Conservation genetics and taxonomic status of the rare Kentucky lady's slipper: Cypripedium kentuckiense (Orchidaceae)

Cypripedium kentuckiense is a recently described rare orchid found in Arkansas (predominantly) and in eight other states. Much debate has focused on whether this taxon should be recognized as a distinct species or considered to be an extreme manifestation of the variability present in the widespread taxon Cypripedium parviflorum var. pubescens. In this study, 12 isozyme loci were analyzed for 14 populations of C. parviflorum var. pubescens and eight populations of C. kentuckiense. These data were used to examine the genetic similarity of these taxa, assess whether isozyme data support the continued recognition of C. kentuckiense as a distinct species, and assess whether a newly discovered disjunct Virginia population of C. kentuckiense is genetically isolated from other C. kentuckiense populations. The isozyme data revealed that the two taxa are very closely related with a high interspecific genetic identity. However, C. kentuckiense populations contain a subset of the variation present in C. parviflorum var. pubescens, and they have expected levels of heterozygosity that are one-quarter that of C. parviflorum var. pubescens populations. Cypripedium kentuckiense also possesses one widespread unique allele and a unique multilocus genotype. These data suggest that C. kentuckiense should be recognized as a distinct species, possibly of recent origin from C. parviflorum. Lastly, the isozyme data support the hypothesis that gene flow between the Virginia population and other populations of C. kentuckiense has been restricted.     

Costs of reproduction in the pink lady's slipper orchid (Cypripedium acaule): defoliation,increased fruit production,and fire

An earlier experiment with the pink lady's slipper orchid demonstrated that plant leaf area was lowered only after successive years of increased fruit production. This result suggested that the cost of reproduction was small in relation to the energy budget of the plant. To test this idea, plants were subjected to experimental hand-pollination treatments to increase fruit set as well as leaf removal treatments to decrease the energy budget of plants. Changes in plant size in years 2 and 3 and, to some extent, rate of flowering, were determined by a combination of initial plant size, leaf removal treatments in year 1, fruit production in year 1, and damage from an unplanned fire in year 2. Plants that had both leaves removed and produced a fruit in 1987 decreased in size in the following 2 years in comparison with other treatment groups. The cost of fruit production was not apparent in plants that had only one or no leaves removed. Plants apparently have to be put into severe physiological stress in order for a cost of reproduction to appear in the following year. The cost of producing one fruit was a decline of plant size in the following year of 30 cm², which is very similar to our previous experiment using a different design. An additional experiment failed to find evidence that these plants increase their photosynthetic rate to compensate for the loss of leaves or the cost of maturing fruit. Published experiments in both the greenhouse and the field that failed to find a cost of reproduction should be reevaluated in terms of the intensity of treatment imposed and the overall energy budget of the plant in field situations.     

Dynamics and viability analysis of transplanted and natural lady's slipper (Cypripedium japonicum) populations under habitat management in South Korea

To assess the effectiveness of conservation‐based transplantation of the endangered orchid (Cypripedium japonicum), we compared the morphology, physiology, stem‐count change, and population viability of natural versus transplanted populations undergoing habitat management (repeated removal of competing understory vegetation) between 2009 and 2015 in South Korea. The restored site had lower transmitted light and soil humidity than the natural site. The natural and transplanted populations differed in leaf morphology and total chlorophyll content (natural: 1.00 ± 0.04, restored: 0.53 ± 0.06). No recruitment occurred during the monitoring period. Population viability tended to decrease in the restored population (λ_G = 0.97, μ = ?0.05, σ² = 0.036) and increase in the natural population (λ_G = 1.07, μ = 0.03, σ² = 0.075). The repeated removal of competing understory vegetation had different effects on leaf traits, abundance, and reproductive properties of the endangered orchids in both populations. Notably, habitat management increased the stem count and flowering rate in natural C. japonicum but did not increase the fruit‐setting rate. Thus, despite repeated habitat management efforts (removal of competing understory vegetation), we conclude that the population viability of transplanted populations of the endangered orchid C. japonicum had poor long‐term viability compared with naturally occurring populations, a difference that is mainly attributed to inappropriate transplant‐site selection.     

