Recurrent polymorphic mating type variation in Madagascan Bulbophyllum species (Orchidaceae) exemplifies a high incidence of auto‐pollination in tropical orchids

The transition from outcrossing to self‐fertilization is one of the most common evolutionary changes in angiosperms. The orchid family exemplifies this evolutionary trend but, because of a general lack of large‐scale surveys on auto‐pollination in orchid taxa, the incidence and modes of auto‐pollination among (sub)tropical orchids remain poorly known. In the present study, we assessed the frequency and mode of auto‐pollination within and among species of a largely monophyletic group of Madagascan Bulbophyllum. The capacity for autonomous fruit set was investigated by bagging experiments in the greenhouse and the field, complemented with detailed floral micromorphological studies of the gynostemium. Our survey comprises 393 accessions, representing at least 78 species, and thus approximately 37% of the species diversity of the genus in the Madagascan region. Our studies revealed that mating type is directly related to gynostemium structure, most often involving the presence or absence of a physical barrier termed ‘rostellum’. As a novel and unexpected finding, we identified eight species of a single lineage of Madagascan Bulbophyllum (termed ‘clade C’), in which auto‐pollinating morphs (selfers), either lacking a rostellum or (rarely) possessing a stigmatic rostellum, co‐exist with their pollinator‐dependent conspecifics (outcrossers). We hypothesize that auto‐pollination via rostellum abortion has a simple genetic basis, and probably evolved rapidly and recurrently by subtle changes in the timing of rostellum development (heterochrony). Thus, species of clade C may have an intrinsic genetic and developmental lability toward auto‐pollination, allowing rapid evolutionary response under environmental, perhaps human‐disturbed conditions favouring reproductive assurance. Overall, these findings should stimulate further research on the incidence, evolution, and maintenance of mating type variation in tropical orchids, as well as how they adapt(ed) to changing environmental conditions. © 2014 The Authors. Botanical Journal of the Linnean Society published by John Wiley & Sons Ltd on behalf of The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 242–258.     

寒兰花器官发生及花发育过程的研究

为了揭示寒兰的成花机理,利用石蜡切片和花芽实体解剖记录了濒危植物寒兰花芽分化和发育的过程,并着重观察唇瓣和合蕊柱早期及中期的发育(在合蕊柱伸长之前)。结果表明:寒兰花芽分化沿着花序轴从下往上可分为4个阶段:花序原基分化,花原基分化,花被片分化和合蕊柱形成。唇瓣分化分为3个阶段:褶片分化,侧裂片分化和色块形成。唇瓣侧裂片和褶片产生较晚,与退化雄蕊可能没有关系。在合蕊柱形成过程中,首先分化出花药,随后分化产生中心皮顶部,侧心皮顶部,并形成花柱道,最终分化出蕊喙和黏盘。     

Floral morphology of southern African Orchideae. I. Orchidinae

The floral morphology of the southern African genera of Orchidaceae-Orchideae-Orchidinae ( Brachycorythis, Schwartzkopffia, Neobolusia, Schizochilus, Holothrix and Bartholina ) is surveyed paying special attention to the gynostemium. Ontogenetic data are provided for a number of species that appear to be essential in formulating a proper interpretation of the gynostemium. The floral architecture is shown to be basically similar to that of the (much better known) European representatives of the subtribe. This, however, does not fully apply to the homology of the lateral gynostemium appendages ("auricles"): In Brachycorythis, Neobolusia and Schizochilus these develop like in Orchis and Dactylorhiza. Their prominent sculptured portions originate from dorsal stamen outgrowths and correspond to filament excrescences. Structures obviously homologous to lateral inner stamens can be recognized in the early ontogeny, but are in the mature flower incorporated in the 'arch' connecting the lip with the gynostemium. In contrast, in Holothrix and Bartholina the gynostemium appendages correspond entirely to staminodes, while the filament excrescences are missing. It is also shown that the 'concave' stigma said to be characteristic of the Orchidinae is in fact ± convex or even pad-like, but is generally positioned in a cavity under the rostellum. The 'erect' anther (the main diagnostic feature of the Orchideae) is reflexed up to 45° in some taxa. Affinities of the genera are briefly discussed. The generic separation of Schwartzkopffia and Neobolusia from Brachycorythis does not appear justified. Neobolusia virginea is obviously misplaced in the respective genus, and eventually merits generic status. The affinities of Schizochilus remain ± obscure at the moment. Bartholina appears to be merely a small group of specialized Holothrix species.     

