Chapter 4 Discussion
4.4 Self-pollination and gender divergence in Daphne
Considering the floral structure of D. arisanensis, I suspect the self-pollinating nature had played an influential role in sexual differentiation. The eight stamens are situated at upper part of the tube and block the opening, and the position of stigma is right beneath the anthers, with its receptive surface directly exposed (Figure 2-4). Despite a slight spatial separation, pollens would fall on stigma spontaneously after anther-dehiscence, which had also been reported in the congeneric D. rodriguezii (Rodríguez-Pérez & Traveset, 2011), D. gnidium (Roccotiello et al., 2009), D. jezoensis (Kikuzawa, 1989) and D. kiusiana var. atrocaulis (in this study), but not in D. laureola (Alonso &
Herrera, 2001). This self-pollinating situation could be further enhanced by pollinator-mediation. Bulk self-pollens would be carried to the stigma when pollinators or their mouth parts enter the floral tube to collect nectar. Presence of tiny insects (e.g., thrips) within flowers would also bring about a similar consequence (Roccotiello et al., 2009).
In D. laureola, Alonso and Herrera (2001) suggested that although selfing within flower was non-spontaneous, it could be simply achieved by the visiting of pollen vectors.
Moreover, considering the simultaneous floral display on a single individual, and the
successively-foraging behavior of pollinators, self-pollination among flowers of same plant (i.e. geitonogamy) should be also frequent (Harder & Barrett, 1995).
Despite the potential benefit from reproductive assurance, self-pollens might clog the stigma and have both physically and physiologically negative effects in sexual reproduction (Barrett, 2002b). Outcrossing pollens have to compete with the self-pollens in receptive surface and germinating resource. In self-compatible species, high inbreeding rate would lead to reduction in genetic diversity of offspring and even inbreeding depression (Charlesworth & Charlesworth, 1987). Though outcrossing rate is guaranteed under self-incompatibility system, presence of self-pollens could still bring about reduction in reproductive success through occupying the available stigmatic space for outcrossing pollens or interfering the pollen-tube growth (Barrett, 2002b). Studies in Narcissus (Barrett et al., 1996; Sage et al., 1999) also indicated that late-acting incompatibility system could operate through degenerating embryo sacs after self-pollination, resulting in discounting of potentially fertile ovules.
From the perspective of male-function, pollens intercepted by self-stigma are no longer available for dispersal, which is also a negative consequence of self-pollination termed pollen discounting (Barrett, 2002b). Thus, the fertility of clogged gynoecia is suspected to be lower in the self-incompatible Daphne, while a high inbreeding rate is expected in the self-compatible species. The former prediction had been verified in the gynodioecious Northern-Japanese D. jezoensis, Kikuzawa (1989) demonstrated that emasculation before anther-dehiscence significantly raised fruit-set in bisexual flowers, which had retrieved the female fecundity from pollen clogging. However, no such effect was detected in the hermaphroditic Mediterranean D. rodriguezii (Rodríguez-Pérez &
Traveset, 2011), fruit-set did not differ between emasculated and non-emasculated flowers followed by hand-pollination. A possible explanation is that pollen-clogging in
self-incompatible Daphne could be partially overcome by a sufficient load of foreign pollens, since each ovary contained only one ovule. Hand-pollination using bulk pollens might not directly reflect the natural condition, especially in areas with severe pollen limitation. The later prediction in self-compatible species was supported in the gynodioecious Mediterranean D. laureola, extremely low outcrossing rate and strong inbreeding depression were both reported in the bisexual flowers (Medrano et al., 2004).
In this study, in vivo pollen germination experiments had revealed the receptivity to self-pollens in male flowers of D. arisanensis, suggesting a probable self-compatible scenario in the ancestor. Polyploidization had been suggested to associate with breakdown of ancestral self-incompatibility and the promotion of further sexual divergence in Lycium and Fragaria (Ashman et al., 2013; Mable, 2004; Miller & Venable, 2000), while chromosome examination had certified the diploid D. arisanensis was not the case. Since direct evidence about ancestral state cannot be obtained, investigations in reproductive biology of the closely related species would be especially valuable (Igic et al., 2008). In Taiwan, the hermaphroditic D. kiusiana var. atrocaulis in Yangmingshan displayed high fecundity under autonomous selfing, nearly half of the bagged flowers set fruits, which might contribute a relative high inbreeding rate in the natural population. Another native hermaphroditic species, D. genkwa, was also supposed to be self-compatible in our preliminary observation. A transplanted individual of D. genkwa in laboratory was video-recorded setting fruits spontaneously without effective pollinators (Appendix Figure A-1), and those seeds also succeeded in germinating. As the most closely related species of D.
arisanensis has not been identified yet, comprehensive studies in the vast diversity of Daphne, especially in China, in which up to 40 endemic species documented (Wang et al., 2007), are fundamental to give a whole picture about reproductive strategies and sexuality evolution in this genus.
Selective pressures for inbreeding avoidance, outcrossing promotion and resources allocation had been addressed for the principal forces driving gender divergence (Barrett, 2002a; Bawa, 1980). The easily self-pollinated floral structure in Daphne might contribute to high inbreeding rate and strong interference between sexual organs. Self-incompatibility would be preferred under this condition but only passively guarantee outcrossing. Spatial or temporal separation of male and female function would benefit more actively in promoting effective pollination and optimizing gender performance (Barrett, 2002a; Bawa, 1980). The coexistence of self-incompatibility and gender dimorphism in the gynodioecious D. jezoensis also supported this differential perspective (Kikuzawa, 1989), since inbreeding avoidance solely could not be interpreted as the main selective pressure driving gender divergence in a preexisted self-incompatible system (Barrett, 2002a, 2002b).
