Floral features,pollination biology and breeding system of Chloraea membranacea Lindl. (Orchidaceae: Chloraeinae)

Background and Aims

The pollination biology of very few Chloraeinae orchids has been studied to date, and most of these studies have focused on breeding systems and fruiting success. Chloraea membranacea Lindl. is one of the few non-Andean species in this group, and the aim of the present contribution is to elucidate the pollination biology, functional floral morphology and breeding system in native populations of this species from Argentina (Buenos Aires) and Brazil (Rio Grande do Sul State).

Methods

Floral features were examined using light microscopy, and scanning and transmission electron microscopy. The breeding system was studied by means of controlled pollinations applied to plants, either bagged in the field or cultivated in a glasshouse. Pollination observations were made on natural populations, and pollinator behaviour was recorded by means of photography and video.

Key Results

Both Argentinean and Brazilian plants were very consistent regarding all studied features. Flowers are nectarless but scented and anatomical analysis indicates that the dark, clavate projections on the adaxial labellar surface are osmophores (scent-producing glands). The plants are self-compatible but pollinator-dependent. The fruit-set obtained through cross-pollination and manual self-pollination was almost identical. The main pollinators are male and female Halictidae bees that withdraw the pollinarium when leaving the flower. Remarkably, the bees tend to visit more than one flower per inflorescence, thus promoting self-pollination (geitonogamy). Fruiting success in Brazilian plants reached 60·78 % in 2010 and 46 % in 2011. Some pollinarium-laden female bees were observed transferring pollen from the carried pollinarium to their hind legs. The use of pollen by pollinators is a rare record for Orchidaceae in general.

Conclusions

Chloraea membrancea is pollinated by deceit. Together, self-compatibility, pollinarium texture, pollinator abundance and behaviour may account for the observed high fruiting success. It is suggested that a reappraisal and re-analysis of important flower features in Chloraeinae orchids is necessary.     

Pollination biology of Cymbidium goeringii (Orchidaceae) in China

The genus Cymbidium comprises three subgenera with ca. 50 species. Interactions between pollinators and plants have been studied in the two subgenera Cymbidium and Cyperorchis, but only a few studies in the subgenus Jensoa have been reported. Here we report on the reproductive characteristics of C. goeringii (in sub-genus Jensoa) in three populations in the southwest of Hunan Province, China, during the winter from December 2005 to March 2006. Floral phenological and morphological features, behavior of visitors, the breeding system, and fruit sets under natural conditions were studied. The flowers of C. goeringii were strongly fragrant, but did not present any rewards to the visitors. The flowering period of C. goeringii in the studied populations lasted about 40 days, and most flowers (about 60%) opened within 30 days. Flowers opened immediately when temperature increased obviously and reached to about 16 ℃. The flowering time of the pollinated flowers, unpollinated flow-ers and the flowers with pollinarium removed were similar. Two bees of Apidae, Apis cerana cerana (honeybee) and Anthophora melanognatha, were observed visiting flowers of C. goeringii, but only the honeybees performed as pollinators. The honeybees mostly visited the orchid flowers at 10:00–17:00 on sunny days with temperature above 10 ℃. A total of only thirteen visits were observed during 20 days of observation, indicating pollinators were rare. Honeybees directly landed on the upper surface of the labellum and inserted their heads into the flowers between column and labellum, while the hind legs trod on the surface of the curved downward mid-lobe of label-lum. When a honeybee landed on the labellum of a flower, the labellum moved up and down slightly. After the honeybee entered the flower further, its head might touch the foot of the column. At this time, the body of the honeybee was parallel with the upper surface of the labellum. Then the honeybee used its front legs to scratch on the callus ridges on the upper surface of the labellum, and its hind legs hooked the edges of the side lobe of label-lum, trying to exit forcibly from the flower. During exiting process, sometimes the honeybee’s body was arched owing to tension. In this case, the surface of the scutellum came into close contact with the viscidium and then pollinaria, together with the anther cap, were removed. When this honeybee visited next flower, the pollinaria would be adhered to the stigma when it arched its body during the exiting process. Three plant species flowered synchronously with C. goeringii in the studied areas, but their flowers were different with C. goeringii in color or shape. Because C. goeringii is rewardless to pollinators, the flowers probably attract visitors by olfactory stimu-lus. Breeding system experiments showed no spontaneous autogamy and pollination success relying on pollinators in C. goeringii. Artificial pollination resulted in 90% fruit set by induced autogamy, 100% by pollinating within clone, and 100% by xenogamous pollination, respectively. These results indicate that C. goeringii is highly self-compatible and the fruit production is pollen limited. Because pollinators are essential for fruit set of C. goeringii in natural habitats, protection of wild honeybee populations or apiculture is likely a simple but effective strategy to maintain the orchid populations.     

