A COMPARATIVE STUDY OF SEED CHARACTERISTICS IN THE CHENOPODIACEAE AND AMARANTHACEAE

Proceedings of the South Dakota Academy of Science, Vol. 83 (2004) 73 A COMPARATIVE STUDY OF SEED CHARACTERISTICS IN THE CHENOPODIACEAE AND AMARANTHACEAE Elke Kuegle and Mark Gabel Biology Department Black Hills State University Spearfish, SD 57799 ABSTRACT The Amaranthaceae and Chenopodiaceae are herbs and shrubs primarily known for their weedy nature. Both families belong to the order Caryophyllales which has characteristic seed shape and embryo position. Numerous characteristics have previously been used to compare the families with differing results. Until this time, no studies have conducted a comprehensive examination of the seed characteristics of the two families. Twenty-five characters were examined in 26 species traditionally placed in the Chenopodiaceae and in 34 species of the traditional Amaranthaceae. Our results indicate that seed characters do not separate the traditional taxonomic division, and thus support recent studies combining the two families. Keywords Amaranthaceae, Chenopodiaceae, seed, cluster analysis, principal component analysis INTRODUCTION The Caryophyllales as described by Cronquist (1981) and Cronquist and Thorne (1994) are nearly synonymous with the Centrospermae of previous authors (Lawrence, 1951; Rodman, 1990). Members of the core Caryophyllales are the Amaranthaceae and Chenopodiaceae which are thought to be closely related, but are traditionally separated. In a study using both phenetic and cladistic analyses of morphological, anatomical, palynological, chemical, and chromosomal data, Rodman et al. (1984) included the Chenopodiaceae and Amaranthaceae in the cohort Amaranthares in the suborder Chenopodiineae. Later (1990), Rodman presented revised and recoded data, but no visual analyses and indicated that the Amaranthaceae are nested within the Chenopodiaceae. A study of trichomes by Carolin (1983) indicated that the Amaranthaceae were contained within the Chenopodiaceae. Rodman (1994) refuted this arrangement indicating that the Amaranthaceae would have had to reduce ovule number to a single basal ovule and then reverse

74 Proceedings of the South Dakota Academy of Science, Vol. 83 (2004) this change to account for the multiovulate gynoecia of Celosia. A study of the Caryophyllales using chloroplast DNA, by Downie and Palmer (1994a) found one major clade of the Caryophyllales (based upon only five taxa) to be composed of the Chenopodiaceae and Amaranthaceae, and the other clade composed of all remaining families. They (1994b) stated that both the Chenopodiaceae and Amaranthaceae appear to be monophyletic. In 1997, Downie et al. conducted a study of chloroplast DNA (OFR2280), and suggested that the Amaranthaceae are polyphyletic with Celosia and Froelichia forming one clade and the Chenopodiaceae and Amaranthus another. Sarcobatus (traditionally Chenopodiaceae) grouped with a clade that included the Phytolacaceae and Nyctaginaceae. Behnke (1997), describing sieve elements and Cuénoud et al. (2002) using nuclear and plastid DNA have shown that Sarcobataceae should be elevated to a family level. The latter authors also indicated a clade of the Chenopodiaceae and a separate well-supported clade composed of genera from the Amaranthaceae. The Chenopodiaceae have traditionally been described as a family of about 100 genera and about 1500 species most commonly found in drier and alkaline or saline habitats (Kuhn et al., 1993; Flora of North America Editorial Committee, 2003). The Amaranthaceae have been delimited as containing about 65 genera and 900 species and are most common in warmer climates (Townsend, 1993; Flora of North America Editorial Committee, 2003). Both families contain numerous herbaceous species and a few shrubs or sub-shrubs and are primarily known for their weedy nature. Historically, the two groups have been considered as separate but related families (Cronquist 1981; Flora of North America Editorial Committee 2003). The Angiosperm Phylogeny Group (1998) included the Chenopodiaceae in the Amaranthaceae. Judd and Ferguson (1999) maintained the separation of the two families, but wrote the separation of the Chenopodiaceae and Amaranthaceae is more or less arbitrary and very probably results in a paraphyletic Chenopodiaceae As exemplified above, numerous characteristics have been used to compare the families with differing results. No studies have ever done a comprehensive examination of the seed characteristics of the two families. It is the purpose of this study to compare the seed characteristics of the traditionally defined Amaranthaceae and Chenopodiaceae to determine if they can help elucidate the relationship among the families, and if they support or refute combining the two families. MATERIALS AND METHODS Seeds from 34 species of Amaranthaceae and 26 species of Chenopodiaceae were freed from the fruit wall and cleaned. A cursory examination of seed coat morphology was conducted using a dissecting microscope. The waxy layer obscuring seed coat cell details of some species, especially Amaranthus, was removed by submerging the seed in a solution of either 50% EtOH or 10% papain for a few minutes. Cross sections were prepared to view the internal anatomy of the

