The origin and speciation of orchids.

Orchids constitute one of the most spectacular radiations of flowering plants. However, their origin, spread across the globe, and hotspots of speciation remain uncertain due to the lack of an up-to-date phylogeographic analysis. We present a new Orchidaceae phylogeny based on combined high-throughput and Sanger sequencing data, covering all five subfamilies, 17/22 tribes, 40/49 subtribes, 285/736 genera, and c. 7% (1921) of the 29 524 accepted species, and use it to infer geographic range evolution, diversity, and speciation patterns by adding curated geographical distributions from the World Checklist of Vascular Plants. The orchids' most recent common ancestor is inferred to have lived in Late Cretaceous Laurasia. The modern range of Apostasioideae, which comprises two genera with 16 species from India to northern Australia, is interpreted as relictual, similar to that of numerous other groups that went extinct at higher latitudes following the global climate cooling during the Oligocene. Despite their ancient origin, modern orchid species diversity mainly originated over the last 5 Ma, with the highest speciation rates in Panama and Costa Rica. These results alter our understanding of the geographic origin of orchids, previously proposed as Australian, and pinpoint Central America as a region of recent, explosive speciation.

Gut microbiota variation of a tropical oil-collecting bee species far exceeds that of the honeybee.

Introduction: Interest for bee microbiota has recently been rising, alleviating the gap in knowledge in regard to drivers of solitary bee gut microbiota. However, no study has addressed the microbial acquisition routes of tropical solitary bees. For both social and solitary bees, the gut microbiota has several essential roles such as food processing and immune responses. While social bees such as honeybees maintain a constant gut microbiota by direct transmission from individuals of the same hive, solitary bees do not have direct contact between generations. They thus acquire their gut microbiota from the environment and/or the provision of their brood cell. To establish the role of life history in structuring the gut microbiota of solitary bees, we characterized the gut microbiota of Centris decolorata from a beach population in Mayagüez, Puerto Rico. Females provide the initial brood cell provision for the larvae, while males patrol the nest without any contact with it. We hypothesized that this behavior influences their gut microbiota, and that the origin of larval microbiota is from brood cell provisions. Methods: We collected samples from adult females and males of C. decolorata (n = 10 each, n = 20), larvae (n = 4), and brood cell provisions (n = 10). For comparison purposes, we also sampled co-occurring female foragers of social Apis mellifera (n = 6). The samples were dissected, their DNA extracted, and gut microbiota sequenced using 16S rRNA genes. Pollen loads of A. mellifera and C. decolorata were analyzed and interactions between bee species and their plant resources were visualized using a pollination network. Results: While we found the gut of A. mellifera contained the same phylotypes previously reported in the literature, we noted that the variability in the gut microbiota of solitary C. decolorata was significantly higher than that of social A. mellifera. Furthermore, the microbiota of adult C. decolorata mostly consisted of acetic acid bacteria whereas that of A. mellifera mostly had lactic acid bacteria. Among C. decolorata, we found significant differences in alpha and beta diversity between adults and their brood cell provisions (Shannon and Chao1 p < 0.05), due to the higher abundance of families such as Rhizobiaceae and Chitinophagaceae in the brood cells, and of Acetobacteraceae in adults. In addition, the pollination network analysis indicated that A. mellifera had a stronger interaction with Byrsonima sp. and a weaker interaction with Combretaceae while interactions between C. decolorata and its plant resources were constant with the null model. Conclusion: Our data are consistent with the hypothesis that behavioral differences in brood provisioning between solitary and social bees is a factor leading to relatively high variation in the microbiota of the solitary bee.

Best in the company of nearby males: female success in the threatened cycad, Zamia portoricensis.

Variation in plant reproductive success is affected by ecological conditions including the proximity of potential mates. We address the hypothesis that spatial distribution of sexes affects female reproductive success (RS) in the dioecious cycad, Zamia portoricensis. Are the frequencies of males, operational sex ratios, and distances to the nearest mate associated with RS in females? We studied the spatial distribution of sexes in two populations in Puerto Rico and compared RS of target females with the number of males and operational sex ratios. Population structure suggests regular successful recruitment. Adults, males, and females were randomly distributed with respect to one another. Reproductive success of females was highly variable, but was higher in neighborhoods with more males than females and generally decreased with increasing distance to the nearest male, becoming statistically significant beyond 190 cm. This possible mate-finding Allee effect indicates that pollinator movement among plants may be limited for this mutually dependent plant-pollinator interaction. Yet being close to male plants is a matter of chance, perhaps a factor generating the high intra-population genetic diversity in Z. portoricensis.

Terrestrial orchids in a tropical forest: best sites for abundance differ from those for reproduction.

