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2Instituto Multidisciplinario de Biología Vegetal, C. C. 495, 5000 Córdoba, Argentina; 3Department of Ecology and Evolutionary Biology, Storrs, Connecticut 06269-3043; 5Casilla 1340, Concepción, Chile; 6Institüt für Botanik, Universität Wien, Rennweg 14, A-1030, Vienna, Austria; 7Department of Plant Biology, Ohio State University, Columbus, Ohio 43210
Received for publication June 26, 1998. Accepted for publication October 27, 1998.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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1 female:1 hermaphrodite. The inconspicuous semipendulous green flowers, usually in mixed-gender inflorescences, do not produce rewards. Hermaphrodite flowers are herkogamous and protogynous. Pollen grains are shed from the extrorse anthers in permanent dry tetrads. There is a mean of 12879 tetrads per hermaphrodite flower. Both flower types bear an average of 18 ovules. The P/O (pollen/ovule) ratios imply facultative or obligate xenogamy, but hand pollinations show that Lactoris is self-compatible. No floral visitors were ever observed, but stigmata of open-pollinated flowers bore tetrads, and 64% of such styles had pollen tubes. Flowers enclosed in large mesh (1 mm) bags bore similar numbers of tetrads and pollen tubes. Thus, we conclude that Lactoris is anemophilous, a syndrome perhaps reflected by the P/O ratio. Low genetic diversity (isozymes and DNA) supports selfing and implies limited distance wind pollen dispersal. The small size of the island, the ± 1000 extant Lactoris plants, coupled with anemophily, self-compatibility, and pendant flower position, have yielded a geitonogamous system with high seed set and low genetic diversity. If inbreeding depression is expressed, it is in seed germination and seedling vigor, for Lactoris is very difficult to cultivate. For this species, effective conservation practices need to focus on habitat preservation and promotion of outcrossing.
Key Words: basal angiosperms • conservation biology • island biology • Lactoridaceae • monoecy • reproductive biology • self-compatibility • wind pollination
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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Anderson, 1995
)—particularly of endangered species where there are very few populations to supply propagules for future generations. Thus, island species could be of considerable importance not just because so many have few populations, but because the few populations are usually very restricted geographically and genetically homogeneous. Close to 65% of the 158 extant flowering plant species of the Juan Fernández archipelago (Chile) are endemic (Stuessy et al., 1997
), with many having very few populations or very few individuals surviving. Furthermore, many of the endemics are threatened by continued foraging by goats, and foraging and habitat destruction by rabbits, as well as the losses of habitat to aggressive non-native species (Stuessy et al., 1997
). In addition to poor adaptation to herbivore resistance, the native flora is also characterized by low fire tolerance (Skottsberg, 1953
), factors that make many species especially vulnerable to human-induced disturbance. A recent paleoecological survey by Simon Haberle (personal communication, Department of Archaeology and Natural History, Australian National University, Canberra) documented that the arrival of humans 400 yr ago led to the reduction of higher altitude vegetation (the primary refuge of the native flora), followed by expansion of non-native species.
One species of particular interest because of its rarity, and potentially basal position in the angiosperms (a "paleoherb" according to Donoghue and Doyle, 1989
), is Lactoris fernandeziana Phil., an endangered species in special need of conservation (Stuessy et al., 1997
). The monotypic family Lactoridaceae is endemic to Robinson Crusoe Island (= "Isla Robinson Crusoe," "Masatierra"), one of the three major land masses comprising the Archipelago Juan Fernández in the Pacific Ocean (667 km west of continental Chile, at latitude 33°37' S). This small herbaceous shrub is mostly restricted to windy fog- and rain-swept mountain forests on steep slopes, where it occurs as an inconspicuous member of the understory (but the canopy over Lactoris is often only at 2–4 m), although it is occasionally found in full sunlight on near-vertical exposed cliff faces. Fortunately for conservation purposes and unfortunately for experimental purposes, the plants grow in these very remote, steep-sided, and exposed high-elevation locations. Once thought to be close to extinction (Carlquist, 1964;
Sanders, Stuessy, and Marticorena, 1982;
Lammers, Stuessy, and Silva, 1986
), our recent botanical expeditions (1991, 1996, 1997) have revealed from tens to hundreds of individuals in relatively inaccessible montane cloud forests of the island located at an altitude of 500 m and above (Crawford et al., 1991,
1994;
Stuessy et al., 1997,
1998
).
