Embryo development in the lady's slipper orchid, Paphiopedilum delenatii, with emphasis on the ultrastructure of the suspensor

BACKGROUND AND AIMS: Owing to large-scale collecting, the lady's slipper orchid, Paphiopedilum delenatii, is under threat of extinction. Asymbiotic germination provides a useful way to re-establish plants in the wild and for commercial propagation. A detailed study of embryo development would provide information on subsequent germination events and aid in the propagation of the species. METHODS: Developing capsules were collected for histochemical and ultrastructural studies by using both light and transmission electron microscopy. KEY RESULTS: The suspensor of this species consists of three vacuolated cells. During the early globular stage of embryo development, structural differentiation occurs, revealing an abundance of smooth endoplasmic reticulum cisternae and wall ingrowths within the suspensor cells. These features are not present in cells of the embryo proper. Furthermore, the results of Nile red staining demonstrate that a cuticular layer is present only in the embryo proper, but absent from the suspensor. Cuticular material is also present in the inner walls of the seed coat, and persists through seed maturation. CONCLUSIONS: The morphological features of the transfer cell and the absence of cuticular material in the suspensor cell wall corroborate the hypothesis that the suspensor is the major nutrient uptake site for the developing embryo in the lady's slipper orchid. The absence of an endosperm and presence of cuticular material in the inner walls of the seed coat enclosing the embryo proper further support the notion that nutrient uptake by the embryo is confined to the micropylar end of the seed through the suspensor.     

Phylogenetics of the slipper orchids (Cypripedioideae, Orchidaceae): Nuclear rDNA ITS sequences

Cypripedioideae (Orchidaceae) have been the subject of numerous taxonomic treatments with conflicting interpretations of relationships among the five genera and the 150–170 species. We have produced nuclear ribosomal ITS nucleotide sequences for nearly 100 slipper orchid species and used parsimony analysis to investigate their relationships. Our results demonstrate that each genus, as currently circumscribed, is monophyletic (Mexipedium andSelenipedium being represented by a single taxon). LikerbcL data, ITS sequences placeMexipedium sister toPhragmipedium. Relationships at the sectional level inPaphiopedilum are largely as described byCribb. However, the division ofPaphiopedilum into subgg.Brachypetalum andPaphiopedilum is not supported; subg.Brachypetalum is paraphyletic to subg.Paphiopedilum. Phragmipedium species are divided into the same three major clades as in the taxonomic scheme ofMcCook. The plicate-leaved genera,Cypripedium andSelenipedium, are successive sister groups to the rest of the subfamily, confirming generally held opinions that they display plesiomorphic characters compared to the conduplicate-leaved genera. A survey of karyotypes in the context of the ITS tree reveals a general trend toward increased chromosome number, probably brought about by centric fission. These data also accord with a previously suggested biogeographic hypothesis of a widespread Northern Hemisphere distribution, followed by range fragmentation due to Miocene cooling.     

Pollination of the Lady's slipper orchid (Cypripedium calceolus) in Scandinavia – taxonomic and conservational aspects

The Lady's slipper orchid Cypripedium calceolus L. is considered one of the most beautiful orchids of Europe. Consequently, the species has suffered from over-collecting and is now critically endangered in many countries. Although pollination success is suspected to influence the long-term survival of Cypripedium calceolus , relatively little is known about the identity of its pollinators in mainland Sweden – a region that comprises the largest European populations. In order to identify which species pollinate eight representative populations in mainland Sweden, we observed and sampled visitors to flowers using a standardized protocol. Specimens were identified and any pollen smear found on their body was examined for the presence of Cypripedium pollen. Nine species were recognized as effective pollen vectors ( Andrena cineraria, A. carantonica, A. haemorrohoa, A. helvola, A. nigroaenea, A. praecox, Colletes cunicularius, Lasioglossum fratellum and L. fulvicorne ), four of them for the first time in Scandinavia. This is the first time that a species of Colletes is reported to carry pollen of Cypripedium in this region. All but one specimens were females. Our results suggest a taxonomically heterogeneous pollinator fauna for Cypripedium calceolus and are discussed in light of the management of this species.     