Suitable habitat for a species is often modeled by linking its distribution patterns with landscape characteristics. However, modeling the relationship between fitness and landscape characteristics is less common. In this study we take a novel approach towards species distribution modeling (SDM) by investigating factors important not only for species occurrence, but also abundance and physical size, as well as fitness measures. We used the Neotropical terrestrial orchid Prescottia stachyodes as our focal species, and compiled geospatial information on habitat and neighboring plants for use in a two-part conditional SDM that accounted for zero inflation and reduced spatial autocorrelation bias. First, we modeled orchid occurrence, and then within suitable sites we contrasted habitat characteristics important for orchid abundance as compared to plant size. We then tested possible fitness implications, informed by analyses of allometric scaling of reproductive effort and lamina area, as well as size-density relationships in areas of P. stachyodes co-occurrence. We determined that orchid presence was based on a combination of biotic and abiotic factors (indicator species, diffuse solar radiation). Within these sites, P. stachyodes abundance was higher on flat terrain, with fine, moderately well-drained soil, and areas without other native orchids, whereas plant size was greater in less rocky areas. In turn, plant size determined reproductive effort, with floral display height proportionate to lamina area (more photosynthates); however, allometric scaling of flower quantity suggests a higher energy cost for production, or maintenance, of flowers. Overall, habitat factors most important for abundance differed from those for size (and thus reproductive effort), suggesting that sites optimal for either recruitment or survival may not be the primary source of seeds. For plots with multiple P. stachyodes plants, size-density relationships differed depending on the size class examined, which may reflect context-dependent population dynamics. Thus, ecological resolution provided by SDM can be enhanced by incorporating abundance and fitness measures.

When stable-stage equilibrium is unlikely: integrating transient population dynamics improves asymptotic methods.

BACKGROUND AND AIMS: Evaluation of population projection matrices (PPMs) that are focused on asymptotically based properties of populations is a commonly used approach to evaluate projected dynamics of managed populations. Recently, a set of tools for evaluating the properties of transient dynamics has been expanded to evaluate PPMs and to consider the dynamics of populations prior to attaining the stable-stage distribution, a state that may never be achieved in disturbed or otherwise ephemeral habitats or persistently small populations. This study re-evaluates data for a tropical orchid and examines the value of including such analyses in an integrative approach. METHODS: Six small populations of Lepanthes rubripetala were used as a model system and the R software package popdemo was used to produce estimates of the indices for the asymptotic growth rate (lambda), sensitivities, reactivity, first-time step attenuation, maximum amplification, maximum attenuation, maximal inertia and maximal attenuation. The response in lambda to perturbations of demographic parameters using transfer functions and multiple perturbations on growth, stasis and fecundity were also determined. The results were compared with previously published asymptotic indices. KEY RESULTS: It was found that combining asymptotic and transient dynamics expands the understanding of possible population changes. Comparison of the predicted density from reactivity and first-time step attenuation with the observed change in population size in two orchid populations showed that the observed density was within the predicted range. However, transfer function analysis suggests that the traditional approach of measuring perturbation of growth rates and persistence (inertia) may be misleading and is likely to result in erroneous management decisions. CONCLUSIONS: Based on the results, an integrative approach is recommended using traditional PPMs (asymptotic processes) with an evaluation of the diversity of dynamics that may arise when populations are not at a stable-stage distribution (transient processes). This method is preferable for designing rapid and efficient interventions after disturbances, and for developing strategies to establish new populations.

Nutrient limitation restricts growth and reproductive output in a tropical montane cloud forest bromeliad: findings from a long-term forest fertilization experiment.

From studies in seasonal lowland tropical forests, bromeliad epiphytes appear to be limited mainly by water, and to a lesser extent by nutrient supply, especially phosphorous. Less is understood about the mineral nutrition of tropical montane cloud forest (TMCF) epiphytes, even though their highest diversity is in this habitat. Nutrient limitation is known to be a key factor restricting forest productivity in TMCF, and if epiphytes are nutritionally linked to their host trees, as has been suggested, we would expect that they are also nutrient limited. We studied the effect of a higher nutrient input on reproduction and growth of the tank bromeliad Werauhia sintenisii in experimental plots located in a TMCF in Puerto Rico, where all macro- and micronutrients had been added quarterly starting in 1989 and continuing throughout the duration of this study. We found that bromeliads growing in fertilized plots were receiving litterfall with higher concentrations of N, P, and Zn and had higher concentrations of P, Zn, Fe, Al, and Na in their vegetative body. The N:P ratios found (fertilized = 27.5 and non-fertilized = 33.8) suggest that W. sintenisii may also be phosphorous limited as are lowland epiphytes. Fertilized plants had slightly longer inflorescences, and more flowers per inflorescence, than non-fertilized plants, but their flowers produced nectar in similar concentrations and quantities. Fertilized plants produced more seeds per fruit and per plant. Frequency of flowering in two consecutive years was higher for fertilized plants than for controls, suggesting that fertilized plants overcome the cost of reproduction more readily than non-fertilized plants. These results provide evidence that TMCF epiphytic bromeliads are nutrient limited like their lowland counterparts.