The discovery of fossil pollen of Lactoridaceae in Cretaceous deposits of Southern Africa dating to 69 myBP suggests that this family may have been a common element in the Cretaceous Gondwana flora (Zavada and Benson, 1987
). Only L. fernandeziana has survived (apparently as a relictual polyploid [Tobe et al., 1993
]) as one of 100 highly restricted endemics on this volcanic oceanic island formed four million yr ago and never connected to a continental area (Stuessy, Sanders, and Silva, 1984
).
Various studies attempting to determine the origin and relationships of this peculiar family have not reached a consensus (Dahlgren and Bremer, 1985;
Crawford, Stuessy, and Silva, 1986;
Lammers, Stuessy, and Silva, 1986;
Donoghue and Doyle, 1989;
Carlquist, 1990;
Qiu et al., 1993;
Tobe et al., 1993;
Doyle, 1994;
Stuessy et al., in press
). Suggested closest relatives include species in the Laurales, Piperales, Magnoliales, or Aristolochiales (Stuessy et al., 1998
).
Most previous work on this species focused mainly on its relationships and placement (e.g., external morphology [Engler, 1887
], anatomy [Carlquist, 1964
], chromosome number [Raven, Kyhos, and Cave, 1971
], phenetics and cladistics [Lammers, Stuessy, and Silva, 1986
], leaf flavonoids [Crawford, Stuessy, and Silva, 1986
], pollen morphology [Erdtman, 1964;
Zavada and Taylor, 1986;
Zavada and Benson, 1987
], ultrastructure of sieve-element plastids [Behnke, 1988
], wood anatomy [Carlquist, 1990
], cladistic and molecular studies [Donoghue and Doyle, 1989;
Brauner, Crawford, and Stuessy, 1992;
Qiu et al., 1993;
Doyle, 1994
], allozyme variation [Crawford et al., 1994
], and embryology and karyotype [Tobe et al., 1993
]). All of these were summarized in a recent paper (Stuessy et al., 1998
). From these studies it is clear that Lactoris is characterized by a mixture of both primitive and specialized traits. Despite the fundamental importance of reproductive biology for systematic and evolutionary studies (Anderson, 1995
), such features have not been central in studies of Lactoris. As with much of the Juan Fernández flora, the Swede Carl Skottsberg (1928)
was the first to shed some insight on the pollination biology of L. fernandeziana. Later, Wiens (in Crawford et al., 1994
and in Stuessy et al., 1998
) added comments based on unpublished data, but no comprehensive study has been done.
In the course of three recent expeditions to Robinson Crusoe Island, we studied L. fernandeziana in the field and examined basic aspects of its reproductive biology. We addressed the following questions: (1) What kinds of flowers does it have and how are they arranged on the plant?, (2) Do the flowers offer rewards?, (3) What is its breeding system?, (4) How is pollination accomplished?, (5) And finally, what are the implications of these data for the assessment of the reproductive strategies of this rare species and its future conservation? As indicated above, the many features of this relictual species place it among the primitive angiosperms (Cronquist, 1981
). Thus, studies of Lactoris could lead to an improved understanding of early angiosperm evolution. Allozyme studies revealed no variation within the species (Crawford et al., 1994
). DNA studies showed only low variation for length in the intergenic spacer and for restriction sites in the 18S–25S genes of rDNA and for the presence of amplified bands (Brauner, Crawford, and Stuessy, 1992
). Consequently, information on reproductive biology is fundamental to designing a program that preserves the genetic potential of this very rare species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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Pollen/ovule (P/O) ratio and pollen viability
In three recent expeditions to the Isla Robinson Crusoe (January 1991, January 1996, and December/January 1997), we fixed buds in 70% ethanol and collected small samples for the study of flower morphology and inflorescence distribution. Buds examined for P/O ratios were near anthesis; thus, pollen was mature but anthers had not dehisced. Pollen quantity was estimated using Anderson and Symon's (1989)
modification of Lloyd's (1965)
technique. A minimum of three buds per specimen were examined. With the aid of a dissecting microscope, all ovules were counted.