Developmental studies in orchid flowers I: Epidendroid and vandoid species

The floral development of 47 epidendroid and vandoid orchids was studied by means of scanning electron microscopy, paying special attention to the early development of the gynostemium (column) and its appendages. The following main conclusions are drawn: the lateral appendages of the adult gynostemium are homologous with the two lateral stamens of the inner whorl; their primordia are present even in species which lack prominent appendages in the adult gynostemium (incorporation of the sta-minodial primordia into the gynostemium during development). Ventral appendages observed in some species are supposed to be vestiges of the adaxial stamens on account of their early initiation. It is confirmed that the rostellum is the upper part of the median stigma lobe and that the lip corresponds to the inner median tepal. The affinities of the epidendroid and vandoid orchids are briefly discussed.     

Evolution,pollination, and systematics of the tribeNeottieae (Orchidaceae)

The various classifications of the orchid tribeNeottieae are reviewed and a new classification is proposed that divides the tribe into three subtribes,Neottiinae, Limodorinae, andCephalantherinae, based primarily on characters of the column (gynostemium). A cladistic analysis illustrates that these three subtribes are more closely related to one another than either is to any other group in subfam.Neottioideae, although there are very few apomorphic characters for the tribe. Pollination biology is also discussed showing links between breeding systems and distribution. There is also a possible role between column and labellum morphology and the emergence of a deceptive pollination syndrome from one of reward.     

Stigma behavior in Mimulus aurantiacus (Scrophulariaceae)

The bilobed stigma of many species in the order Scrophulariales closes in response to touch by an animal pollinator. In hummingbird-pollinated bush monkey flower, Mimulus aurantiacus (Scrophulariaceae), closure is rapid, occurring within seconds of tactile stimulus. We investigated the proximate causes of stigma closure and subsequent reopening in M. aurantiacus, as well as potential costs and benefits of stigma closure for female fitness. Stigma closure is elicited by both touch and pollen, but closure in response to pollen is much slower, requiring 0.5–1.5 h. Stigmata reopen within 2.5–4.5 h if touch, but no pollen, is applied. Upon receipt of pollen, most stigmata remain closed for the remaining lifetime of the flower, even if less pollen is received than is needed for full seed set. Those stigmata that do reopen after pollination generally require between 20 and 28 h to do so, much longer than for unpollinated stigmata. Reopening after pollination appears to be a response to low seed set rather than to low pollen load. Natural pollination of stigmata manipulated to prevent closure shows that closure does not increase capture of pollen or seed set. In fact, closure reduces the average pollen load received by flowers. Despite this, there is no evidence that stigma closure has any negative effect on female fitness in terms of seed set or germinability. Hypotheses for the adaptive significance of stigma closure are discussed. Understanding proximate causes of stigma closure and reopening is essential in the evaluation of these hypotheses.     

More than meets the eye: a morphological and phylogenetic comparison of long‐spurred,white‐flowered Satyrium species (Orchidaceae) in South Africa

Superficial similarities among unrelated species are often a result of convergent evolution and can cause considerable taxonomic confusion. A case in point is Satyrium eurycalcaratum , described here as a new species, which has been confused with several other Satyrium spp. with similar long‐spurred, white flowers. A phylogenetic analysis, based on molecular data, indicated that S. eurycalcaratum is not closely related to any of the species with which it has been previously confused. A comparative analysis of morphological characters in the seven South African Satyrium spp. with long‐spurred, white flowers showed that each of these, including S. eurycalcaratum , is characterized by a unique combination of traits. Despite the similarity in pollination syndrome characters, such as spur length and flower colour, variation in rostellum structure was particularly pronounced and four distinctive forms were present. There was no phylogenetic signal in patterns of interspecific rostellum variation, as some closely related species had different rostella, whereas some distantly related species shared similar rostellum structures. We therefore conclude that the use of rostellum traits in conjunction with phylogenetic evidence can resolve species delimitations among orchid species that share the same pollination syndrome. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 166 , 417–430.     

The gynostemium of Hemipiliopsis purpureopunctata and Senghasiella glaucifolia, two taxonomically disputed species of Habenariinae (Orchidaceae)

The gynostemium structure and ontogeny of two taxonomically disputed orchids, Hemipiliopsis (= Habenaria ) purpureopunctata and Senghasiella (= Habenaria ) glaucifolia , are described and illustrated by scanning electron micrographs. The early gynostemium ontogeny of Hemipiliopsis purpureopunctata is shown to be fundamentally similar to that of the species of the tribe Orchideae that have been previously studied. This includes the initiation sequence of sepals, petals and lip, form and orientation of anthers, three-lobed condition of median carpel apex, and presence of auricles and basal bulges. During the later developmental stages some differences occur. The stigma processes of Senghasiella glaucifolia are united into a tongue-shaped organ, and the lateral rostellum lobes of Hemipiliopsis purpureopunctata protrude forwards with their viscidia positioned above the spur-mouth. Based on gynostemium characters, the generic rank of Hemipiliopsis was confirmed, but that of Senghasiella was not supported.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 147 , 191–196.     

