CHAPTER 3

Sexual Reproductive Biology

Dwarf mistletoes reproduce only from seeds. The shoots occurring on widely divergent branch systems oflarge, systemic witches' brooms might be consid­ered clonal ramets of the founder individual, but this represents only an increase in the size and reproduc­tive output of the founder individual. New genets can only be established by seed.

Stages of sexual reproduction in seed plants can be conveniently divided into two categories:

1. Pre-dispersal events, beginning with pollina­tion and culminating with viable seed produc­tion; these processes occur in the maternal environment but are primarily controlled by the genetic constitution of the zygotic genome.

2. Post-dispersal events, beginning with seed dis­persal and culminating with successful repro­duction of the progeny; these events occur in the ambient environment and are controlled by selective forces in the physical and biotic envi­ronments (Wiens and others 1987).

The pre-dispersal reproductive process includes several discontinuous phases: pollination; pollen ger­mination and pollen tube growth; and fertilization and seed development (embryo and endosperm). Post­dispersal phases of dwarf mistletoe reproduction are discussed in chapter 2.

Pollination Controversy has surrounded the pollination biolo­

gy of Arceuthobium through much of this century because the genus exhibits floral characteristics typical of both insect-pollinated (entomophilous) and wind-pollinated (anemophilous) flowers. Interestingly, much the same controversy surrounds the mode of pollination in the European mistletoe, Viscum album. Heinricher (1915a) speculated that the floral characteristics of Arceuthobium favored ento­mophily, but he later modified his view after observ­ing that greenhouse plants set seed in the absence of insects (Heinricher 1920).

Sexual Reproductive Biology

Characteristics of Arceuthobium that favor ento­mophily include: (1) sessile anthers; (2) spined pollen; (3) clustered pollen bound together by mucous strands; (4) relatively low pollen production (over 11,000 grains per flower) in comparison to that of many wind-pollinated plants (cf. 50,000 grains per rye floret); (5) non-plumose stigma; (6) nectar production in staminate flowers and stigmatic exudate in pistillate flowers (figs. 2.7 and 2.8); and (7) faint odor produc­tion by both pistillate and staminate flowers.

Features indicating anemophily include: (1) exposed anthers; (2) pollen size in the general range of wind-pollinated species (10 to 60 !Jm, although pollen-clustering must upset this size and weight rela­tionship); (3) long-distance transport of pollen (sever­al kilometers) and its common occurrence in the fossil palynological record; (4) localized, dense population structure; (5) unisexual flowers; (6) single-"ovule" ovaries; (7) flowering periods temporally separated from those of their wind-pollinated hosts (the more abundant host pollen would likely saturate the mistle­toe stigmas if they flowered simultaneously); (8) tem­poral partitioning of flowering periods when two dwarf mistletoe species co-occur in the same habitat (see discussion of sympatry in chapter 5); and (9) sex­ual dimorphism in which staminate plants are open and spreading (thereby favoring the release of pollen with minimum filtration effect from branches of the staminate plant) and pistillate plants are compact and densely branched (figs. 3.1 and 3.2). Other aspects of sexual dimorphism are discussed in chapter 14.

Pollination biology in Arceuthobium americanum has received greater study than other dwarf mistletoes (Coppola 1989; Gregor and others 1974; Penfield and others 1976; Stevens and Hawksworth 1984; Gilbert and Punter 1984, 1990, 1991). These studies generally. indicated that both insects and wind contributed to pollination success, but Gregor and others (1974) sug­gested that entomophily predominated. Penfield and others (1976) reported that large numbers of generalist insects (over 200 species), especially various flies and ants, carried pollen and played a role in the pollination of three dwarf mistletoes in Colorado. For A. ameri­canum, Formicafusca (silky ant) was considered the most important pollinator and Philygria debilis (a gnat)

15

This file was created by scanning the printed publication.Errors identified by the software have been corrected;

however, some errors may remain.

Figure 3.1-$taminale plant of Arcewhobfum nfgmm illustrating the typical panem of sexual dimorphism, i.e., the open spreading branch habit of staminate plantS.

second-most important. Other flies were involved in pollination at different stages of the flowering period. Penfield and others (1976) also nQ(ed that pollen was commonly transferred by wind to distances of 12 m and occasionally as far as 150 m. Coppola (1989) found that pollen in a Colorado population was wind­dispersed as far as 512 m; Gilbert and Punter (1984) in Manitoba obsetved a maximum distance of pollen dis­persal of 400 m.