The plants bear both female (F) and hermaphroditic (H) flowers. Thus, the standard method of determining the P/O ratio had to be modified. Buds were sampled from nine specimens representing an unknown number of plants (some specimens were taken from the same plants). The pollen and ovule numbers were estimated/counted from a minimum of three F and three H buds per specimen. From a total of 22 specimens, the number of F and H flowers was counted (1115 and 933, respectively). There are two P/O ratios that can be calculated. Either of these can use the tetrad number or the pollen number for P, the former if one wishes to know the number of "delivery units" and the latter when considering the individual gamete "packages" (i.e., pollen grains for the male gametes and ovules for female gametes). Obviously, the P/O will be 0 for the F flowers (no anthers). For H flowers, it will be the number of tetrads or pollen grains (4 x tetrads) divided by the number of ovules. However, it is also of interest to know the P/O ratio per flower on a plant (i.e., the P/O when all the H and F flowers are combined). We calculated this value using the known ratio of H flowers to F flowers. The ratio was derived from the large sample of plants we measured as follows: pollen number x percentage of H flowers on a plant, and ovule number x one (because both H and F flowers bear ovules).
Pollen viability was estimated as the percentage stainability of 100 grains from each of ten flowers using aniline blue in lactophenol (Hauser and Morrison, 1964)
.
Experimental crosses
Lactoris fernandeziana Phil. seeds are difficult to germinate, and the plants very difficult to grow. As a consequence, we performed all experimental manipulations on naturally occurring populations on Isla Robinson Crusoe. Given the relatively inaccessible habitat, some of the manipulations were difficult.
Branches with unopened flowers were tagged and bagged with nylon net bags either with 0.3 x 0.3 mm openings (fine mesh bags) or 1 x 1 mm openings (large mesh bags). The former excluded visitors and likely most airborne pollen; the latter excluded larger potential floral visitors, but allowed the passage of airborne pollen. Three days later, after the flowers had opened, the field sites were revisited and self (geitonogamous) crosses were performed in female and hermaphroditic flowers in the fine mesh bags by applying pollen from recently opened anthers (using the anthers themselves as pollen applicators) to stigmata on the same plant. After 48 h, the field sites were visited a third time, and the pollinated flowers were collected and fixed in 70% ethanol for fluorescence analysis of pollen tube growth.
Fluorescence
Gynoecia were softened with 8 mol/L NaOH for 1 h at 60°C in a water bath, rinsed, and stained in aniline blue-0.1 NK3PO4 for 2 h (Martin, 1959
). The carpels were dissected from the flower in glycerin on glass slides, separated from each other, and flattened. Pollen tubes were examined under an epifluorescence microscope.
Floral anatomy
Flowers were fixed in 70% ethanol, dehydrated in an ethyl alcohol-xylol series, and embedded in Paraplast (Oxford Labware, St. Louis, Missouri). Serial cross- and longi-sections were cut at 10 µm, mounted serially, and stained with safranin-fast green-hematoxylin and observed in a compound microscope.
Floral visitors
Around 100 plants were observed in the field for more than 50 h, during 1991, 1996, and 1997 in the sites given in the Appendix. Periods of observation ranged from 10 min to 1 h and were done during daylight hours (from 1000 to 1600).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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94 µm ± 13.5 in length (N = 10, from three specimens). Upon anthesis, the papillae are turgid (Fig. 1A, C, E), but later shrivel (Figs. 1F, 2A, C).
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= 2.36 ± 0.10 and 2.44 ± 0.36 mm, respectively). However, there are significant differences in total flower length (from sepal attachment to the pedicel to the end of pistil): female = 2.7 ± 0.16 mm, hermaphrodite = 3.5 ± 0.24 mm (t = 4.1263**, P 
0.05).
Flower sex and distribution
Lactoris normally has abundant flowers, although our general impression is that there are fewer flowers on plants growing in deep shade. We studied 16 dried specimens (collected in 1990, 1991, and 1996) from several different populations, and in 1997, more than 50 live plants from at least five different populations and found that each genet always has both flower types, female and hermaphrodite. That is, this species is gynomonoecious, not polygamomonoecious or polygamodioecious, as others have cited.