Genetic diversity and structure of the endangered lady's slipper orchid Cypripedium japonicum Thunb. (Orchidaceae) in Japan

Cypripedium japonicum Thunb. (Orchidaceae), once a common perennial herb, is now designated as endangered throughout most of its distribution due to habitat destruction and fragmentation, and the impacts of horticultural collection. We investigated the genetic characteristics of this species for conservation purposes, using microsatellite markers to examine the genetic diversity and structure of 15 native and 5 ex situ populations in Japan. The results imply that although allelic variation is low in Japanese C. japonicum, sexual reproduction by seed, as well as clonal propagation, may occur in some populations. Both native and ex situ populations were found to be genetically differentiated, indicating that some populations may have experienced recent population declines, genetic fragmentation, or bottlenecks. The degree of genetic drift from the putative ancestral population, inferred through STRUCTURE analysis, was more pronounced in northern populations than in southern populations. Some of the ex situ conserved populations exhibited a low degree of differentiation from ancestral native populations. Our results imply that conservation of C. japonicum in Japan is best supported by maintaining individual populations and their unique genetic characteristics.     

Pollination of a slippery lady slipper orchid in south-west China: Cypripedium guttatum (Orchidaceae)

Cypripedium guttatum was studied in north-west Yunnan at 3490 m a.s.l. The flowers are rewardless 'kettle traps'. The structure of the lip, where pollinators are temporarily kept prisoner, and the method of their capture, are unusual in being Paphiopedilum - rather than Cypripedium -like. The deceptive orchid does not mimic any of the diverse flowers concurrently blooming in the habitat, all being visited by the polylectic pollinators of C. guttatum , viz . Lasioglossum virideglaucum, L. clypeinitens and L. sauterum , besides two additional probable pollinators and four non-pollinating visitors (all Halictidae; three new species). The bees got caught when they tried to climb onto the staminode and their forelegs slid down its slippery downward ridges, causing them to tumble to the pouch bottom. To leave, they had to climb a tunnel leading past the stigma to the anthers where a pollen smear was acquired while extruding themselves from the narrow exit. The similarities with myiophylous Paphiopedilum are discussed in view of the possibility that they may foreshadow evolutionary transitions between melittophily and myiophily found in slipper orchids.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 148 , 251–264.     

Floral biology and histochemical analysis of Vanilla edwallii Hoehne (Orchidaceae: Vanilloideae): an orchid pollinated by Epicharis (Apidae: Centridini)

The genus Vanilla is the most diverse in Vanilloideae, with ca 90 species distributed among tropical regions. Despite their economic importance, studies on pollination of Vanilla are very scarce and data on pollinators of species endemic to Brazil are lacking. Based on fieldwork and laboratory investigations, the floral biology of V. edwallii was studied. The pollinators and pollination process were recorded at the Serra do Japi reserve, state of São Paulo, southeastern Brazil, and the presence of floral reward was also investigated. Vanilla edwallii blooms in summer. The lateral inflorescences produce up to four pale green flowers. The white labellum is united to the base of the column forming a mentum. In the studied population V. edwallii is pollinated by Epicharis (Hoplepicharis) affinis, where the males exhibit a territorial behavior, defending flowers from other possible flower visitors. The pollen is deposited on the scutellum of bees when they abandon the flower. The mentum region is dry, suggesting no nectar production. The only secretory structures are osmophores dispersed on the inner surface of the lip responsible for production of a sweet fragrance, which together with color and morphology of flowers is related to bee attraction. The labellum is rich in mucilaginous cells, while the mucilaginous substance is retained inside the cells. The histochemical analysis also detected the presence of phenolic compounds and starch concentrated mainly at the adaxial surface of the lip and around the vascular bundles.     