Insect pollination and floral mechanisms in South African species ofSatyrium (Orchidaceae)

The morphologically diverse flowers in the genusSatyrium reflect adaptations to a wide range of pollinators. Several recently discovered pollination systems inSatyrium are described and illustrated here; these include pollination by solitary bees, carrion flies, butterflies and moths. Two basic types of floral mechanism are recognised inSatyrium: (1) Species pollinated by lepidoptera and birds have long floral spurs and plate-like viscidia seated in lateral rostellum notches; these viscidia become attached to the proboscis or bill of the pollinator. (2) Species pollinated by flies and bees have relatively short floral spurs and globose viscidia seated in terminal rostellum notches; these viscidia become attached to the face, thorax or eyes of the pollinator.     

Autonomous self‐pollination in the nectarless orchid Pogonia minor

The pollination biology of the nectarless orchid Pogonia minor was investigated in central Japan. The investigation revealed that the solitary flowers failed to attract pollinators, while high rates of fruit set were observed in the natural population. Comparable levels of fruit set were obtained in bagged, artificial self‐pollinated and artificial cross‐pollinated plants, indicating that the species is not pollinator‐limited for fruit set under natural conditions. Autonomous self‐pollination in P. minor resulted from a reduced rostellum, which allowed contact between the pollinia and the stigma. Self‐pollination is thought to be an adaptive response that provides reproductive assurance under conditions of pollinator limitation. Since pollen limitation is generally known to be frequent among deceptive orchids, strong pollen limitation is probably a driving force in the autonomous self‐pollination mechanism in the nectarless orchid P. minor.     

Floral morphology and ontogeny in Orchidaceae subtribe Disinae

Floral morphology and ontogeny in Orchidaceae subtribe Disinae. The flower structure and development of 24 species of the orchid subtribe Disinae are described and illustrated by drawings and scanning electron micrographs with special attention being paid to the gynostemium. The morphogenesis of this subtribe is fundamentally similar to that of the closely related tribe Orchideae. This includes the initiation of the auricles on the anther base in a dorsolateral position, and hence their interpretation as being mere appendages of the filament. The keel connecting the petals and the gynostemium plus its protrusion is considered homologous to the inner lateral staminodeS. Presumed vestiges of the adaxial staminodes were detected in one specieS. A peculiarity of the Disinae is that the entire apex of the median carpel develops into the rostellum, whereas its stigmatic portion emerges from the median carpel below the rostellum in later stages. The main diagnostic feature of the group is the reflexed position of the mature anther. However, it is shown here that this anther movement occurs in the later stages and that the initial anther is erect.     

Temporal variation in pollinarium size after its removal in species of Bulbophyllum: A different mechanism preventing self-pollination in Orchidaceae

InBulbophyllum involutum andB. ipanemense (Orchidaceae), two closely related species, shortly after removal the pollinarium has a diameter of approximately twice that of the entrance of the stigmatic cavity, requiring a mean time of 105 to 135 minutes to shrink in width and allow pollination. Because the pollinators of these species remain for some minutes in the same flower after removing the pollinia this mechanism, previously unknown inOrchidaceae, may be very important in preventing self-pollination. This mechanism does not occur inB. weddellii, and the pollinator does not remain in the flower after removing the pollinia. The smaller diameter of the stigmatic cavity inB. involutum reduces by 50% the chances of interspecific pollination withB. weddellii, and interspecific crossing is strictly unidirectional. This is important in maintaining isolation between these sympatric species, which share the same pollinators and have synchronized flowering.     