Flowers are either solitary or borne in small cymose axillary inflorescences (monochasia) composed of two to three flowers (Fig. 1B, Table 1). The inflorescences are subtended by short papery bracts similar to leaf stipules. When three flowers are found, they sometimes represent true three-flowered inflorescences (thus, with one subtending bract), but in most instances they consist of two inflorescences each subtended by a bract: one of one flower, and the second of two flowers (Fig. 1B). We analyzed 1156 inflorescences from 22 specimens for a total of 2048 flowers. Overall, the percentage of female (F) vs. hermaphrodite (H) flowers showed a slight excess of female flowers (F = 54%: H = 46%, Table 2), a difference that, when analyzed with a t test, is not significant (t = 0.49, P 0.05). A chi-square test indicated that there are significant differences from the expected ratio of 1:1 (
2 = 41.401, P 0.01). However, only three of the 22 samples, those with comparatively few hermaphrodite flowers (listed at the end in Table 2) were responsible for this result. When these three samples are removed, the 2 test is not significant.
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Ovules, pollen, and P/O ratio
Ovule counts revealed that there is no significant difference in the number of ovules in female vs. hermaphroditic flowers ( = 17.2 ± 1.37 and 18.1 ± 1.38, respectively, or 17.7 per flower). The P/O ratio of female flowers is obviously 0; there are no pollen-bearing anthers. The P/O ratio of hermaphroditic flowers is 712 tetrads/ovule or 2848 pollen grains per ovule. For a plant as a whole, taking into account the ratio of 54% F flowers and 46% H flowers, the P/O per flower (F and H flowers combined) would be: 46% x 712 (or 2848)/1 x 17.7, which results in 335 tetrads/ovule or 1339 pollen grains/ovule, respectively.
Breeding system
Pollen viability is very high ( = 96% ± 2.08, N = 1000). Artificial self-pollinations (geitonogamous crosses, N = 123) performed with female (N = 90) and hermaphroditic (N = 33) flowers indicate that the species is self-compatible (91 and 86% crosses successful, respectively). All four pollen grains in the tetrad can germinate (Fig. 2D). Pollen tubes often were observed to reach the ovules (Fig. 2B), and, although we could not follow later development, we assume that they produce normal fruit and seed set. Based on our observations, and those of others (e.g., Skottsberg, 1928
; D. Wiens in Crawford et al., 1994
), plants in the field have very high fruit set. Given the pollen tube growth data cited above, the observation of maturing fruits in flowers with dried anthers and the abundance of the fruit set, we assume that both female and hermaphroditic flowers set fruit easily and equally. From studies in January 1996, we observed that each follicle bears one to six mature (with endosperm) seeds ( = 3.6 ± 1.50, N = 25). The dark brown seeds are small and light, and dispersal may be carried out by wind, gravity, or water (the habitats are all on very steep slopes).
Pollination
The hermaphroditic flowers are protogynous, with whitish stigmatic papillae prominent on the female-phase flowers, i.e., after anthesis (Fig. 1C, E). The anthers are much shorter than the pistils and open apparently after the stigmata are no longer receptive (the papillae are no longer prominent [Figs. 1F, 2A, C]; however, no specific receptivity tests were done). Thus, the flowers are dichogamous. The anthers open extrorsely by longitudinal slits (Fig. 1F) and are positioned 1.6 mm from the inward- or downward-facing stigmata (the flowers are usually pendant below the branches, resulting in the stamens effectively being held above the stigmata). Thus, the flowers are also herkogamous, but positioned such that gravity could move pollen tetrads from stamens to stigmata.
The flowers offer no reward: they do not produce nectar, oils, nor extraordinary amounts of pollen. Furthermore, their size, color, and position are not attractive to visitors. In 50+ h of field observation we never observed floral visitors. Nevertheless, 64% of open-pollinated control flowers have tetrads (Fig. 2A, C) and germinating pollen grains on their stigmata (N = 30 flowers from two individuals). Furthermore, the number of tetrads per carpel ( = 4.9 in one individual and 6.9 in the other, grand mean = 5.9 ± 4.94, range = 0–10/stigma) is more than sufficient to fertilize the 4–8 ovules/ovary. In addition, these counts were made on much-handled, alcohol-preserved flowers. This means that other tetrads may have been present, but were abraded off stigmata during handling.
In addition to Lactoris pollen, we found pollen grains in monads from at least three different species on the stigmata (Fig. 3B showing one of these). This fact indicates that the pollen of several species in the understory is distributed abiotically and that Lactoris stigmata can collect various windborne pollen.