The pollination ofStenorrhynchos lanceolatus (Aublet) L. C. Rich. (Orchidaceae: Spiranthinae) by hummingbirds in southeastern Brazil

Hummingbird pollination is documented for a natural population ofStenorrhynchos lanceolatus Aublet. L. C. Rich. occurring in Rio de Janeiro State, southeastern Brazil. At the study site the plants are pollinated byPhaethornis eurynome (Phaethorninae),Thalurania glaucopis (females only) andLeucochloris albicollis (Trochilinae). The plants offer nectar as a reward and the pollinaria become stuck to the surface of the hummingbird's bill while it is probing the flowers. The orchid population received a few (0–4) hummingbird visits per day, with about 83% of the flowers being pollinated. In spite of the low frequency of visits, the granular structure of the pollinarium plus the behaviour of the most frequent pollinators, which tend to visit all the fresh-looking flowers of each inflorescence, a very high fruiting success was promoted. Experimental evidence suggests that the pollinaria may remain up to 6.30 hours on the hummingbird's bill, enhancing the chances of cross-pollination and long-distance pollen flow.     

The pollination of Habenaria rhodocheila (Orchidaceae) in South China: When butterflies take sides

Habenaria is one of the largest terrestrial genera in the family Orchidaceae. Most field studies on Habenaria species with greenish–white and nocturnal scented flowers are pollinated by nocturnal hawkmoths and settling moths. However, H. rhodocheila presents reddish flowers lacking a detectable scent and fails to fit the moth pollination syndrome. We investigated the pollinators, breeding system, and functional traits of H. rhodocheila in South China and found that two diurnal swallowtail butterflies Papilio helenus and Papilio nephelus (Papilionidae) were the effective pollinators. When butterflies foraged for nectar in the spur, the pollinia became attached between the palpi. A triangular projected median rostellar lobe was found at the entrance (sinus) of the spur of H. rhodocheila. This lobe divided the spur opening into two entrances forcing butterflies to enter their proboscides through the left or right side. When the projection of median rostellar lobe was removed, the site of pollinium attachment changed to the eyes of the butterflies, leading to a higher rate of pollinium removal but lower rate of pollinium deposition. Our quartz glass cylinder choice experiment suggested that visual rather than olfactory cues provided the major stimuli for butterflies to locate these flowers. Hand pollination experiments suggested this species was self‐compatible but pollinator‐dependent. However, the proportion of seeds with large embryos produced in self‐pollinated fruits was significantly lower than in cross‐pollinated fruits, indicating a significant inbreeding depression. Unlike many other orchid species, fruit set was higher than rates of pollinium removal, indicating a high level of pollination efficiency in a species with friable pollinia. Shifts from moth to butterfly pollination in the genus Habenaria parallel other orchid lineages providing insights into the potential for pollinator‐mediated floral trait selection.     

The pollination mechanism in the 'Pelexia alliance' (Orchidaceae: Spiranthinae)

The biological function of the wedge–like, dorsally adhesive viscidium of the neotropical orchid genera Cyclopogon, Pelexia and Sarcoglottis (grouped in the so-called 'Pelexia alliance') is elucidated by the study of the pollination biology of three species occurring in Sao Paulo State, southeastern Brazil. Cyclopogon congestus is pollinated by the bee, Pseudoaugochloropsis graminea (Halictidae), Pelexia oestrifera by workers of Bombus (Fervidobombus) atratus (Apidae) and Sarcoglottis fasciculata by males and females of Euglossa cordata (Apidae: Euglossini). These three species offer nectar as a reward and are self-compatible, though they need pollinators to set fruits. In spite of the difference in flower sizes and in their pollinators' taxonomic groups, the pollination mechanism is essentially the same for these species. The pollinarium adheres to the ventral surface of the bee labrum. The viscidium needs to be dorsally pressed in order to liberate a glue which fixes the pollinarium to a bee. Pollination is achieved by the interaction of the orchid column and the mouthparts of the bees. Fivation to the ventral surface of the labrum is advantageous for the orchid, since it is a difficult place for the bees to clean. Another advantage is that, since the labrum is articulated, when the bees fold and close their mouthparts, the pollinarium remains protected under the bee's head, thus reducing the risks of pollen loss. Since the wedge-like, dorsally adhesive viscidiurn is a characteristic feature of Cyclopogon, Pelexia and Sarcoglottis , it is suggested that some kind of phylogenetic constraint may exist, impeding the occurrence of pollinators other than bees in these orchid genera. All other flower-visiting animals lack the labrum-like structure needed to fix the pollinarium.     