Stigma closure and re-opening in Oroxylum indicum (Bignoniaceae): Causes and consequences

The study of plant responses to touch, particularly the responses of leaves, stems, and roots, has a long history. By contrast, floral responses are relatively unexplored. Stigma closure is common in the Bignoniaceae, but the factors influencing it are not well understood. We investigated factors influencing stigma closure and reopening and its effects on pollen receipt in seven Oroxylum indicum trees near Hat Yai, Thailand. The effects of pressure, conspecific and heterospecific pollen, and pollen load (the amount of pollen deposited) on stigma behavior were examined in 270 flowers (of the total 430 flowers evaluated in the entire study). Pressure alone resulted in faster closure than did conspecific pollination and faster reopening than did heterospecific pollination. Stigmas never reopened after conspecific pollination. Pollen load had no effect on stigma behavior. Stigmas discriminated between conspecific and foreign pollen; they reopened only after pollination with the latter. A manipulative experiment revealed that stigma closure did not affect the number of conspecific pollen grains received. We also counted pollen tubes in styles that were either hand-supplemented with outcross conspecific pollen or open-pollinated. Pollen tube numbers were highest after light pollination (～900 grains), indicating that interference among pollen grains may occur after pollination with very heavy loads (>6000 grains). Possible fitness consequences of these responses are discussed.     

Pollination mechanism in southern Brazilian orchids which are exclusively or mainly pollinated by halictid bees

The pollination process is documented in three sympatric orchids found in Curitiba, Paraná State, southern Brazil. InCampylocentrum aromaticum (Epidendroideae: Angraeciinae) andPrescottia densiflora (Spiranthoideae: Prescotiinae) halictid bees act as main pollinators with minor contributions from other insects, such as Syrphid flies. Because of the pollinarium and column structure inCyclopogon diversifolius. (Spiranthoideae, Spiranthinae), halictid bees are the only insects capable of performing pollination. In the three studied species pollinarium removal and deposition are achieved by interaction between the column and bee's mouth parts. Morphological and ecological flower features in agreement with this pollination mode are discussed and pollination by halictid bees is suggested to occur in a number of South American orchids.     

蝴蝶兰花发育的分子生物学研究进展

蝴蝶兰花非常独特且高度进化,如萼片瓣化、瓣片特化为唇瓣、雌雄蕊合生成合蕊柱及子房发育须由授粉启动等,是单子叶植物花发育研究的理想材料。近年来蝴蝶兰花发育分子生物学取得了重要进展。该文就近年来国内外有关蝴蝶兰开花转换及花器官发育相关基因研究以及B类基因与兰花花被的进化发育关系方面的研究进展进行综述。研究表明:MADS基因在蝴蝶兰开花转换及花器官发育过程中起重要作用,推测其中的DEF(DE-FICIENS)-like基因早期经过2轮复制,形成了4类不同的DEF-like基因,进而决定兰花花被属性。蝴蝶兰花发育分子生物学的深入研究,将极大地利于通过基因工程手段提高蝴蝶兰花品质如花色改良及花期调控等,推动分子育种进程。     

Developmental studies in orchid flowers II: Orchidoid species

The floral development of 11 species of Orchidoideae (sensu Rasmussen 1985) was studied by means of SEM, paying special attention to the early development of the gynostemium and its appendages. In contrast to the staminodes found in epidendroid and vandoid orchids, the 'auricles' of the tribe Orchideae are developed on the dorsal side of the fertile anther and therefore are not interpreted as staminodes. Presumed vestiges of the staminodes corresponding to those of the Epidendroideae and Vandoideae are differentiated in early developmental stages, but remain inconspicuous structures later on. The three-lobed rostellum originates entirely from the median carpel. The outstanding systematic position of the tribe Orchideae is briefly discussed.     

Spiranthes ovalis var. Erostellata (Orchidaceae), a new autogamous variety from the Eastern United States

Examination of flowers from herbarium specimens ofSpiranthes ovalis revealed two kinds of column structure. Some plants, including the type, have flowers with a normally developed rostellum and viscidium; other plants that have flowers in which these structures are entirely lacking are here described asSpiranthes ovalis var.erostellata. Self-pollination in the new variety was proven by pollinia removal and observation of plants in insect-proof cages. The var.erostellata has a somewhat smaller perianth with sepals 3.5–5 instead of 4–6.1 mm long and is more common and more widespread than the typical variety.     