Experimental studies with large mesh bags showed the same percentage of flowers with pollen tubes (60%) as with the controls. In addition, the average mean number of tetrads per stigmata ( = 5.6 ± 6.58 tetrads) in the large mesh bags is not statistically different from tetrads on stigmata of control flowers.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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indicated that it has "apparently truly hermaphrodite" flowers, "apparently female" and "apparently male" on the same plant, i.e., the species is polygamomonoecious. He described the male flowers as having "six fully developed stamens and rudimentary ovaries"; however, broader experience with Lactoris flowers and a study of the specimen he cited (K) show that the "apparently male" flower is more likely a hermaphroditic flower with somewhat smaller carpels rather than "rudimentary ovaries." Later reports cite Lactoris as either polygamomonoecious (Engler, 1888
; Hutchinson, 1964
; Muñoz Pizarro, 1966
; Kubitzki, 1993
), gynomonoecious (Skottsberg, 1928
, 1953
; Takhtajan, 1997
), or polygamodioecious (Cronquist, 1981
; Lammers, Stuessy, and Silva, 1986
; Zavada and Taylor, 1986
; Brauner, Crawford, and Stuessy, 1992
; Crawford et al., 1994
; Mabberley, 1997
), although some of these studies did not focus on the flowers and others did not study specimens. Our conclusions agree with Skottsberg's (1928
, 1953)
and Takhtajan's (1997)
observations. Analyses of more than 80 individuals from several populations confirm that the species is gynomonoecious. As with most species, however, there is floral variation; here, a small percentage of hermaphrodite flowers have only three stamens instead of six, and we have observed abnormal hermaphrodite flowers with different numbers and sizes of carpels or stamens.
Richards (1997)
indicated that 3% of the angiosperms are gynomonoecious. Gynomonoecy is widespread only in species with complex coadapted inflorescences in which there is specialization for pollination, such as for some entomophilous Asteraceae and Apiaceae. This syndrome does not characterize Lactoris. Gynomonoecy seems to be equally rare on islands: 3.6% of the genera in the Hawaiian flora are reported as such (Carlquist, 1974
), and in the Juan Fernández only two other species (Chenopodium crusoeanum Skottsb. and C. nesodendron Skottsb.) are known to be gynomonoecious.
Female flowers of Lactoris are significantly smaller than perfect flowers. This phenomenon has been observed in many gynodioecious and gynomonoecious species (Darwin, 1877
; Baker, 1948
; Delph, 1996
), and in some fully dioecious species (Baker, 1948
; Bawa and Opler, 1975
; Delph, 1996
). Flower size dimorphism can affect pollinator visitation (cf. Delph, 1996
); that would not seem to apply here where no pollinators have been recorded. Furthermore, the flower size features cited as different in Lactoris are only indications of total flower length, not real differences in the size of the "attractant unit" as is the case in entomophilous species. Perhaps the size difference simply reflects a developmental constraint in the sense that the hermaphroditic flowers must be larger to accomodate the androecium as well as the gynoecium.
The proportion of female and hermaphroditic flowers is similar among plants of Lactoris fernandeziana. In some other diclinous species, for example in andromonoecious Solanum hirtum, studied carefully by Diggle (1993)
, subsequent floral sex expression of flowers produced later responds to the degree of pollination and fruit set of flowers produced earlier. In this Solanum a larger number of fruits developing on first-opening flowers results in the subsequent production of a higher proportion of staminate flowers. The environment might be less significant in effecting differences in sex expression in wind-pollinated species like L. fernandeziana because most of the flowers open simultaneously, i.e., there is not a protracted period of flowering, or sequential gender-based flowering, where there might be an opportunity and advantage to temporal alterations of gender/sex ratio adjustment.
Breeding system
Even with a nondirectional pollination system like anemophily, gynomonoecy increases the opportunity of outcrossing over a purely hermaphrodite-flowered system. Gynomonoecy in Lactoris could also represent a feature carried over from ancestors. However, lacking a consensus on the closest relatives of Lactoris (e.g., see Stuessy et al., 1998), such hypotheses are difficult to make. Unisexual flowers and syndromes of monoecy and dioecy do occur in a number of families of the Magnoliales, Piperales, and Laurales (Cronquist, 1981
).