The pollination mechanism in Trigonidium obtusum Lindl (Orchidaceae: Maxillariinae): sexual mimicry and trap-flowers

The pollination process in Trigonidium obtusum Lindl. (Epidendroideae: Maxillariinae) is documented. The flowers are pollinated by sexually excited drones of Plebeia droryana (Meliponinae). When attempting to copulate either with sepals or petals, these bees slip on the waxy perianth surface and become trapped in the funnel-like flower tube. Bees trying to escape from the flowers may instead access the space between the column and lip, fixing the pollinarium on their scutellum. Pollinarium-bearing bees may pollinate the flowers when repeating the above-mentioned steps, leaving pollinia on the concave stigmatic surface, thus effecting pollination. Recently removed pollinaria are too broad to enter the stigma but they begin to dehydrate and within 40 min of removal are small enough to fit the stigmatic cavity. This mechanism prevents insect-mediated self-pollination and promotes cross-pollination. Preliminary evidence based on experiments with cultivated plants suggests that they are self-compatible but that fruit set is pollinator-dependent. The data obtained are discussed in a phylogenetic context. It is suggested that the pseudocopulatory syndrome in Trigonidium could have evolved from rewardless (food advertising) ancestors. Pseudocopulation in the context of the long flowering period of this orchid species (about 7 months) is understandable since the eusocial Plebeia bees produce fertile individuals several times a year.     

A new species of Hapalorchis (Spiranthinae,Orchidaceae) from Colombia

Hapalorchis bicornis, a new species from Colombia, is described, illustrated and placed within the key for the determination of Colombian Hapalorchis species. The information about the distribution and ecology of the new species as well as brief taxonomic notes are provided.     

On the low reproductive success of two Cyrtopodium species (Orchidaceae: Cyrtopodiinae): The relative roles of biotic and abiotic pollination

Rewardless orchid species are pollinated by deception and have reduced reproductive success. Those species that present self-compatibility followed by alternative mechanisms of autogamy may compensate for such episodic allogamy events. This is the case for rain-assisted self-pollination, a rare mechanism in Orchidaceae. In this study, we investigated the reproductive biology of two terrestrial species: Cyrtopodium hatschbachii and C. paludicolum. We studied their reproductive systems, examined pollinators and male and female successes, and ascertained if rain promotes self-pollination. Both species are allogamous but self-compatible. However, natural fruit set is low, not exceeding 1.40% during 4 years of observation. This may result from the reduced frequency of pollinator visitation followed by a high pollinator specificity, because each species was pollinated by a single specialist bee. Although spontaneous selfing did not occur, we verified fruits formed by rain action. The evaporation of the water droplet that accumulates on stigma helps the pollinarium to slip back, promoting selfing. This occurred in approximately 10% of C. hatschbachii fruits, but in C. paludicolum it was recorded only once during three observation years. This indicates that the phenomenon happens at random and may vary temporally. Nevertheless, rain may confer additional fruit set, especially under circumstances of pollinator limitation. Overall, we confirmed deception as a strategy for this understudied genus. In addition, we report a further two cases of rain-assisted self-pollination for Cyrtopodium, for species from the center of diversity of the genus, indicating that it may be more widespread than previously thought.     