Nuclear cytoplasmic domains,microtubules and organelles in microsporocytes of the slipper orchid Cypripedium californicum A. Gray dividing by simultaneous cytokinesis

Microsporocytes of the slipper orchidCypripedium californicum A. Gray divide simultaneously after second meiosis. The organization and apportionment of the cytoplasm throughout meiosis are functions of nuclear-based radial microtubule systems (RMSs) that define domains of cytoplasm - a single sporocyte domain before meiosis, dyad domains within the undivided cytoplasm after first meiosis, and four spore domains after second meiosis. Organelles migrate to the interface of dyad domains in the undivided cytoplasm after first meiotic division, and second meiotic division takes place simultaneously on both sides of the equatorial organelle band. Microtubules emanating from the telophase II nuclei interact to form columnar arrrays that interconnect all four nuclei, non-sister as well as sister. Cell plates are initiated in these columns of microtubules and expand centrifugally along the interface of opposing RMSs, coalescing in the center of the sporocyte and joining with the original sporocyte wall at the periphery to form the tetrad of microspores. Organelles are distributed into the spore domains in conjunction with RMSs. These data, demonstrating that cytokinesis in microsporogenesis can occur in the absence of both components of the typical cytokinetic apparatus (the preprophase band of microtubules which predicts the division site and the phragmoplast which controls cell-plate deposition), suggest that plant nuclei have an inherent ability to establish a domain of cytoplasm via radial microtubule systems and to regulate wall deposition independently of the more complex cytokinetic apparatus of vegetative cells.     

Evolution and biogeography of the slipper orchids: Eocene vicariance of the conduplicate genera in the Old and New World Tropics

Intercontinental disjunctions between tropical regions, which harbor two-thirds of the flowering plants, have drawn great interest from biologists and biogeographers. Most previous studies on these distribution patterns focused on woody plants, and paid little attention to herbs. The Orchidaceae is one of the largest families of angiosperms, with a herbaceous habit and a high species diversity in the Tropics. Here we investigate the evolutionary and biogeographical history of the slipper orchids, which represents a monophyletic subfamily (Cypripedioideae) of the orchid family and comprises five genera that are disjunctly distributed in tropical to temperate regions. A relatively well-resolved and highly supported phylogeny of slipper orchids was reconstructed based on sequence analyses of six maternally inherited chloroplast and two low-copy nuclear genes (LFY and ACO). We found that the genus Cypripedium with a wide distribution in the northern temperate and subtropical zones diverged first, followed by Selenipedium endemic to South America, and finally conduplicate-leaved genera in the Tropics. Mexipedium and Phragmipedium from the neotropics are most closely related, and form a clade sister to Paphiopedilum from tropical Asia. According to molecular clock estimates, the genus Selenipedium originated in Palaeocene, while the most recent common ancestor of conduplicate-leaved slipper orchids could be dated back to the Eocene. Ancestral area reconstruction indicates that vicariance is responsible for the disjunct distribution of conduplicate slipper orchids in palaeotropical and neotropical regions. Our study sheds some light on mechanisms underlying generic and species diversification in the orchid family and tropical disjunctions of herbaceous plant groups. In addition, we suggest that the biogeographical study should sample both regional endemics and their widespread relatives.     

Duration of prepupal summer dormancy regulates synchronization of adult diapause with winter temperatures in bees of the genus Osmia

Osmia (Osmia) bees are strictly univoltine and winter as diapausing adults. In these species, the timing of adult eclosion with the onset of wintering conditions is critical, because adults exposed to long pre-wintering periods show increased lipid loss and winter mortality. Populations from warm areas fly in February-March and are exposed to longer growth seasons than populations from colder areas, which fly in April-May. Given their inability to produce an extra generation, early-flying populations should develop more slowly than late-flying populations and thus avoid the negative consequences of long pre-wintering periods. In this study we compare the development under natural and laboratory conditions of phenologically-distinct populations in two Osmia species. Early-flying populations took ～2 months longer to develop than late-flying populations. Differences between populations in larval and pupal period duration were very small, whereas the prepupal period was much longer in early-flying populations. In contrast to the larval and pupal stages, the prepupal stage showed a non-linear response to temperature, was strongly affected by thermoperiod, and exhibited minimum respiration rates. Coupled with other lines of evidence, these results suggest that the prepupal period in Osmia corresponds to a summer diapause, and its duration may be under local selection to synchronize adult eclosion with the onset of winter temperatures. We discuss the implications of our results relative to current expectations of global warming.     