Pollination of Cypripedium plectrochilum (Orchidaceae) by Lasioglossum spp. (Halictidae): the roles of generalist attractants versus restrictive floral architecture

The pollination of Cypripedium plectrochilum Franch. was studied in the Huanglong Nature Reserve, Sichuan, China. Although large bees (Bombus, Apis), small bees (Ceratina, Lasioglossum), ants (Formica sp.), true flies (Diptera) and a butterfly were all found to visit the flowers, only small bees, including three Lasioglossum spp. (L. viridiclaucum, L. sichuanense and L. sp.; Halictidae) and one Ceratina sp., carried the flower's pollen and contacted the receptive stigma. Measurements of floral architecture showed that interior floral dimensions best fit the exterior dimensions of Lasioglossum spp., leading to the consistent deposition and stigmatic reception of dorsally-placed, pollen smears. The floral fragrance was dominated by one ketone, 3-methyl-Decen-2-one. The conversion rate of flowers into capsules in open (insect) pollinated flowers at the site was more than 38%. We conclude that, while pigmentation patterns and floral fragrance attracted a wide variety of insect foragers, canalization of interior floral dimensions ultimately determined the spectrum of potential pollinators in this generalist, food-mimic flower. A review of the literature showed that the specialised mode of pollination-by-deceit in C. plectrochilum, limiting pollinators to a narrow and closely related guild of 'dupes' is typical for other members of this genus.     

Confronting assumptions about spontaneous autogamy in populations of Eulophia alta (Orchidaceae) in south Florida: assessing the effect of pollination treatments on seed formation,seed germination and seedling development

The breeding system of the terrestrial orchid Eulophia alta was investigated in south Florida where it has previously been reported as an auto‐pollinated species. The effect of breeding system on seed viability and germinability and seedling development was also investigated. Incidences of spontaneous autogamy in E. alta were rare at the study site, resulting in only 7.1% of observed flowers forming capsules. In addition, hand pollination resulted in significantly greater capsule formation when flowers were subjected to induced autogamy (46.4%), artificial geitonogamy (64.3%) and xenogamy at both short (pollen source 10–100 m away; 42.9%) and long (pollen source > 10 km away; 67.9%) distances. Pollen source had little effect on seed viability and germinability or seedling growth rates. However, seed resulting from spontaneous autogamy developed more slowly than seed originating from the other treatments. These data indicate that spontaneous autogamy is rare in E. alta and that naturally forming capsules may be the result of unobserved pollination events. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161 , 78–88.     

Ecological factors affecting the fitness of the threatened orchid Anacamptis robusta (Orchidaceae): Habitat disturbance,interactions with a co‐flowering rewarding orchid and hybridization events

• The food‐deceptive species Anacamptis robusta is threatened in the Balearic Islands, and its habitat has recently been transformed through human disturbance. This study investigated how human disturbance affects the reproductive output of A. robusta and how its fitness is affected by competition with rewarding relatives, fungal infections and hybridization processes.
• To evaluate the impact of habitat loss on plant fitness, data on reproductive measures were obtained in two well‐conserved subpopulations and the unique disturbed subpopulation. Photo‐trapping cameras were installed to determine the floral visitation rate. All flowering individuals in 2019 were georeferenced using differential GPS to examine the influence of geospatial patterns on the reproductive success of A. robusta. In addition, hand‐pollination treatments were performed to evaluate the hybridization between A. coriophora and A. robusta and the origin of A. × albuferensis.
• The human‐disturbed subpopulation of A. robusta had a lower fruit set success than the subpopulations in well‐conserved areas. The presence of A. coriophora is negatively affecting the reproductive output of A. robusta. Moreover, A. robusta can only act as the pollen donor during hybridization.
• The complexity of the ecological system, which is enhanced by the strong pollinator dependence of the threatened species, must be considered when making conservation decisions. Although human disturbance directly affects plant population stability, other ecological issues must be considered, such as pollinator interaction, interspecific competition for pollinators, fungal infection and hybridization events.