In light of several reproductive features of Lactoris (i.e., gynomonoecy, protogyny, herkogamy, absence of visitors), Skottsberg (1928)
suggested the possibility of apogamy for Lactoris. However, our open pollination data indicate that the flowers reproduce sexually and presumably that pollination and fertilization take place normally. Furthermore, Tobe et al. (1993)
found no embryologic evidence that Lactoris is agamospermous.
Our experimental studies indicate that L. fernandeziana is self-compatible: geitonogamous pollinations produce tubes that reach the ovaries in both hermaphrodite and female flowers. In addition, single plants cultivated at the Royal Botanical Gardens at Kew set seed without manipulation, further confirming self-compatibility (T. Hall, personal communication, Royal Botanic Gardens at Kew). Autogamy is facilitated by self-compatibility and hindered by herkogamy and protogyny. This combination leads primarily to geitonogamous pollinations. Such pollinations may be more successful if the pollen transfer occurs in multigrain units, such as tetrads, as opposed to monads (Kress, 1981
). Thus, the successful transfer of only 4–5 tetrads allows for full fertilization. The high seed set of the species, also reported by Skottsberg (1928)
and Wiens (>70%; in Crawford et al., 1994
), may be explained in this way. Wiens (in Crawford et al., 1994
) also commented that plants in shaded habitats set somewhat fewer seeds than those in full sunlight; although overall flowering is lower in the shade, we did not observe lower seed set.
Genetically, the potential disadvantage of facultative geitonogamy is that all, or at least more, of the resultant seeds will be full sibs. Allozyme analyses demonstrate that there is great uniformity among the extant populations, as for most of the endemics studied on the island (cf. Crawford et al., 1994
). Among the factors suggested to account for low allozymic diversity in rare endemic species (Hamrick et al., 1991
), founder effect, genetic drift associated with small population sizes, and a selfing breeding system, seem to apply in Lactoris. At present, most presumed populations sizes in Lactoris are small (10–20 plants; Crawford et al., 1994
; Stuessy et al.,
1998; our data) and there are ± 1000 living plants. Three length variants of nuclear ribosomal DNA occur in populations throughout the island, and each population studied was fixed for one of two variants (Brauner, Crawford, and Stuessy, 1992
). According to these authors, it is likely that the variants were present in the ancestor of the species, but which variant was fixed in populations may be dependent on founder effects and/or drift following the establishment of small populations. Random Amplified Polymorphic DNA (RAPD) variation was low but present (Brauner, Crawford, and Stuessy, 1992
), indicating that genetic changes have occurred within single populations of Lactoris.
The P/O ratios obtained with the total number of pollen grains and the number of tetrads are within the range established by Cruden (1977)
for obligate and facultative xenogamy, respectively. Given the species is self-compatible, the P/O values may not be real indicators of reproductive behavior. Instead, the P/Os may be high (in fact very high for a self-compatible, mostly geitonogamous species) as an indication of dependence on the abiotic forces of the wind to distribute outcross pollen among flowers. That is, in this species, any opportunity for even a small percentage of outcrossing will depend on anemophily (see more below). The higher pollen to ovule ratio is obviously advantageous given the relative imprecision of anemophily for pollen delivery.
Pollination
Skottsberg (1928)
cited no visitors, but he suggested that "perhaps small beetles or flies are the pollinators." Carlquist (1964)
mentioned that "the presence of nectary areas cannot be established unequivocally from dried material, but small cells with staining reactions suggestive of nectary cells were observed at the base of stamens and perianth segments." In our extensive field observations, we did not detect either visitors or nectar in any flower, and subsequent limited histological studies have failed to reveal any structures that resemble nectaries. Wiens (in Crawford et al., 1994
) mentioned that "although pollinators have not been studied in detail during the height of flowering, Lactoris appears to have few visitors," but did not elaborate.
From our data and experiments, we conclude that pollination is accomplished through wind and/or rain, two frequent agents on the island. Wind pollination is the more likely, and is generally a significant reproductive mode for remote island floras and early successional systems (Carlquist, 1966
; Whitehead, 1969
, 1983
; Regal, 1982
; Barrett, 1995
). Although anemophily is less common in the Galápagos islands (McMullen and Close, 1993
), it does characterize important percentages of species in Hawaii, Juan Fernández, and New Zealand floras (Carlquist, 1974
). Anemophily increases with both latitude and elevation (Whitehead, 1983
), which could be some explanation of its paucity on the near-equatorial Galápagos Islands. Self-compatibility, monoecy (Niklas, 1985
), and grouping of flowers to branch tips are all features common to wind pollination. Niklas (1985)
also notes that nonclonal species (such as Lactoris) benefit from near monotypic populations.