Survival dynamics of reef coral larvae with special consideration of larval size and the genus Acropora

Temporal dynamics of larval survival were examined in vitro in four broadcast-spawning reef coral species, Acropora hyacinthus, A. japonica, A. solitaryensis, and Goniastrea pectinata. Larval size was treated as an important characteristic that may relate to larval lifespan. Two patterns were observed in larval survival dynamics between the three Acropora species (mean initial larval size; 0.05-0.08 mm(3)) and G. pectinata (0.02 mm(3)), based on the timing of a sharp drop in larval survival rates (ca. > 50% reduction over a 1-2 week period). Consequently, the majority of larvae of the three Acropora species had a lifespan of less than 2-3 weeks, whereas those of G. pectinata were extended a further 2-3 weeks despite the smaller larval size. No significant relationship was detected between the initial larval size and larval lifespan in any of the four reef coral species. These results suggest that (1) larval dispersal of spawning Acropora species may be on a more local scale than that of G. pectinata and most other reef coral species previously reported, and (2) larval size is not a good estimator of larval lifespan in reef coral species.     

Genetic differentiation in plants of the genus Cypripedium from Russia inferred from allozyme data

Ten gene loci of nine enzyme systems (PGI, 6-PGD, NADHD, SKDH, GDH, PGM, DIA, ADH, GOT-1, and GOT-2) were analyzed in Cypripedium calceolus, C. macranthon, C. shanxiense, and C. ventricosum plants from the south of the Russian Far East. Alleles of loci 6-PGD, NADHD, GDH, ADH, GOT-1, and PGIproved to be diagnostic for C. calceolus and C. macranthon. Plants of C. shanxiense from Primorye and Sakhalin Island were monomorphic at all of the loci examined, and their allelic structure can be regarded as diagnostic for the species. The allelic structure for fragments of the C. calceolus population from the western and eastern parts of the species range differed in two loci, PGl and SKDH: alleles absent in C. calceolus plants from the western part of the range occurred at a high frequency in the plants of this species from the eastern part of the range (28 and 55 plants or 41% and 68%, respectively). These alleles were found in C. shanxiense. The genetic structure of C. shanxiense was similar to that of C. calceolus from the eastern part of the range, i.e., the region when these species are sympartic. The additional alleles in C. calceolus from the eastern part of the range might have appeared as a result of hybridization with C. shanxiense. Our results indicate that C. calceolus plants occuring on the territory of Russia form two groups that represent two different units of genetic diversity preservation. We suggest that C. x ventricosum plants in southern Primorye were formed by hybridization between C. macranthon and C. calceolus x C. shanxiense hybrids. Thus, they differ from plants inhabiting the Urals and West Siberia, which originated by hybridization between C. macranthon and C. calceolus. The population of C. x ventricosum presumably also consists of two plant groups differing in genetic structure, which should be regarded as two different units of preservation of this taxon.     

Study of the inheritance of seed primary dormancy and the ability to enter secondary dormancy in Petunia: influence of temperature, light and gibberellic acid on dormancy

Abstract. The germination behaviour of two Petunia hybrida lines. M₃₀ and Th₇, and their reciprocal hybrids was studied. Two sets of experimental conditions appeared helped to distinguish between dormant and non-dormant parental lines: (1) 25 and 35 °C in the dark, in the latter case after 2 months of dry storage at 20 °C; (2) 35 and 40 °C in the light. Photosensitivity was tested in the first case and sensitivity to GA₃ in the second case. The predominance of paternal control over dormancy was evident. A maternal or tegumentary control of photosensitivity and of sensitivity to GA₃ was also shown. Transferring the seeds, originally imbibed in conditions expressing primary dormancy, to conditions which previously supported their germination, allowed us to show that secondary dormancy could be easily induced when a deeper primary dormancy had already developed in the seeds.     

Survival of parent and brood adult bark beetles, Ips typographus, in relation to size, lipid content and re-emergence or emergence day

ABSTRACT. This study investigated a possible trade-off between bark beetle reproductive effort and future survival. Parent adult Ips typographus (L.) (Coleoptera: Scolytidae) were collected when they re-emerged from a laboratory culture after completing their first brood and held at constant temperatures of 4 or 25^oC. As a comparison, emerging brood adults were collected and held at 25^oC. The longevity of the beetles was related to when they re-emerged or emerged (measured in days since parent introduction) and to their size (pronotal width), fresh weight and lipid content at collection, by multiple regression analysis. The expression (fresh weight x pronotal width ^-2.6), used as an estimate of lipid content of the living beetles, was significantly correlated (r=0.67, P<0.001) with the actual (extracted) lipid content. At 25^oC, fat content was the variable most strongly correlated with survival time in all beetle groups except parent females. In both parent and brood females, re-emergence or emergence day, respectively, contributed significantly to the explanation of survival time, whereas pronotal width and fresh weight never contributed significantly to the regression equation. At 4^oC, fat content was not estimated but, re-emergence day was negatively correlated with survival time. In conclusion, beetles with high fat content and re-emerging or emerging early have longer expected survival than beetles leaving the log later or containing less fat. This means that females laying a large first brood might suffer a somewhat higher mortality than females laying a smaller brood.     