For Arceuthobiurn vaginaturn subsp. cryptopo­dum, the parasitic wasp Copidosoma bakeri and the flea beetle Phyllotreta lewisii were judged to be the most important pollinators; the ants Formica/usca and F hernmorhOidalis and flies of the genera Bradysia and Hylemya were also reported to be common polli­nators (penfield and others 1976). Vasquez (1991) studied pollination inA. vaginatum subsp. vaginaturn in Mexico and concluded that anemophily predomi­nated.

InArceuthobium cyanocarpum, the primary polli­nators were wasps (Copidosoma bakert), flies (Hylemya spp.), and a beetle (Hoppingiana sp.). Ants

16

Chapler 3

Figure 3.2 -Pistillate plant of Arcellthobfum nigrnm illustrating the typical pattern of sexual dimorphism, Le., the compact and dense branch habit of pistillate plants.

were less important for pollination of A. cyanocarpum than for A. americanum and A. vaginatum subsp. cryptopodum.

Player (1979) studied the pollination biology of Arceuthobium douglasit in Utah and A. strictum in Durango, Mexico, and concluded that these species (and the genus as a whole) were fundamentally wind­pollinated. He based this conclusion primarily on the general lack of insect visitation and the abundance of airborne pollen from the populations of A. douglasii he studied.

Baker and others (1985), however, suggested that insects played the primary role in the pollination of Arceuthobium pusillum. They enclosed mistletoe plants with a relatively large mesh screen (4 mm) that should have reduced but not precluded insect visita­tion, yet only slightly interfered with transport of pollen by wind. Fruit set by these enclosed plants was Significantly reduced. Airborne pollen was also much less prevalent in populations of A. pusillum than in A. douglash

Sexual Reproductive Biology

Chapter 3

Aspects of the pollination biology of dwarf mistle­toes that deserve further comment include nectar pro­duction and anther movement. The flowers of dwarf mistletoes must be among the smallest that produce nectar. The staminate flowers (1 to 4 mm across) pos­sess a nectary (the "central cushion" of earlier termi­nology), and the smaller pistillate flowers (0.5 to 1.5 mm across) produce a stigmatic exudate. The quantity of nectar secreted by staminate flowers is especially minute, and the nectary rarely produces more than a glistening moist layer over its surface (fig. 2.7). In contrast, the stigmatic exudate under condi­tions of high humidity forms large droplets that are many times the size of the stigma (fig. 2.8). In absolute terms, however, the quantity of stigmatic exudate is still minute. In Arceuthobium abietinumJ the exudate is highly concentrated (58 to 92% solids) and com­prised of 48% sucrose, 39% fructose, and 11% glucose (Brewer and others 1974). InA. americanum the stig­matic exudate is 50 to 65% sugars, but the staminate flowers produce significantly less concentrated nectar (19%) (Gilbert and Punter 1990). The importance of this difference, if any, is not readily apparent. Generally, nectar with a high sugar concentration is typical of fly­pollinated flowers (Faegri and van der Pij11966).

Pollen is likely depOSited on the stigmatic exudate, and the exudate may therefore serve multiple func­tions: (1) pollinator attractant, (2) pollen adhesive, and (3) stimulant for pollen germination (Heinricher 1915a). Jones and Gordon (1965) and Hudson (1966) report that the pollen grains of Arceuthobium ameri­canum andA. douglasii are held in place by the stig­matic secretions. They also comment that there was no marked increase in insect visitation during the peri­od of exudate secretion. This observation offers strong su pport for wind pollination in A. douglasii as argued by Player (1979). As will be discussed later, the pollen of A. americanum requires an unusually high concen­tration of sucrose (20%) for optimal germination (Gilbert and Punter 1991). The high sugar content of the stigmatic exudate might, therefore, be important primarily as an aid to germination of the pollen grains and only of incidental importance as an attractant for opportunistic foraging insects. The large number of taxonomically diverse insects (over 200 species) known to visit dwarf mistletoe flowers exhibit no com­mon characteristics to suggest that there are any co­evolved features between themselves and the dwarf mistletoes that they may occasionally pollinate.