As suggested by Skottsberg (1928)
, in L. fernandeziana "the possibility of wind pollination is not completely ruled out, but almost all of the characteristics of anemophiles are absent." Indeed, despite a number of anemophilous features (reduced perianth, relatively large stigmatic surface, absence of bright color, simultaneous blooming of flowers, dry unornamented pollen, monoecy, absence of reward, gregariousness of the plants—Faegri and van der Pijl, 1979
; Whitehead, 1983
; Niklas, 1985
; Proctor, Yeo, and Lack, 1996
), in other traits, Lactoris is less typical (relatively low P/O, included anthers, stigma not feathery, flowers hidden and not entirely condensed on the branches, pollen in tetrads, larger number of ovules/ovary). The pollen structure of Lactoris is unusual for wind-pollinated plants, although not unique. Tetrads of Typha latifolia are transfered by wind (Cox, 1991
) and are larger than those of Lactoris, measuring 40–50 µm in diameter (Erdtman, 1952
). Tetrads were also found to be wind dispersed in a dioecious species of Ericaceae from southern Chile (Arroyo and Squeo, 1987
). The size of Lactoris tetrads falls well within the range of anemophilous pollen (20–60 µm), and they are much smaller than many fern spores that are also dispersed by wind. The wet dense overstory forest (at least for some populations) could make pollen movement by wind as difficult as it is in some rain forest anemophiles (reviewed by Whitehead, 1983
). However, there is precedence for wind pollination in other understory species (Corner, 1952
; Zapata and Arroyo, 1978
; Bawa and Crisp, 1980
). Also, plants in many of the Lactoris populations do not grow under a forest canopy, and are much exposed to the strong, predictable, and constant wind coming off the South Pacific. Finally, the bottom line in terms of pollination is that there simply are few significant biotic pollinators on the island (other than the two species of hummingbird—Colwell, 1989
), and no floral visitors have been documented for Lactoris.
The position of the flowers and inflorescences is important in pollination. For the hermaphroditic flowers, the pendant geometry puts anthers bearing the dry pollen above stigmata. Thus, gravity could account for intrafloral pollen transfer if not prevented by dichogamy. In terms of flowers overall, 69% are either hermaphroditic, or are borne in two- or three-flowered inflorescences where at least one of the flowers is hermaphroditic (Table 1). Further, for inflorescences of more than one flower, 93% bear at least one hermaphroditic flower. Thus, hermaphroditic flowers are next to each other, or more to the point, female flowers are next to hermaphrodites in virtually all instances. Furthermore, examination of the distribution of the single female flowers, or of the few all-female inflorescences, shows that they are often near other inflorescences that contain at least one hermaphroditic flower. Intra-inflorescence distances are usually very small (averaging 5.8 ± 1.26 mm, for a sample of 25 inflorescences). The planar branch structure creates a microenvironment between the flexible branches in which pollen could be moved very easily among the flowers. In sum, there are pollen sources immediately adjacent to almost all of the flowers in an inflorescence. The dry pollen can fall from the large openings in the anthers directly onto the stigmata within the same flowers (though there may be sufficient protogyny or herkogamy to prevent that) or onto adjacent female or hermaphroditic flowers. In addition, most of the flowers are open at about the same time, i.e., flowering is simultaneous. This is a feature characteristic of, and obviously, conducive to, effective wind pollination (Whitehead, 1983
). Our experimental studies showed that unmanipulated flowers from which insects had been excluded by large mesh bags did have tetrads on stigmata. Thus, wind (or rain) may be the abiotic (and only) means of pollen transfer.
Most of the pollination probably represents geitonogamous selfing. As suggested above, some autogamy might occur because of the pendant nature of the flowers, i.e., the floral orientation putting stigmata below anthers may overcome their spatial separation. In either case, the result would be genetically identical seeds. Given that Lactoris grows on steep slopes, it is also possible that a certain degree of xenogamy might occur, if wind gusts (or rain drops) in the understory carry tetrads off to more distant plants. Even if the pollen gets lifted high enough up into the air to be carried to other parts of the island, the distances are not great, certainly not as great as for typical wind-pollinated species (Proctor, Yeo, and Lack, 1996
). However, the available data on genetic diversity (Brauner, Crawford, and Stuessy, 1992
; Crawford et al., 1994
) indicate homogeneity higher than that of typical wind-pollinated species (Kress, 1981
).