     

Life-history evolution in guppies viii: the demographics of density regulation in guppies (poecilia reticulata)

In prior research, we found the way guppy life histories evolve in response to living in environments with a high or low risk of predation is consistent with life-history theory that assumes no density dependence. We later found that guppies from high-predation environments experience higher mortality rates than those from low-predation environments, but the increased risk was evenly distributed across all age/size classes. Life-history theory that assumes density-independent population growth predicts that life histories will not evolve under such circumstances, yet we have shown with field introduction experiments that they do evolve. However, theory that incorporates density regulation predicts this pattern of mortality can result in the patterns of life-history evolution we had observed. Here we report on density manipulation experiments performed in populations of guppies from low-predation environments to ask whether natural populations normally experience density regulation and, if so, to characterize the short-term demographic changes that underlie density regulation. Our experiments reveal that these populations are density regulated. Decreased density resulted in higher juvenile growth, decreased juvenile mortality rates, and increased reproductive investment by adult females. Increased density causes reduced offspring size, decreased fat storage by adult females, and increased adult mortality.     

Minority cytotypes in European populations of the Gymnadenia conopsea complex (Orchidaceae) greatly increase intraspecific and intrapopulation diversity

Background and Aims

Patterns of ploidy variation among and within populations can provide valuable insights into the evolutionary mechanisms shaping the dynamics of plant systems showing ploidy diversity. Whereas data on majority ploidies are, by definition, often sufficiently extensive, much less is known about the incidence and evolutionary role of minority cytotypes.

Methods

Ploidy and proportions of endoreplicated genome were determined using DAPI (4'',6-diamidino-2-phenylindole) flow cytometry in 6150 Gymnadenia plants (fragrant orchids) collected from 141 populations in 17 European countries. All widely recognized European species, and several taxa of less certain taxonomic status were sampled within Gymnadenia conopsea sensu lato.

Key Results

Most Gymnadenia populations were taxonomically and/or ploidy heterogeneous. Two majority (2x and 4x) and three minority (3x, 5x and 6x) cytotypes were identified. Evolution largely proceeded at the diploid level, whereas tetraploids were much more geographically and taxonomically restricted. Although minority ploidies constituted <2 % of the individuals sampled, they were found in 35 % of populations across the entire area investigated. The amount of nuclear DNA, together with the level of progressively partial endoreplication, separated all Gymnadenia species currently widely recognized in Europe.

Conclusions

Despite their low frequency, minority cytotypes substantially increase intraspecific and intrapopulation ploidy diversity estimates for fragrant orchids. The cytogenetic structure of Gymnadenia populations is remarkably dynamic and shaped by multiple evolutionary mechanisms, including both the ongoing production of unreduced gametes and heteroploid hybridization. Overall, it is likely that the level of ploidy heterogeneity experienced by most plant species/populations is currently underestimated; intensive sampling is necessary to obtain a holistic picture.     

Dispersal and population structure of two sympatric species of the mediterranean land snail genus Mastus (Gastropoda, Pulmonata, Enidae)

We studied the population dynamics of Mastus by investigating the effects of population structure, spatial ecology and biology of reproduction on the genetic diversity of two sympatric Mastus species endemic to the island of Crete. Over a period of 27 months, we carried out both mark–recapture and random quadrat sampling techniques in order to assess the dispersal trends, the aggregation patterns, the neighbourhood size and the habitat preferences of these species. There were 154 recorded movements for M. butoti and 114 for M. cretensis . Mean monthly dispersal was estimated at d  = 0.5 m for M. butoti and d  = 1 m for M. cretensis . Both species showed a random dispersal pattern but tended to aggregate in the field. Their populations were found to be highly structured owing to their highly parsimonious dispersal behaviour and the very low population densities, estimated at D  = 2.07 ± 0.16 and D  = 0.73 ± 0.16 individuals m⁻² for M. butoti and M. cretensis , respectively. The neighbourhood size did not exceed 150 individuals for either species. The habitat occupied by each species changed during the active season, but both the immature and the adult individuals of each species seemed to prefer the same habitats throughout the active season. Partial population activation during the active season was observed in both species. We conclude that the population structure, the partial population activation and the species-specific reproductive strategies have a profound effect on maintaining the genetic diversity of the genus' populations.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 83 , 131–144.     