Proceedings of the South Dakota Academy of Science, Vol. 83 (2004) 75 seeds by cryofracture or by microtome sectioning after embedding the seeds in paraffin or L.R. White resin. Seeds were mounted on a carbon stub for detailed examination with the scanning electron microscope (SEM). Images obtained by the SEM and dissecting microscope observations were then used to establish a matrix, with 25 distinct characters. To measure the phenetic relationships of the two families, a cluster analysis using a similarity matrix of the seed characters was used to construct a dendrogram or phenogram. The same data was used to perform principal component analysis (PCA) by calculating correlation coefficients among the seed characters. Both analyses used NTSYS 2.11S (Rohlf, 2003). RESULTS Results of the measurement of 25 seed characters of 60 species (65 specimens) are shown in Table 1. These data were then used in cluster analyses and principal component analyses. The cluster analysis (Fig. 1) indicates that seed characters do not provide a clear distinction between the Amaranthaceae and Chenopodicaceae. Three major groups were delimited, one with primarily Amaranthus (traditional Amarathaceae), Iresine (Amaranthaceae), and two species of Suadea (Chenopodiaceae). Figure 1. Cluster analysis (resulting in a dendrogram or phenogram) of 25 characters from 65 seed samples of the traditional Amaranthaceae and Chenopodiaceae. The figure was derived from a standardized similarity matrix by computing and clustering distance coefficients. The first letter of each label refers to traditional placement (A = Amaranthaceae, C = Chenopodiaceae), while the second and third letters refer to the genus. Numbers refer to sample (Table 1). Axis aspect has not been preserved to allow easier viewing.

76 Proceedings of the South Dakota Academy of Science, Vol. 83 (2004) The second group was composed mostly of Atriplex (Chenopodiaceae), with Gomphrena (Amaranthaceae), one Celosia (Amaranthaceae), two Amaranthus species and another Suadea (Chenopodiaceae). The third group included most of the Chenopodium (Chenopodicaceae), as well as Kochia (Chenopodiaceae), Celosia (Amaranthaceae) and several species of less well-represented genera. The PCA very broadly separated the two families (Fig. 2), but there were numerous genera which were traditionally members of one family which were placed within a group of the other family. Examples of the misplaced genera include Suaeda (100) (Chenopodiaceae) Chenopodium 83 and Gomphrena (Amaranthaceae). The PCA supported the placement of the genera in the cluster analysis (Fig. 1). While the details of the PCA presented in Fig. 2 appear rather obscured by overlapping labels, it should be noted that the NTSYS software (Rohlf, 2003) allows the user to freely move the model in apparent three dimensional space, thus allowing a visualization of each datum point. While the analyses of the seed characteristics broadly separate the two families, there are numerous outlying species that do not group as expected. Figure 2. Principal Component Analysis of 25 characters from 67 seed samples of the traditional Amaranthaceae and Chenopodiaceae. The figure was derived from a standardized correlation matrix and presents orthogonal coordinate axes such that points have variance in as few dimensions as possible. The first letter of each label refers to traditional placement (A = Amaranthaceae, C = Chenopodiaceae), while the second and third letters refer to the genus. Numbers refer to sample (Table 1).