Although there is some support for anemophily as primary in some early angiosperms (Meeuse, 1972
), the current view is that wind pollination is derived from insect pollination—and many times (Cronquist, 1988
). If the primitive angiosperms to which Lactoridaceae is related (Piperales, Magnoliales, Aristolochiales, Laurales; Stuessy et al., 1998
) are presumed to be insect pollinated (Crepet, 1983;
Gottsberger, 1988
), we would conclude that anemophily is derived. That is speculative, given that there is still much to learn about the reproductive biology of Lactoris as well as that of its closest relatives. However, other wind-pollinated species and the ecological conditions favoring anemophily seem to have been present at the time, sometime prior to the Maestrichian, and place, perhaps in Western Gondwana, of Lactoris diversification (Lammers, Stuessy, and Silva, 1986
).
Berry and Calvo (1989)
suggested that the shift from insect to wind pollination in Espeletia could be related to low pollinator availability at high elevations. None of the elevations on the Juan Fernández are high in the absolute sense. However, Lactoris grows near the highest, most exposed, ridge tops on Isla Robinson Crusoe. Furthermore, by virtue of being a small, but abrupt land mass in a very vast ocean, many habitats are buffeted by constant and strong winds, and as unsuitable for small biotic (insect) pollinators as any continental montane habitat. And, to reiterate, no biotic pollinators have ever been reported for Lactoris.
Conservation biology
Lactoris fernandeziana is rare, but considerably less so than previously thought (Stuessy et al., 1998
). If our conclusions are correct, it is self-compatible and mostly geitonogamous. This makes effective pollination easy. The high seed set seems to support this conclusion. It is also wind pollinated. It is subject to the unfortunate habitat destruction and pervasive invasion of exotics that is plaguing many of the Juan Fernández native species, but, by virtue of anemophily, it is not subjected to the ramifications of habitat degradation that would also reduce biotic pollination services (e.g., Buchmann and Nabhan, 1996
). Again, the high seed set seems to bear out effective wind pollination. With a small island, relatively few plants growing in clumped populations, wind pollination, and self-compatibility, it is not surprising that the isozyme and molecular studies indicate a great deal of genetic homogeneity. Thus, this species benefits from a reproductive system that facilitates high seed set. In spite of this, like many rare species (Hamrick et al., 1991
), Lactoris is relatively homogeneous. From the seed set data, it does not seem that inbreeding depression is being expressed in terms of limiting seed set. However, seed germination is not easy, and cultivation of the plants from seed is almost impossible (Stuessy et al., 1997
), even on Robinson Crusoe Island in the CONAF greenhouses (I. Leiva, personal communication, CONAF, Isla Robinson Crusoe, Chile), where plants have not yet been successfully grown to maturity. Furthermore, even with good seed germination, young seedlings perish of unknown causes relatively early (Stuessy et al., 1997
). These phenomena may reflect exacting growth or soil requirements, or they may be manifestation of inbreeding depression. There is one report of seedlings in the wild (D. Crawford, personal communication, Ohio State University). However, the plants do grow luxuriantly in their native habitats, so either seedling establishment is effective and/or plants are long lived. Although Lactoris is not in immediate danger of extinction, it is confined to one small place in the world. In the Cretaceous, it was presumed to be more common among the Gondwana flora (Zavada and Benson, 1987
), but for unknown causes has all but disappeared. Perhaps the balance between reproductive success and environmental factors is delicate and can be easily broken, a situation that could be repeated once again in its last island refuge, ending with the extinction of this species.
What we have learned about the reproductive biology means that features of the reproductive system do not stand in the way of successful propagation. Thus, conservation efforts should be focused on identifying appropriate growing conditions, and perhaps on vegetative propagation. Increasing population size by cuttings (an easier method of propagating this species, T. Hall, personal communication, Royal Botanic Gardens at Kew), and perhaps tissue culture techniques may facilitate broadening the base of living representatives in the Juan Fernandez Islands and elsewhere.
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APPENDIX |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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