Gilbert and Punter (1990) discovered that the anthers of Arceuthobium americanum and to a lesser extent those of A. pusillum open in response to high temperatures and low humidity, and they close when the reverse conditions prevail. Although temperature

Sexual Reproductive Biology

increases would also stimulate insect activity, the lower humidity requirement argues against ento­mophilous pollination. The small insects (gnats, flies, wasps, and ants) that are commonly associated with dwarf mistletoe flowers should be particularly sensi­tive to low humidities. Small insects are able to forage at higher-than-optimal temperatures only if the humid­ity is also correspondingly high (D. Feener, personal communication). The combination of high tempera­tures and low humidity necessary for anther opening according to Gilbert and Punter (1990) are known to be the most favorable conditions for the airborne dis­tribution of pollen (Whitehead 1969). Because of this dual requirement, it seems most probable that anther closing and opening enhance the possibilities of wind pollination. Such an apparently major structural modi­fication would strengthen the case that at least these species of Arceuthobium are basically adapted for wind pollination. The occurrence of this character in other species has not yet been investigated, but inas­much as the extent of anther opening and closing is variable between A. americanum and A. pusillumJ this feature might serve as an indicator for anemophily or entomophily among other dwarf mistletoes.

Pollen Germination and Pollen Tube Growth

Pollen germination and subsequent pollen tube growth have received less study than any other phase of sexual reproduction in Arceuthobium. Pollen ger­mination has been investigated only for A. ameri­canum in Manitoba, Canada (Gilbert and Punter 1991). This species exhibited some unusual characteristics of pollen germination. The most noteworthy of these are the high concentrations of sucrose (20%) necessary for optimal germination and the failure of pollen to respond to known germination stimulants such as boric acid; salts of Ca, Mg, and K; and macerates of pis­tillate flowers. Optimal temperature for germination in vitro was 30° C. Germination percentages, however, were typically less than 30%. Germination of pollen originating from different dwarf mistletoe plants also varied significantly. In general, as the flowering sea­son progressed, percent germination increased; pre­sumably, this change was due to increased air temper­atures. Thus, at least in this region, most fertilization likely occurs toward the end of the flowering period. As Gilbert and Punter (1991) point out, this also may explain why the anthers open and close in relation to temperature. Pollen in A. americanumJ however, remains viable for long periods; it forms in August or September and is not dispersed until early spring.

17

Hudson (1966) observed pollen tubes in the stylar canals of Arceuthobium americanum in April, but he also reported that embryo sac meiosis (megasporo­genesis) did not occur for almost 2 months (late May) and that fertilization was delayed until June. Thus, the pollen tube must grow for at least 2 months before it reaches and penetrates the embryo sac. Such an inter­val is inordinately long among flowering plants; the time between pollination and fertilization is usually about 48 hours. Hudson's (1966) observations dis­agree with Dowding's (1931a) earlier report that fertil­ization in A. americanum occurs within "a few days" of pollination. Hudson's (1966) assertions are also in marked contrast to observations for A. douglasii and A. pusillum that the interval between pollination and fertilization is "a few days" Oones and Gordon 1965, Tainter 1968). The sperm of A. oxycedri (a fall-flower­ing species) are reported to overwinter in the pollen tube in contact with the embryo sac; they are released the following spring when growth is resumed and fer­tilization occurs Oohnson 1888).

Arceuthobium americanum, A. douglasii, and A. pusillum are all indirect flowering species-their flowers develop in the autumn and overwinter as mature buds. These mistletoes are among the earliest to initiate anthesis the following spring (March or April). Although pollen grain meiosis (microsporogen­esis) occurs in the autumn, megasporogenesis is delayed until spring.

The reasons for the asynchrony in pollen grain and embryo sac development and the long period of pollen tube growth are not immediately evident. Interestingly, the conifer hosts of the dwarf mistletoes also require a year for their pollen tubes to reach the embryo sac. Furthermore, fruit development in dwarf mistletoes parallels Jhat of conifers; both groups require about 12 to 18 months for seed maturation. Whether these unusual similarities in phenology between host and parasite are merely coincidental-or are evolutionarily significant-requires clarification.

Embryo Sac Development, Fertilization, and Fruit Maturation

Because all mistletoes lack ovules, technically they do not possess true "seeds" or "fruit." This anomaly has been the subject of considerable study since its dis­covery in the middle of the last century. For summa­tions of our current understanding of this phenome­non, see Bhatnagar andJohri (1983) for Loranthaceae and Bhandari and Vohra (1983) for Eremolepidaceae and Viscaceae.

18

Chapter 3

Embryo sacs in dwarf mistletoes arise from a mound of tissue at the base of the ovary, usually termed the ovarian papilla or mamelon (fig. 3.3A). The papilla has no integument and is not part of the mature fruit, having been crushed by the developing endo­sperm mass (fig. 3.3B-E). Two megasporocytes initiate development of the embryo sacs in the papilla. In Arceuthobium americanum, however, one mega­sporocyte is possibly arrested in development at the 4-nucleate stage (Hudson 1966; C. L. Calvin, unpub­lished data). In A. douglasii, after one megasporocyte is fertilized, the other degenerates, without being fer­tilized Oones and Gordon 1965). The sequence of events in early embryo sac development requires fur­ther study.

