome complement (i.e., the presence or absence of a given trichome type on a given organ). Eight of nine taxa in which multiple populations were examined displayed at least one difference (out of the possible six trichome characters above) among their populations. Five of these taxa displayed two or more such differences among their populations. Variability in complement is also common within populations. Of the to- tal 34 populations examined, 28 display at least one difference in complement among their individuals, and 21 display two or more. Because of this variability, trichome complement of disk florets is a phylogenetically uninformative character if used in a parsimony analysis. Species boundaries and delimitation—In light of this, it is evident that trichome complement alone would be of little use in species delimitation. Encelia farinosa has three different varieties (E. farinosa var. farinosa, var. phenicodonta and var. radians). Var. radians differs in trichome complement from the other two by three characters. But PCA on counts of the different trichome types shows the three varieties clustering tightly together, which is concordant with other data supporting the contention that they are conspeci- fic. In the principal components analysis (above), most of the observations form relatively small clusters representing their respective species, concordant with existing species boundaries. Evolutionary implications—This analysis provides addi- tional support for the hybrid origin of E. asperifolia and E. virginensis: both cluster between their respective putative parents (above). The two major clades within Encelia cluster differently in PCA (above). MANOVA supports the distinctness of the two clades. Conclusions

§ The two phylogenetically useful trichome types found on leaves of Encelia are absent or rare on the florets. § Extensive intra-taxon and intra-population variability in trichome complement makes these characters unfit for use in phylogenetic (cladistic parsimony) analysis, and species delimitation. § Trichome counts, when analyzed with multivariate statistical techniques (i.e. PCA and MANOVA), prove concordant with: § Existing species boundaries § The hypothesis of hybrid origin for two of the species § The existence of two major clades in Encelia. Literature Cited

Charest, N. A. 1988. A scanning electron microscopic study of peduncle, phyllary, and paleae trichomes of Encelia (Asteraceae, Heliantheae). M.S. Thesis, California State Polytechnic University, Pomona, vii + 83p. Clark, C. 1998. Phylogeny and adaptation in the Encelia alliance (Asteraceae, Heliantheae). Aliso 17(2): 89-98. Nishida, J., and C. Clark. 1988. Scanning electron microscopic study of trichomes of Geraea (Asteraceae, He- liantheae). Amer. J. Bot. 75(6), Part 2, pp. 196-197. Sanders, D. L., and C. Clark. 1987. Comparative morphology of the capitulum of Enceliopsis (Asteraceae: He- liantheae). Amer. J. Bot. 74(7): 1072-1086.