Proceedings of the South Dakota Academy of Science, Vol. 83 (2004) 77 DISCUSSION It is obvious from Figs. 1 and 2 that seed characters do not clearly separate the Amaranthaceae and Chenopodiaceae, thus supporting the hypotheses of Downie et al. (1997) and the Angiosperm Phylogeny Group (1998). Frequently the taxa which were placed unexpectedly or misplaced are problematic genera discussed by other authors (Suaeda, Judd and Ferguson, 1999). Of the 76 characters employed by Rodman (1994) in a study of the Caryophyllales, only seven differentiated the Amaranthaceae from the Chenopodiaceae. The seven characters, presence of: embryo chlorophyll, arillate seeds, myricetin, 6-hydroxyflavenol, flavonol sulfate, 6-hydroxyflavones, and type of leaf wax were combined with our dataset. The two families unsurprisingly were separated. The validity of selecting only variant characters in such an analysis is questionable. It should also be noted that Rodman (1994) was analyzing family positions in an order, and did not include data for species or genera. It is notable that some species are exceptions to the generalizations about families. One example is the presence of an aril on the seed of Chamissoa altissima which is a member of the Amaranthaceae (Townsend, 1993). We did not include Sarcobatus (traditionally in the Chenopodiaceae) in our study. The genus has been elevated to family status (Sarcobataceae) by Behnke (1997) or may be closely related to the Nyctanginaceae (Cuénoud et al., 2002). It is important to remember that these are phenetic data, and we make no conclusions about the primitive or derived nature of the character states. We can conclude, however, that seed characters alone do not support the maintenance of two separate families. The seed characters studied, showed no clear distinction between the Chenopodiaceae and Amaranthaceae. This could be due to variability within these two groups or to the characteristics selected. ACKNOWLEDGEMENTS The authors wish to thank the curators of ISC, MO, and BHSC. The BHSU Scanning Electron Microscope Facility was used in this study. The National Geographic Society is thanked for a grant to MG. The Nelson Scholarship Committee (BHSU) and BRIN (NIH) are acknowledged for a grant to EK. LITERATURE CITED Angiosperm Phylogeny Group. 1998. An ordinal classification for the families of flowering plants. Annals of the Missouri Botanical Garden 85:531-553. Behnke, H. D. 1997. Sarcobataceae a new family of Caryophyllales. Taxon 46:495-507. Carolin, R.C. 1983. The trichomes of the Chenopodiaceae and Amaranthaceae. Bot. Jahrb. Syst. 103:451-466.