Embryo sac development in Arceuthobium con­forms to the Allium or bisporic type (Hudson 1966, Tainter 1968, Bhandari and Nanda 1968, Bhandari and Vohra 1983). In this system, only one of the two dyads produced by the first meiotic division survive. One degenerates rapidly, and the surviving, functional dyad completes meiosis and produces two haploid megaspore nuclei. These megaspore nuclei undergo two successive mitotic divisions that ultimately result in an 8-nucleate embryo sac. The Allium-type embryo sac is otherwise typical of the common Polygonum type at maturity, and the embryo sac is characterized by 3 antipodals, 2 polar nuclei, and 2 synergids sur­rounding the somewhat larger egg. Because the Polygonum-type of embryo sac has a monosporic ori­gin, all cells are genetically identical, whereas bisporic embryo sacs should contain cells of two genetically distinct origins. The significance of this difference, if any, is not immediately apparent. Variant 7-nucleate embryo sacs are reported in A. americanum (Dowding 1931a) and inA. campylopodum (Cohen 1970), but these observations have not been con­firmed (Bhandari and Vohra 1983).

Double fertilization and the formation of a triploid endosperm is common to all Viscaceae (Bhandari and Vohra 1983). There is apparently interspecific varia­tion with respect to whether a primary diploid endosperm nucleus is formed or whether the two polar nuclei fuse with the sperm independently (Bhandari and Vohra 1983). Hudson (1966) reports an interesting anomaly in Arceuthobium americanum wherein the egg lies above and to the side of the syn­ergids instead of between them, as is typical. If cor­rect, this raises interesting questions as to the function of the synergids in dwarf mistletoes. Typically, sperm are released from the pollen tube, de·posited into one of the adjacent synergids, and transferred to the egg by the synergid. The lack of juxtaposition in this case would appear to preclude this process. The role the

Sexual Reproductive Biology

Chapter 3

Figure 3.3 -Longitudinal sections through ovaries of flowers (A) and developing fruit (B-F) of Arceuthobium cyanocarpum (A-D) and A. ameri­canum (E,F). A: mamelon (m) with 2 embryo sacs (g) and fruit wall (I), x 100. B: cellular endosperm (en) in mamelon (m), x 100. C: same speci­men as B but at a different focal plane, embryo (e), mamelon (m), and fruit wall (I), x 100. D: enlarged view of specimen seen in C showing two­celled embryo (e) and endosperm (en), x 250. E: globular embryo (e) in endosperm (en), note crushed mamelon (cm), x 100. F: same develop­ment stage as E, but with necrotic, aborted embryo (n) surrounded by necrotic endosperm and fruit wall (I), x 100. (c. L. Calvin)

synergids in fertilization requires further study in dwarf mistletoes.

Endosperm formation is cellular in Arceuthobium, as is typical of plants with bisporic embryo sac devel­opment. Soon after fertilization, the embryo sac is sep­arated transversely into two chambers; one includes the zygote and disintegrating synergids and the other contains the fertilized primary endosperm nucleus and degenerating antipodals. This latter portion of the sep­arated embryo sac then develops a lateral extension or "haustorium" that elongates downward toward the base of the papilla, where it obtains nutrition from maternal tissue for the development of the endosperm. Endosperm production around the zygote continues for some time prior to the initiation of embryogenesis. No suspensors are formed, and embryonic develop­ment continues in the cellular endosperm until autumn. By then, however, embryogenesis has not progressed beyond the globular stage, and the embryo overwinters at this stage in the relatively massive endosperm. Fruit development is resumed the follow­ing spring; embryogenesis and endosperm formation

Sexual Reproductive Biology

are complete by middle to late summer of the year fol­lowing fertilization. Knutson (1984) states that seeds approximately double in size from mid-July to mid­August.

The mature seed of dwarf mistletoes is a hypocoty­lar cylinder that has a highly meristematic radicular apex (without root cap) at one end and a pair of minute, vestigial cotyledons at the other. The mature fruit consists of a thick-walled pericarp with a layer of viscin cells and a parenchymatous zone surrounding the seed (see fig. 2.3 and fig. 10.9). With the exceptions of Arceuthobium pusillum and several tropical species CA. abietis-religiosae, A. aureum subsp. aureum, A. hawksworthii, A.junipenprocerae, and A. nigrum), dwarf mistletoes typically require about 12 to 18 months to complete fruit development.