Competition, niche specialization and the evolution of brain size in the genus Peromyscus

Previous studies of relative brain size in mammals have suggested an association with complex habitats and with low reproductive rate. In order to examine the causal relationships more thoroughly, a detailed examination of relative brain size variation in the genus Peromyscus was undertaken. Endocranial volumes were used to estimate brain weight for 32 species including 161 subspecies, and relative brain size calculated as the species deviation from the allometric relationship between brain and body size. The intrageneric allometric coefficient was higher than most values previously reported from low taxonomic levels, but intraspecific coefficients were generally lower than this.
Island species, and relict species isolated on mountain tops, which may be ecological 'islands', had consistently small relative brain sizes, but peninsular species were large brained. Among the remaining species there were significant correlations between litter size and relative brain size, and between the number of competitor species and relative brain size. Species with many competitor species have relatively large brains and small litters. It is concluded that the nature of the geographical distribution, the pattern of species formation and habitat complexity all influence relative brain size in existing forms.     

Competition,niche specialization and the evolution of brain size in the genus Peromyscus

Previous studies of relative brain size in mammals have suggested an association with complex habitats and with low reproductive rate. In order to examine the causal relationships more thoroughly, a detailed examination of relative brain size variation in the genus Peromyscus was undertaken. Endocranial volumes were used to estimate brain weight for 32 species including 161 subspecies, and relative brain size calculated as the species deviation from the allometric relationship between brain and body size. The intrageneric allometric coefficient was higher than most values previously reported from low taxonomic levels, but intraspecific coefficients were generally lower than this. Island species, and relict species isolated on mountain tops, which may be ecological ‘islands’, had consistently small relative brain sizes, but peninsular species were large brained. Among the remaining species there were significant correlations between litter size and relative brain size, and between the number of competitor species and relative brain size. Species with many competitor species have relatively large brains and small litters. It is concluded that the nature of the geographical distribution, the pattern of species formation and habitat complexity all influence relative brain size in existing forms.     

Genome size and environmental factors in the genus Pinus

Nuclear 1C DNA content in haploid megagametophyte tissue of 18 North American and one exotic Pinus species was determined using scanning microspectrophotometry. The nuclear DNA content in root meristematic cells of Zea mays L. ssp. mays, inbred line Va35 (4C = 10.31 pg) was used as a standard. DNA content measured by microspectrophotometry was verified using laser flow cytometry with two additional standards, Hordeum vulgare cv. Sultan (2C = 11.12 pg) and P. eldarica (2C = 47.30 pg). DNA values obtained by both methods were significantly correlated (r = 0.987). The 1C nuclear DNA content ranged from 21 pg to 31 pg. The ratio of DNA content in embryo tissue of P. eldarica to that in megagametophyte tissue was 1.72 by scanning microspectrophotometry and 1.74 by laser flow cytometry. To date, this is the most comprehensive data set available for North American Pinus species. Relationships between genome size of 18 North American Pinus species and climatic factors and indices of growth were investigated using regression and correlation analyses. Positive correlations were observed between nuclear DNA content and growth indices, minimum seed-bearing age, and seed dimensions. Strong negative correlations were observed between nuclear DNA content and two climatic factors, the lowest mean annual and monthly precipitation (excluding January) and the highest mean monthly spring air temperature. These correlations suggest that the large genome size and its variation in Pinus are adapted responses to the habitats of these species.     

Effects of temperature and season on egg size, hatchling size and adult size in Notiophilus biguttatus

1. Temperature-induced egg size variation and its effects on successive life stages in the carabid beetle Notiophilus biguttatus were examined.
2. In the laboratory, across temperature regimes, egg size and number were inversely related; number of eggs and total egg mass were higher, but egg size was smaller at high temperatures.
3. Food intake rate was shown not to be involved in the temperature effect on egg size.
4. Within the higher temperature regimes, among females, egg size was negatively correlated with number of eggs and with total egg mass.
5. Data on egg mortality and egg development time did not explain why at low temperature eggs were larger than at high temperature.
6. Larvae hatching from eggs produced at a low temperature were heavier than larvae from eggs produced at a high temperature, irrespective of temperature during development.
7. In a prolonged outdoor experiment (January – July), encompassing the main breeding period of N. biguttatus , egg size decreased and both egg production rate and total egg mass increased in the course of the experiment.
8. In the field, an effect of seasonal change in temperature on adult body size was found; teneral beetles that had their juvenile period early in the season were larger than those that had their juvenile period later in the season.
9. The results of the study suggest a mechanistic explanation in which the egg size response to temperature follows from a difference in temperature sensitivity between two processes in oogenesis; adaptiveness of the temperature response was not clarified.     