Reproductive biology and conservation genetics of Goodyera procera (Orchidaceae)

Goodyera procera is an endangered terrestrial orchid in Hong Kong. Information on its reproductive biology and pattern of genetic variation is needed to develop efficient conservation strategies. Pollination experiments showed that the species is self-compatible, but dependent on pollinators for fruit set. Bagged plants produced no fruits. Artificial pollinations resulted in 92% fruit set through selfing, 94% with geitonogamous pollination, and 95% following xenogamous pollination. Fruit set in the open-pollinated control was 75% at the same sites. Allozyme electrophoresis and random amplified polymorphic DNA (RAPD) were used to evaluate genetic variation and structure of 15 populations of Goodyera procera. Despite its outbreeding system, allozyme data revealed low variation both at the population (P = 21.78%, A = 1.22, and H = 0.073) and species (P = 33%, A = 1.33, and H = 0.15) levels, in comparison with other animal-pollinated outbreeding plant species. However, RAPD variation was relatively high (P = 55.13% and H = 0.18 at the population level, and P = 97.03% and H = 0.29 at the species level). G_ST estimates indicated high levels of genetic differentiation among populations (G_ST = 0.52 and I = 0.909 ± 0.049 based on allozyme data, and G_ST = 0.39 and I = 0.859 ± 0.038 based on RAPD data), much above the average for outcrossing species, suggesting that gene flow was limited in this species. Based on these data, suitable strategies were developed for the genetic conservation and management of the species.     

姜科、闭鞘姜科植物繁育系统与传粉生物学的研究进展

综述了姜科Zingiberaceae植物繁育系统和传粉生物学方面的研究及其进展。姜科有50余属1500多种,是一个泛热带分布的、动物传粉的单子叶植物大科。姜科植物中存在着雌花两性花异株、雄花两性花同株、花柱卷曲性、雄性先熟、自交不亲和等多种性表达方式和花部机制。姜科植物重要的传粉动物包括各种蜂类、天蛾、蝴蝶、鸟类等,不同的传粉动物对应不同的花部特征。在相对有限的研究中,姜科植物展现出了一些独特的传粉和繁育机制,在豆蔻属Amomum、山姜属Alpinia等植物中发现的花柱卷曲性被认为是植物界中一种独特的促进异交的行为机制,在对黄花大苞姜Caulokaempferia coenobialis的研究中发现了植物界中一种全新的花粉滑动自花传粉机制,这些新的研究和发现丰富了我们对姜科植物传粉和繁育系统多样性的认识。本文提出了今后姜科植物繁殖生物学研究的建议:对更多未知姜科植物类群的传粉和繁育系统进行研究;从系统发育的角度开展姜科不同类群植物的传粉和繁育系统的演化研究;对花柱卷曲性这一独特的性多态现象开展全面深入的研究。     

Biotic versus abiotic pollination in Oeceoclades maculata (Lindl.) Lindl. (Orchidaceae)

Geographical variation in the reproductive biology of widespread species often occurs at their distributional boundaries. We sought to determine whether such variation has occurred in an invasive orchid, Oeceoclades maculata, across its naturalized range. We compared its reproductive biology in a Brazilian population with that published for a population on the Caribbean island of Puerto Rico. In the state of São Paulo, O. maculata flowers between December and February, at the height of the rainy season. Similar fruit sets were observed in manual self (76%) and cross (70.4%) pollination treatments. The fruit set of plants protected from both pollinators and rainfall was 6.1%, whereas plants exposed only to rainfall had a fruit set of 41.4%, slightly less than the controls (48.3%). Like the Puerto Rico population, reproduction is primarily through rain‐assisted autogamy, but unlike observations made on the island, outcrossing can eventually occur. We observed two butterfly species (Heliconius ethilla narcaea and Heliconius erato phyllis) pollinating O. maculata. Secretory epidermal cells and trichomes of the spur lumen produced 0.7 µL of 25% (sucrose equivalents) nectar per flower each morning, which was stored in a dilated basal portion of the spur and reabsorbed by the afternoon. Thus, geographical variation in reproductive biology exists across the broad invasive range of O. maculata.     