Pre-dispersal embryo survivorship in dwarf mistle­toes averages around 50%, but the variance is high (Wiens, unpublished data). Typically, 80 to 90% of the fruits survive the first summer's growth, at which time the embryos are only at the globular stage of develop­ment. During the second summer's growth and prior to

19

dispersal, an additional 30-40% of the embryos die (fig. 3.3F). These rates of first and second year survivorship have been confirmed in Arceuthobium americanum by Gilbert and Punter (1984) and Gilbert (1988).

Wiens and others (1987) attributed most of this loss to genetic lethals. Reduced seed set should not be due to resource limitations because dwarf mistletoe plants are resource sinks with relatively large reserves provid­ed by the host.

Exceptional Characteristics of Fruits and Seeds

Several exceptional aspects of seed and fruit devel­opment in Arceuthobium are absence of true seeds, chlorophyllous endosperm and embryos, stomata on the growing hypocotyl, lack of a root cap, and occa­sional polyembryony. Virtually all of these characteris­tics are likely related, directly, or indirectly to the evo­lution of the parasitic habit.

Loss of the integument in A rceuthobium is perhaps the result of extreme evolutionary specialization of the seed for explosive dispersal. However, all other mistletoes also lack an integument and the majority of these are bird dispersed. Therefore, the ultimate evo­lutionary cause of integument loss is perhaps due to other factors. Another possible explanation is that the seed must be energetically self-sufficient for long peri­ods before it infects the host. Thus, both the endosperm and embryo are chlorophyllous, and at least the hypocotyl possesses stomata. A testa would likely hinder the photosynthesis that occurs at low rates in the seedling (Tocher and others 1984).

Arceuthobium seeds have no innate dormancy systems. In temperate regions, growth of the hypocotyl is retarded through periods of unfavorable environments, but the radicular apex resumes growth whenever favorable conditions return.

The viscin coat may have importance beyond its adhesive qualities. It has a high water retention capaci­ty, and it may playa role in reducing desiccation and infection by fungal pathogens (Knutson 1984).

The existence of multiple embryo sacs and the general occurrence of a single seed has prompted the assumption that there is competition for survival among embryo sacs or their developing embryos. Either one embryo sac may be arrested at the 4-nucle­ate stage (as in Arceuthobium americanum) or only one embryo sac may be fertilized and the other degen­erates soon thereafter (as in A. douglasiz). Because

20

Chapter 3

polyembryony does occur in dwarf mistletoes, limits on fertilization and development can not be absolute. In many species of Viscum, polyembryony is common, and the occurrence of 1 to 3 embryos per seed is the rule (Wiens, unpublished data). Therefore, competi­tion among developing embryo sacs or embryos is possible.

Polyembryony in Arceuthobium, however, is rare. Hawksworth (1961 b) reported that about 1 % of the seeds of Arceuthobium americanum and A. vagina­tum subsp. cryptopodum contained two embryos and two endosperms. Weir (1914) found somewhat higher levels of polyembryony in A. vaginatum subsp. cryp­topodum (15%) andA. douglasii (13%), but his sample sizes were small. He also indicated that both germinat­ing embryos in di-embryonic seeds grew "vigorously" upon germination and that infection should occur if penetration could be effected. The di-embryonic seeds were no smaller than the mono-embryonic seeds. Hawksworth (1961b) suggested that di-embry­onic seeds provided a possible mechanism whereby new populations of a dioecious species could become established from a single seed, provided, of course, the embryos were of different sexes.

Sex Ratios All dwarf mistletoes are obligately dioecious

plants, and there is no evidence that bisexual flowers or unisexual flowers of the opposite sex occur in even low frequencies. Other than for Arceuthobium tsug­ense, sex ratios in Arceuthobium have not been exten­sively studied; table 3.1 summarizes available informa­tion. For those species of dwarf mistletoe (and other dioecious mistletoes) with a skewed sex ratio, the bias generally favors female plants. This is certainly the case for A. tsugense subsp. tsugense, which has a signif­icantly female-biased sex ratio of approximately 3:2 in populations from Alaska, Washington, and Oregon (Wiens, Hawksworth, Shaw, and Hennon, unpub­lished data). Although fewer data are available, A. tsug­ense subsp. mertensianae in Washington and Oregon has a similar sex ratio.

Female-predominant sex ratios are typical ofvari­ous African and European species of Viscum (Wiens and Barlow 1979), but Arceuthobium tsugense is the first clear example of a dwarf mistletoe characterized by a female-biased sex ratio over a broad geographical distribution. The sex ratios of other dwarf mistletoes should be examined.

Sexual Reproductive Biology

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