Clutch size and the costs of incubation in the house wren

Trade-offs in the allocation of finite resources among differentstages of a breeding attempt as well as between different reproductiveevents should shape the evolution of life-history traits. Toinvestigate the effects of incubation effort on within-broodand between-brood trade-offs in house wrens (Troglodytes aedon),we manipulated the clutch size that females incubated. We isolatedeffects of incubation by reversing the manipulation at hatchingto allow all parents to provision their natural brood sizes.Females that incubated enlarged clutches had longer incubationperiods than control females, both early and late in the season,suggesting that the experimental treatment increased incubationeffort. Contrary to predictions, however, increased incubationeffort did not adversely affect the allocation of effort tonestling provisioning. Rather, in the early season, but notin the late season, females that incubated enlarged clutchesappeared to allocate more effort to nestling provisioning, producingheavier and larger fledglings than control females. Althoughfemales with enlarged early-season clutches consequently lostmore mass than control females, this was likely an adaptiveresponse to reduce wing loading in anticipation of high provisioningdemands. There were no treatment-related differences in fledglingmass or size, or in female mass loss, in the late season. Thus,elevated incubation demands negatively affected a fitness-relatedtrait (duration of incubation) that may constrain clutch sizebut not the allocation of resources to subsequent stages ofthe same breeding event or to subsequent breeding events. Wesuggest that environmental conditions may mediate clutch-sizeeffects on trade-offs in allocation of resources between incubationand nestling provisioning.     

Taxonomic and functional characterisation of fungi from the Sebacina vermifera complex from common and rare orchids in the genus Caladenia

The terrestrial orchid genus Caladenia contains many species which are threatened with extinction. They have highly specific associations with Sebacina vermifera and closely related fungi, and conservation of these terrestrial orchids, in part, relies on symbiotic propagation to produce plants for reintroduction and ex situ conservation collections. However, little is known of the diversity of mycorrhizal fungi associating with natural populations. Here, restriction fragment polymorphism analysis, internal transcribed spacer and nuclear large subunit sequencing and symbiotic seed germination were used to investigate the taxonomic and functional diversity of fungal isolates from single populations of six endangered Caladenia species and one common species across the same biogeographic range. Fifty-nine fungal isolates were collected for investigation including ten isolates from the six endangered species Caladenia audasii, Caladenia amoena, Caladenia sp. aff. fragrantissima (Central Victoria), Caladenia sp. aff. patersonii, Caladenia rosella and Caladenia orientalis and 49 isolates from six populations of the common species Caladenia tentaculata. While the common species associated with three distinct S. vermifera-like taxa, the six endangered species were restricted to one of these fungal taxa. No direct relationship between the taxonomic identity of the fungi and their ability to stimulate seed germination was observed; however, the majority of the fungi isolated from the Caladenia species were capable of germinating seed in vitro, indicating their mycorrhizal status and potential for symbiotic propagation in conservation programmes.     

The relationship between seed dormancy,seed size and weediness,in Crepis tectorum (Asteraceae)

Summary I examined the germination characteristics of weed and outcrop populations of Crepis tectorum to test the hypothesis that the presumably more ephemeral weed habitat favors the highest levels of seed dormancy. The winter annual habit characterizing most plants of this species was reflected in a rapid germination of seeds sown in late summer. A slightly higher fraction of surface-sown seeds of weed plants delayed germination. Buried seeds of weed plants also survived better than seeds produced by plants in most outcrop populations, supporting the idea that weediness favors seed dormancy and a persistent seed bank. However, the differences in seed dormancy between the two ecotypes were small and not entirely consistent. Furthermore, high levels of seed dormancy were induced during burial in the outcrop group, suggesting that there is a potential for a dormant seed population in this habitat as well. Demographic data from one of the outcrop populations verified the presence of a large between-year seed bank. Possible environmental factors favoring seed dormancy in outcrop populations are discussed. The unusually large seeds of weedy Crepis contrasts with the relatively small difference in seed dormancy between the two ecotypes.