短柄五加开花及传粉生物学研究

本文对短柄五加（Ｅｌｅｕｔｈｅｒｏｃｏｃｃｕｓｂｒａｃｈｙｐｕｓ）的花期物候、开花式样、访花昆虫种类、访花行为及访花频率进行了观察,对花粉活力、柱头可授性、花粉胚珠比以及传粉效果和繁育系统做了检测。初步结果表明,短柄五加以根茎进行无性繁殖,１个居群为１个无性系。虽自交亲和,但昆虫的访花活动可显著提高座果率。昆虫传粉主要在无性系内异花间进行,属于广义的自花传粉。传粉者为多种不同的昆虫,包括马蜂和胡蜂在内的蜂类、蝇类和甲虫。其花期物候、花粉活力、柱头可授期和蜜汁分泌期等特征使其能够适应环境,从而获得较高的座果率。平均座果率为６５％,饱满种子率为４５．５％。自然条件下未发现实生苗,广义的自花传粉导致的种子质量低下可能是其原因,也是限制短柄五加分布范围的重要因素。     

Reproductive biology and pollination of Utricularia reniformis A.St.‐Hil. (Lentibulariaceae)

Utricularia reniformis is an endemic Brazilian carnivorous plant, most common in high‐altitude grasslands. Knowledge of the reproductive biology of U. reniformis is essential for planning conservation strategies, but it is currently poorly understood. Thus, we studied the floral morphology, floral biology, breeding system and pollination of this species. U. reniformis produces and stores nectar in the flower spur, a classic feature of bee‐pollinated flowers, and we recorded Xylocopa sp. and Bombus sp. as pollinators. Moreover, although it is self‐compatible it is an obligate animal‐pollinated species, as the sensitive stigma avoids self‐pollination. However, in natural conditions reproductive success is low due to the rarity of visits from pollinators. We suggest that the low reproductive success caused by a deficit of pollinators may affect gene flow, causing loss of genetic diversity in U. reniformis.     

The pollination ecology ofBletilla striata (Orchidaceae)

Anthecological observations of an orchidBletilla striata were carried out in Kobe, Japan. The purplish pink flowers were visited by 26 species of insects belonging to Hymenoptera, Diptera and Lepidoptera, although the flower did not offer food rewards of any kind. Candidates for pollination were seven species of aculeate Hymenoptera with a body size that well fitted the space in between the column and labellum. The most effective candidate was the male of the longhorn beeTetralonia nipponensis, judging from its abundance and legitimate intrafloral behavior. The female ofT. nipponensis and the other six species also contributed to promote out-crossing, but to a lesser extent than the maleT. nipponensis, since they occasionally exhibited illegitimate intrafloral behaviors for pollination and also, their flower visitation rate was lower.     

Pollination biology of four sympatric species of Habenaria (Orchidaceae: Orchidinae) from southern Brazil

The pollination process and breeding system of the sympatric Habenaria johannensis, H. macronectar, H. megapotamensis and H. montevidensis was documented for native populations from Rio Grande do Sul, Brazil. All species investigated offer a nectar reward (mean values of total sugars ranging from 18 to 26%) concealed in a spur. Habenaria montevidensis is pollinated by butterflies (Hesperiidae, especially of the genus Urbanus) that carry pollinaria on their eyes; the other three species are pollinated by Sphingidae. Habenaria johannensis is pollinated by the moths Manduca rustica and M. sexta that carry the pollinaria at the base of the proboscis. Habenaria macronectar is pollinated by the moths Eumorpha labrusca and M. cf. lucetius, and these bear pollinaria between the palpi. Habenaria megapotamensis is pollinated by moths of M. cf. lucetius that bear the pollinaria on the proboscis. All species studied are self‐compatible, but pollinator dependent. They also displayed high reproductive success (ranging from 69.48 to 97.40%) and male efficiency factors slightly higher than 1, suggesting that at least one flower was pollinated for each flower acting as pollen donor. At the study sites, the investigated Habenaria spp. are isolated (in terms of pollination) by a set of factors that includes differing floral morphologies, different pollinators and/or different pollinarium placement on the pollinator. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, ??, ??–??.     

Biosystematics of the genus Piperia Rydb. (Orchidaceae)

Piperia is a North American segregate of Habenaria consisting of 5 taxa (4 species and 1 subspecies). The genus is defined and differentiated from other North American genera of the Habenaria alliance by its morphology and pollination mechanism. All taxa have n = 21 chromosomes. Thin-layer chromatography of fresh leaf extracts and a morphological assessment revealed discrete, but variable, taxa. All species are partially interfertile; however, a high degree of sympatry suggests these species are reproductively isolated. One new combination is proposed: P. elongata subsp. michaelii.     