78 Proceedings of the South Dakota Academy of Science, Vol. 83 (2004) Cronquist, A. 1981. An integrated system of classification of flowering plants. Columbia University Press, New York. Cronquist, A. and R.F. Thorne. 1994. Nomenclatural and Taxonomic History. pp 5-25 in Behnke, N.D. and T.J. Mabry (eds.) Caryophyllales: Evolution and Systematics. Springer-Verlag, Berlin. Cuénoud, P., V. Savolainen, L.W. Chatrou, M. Powell, R. Grayer and M.W. Chase. 2002. Molecular phylogenetics of Caryophyllales based on nuclear 18S rdna and plastid rbcl, atpb and matk DNA sequences. American Journal of Botany 89:132-144. Downie, S.R. and J.D. Palmer. 1994a. Phylogenetic relationships using restriction site variation of the chloroplast DNA inverted repeat. In Caryophyllales: Evolution and systematics N.D. Behnke and T.J Mabry (eds.) p. 223-233. Springer-Verlag, Berlin. Downie, S.R. and J.D. Palmer. 1994b. A chloroplast DNA phylogeny of the Caryophyllales based on structural and inverted repeat restriction site variation. Systematic Botany 19: 236-252. Downie S.R., D.S. Katz-Downie and K.J. Cho. 1997. Relationships in the Caryophyllales as suggested by phylogenetic analysis of partial chloroplast DNA ORF2280 homolog sequences. American Journal of Botany 84: 253-273. Flora of North America Editorial Committee. 2003. Flora of North America. Volume 4: Caryophyllales. Oxford, New York. Judd, W.S. and T.K. Ferguson. 1999. The genera of Chenopodiaceae in the southeastern United States. Harvard Papers in Botany 4:365-416. Kuhn, U., V. Bittrich, R. Carolin, H. Freitag, I.C. Hedge, P. Uotila and P.G. Wilson. 1993. Chenopodiaceae. In The families and genera of vascular plants, vol 2, Magnoliid, hamamelid and caryophyllid families, K. Kubitzki, J.G. Rohwer and V. Bitterich (eds.) p. 253-281. Springer-Verlag, Berlin. Lawrence, G.H.M. 1951. Taxonomy of Vascular Plants. MacMillan, New York. Rodman, J.E. 1990. Centrospermae revisited. Part I. Taxon 39:383-393. Rodman, J.E. 1994. Cladistic and phenetic studies. In Caryophyllales: Evolution and systematics N.D. Behnke and T.J Mabry (eds.) 279-301. Springer- Verlag, Berlin. Rodman, J.E., M.K. Oliver, R.R. Nakamura, J.U. McClammer, Jr. and A.H. Bledsoe. 1984. A taxonomic analysis and revised classification of Centrospermae. Syst. Bot. 9:297-323. Rohlf, J. 2003. NTSYS 2.11S. Exeter Software, Setauket, NY. Townsend, C.C. 1993. Amaranthaceae. In The families and genera of vascular plants, vol 2, Magnoliid, hamamelid and caryophyllid families. K. Kubitzki, J.G. Rohwer and V. Bitterich (eds.) 70-91. Springer-Verlag, Berlin.

Proceedings of the South Dakota Academy of Science, Vol. 83 (2004) 79 Table 1. Table of species of seeds studied and characters determined. The letters preceding the species names refer to traditional placement of the species. A = Amaranthaceae, C = Chenopodiaceae. Character number descriptions are found at the end of the table. Number following species name refers to sample number.

80 Proceedings of the South Dakota Academy of Science, Vol. 83 (2004) Table 1 continued. Table of species of seeds studied and characters determined. The letters preceding the species names refer to traditional placement of the species. A = Amaranthaceae, C = Chenopodiaceae. Character number descriptions are found at the end of the table. Number following species name refers to sample number.

Proceedings of the South Dakota Academy of Science, Vol. 83 (2004) 81 Table 1 continued. Table of species of seeds studied and characters determined. The letters preceding the species names refer to traditional placement of the species. A = Amaranthaceae, C = Chenopodiaceae. Character number descriptions are found at the end of the table. Number following species name refers to sample number. Table 1 character descriptions: 1. Seed coat glaucous, 2. Margin ridged, 3. Surface tuberculate or papillate, 4. Surface granular, 5. Surface pitted, 6. Surface very smooth with a glassy luster, 7. Calyx and/or funiculus persistent, 8. Radial striation visible at magnification of up to 65x, 9. Fine reticulate pattern visible at magnification of up to 65x, 10. Cell shape of seed coat at margin elongated (1) or squamous (0), 11. Cell shape of seed coat center elongated (1) or squamous (0), 12. Cell shape of seed coat at hilum elongated (1) or squamous (0), 13. Small sulcus at hilum indicating folded embryo, 14. Notch at one point on margin evident, 15. If notch present in 14, measurement was obtained for length of notch: 1/10 mm and larger (1), 1/10 mm and smaller (0), 16. Tip protruding, 17. Hilum in a notch formed by an extension (1), without extension (0), 18. Seed scar depressed and round (0), oval (1), or triangular (2) in shape, 19. Surface indicating the inner embryo, 20. Several prominent veins and/or ridges running lengthwise on seed coat, 21. Scar at center of seed, 22. Outline of seed elliptical (1) or circular (0), 23. End of seed obviously truncated, 24. Seed coat color black (1), other (0), 25. Length/width ratio of seed.