The interplay of pollination,costs of reproduction and plant size in maternal fertility limitation in perennial <Emphasis Type="Italic">Paeonia officinalis</Emphasis>

Although several factors can limit female fertility in perennial plants, rarely have they been jointly studied in a single species over several years. In this study we experimentally manipulate seed production and simultaneously analyse the potential contribution of pollen limitation, costs of reproduction and plant size to variation in seed output over a 3-year period in the perennial herb Paeonia officinalis, in southern France. Since this rare species is threatened by forest closure in many sites we also examine the causes of female fertility variation in relation to habitat closure (open habitat vs. woodland). P. officinalis has a partial self-incompatibility system and only very low ability for autonomous self pollination in the absence of pollinators. However, supplementary pollination of individual plants in three consecutive years did not significantly increase seed production above natural levels. Forest closure was associated with a decline in ovule and seed production, which again was not due to pollen limitation since supplementary pollination had no significant effect on seed set in the woodland habitat. Comparison of the maternal fertility of plants which were previously excluded from reproduction with those which were hand pollinated to maximise seed set in two previous years produced no evidence that seed production in year three is limited by costs associated with prior reproduction. Likewise, flowering probability was not related to prior seed production but was however positively related to plant size. The absence of any influence of pollen limitation or prior reproduction on seed production suggests that sub-maximal seed production in long-lived perennial herbs may be part of a size-dependent strategy that maximises life-time seed production and fitness without compromising survival.     

Histological and Micro-CT Evidence of Stigmatic Rostellum Receptivity Promoting Auto-Pollination in the Madagascan Orchid Bulbophyllum bicoloratum

Background

The rostellum, a projecting part of the gynostemium in orchid flowers, separates the anther(s) from the stigma and thus commonly prevents auto-pollination. Nonetheless, as a modified (usually distal) portion of the median stigma lobe, the rostellum has been frequently invoked of having re-gained a stigmatic function in rare cases of orchid auto-pollination. Here it is shown that a newly discovered selfing variant of Madagascan Bulbophyllum bicoloratum has evolved a modified rostellum allowing the penetration of pollen tubes from in situ pollinia.

Methods

Gynostemium micro-morphology and anatomy of selfing and outcrossing variants of B . bicoloratum was studied by using light and scanning electron microscopy and histological sections. Pollen tube growth in the selfing variant was further observed via X-ray computed microtomography (micro-CT), providing 3D reconstructions of floral tissues at a micron scale.

Findings

Selfing variants possess a suberect (‘displaced’) rostellum rather than the conventional, erect type. Very early in anthesis, the pollinia of selfers are released from the anther and slide down onto the suberect rostellum, where pollen tube growth preferentially occurs through the non-vascularized, i.e. rear (adaxial) and (semi-) lateral parts. This penetrated tissue is comprised of a thin layer of elongate and loosely arranged cells, embedded in stigmatic exudates, as also observed in the stigmatic cavity of both selfing and outcrossing variants.

Conclusions

Our results provide the first solid evidence of a stigmatic function for the rostellum in orchid flowers, thereby demonstrating for the first time the feasibility of the micro-CT technique for accurately visualizing pollen tube growth in flowering plants. Rostellum receptivity in B . bicoloratum probably uniquely evolved as an adaptation for reproductive assurance from an outcrossing ancestor possessing an erect (non-receptive) rostellum. These findings open up new avenues in the investigation of an organ that apparently re-gained its ‘primordial function’ of being penetrated by pollen tubes.     

Floral closure in response to temperature and pollination in Gentiana straminea Maxim. (Gentianaceae), an alpine perennial in the Qinghai-Tibetan Plateau

Floral closure may be induced by pollination and various other factors, but is rarely studied comprehensively. Different kinds of floral closure should have various effects on reproductive fitness of plants. Two contrasting types of floral closure were observed in the flowers of Gentiana straminea Maxim. in the eastern Qinghai-Tibetan Plateau. The first type occurred prior to pollination during both gender phases, in response mainly to decreasing air temperatures. Flowers closed when decreasing temperatures approached 20°C and subsequently began to reopen the following day during mid-morning when air temperatures warmed to approximately 13–15 °C. This kind of floral closure can protect pollen grains on either stamens or stigmas, increasing fitness of both male and female. Following pollination, permanent floral closure occurred, although there was a delay between the dates of pollination and permanent closure, during which flowers continued to show temporary closure in response to low temperature episodes. The time required for permanent, pollination-induced closure varied according to the age of the gender phase, including a prolonged time before closure if pollination occurred early in the female phase. The retaining of permanent closed flowers increased both approaching (to inflorescences) and visiting (to unpollinated flowers) frequencies of individual plants when with fewer open flowers and the persisting corolla is further beneficial for seed sets of these pollinated flowers. Thus, two separate types of floral closure, one in response to environmental cues and the other in response to the age of each gender stage, appeared to have a strong influence on reproductive fitness in this species. These results revealed a different adaptive strategy of alpine plants in the sexual reproduction assurance in addition to the well-known elevated floral longevity, dominant role of more effective pollinators and increased reproduction allocation in the arid habitats.