Plant P1. Syst. Evol. 196:89-98 (1995) Systematics and Evolution '© Springer-Verlag 1995 Printed in Austria Microgametophytic plastid nucleoid content and reproductive and life history traits of tribe Trifolieae (Fabaceae) R. N. KEYS, S. E. SMITH, H. LLOYD MOOENSEN, and E. SMALL Received August 2, 1994; in revised version September 28, 1994 Key words: Leguminosae, Medicago, alfalfa. - Microgametophyte, biparental inheri- tance, DAPI. Abstract: Microgametophytic plastid nucleoids were quantified for 18 species represent- ing the four core genera of the tribe Trifolieae (Fabaceae), Medicago, Melilotus, Trigonel- la, and Trifolium. Generative cells of all taxa contained nucleoids, establishing that bipa- rental plastid inheritance is common in the Trifolieae. Nucleoid number and volumes of pollen grains and generative cell nuclei differed among taxa. Nucleoid number was posi- tively correlated with pollen grain and generative cell nuclear volumes, flower size and style length. These relationships disappeared after adjusting nucleoid number for pollen grain and generative cell nuclear volumes. Adjusted nucleoid numbers provided no evi- dence to support hypotheses that plastid content is associated with ploidy level, mating system, perenniality or size of the reproductive apparatus. Advances in cytology and molecular biology have stimulated interest in describ- ing the mode of organelle inheritance in plants. Data from a number of studies have shown that the assumption of strict maternal inheritance of plastids in angio- sperms is not justified (review in HARRIS • INGRAM 1991). Using the DNA fluo- rochrome DAPI (4'-6-diamidino-2-phenylindole), CORRIVEAU & COLEMAN (1988) analyzed generative or sperm cells from 235 angiosperm species for the presence of plastid nucleoids (DNA aggregates) and found cytological evidence for poten- tial paternal inheritance of plastids in 43 species from 26 genera. Such cytological data generally correlate closely with available genetic data on plastid DNA in- heritance (CoRRIVEAU& al. 1990, HARRIS & INCRAM 1991). Plastid inheritance has been studied extensively in the widely cultivated taxon Medicago sativa L. (Fabaceae), which exhibits biparental inheritance of plastids with a strong paternal bias (SMITH & al. 1986). Genotypic differences have been observed in the extent of plastid transmission as both a maternal and paternal parent (SMITH 1989), and cytological studies using serial ultrathin sections or DAPI- stained whole cells have also demonstrated the existence of plastids and plastid nucleoids within generative (ZHu & al. 1990, SHI & al. 1991) and sperm cells (ZHu & al. 1993). These studies have also shown that the number of plastid nucleoids 90 R.N. KEys & al.: in these cells is proportional to, but less than the number of plastids as determined from electron microscopic observation of serial ultrathin sections. Very little research has addressed possible functions of plastids either with or without DNA within the microgametophyte. The observation that many conifers exhibit strongly paternal plastid inheritance (SuTvON & al. 1991, NEALE ~1; al. 1991) as well as temporal separation between pollination and fertilization (WmLsON & BURLEY 1983) has led to the suggestion that plastids within the male gametophyte may play a functional role in reproductive success perhaps by influencing micro- gametophyte survival. Similarly, organelle DNA has been visualized within the cytoplasm of vegetative cells of pollen and microgametophytes from three genera in the Orchidaceae in which pollen is very long-lived (ALBERT & al. 1989). These observations indicate that plastids could provide an additional source of energy and/or essential metabolites to pollen, microgametophyte, or embryo (CRuzAN 1990). If this is the case, quantitative differences in plastid content might be expected to be related to microgametophyte or embryo performance, but the amount of DNA in the plastids would be unimportant. Alternatively, these organelles may produce RNA or protein important in the development of these organs (WILLSON& BURLEY 1983, CORRIVEAU• COLEMAN 1991), SO that a certain minimum amount of functional DNA within plastids would then be required. Ontogenetic factors may also determine the presence of DNA-containing organelles in microgametophytes. CORglVEAU& COLEMAN(1991) showed that nucleic acids were present in both mito- chondria and plastids of generative and vegetative cells of six angiosperm taxa until pollen maturation. After that, nucleic acids were observed only in plastids in the generative cells and only in species in which plastids are inherited biparental- ly. Attempts to describe the possible evolutionary significance of the mode of plastid inheritance, and the use of such information in systematic