The energetics of asexual reproduction: Pedal laceration in the symbiotic sea anemone Aiptasia pulchella (Carlgren, 1943)

Asexual reproduction of the symbiotic sea anemone Aiptasia pulchella (Carlgren, 1943) was studied in the context of energy budgets determined under a variety of light and feeding conditions. Continuous darkness significantly increased rates of pedal laceration (the method of asexual reproduction), while different feeding rates had no significant effect. Digestive assimilation efficiency averaged 63% for whole brine shrimp, and conversion efficiencies (assimilated energy into biomass) averaged 31%. There were no significant differences between the conversion efficiencies of energy obtained from light or food. The index of reproductive effort (RE) for pedal laceration was very low, ranging from 0.004 to 0.044. This low RE associated with a substantial rate of asexual reproduction may, in part, explain the widespread success of certain sea anemones.     

Size and fecundity variation in populations of Poecilia vivipara Block & Schneider (Teleostei; Poeciliidae) inhabiting an environmental gradient

The reproductive variables [female size (geometric centroid size), fecundity and offspring size (longest diameter of the embryo)] of the livebearer Poecilia vivipara in the quaternary lagoons of northern Rio de Janeiro State varied significantly among sites and were partially associated within and among sites. Mean female size was directly related to salinity. Fecundity (number of offspring) was directly related to female size both within and among sites. The female size × fecundity relationship, however, was not constant among sites. Offspring size was partially and directly associated with female size and salinity. The relationship between fecundity and female size was positively related with fecundity, suggesting a non-linear relationship of these variables when the total sample was considered. The results suggest that life-history differences among sites were more related to different predation regimes than to simple effects of plasticity caused by increased growth in saltwater environments.     

Pollen and nectar as a reward in the basal epidendroid Psilochilus modestus (Orchidaceae: Triphoreae): A study of floral morphology, reproductive biology and pollination strategy

Psilochilus modestus Barb. Rodr. is a basal epidendroid orchid occurring in both the semi-deciduous and Atlantic rain forests of the state of São Paulo, southeastern Brazil. This species presents a perfect flowering synchrony within populations, since all the mature buds of each plant open simultaneously in the morning hours of the same day. These flowers are available only for 1 day and are pollinated by several species of small solitary and social native bees. These bees exploit both pollen and nectar as a reward. The bees collecting pollen promote a higher fruit set and perform mainly self-pollination while those collecting nectar, which are less numerous, are responsible for an increase in cross-pollinations. P. modestus is self-compatible but pollinator dependent. Natural fruit set (open pollination) is low when compared with the numbers obtained under manual self- and cross-pollination. Low fruit set in natural conditions is related to deficient pollen transfer, and pollinator inefficiency seems to be the main factor. Some factors, such as the small amount of nectar produced, the low number of flowers per inflorescence and their availability for 1 day only added to the perfect flowering synchrony seem to be responsible for the increase of cross-pollinations. The offering of both pollen and nectar as a reward can represent a transitional condition in basal Epidendroideae. Based on floral morphology, reward production and pollinator behavior, the relationship of P. modestus with the basal and most derived groups within Orchidaceae is discussed.     