A specialised pollination system using nectar‐seeking thynnine wasps in Caladenia nobilis (Orchidaceae)

• Caladenia is a diverse Australian genus that is exceptional among orchids in having both species pollinated by food‐seeking and sexually deceived insects. Here, we investigated the pollination of Caladenia nobilis, a species predicted to be food‐deceptive due to its large, cream‐coloured and apparently nectarless flowers.
• Pollinator observations were made using experimental clumps of flowers. Measurements of floral colour were undertaken with a spectrometer, nectar was tested using GC‐MS, and reproductive success was quantified for 2 years.
• While C. nobilis attracted nine species of insect, only males of the thynnine wasp Rhagigaster discrepans exhibited the correct size and behaviour to remove and deposit pollen. Male R. discrepans attempted to feed from the surface of the labellum, often crawling to multiple flowers, but showed no evidence of sexual attraction. Most flowers produced little or no nectar, although some may provide enough sucrose to act as a meagre reward to pollinators. Floral colouration was similar to a related Caladenia species pollinated by sexual deception, although the sexually deceptive species had a dull‐red labellum. Reproductive success was generally low and highly variable between sites and years.
• In addition to most visitors being of inappropriate size for pollinia removal, the lack of response to the orchid by several co‐occurring species of thynnine wasp suggests filtering of potential pollinators at the attraction phase. Our discovery of a pollination strategy that may be intermediate between food deception and food reward raises the question, how many putatively rewardless orchids actually produce meagre amounts of nectar?

     

Evolutionary disruption in the pollination system of Vanilla (Orchidaceae)

• Pollination of the pantropical Vanilla has been linked to melittophily and food deception.
• Here we investigated the role of flower traits on the reproduction of Neotropical Vanilla. We also studied the evolution of pollination systems in order to understand the origin of production of flower resources and the diversification of pollinators in this orchid genus. Our study was founded on data of adaptations in flower morphology, production of resources, scent release, pollinators and breeding systems of Vanilla and presenting new data on reproductive biology of V. palmarum. Data on reproductive biology of Vanilla were mapped onto a phylogeny to address our queries on the evolution of pollination systems in this genus.
• Vanilla palmarum shows a mixed mating system, with its facultative autogamous flowers being pollinated by hummingbirds. Its yellow flowers are scentless and produces nectar. Mapping of the pollination system onto trees resulted in one origin for bird pollination and at least two origins for autogamy in Vanilla. Nectar secretion has a single origin in the Neotropical thick-leafed lineage.
• Bird pollination of Vanilla is shown for the first time. The origin of ornithophily within a bee-pollinated clade is supported by flower morphology. Floral transitions to ornithophily have been favoured by the occupation of a distinct niche from that of the other thick-leafed Vanilla species. Despite its specialized pollination, V. palmarum is autogamous. A mixed mating system can promote reproductive assurance in the case of a decline in pollinator populations, or in areas where pollinator services are irregular or absent.

     

Genetic variation in three species of Epipactis (Orchidaceae): geographic scale and evolutionary inferences

The breeding system is expected to strongly influence the genetic structure of plant populations. In the present study, isozyme variation is documented in Danish populations of three species of Epipactis , varying in breeding system from allogamy to obligate autogamy. The allogamous and widespread E. helkborine subsp. helkborine shows high levels of polymorphism. Most of the genetic variation is found within local populations. A hierarchical analysis indicates significant among-population differentiation, but no regional differentiation in E. helkborine is apparent. This may be due to higher levels of gene flow in the past, before forest was fragmented. The ecotype from coastal dunes, E. helleborine subsp. neerlandka , does not differ from E. helkborine subsp. helkborine in any of the examined loci, but it has a significant population inbreeding coefficient that can probably be explained by higher levels of geitonogamy and the possibility of spontaneous autogamy. The entomophilous E. purpurata and the obligately autogamous E. phylhmthes are monomorphic at all loci examined. Several factors, including a founder effect at the time of colonization, high levels of geitonogamy, as well as habitat specialization combined with erratic flowering may have contributed to the lack of variation in E. purpurata. The lack of variation in the autogamous E. phyllanthes is probably due to inbreeding. It is proposed diat autogamy in Epipactis may in some cases have evolved through paedomorphosis of allogamous flowers and that the occurrence of local breeding groups may have facilitated the speciation process.