Sexual dimorphism in Daphne would evolve when the environment was no more suitable for the ancestral pollinating condition (either compatible or self-incompatible), or just substituting it with the more effective reproductive strategy.
Considering the coexistence of morphologically bisexual and male-sterile plants in D.
arisanensis, the evolution toward dioecism probably had undergone gynodioecy pathway.
In the theoretical model, male-sterile mutant (i.e. female) could spread in the ancestral hermaphroditic population when the benefit of relieving from self-pollination and the increased seed fitness compensate the loss of paternal output (Charlesworth &
Charlesworth, 1978). The increased frequency of females in population would further exert selective pressure for enhanced male function in other bisexual plants, thus different degrees of allocation to maleness in the bisexual flowers (gynodioecy) or even female-sterile individuals (subdioecy) could be presented at the same time (Spigler & Ashman, 2012). This intermediate stage could be temporally stable before transition to fully
differentiation, and the relative maleness of bisexual flowers in gynodioecious population, or the relative fertility of purely male plants in subdioecious population, could be interpreted as the evolutionary position toward dioecy (Spigler & Ashman, 2012). It is noteworthy that pollinator service must be sufficient enough to maintain the fecundity advantage in females at the first step, because reproduction in the unisexual mostly rely on outcrossing. Severe pollen limitation might retard and even drive backwards the gender-diversifying progress (Alonso & Herrera, 2011; McCauley & Taylor, 1997).
Therefore, geographical intraspecies variations in gender divergence might exist among populations and represent a complex hermaphrodite-gynodioecy-dioecy continuum, which had also been demonstrated in D. laureola (Alonso & Herrera, 2011; Cuevas et al., 2014).
Despite having morphologically bisexual flowers, the functional sexual system of extant D. arisanensis in studied populations had already evolved to predominant dioecy.
The rarely restored female function in male plants (i.e. feminization) seemed to be restricted in feminization event, which was unlikely a simple remnant of bisexual flowers during the gynodioecy pathway. It would be controversial in directly referring the ecological factors of extant populations to the ancestral condition. However, considering the great variations in floral traits among populations, I’m also wondering whether the extant dioecy system could be actually diversified in Taiwan, specifically with existence of the relatively ancestral gynodioecy or subdioecy state. Indeed, I had examined a confusing specimen of D. arisanensis from Peitawushan (北大武山, Pingtung County), which seemed to have fruit-setting bisexual flowers (C.H. Chen 567, HAST). Although this could be a mistake of examining the floral morph in a dried specimen, and it had not been confirmed with field observations yet, there might still exist hermaphroditic individuals and even populations in Taiwan. If so, comparative investigations into the
mating systems, and the post-pollination ontogeny of embryos between the bisexual and male flowers will be especially helpful to elucidate the evolutionary history in gender divergence of D. arisanensis.
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APPENDIX
Table A-1. Record of nocturnal pollinator observations.
Each framed-cell represents a successfully recorded individual. “None” means no flower visitor was found during the period. “Unidentified”
represents a moth individual without taxonomic identification.
Population Date Period
Menophra sp. x2 Paradarisa comparataria rantaizanensis x2 Cyana formosana Menophra sp.
Psyra conferta Tanaoctenia haliaria Menorpha sp. (unidendified)
(unidendified) (unidendified) (unidendified)
2016/1/26 19:30-21:30 (none)
2016/2/17 18:30-20:30 (none)
2016/3/7 19:30-22:00 Loxaspilates arrizanaria Orthosia alishana Orthosia reticulata fuscovestita
2016/4/1 19:30-22:00 (none)
2016/4/19 19:00-21:00 (none)
Harutalcis fumigata x2 Loxaspilates arrizanaria Loxaspilates montuosa Paradarisa comparataria rantaizanensis x2
Geometridae Geometridae Geometridae
2017/2/20 20:00-22:00 Harutalcis fumigata (unidendified) (unidendified) (unidendified)
2017/2/14 18:30-21:00 (unidendified)
Table A-2. Chromosome numbers in genus Daphne.
Data are gathered from Flora of Japan and Index to Plant Chromosome Numbers (IPCN) website.
Table A-3. A selection of Daphne arisanensis specimens.
Some of the flowering D. arisanensis specimens are listed here to represent the variations in flowering time. Symbol “+’ means specimen fits with the description and “-” means non-fitting. Presence of “large flowers” and “linear leaves” was judged on the basis of a typical size in Guanyuan population, which may rely on personal experiences. Ambiguous conditions are left unnoted. Herbaria: HAST = Biodiversity Research Center, Academia Sinica, Taipei; TAI = National Taiwan University, Taipei; TAIF = Taiwan Forestry Research Institute, Taipei; TNM = National Museum of Natural Science, Taichung.
Leaves Locality Collection No. Herbarium
JAN 1986/1/27 - - - Pingtung S. F. Huang 3360 TAI
Figure A-1. Continuous video-recording of Daphne genkwa inflorescences.
(A) Flower buds on recording-day 0. (B) Day 1. (C) Day 2. (D) Day 3. (E) Day 5.
(F) Developing fruits on Day 7. (G) Day 11. (H) Day 15. Arrowheads indicate the swollen ovaries.