Life span and reproductive cost explain interspecific variation in the optimal onset of reproduction

Fitness can be profoundly influenced by the age at first reproduction (AFR), but to date the AFR–fitness relationship only has been investigated intraspecifically. Here, we investigated the relationship between AFR and average lifetime reproductive success (LRS) across 34 bird species. We assessed differences in the deviation of the Optimal AFR (i.e., the species‐specific AFR associated with the highest LRS) from the age at sexual maturity, considering potential effects of life history as well as social and ecological factors. Most individuals adopted the species‐specific Optimal AFR and both the mean and Optimal AFR of species correlated positively with life span. Interspecific deviations of the Optimal AFR were associated with indices reflecting a change in LRS or survival as a function of AFR: a delayed AFR was beneficial in species where early AFR was associated with a decrease in subsequent survival or reproductive output. Overall, our results suggest that a delayed onset of reproduction beyond maturity is an optimal strategy explained by a long life span and costs of early reproduction. By providing the first empirical confirmations of key predictions of life‐history theory across species, this study contributes to a better understanding of life‐history evolution.     

Insights of the life history in the porcellanid crab Petrolisthes armatus (Gibbes, 1850) (Crustacea: Anomura: Porcellanidae) from the Southwestern Atlantic coast

Petrolisthes armatus (Gibbes, 1850) life history was evaluated based on growth and reproduction. Specimens were captured at Ubatuba (Brazil), identified, sexed and measured: carapace (CL, length; CW, width), larger cheliped propodus (PL, length; PW, width; and PH, height), and 2th and 5th abdominal somite (AW₂ and AW₅, width). Relative growth was studied using CL as independent variable and its relation with other variables (dependents), to estimate puberty size and morphological maturity. Fecundity was represented by number of eggs vs. body size CL, with a better fit using a power function. Considering the 257 specimens analyzed, males were larger than females, and the overall sex ratio was 1:1, with some differences among size classes. Maturity (puberty size) was revealed by: AW₂ × CL (males: 8.6 mm CL; females: 7.6 mm CL); and AW₅ × CL (7.1 and 7.6 mm CL, respectively). Reproduction was continuous, but more intense in rainy season and recruitment occurring in dry season. Fecundity of this species was 228 ± 163 eggs, with better fit by a power function (R² = 0.72). Maturity size and growth differed when a pristine area (Ubatuba) was compared with a polluted area (São Sebastião), in the same Brazilian area (São Paulo State).     

Cost of reproduction in a spring ephemeral species, Adonis ramosa (Ranunculaceae): carbon budget for seed production

BACKGROUND AND AIMS: Spring ephemerals have a specific life-history trait, i.e. shoot growth and sexual reproduction occur simultaneously during a short period from snowmelt to canopy closure in deciduous forests. The aim of this study is to clarify how spring ephemerals invest resources for seed production within a restricted period. METHODS: In order to evaluate the cost of reproduction of a typical spring ephemeral species, Adonis ramosa, an experiment was conducted comprising defoliation treatments (intact, one-third and two-thirds leaf-cutting) and fruit manipulations (control, shading and removal) over two growing seasons. In addition, measurements were made of the movements of carbon assimilated via (13)C tracing. KEY RESULTS: Survival rate was high irrespective of treatments and manipulations. The proportion of flowering plants and plant size decreased as a result of the defoliation treatments over 2 years, but the fruit manipulations did not affect flowering activity or plant size. Seed set and seed number decreased as a result of fruit shading treatment, but the defoliation treatments did not affect current seed production. Individual seed weight also decreased in the second year due to fruit shading. The (13)C tracing experiment revealed that young fruits had photosynthetic ability and current photosynthetic products from leaves were mainly transferred to the below-ground parts, while translocation to fruit was very small even when fruit photosynthesis was restricted by the shading treatment. CONCLUSIONS: Current foliage photosynthetic products are largely stored in the below-ground parts for survival and future growth, and about one-third of the resources for seed production may be attained by fruit photosynthesis. Therefore, the trade-off between current seed production and subsequent growth is weak. The cost of seed production may be buffered by sufficient storage in the below-ground organs, effective photosynthesis under high irradiation and self-assimilation ability of fruits.