research, have been hampered by lack of studies that have systematically sampled taxa. This study uti- lized a systematic approach to study plastid inheritance in the tribe Trifolieae (Fabaceae), that includes four closely related genera, Medicago, Trigonella, Melilotus, and Trifolium. Parochetus and Ononis are sometimes included in the tribe, but doubtfully so (SMALL 1987, 1988). The genus Medicago presents an uncommon opportunity to address questions related to plastid inheritance and reproductive and life history traits. The genus contains about 85 species (SMALL & JOMPHE 1989) of annual or perennial herbs or shrubs that exhibit an irreversible explosive pollination mechanism (SMALL & al. 1987). The majority of perennial Medicago taxa are polyploid, outcrossing, and entomophilous, while most annual species are diploid and strongly autogamous (HEYN1963). A substantial difference in time between pollen dehiscence and fertilization would be expected between autogamous and outcrossing, entomophilous species. Although considerably less than the time lag between pollination and fertilization in conifers, the time lag and environmental stress involved in entomophily should permit testing of hypotheses regarding plastid contribution toward pollen survival in angiosperm genera. The objectives of this research were to 1) described plastid content (as DAPI- staining plastid nucleoids) in generative cells from 18 species representing ten sections of Medicago and, less intensively, the three other core genera in the Tri- folieae; and 2) determine whether plastid nucleoid content is associated with Microgametophytic plastids and life history traits 91 reproductive biology and life history traits in these taxa. In an attempt to elucidate the role of plastids in reproductive biology within this tribe, we tested the specif- ic hypotheses that microgametophytic plastid nucleoid content 1) does not differ between outcrossing and autogamous species, 2) does not differ among plants with different ploidy levels, 3) does not differ between annual and perennial spe- cies, and 4) is unrelated to size of the reproductive apparatus. Material and methods Plant material. Accessions of 14 species of Medicago from 10 sections as well as one species of Trigonella, one of Melilotus, and two of Trifolium were utilized (Table 1). Flowers for pollen studies were collected in Tucson, Arizona, from greenhouse-grown plants of each accession except for M. arborea, which was collected from field-grown plants. Pollen collection, germination and cytology. Pollen tubes were grown using a modi- fication of method (iv) described by CORRIVEAU & COLEMAN (1988). Pollen from five to eight flowers was placed in a depression well to which 100 ~1 of germination medium (15% sucrose, 0.16 mM H3BO3, 0.20 mM KNO3, and 0.13 mM Ca(NO3)2 • 4H20) were added. Germination was allowed to proceed for 2.5-3 h at 22-27 °C in a petri plate con- Table 1. Description of plants used in analysis of plastid nucleoid content within generative cells Taxon (ploidy, chromosome number) Section ~ Accessionb Habitc Mating system Medicago arborea L. (4x = 32) Dendrotelis PI 330671 P-shrub Cross-pollinated M. sativa L. (4x = 32) Medicago cv. Malone P-herb Cross-pollinated M. sativa L. (2x = 16) d " (M) 225467 P-herb Cross-pollinated M. cancellata M. B~EB. (6X = 48) " PI 440491 P-herb Cross-pollinated M. carstiensis WULF. (2 × = 16) Carstiensae (M) 47 P-herb Self-pollinated M. truncatula GAERTN. (2X = 16) Spirocarpos PI 292434 A-herb Self-pollinated M. scutellata (L.) MmLER (2n = 30) " PI 197821 A-herb Self-pollinated M. polymorpha L. (2x = 16) " (A) LONG A-herb Self-pollinated M. lupulina L. (2x = 16) Lupularia (C) M-1231 A-herb Self-pollinated M. heyniana GREETER (2X = 16) Heynianae (C) M-839 A-herb Self-pollinated M. radiata L. (2x = 16) Hymenocarpos (C) M-225 A-herb Self-pollinated M. ruthenica (L.) LEOEBOUR(2X = 16) Platycarpae (M) 2139-4x2082-8 P-herb Cross-pollinated M. edgeworthii SIRJAEV (2X = 16) " (C) M-957 P-herb Cross-pollinated M. biflora (Griseb.) E. SMALL(2X = 16) Lunatae (C) 157 A-herb Self-pollinated M. monantha (C. A. MEYER) TRAUTV. Buceras (C) T-133 A-herb Self-pollinated (2x = 16) Trigonellafoenum-graecum L. (2n = 16) ..... PI 138685 A-herb Self-pollinated Melilotus officinalis (L.) PALLAS(2n = 16) ..... (A) MNLE P-herb Cross-pollinated Trifolium repens L. (2n = 4x = 32) ..... (A) DLAW P-herb Cross-pollinated T. pratense L. (2n = 2x =16) ..... cv. Kenstar P-herb Cross-pollinated a For Medicago species only; following SMALL& JOMeHE 1989 b Collection designations: PIs - USDA Plant Introduction Collection, Pullman, WA; (M) - T. J. McCoY, Dep. of Plant and Soil Sciences, Montana State University, Bozeman, MT; (A) - S. E. SMITH,Dep. of Plant Sciences, Uni- versity of Arizona,