Riding across the selection landscape: fitness consequences of annual variation in reproductive characteristics.

Evolutionary models estimating phenotypic selection in character size usually assume that the character is invariant across reproductive bouts. We show that variation in the size of reproductive traits may be large over multiple events and can influence fitness in organisms where these traits are produced anew each season. With data from populations of two orchid species, Caladenia valida and Tolumnia variegata, we used Bayesian statistics to investigate the effect on the distribution in fitness of individuals when the fitness landscape is not flat and when characters vary across reproductive bouts. Inconsistency in character size across reproductive periods within an individual increases the uncertainty of mean fitness and, consequently, the uncertainty in individual fitness. The trajectory of selection is likely to be muddled as a consequence of variation in morphology of individuals across reproductive bouts. The frequency and amplitude of such changes will certainly affect the dynamics between selection and genetic drift.

Oeceoclades maculata, an alien tropical orchid in a Caribbean rainforest.

BACKGROUND AND AIMS: Undisturbed forest habitat can be relatively impenetrable to invasive, non-native species. Orchids are not commonly regarded as invasive, but some species have become invasive and these generally depend on habitat disturbance. One of the most aggressive orchids is Oeceoclades maculata, a terrestrial species with remarkable ecological amplitude. Originally from tropical Africa, it is now widespread in the neotropics. By associating its local distribution with land-use history and habitat characteristics, it was determined whether O. maculata is dependent on habitat disturbance. It was also investigated whether this exotic orchid occupies the same habitat space as two sympatric native species. METHODS: Six 10 m x 500 m transects were censused in June 2007 on the 16-ha Luquillo Forest Dynamics Plot, located in the Luquillo Mountains, Puerto Rico. The plot had been mapped for historical land use, topography and soil type. KEY RESULTS: Oeceoclades maculata was the most abundant of three orchid species surveyed and was found in all four historical cover classes. In cover class 3 (50-80 % forest cover in 1936), 192 of 343 plants were found at a density of 0.48 plants per 5 x 5 m subplot. Over 93 % of the 1200 subplots surveyed were composed of Zarzal or Cristal soil types, and O. maculata was nearly evenly distributed in both. The orchid was most common on relatively flat terrain. The distribution and abundance of two sympatric orchid species were negatively associated with that of the invasive species. CONCLUSIONS: Oeceoclades maculata does penetrate 'old growth' forest but is most abundant in areas with moderate levels of past disturbance. Soil type makes little difference, but slope of terrain can be important. The negative association between O. maculata and native species may reflect differences in habitat requirements or a negative interaction perhaps at the mycorrhizal level.

Widespread mycorrhizal specificity correlates to mycorrhizal function in the neotropical, epiphytic orchid Ionopsis utricularioides (Orchidaceae).

Tropical orchids constitute the greater part of orchid diversity, but little is known about their obligate mycorrhizal relationships. The specificity of these interactions and associated fungal distributions could influence orchid distributions and diversity. We investigated the mycorrhizal specificity of the tropical epiphytic orchid Ionopsis utricularioides across an extensive geographical range. DNA ITS sequence variation was surveyed in both plants and mycorrhizal fungi. Phylogeographic relationships were estimated for the mycorrhizal fungi. Orchid functional outcomes were determined through in vitro seed germination and seedling growth with a broad phylogenetic representation of fungi. Most fungal isolates derived from one clade of Ceratobasidium (anamorphs assignable to Ceratorhiza), with 78% within a narrower phylogenetic group, clade B. No correlation was found between the distributions of orchid and fungal genotypes. All fungal isolates significantly enhanced seed germination, while fungi in clade B significantly enhanced seedling growth. These results show that I. utricularioides associates with a phylogenetically narrow, effective fungal clade over a broad distribution. This preference for a widespread mycorrhizae may partly explain the ample distribution and abundance of I. utricularioides and contrasts with local mycorrhizal diversification seen in some nonphotosynthetic orchids. Enhanced orchid function with a particular fungal subclade suggests mycorrhizal specificity can increase orchid fitness.

Differences in mycorrhizal preferences between two tropical orchids.

Orchids parasitize their mycorrhizal fungi and are dependent on them for seed germination. Controversy reigns over how specific the mycorrhizal association is in tropical species. Although there is little experimental evidence to support any viewpoint, some variation is known to exist. We compared mycorrhizal specificity and performance in two phylogenetically related epiphytic orchids from Puerto Rico, Tolumnia variegata and Ionopsis utricularioides (Oncidiinae) by integrating two techniques: phylogenetic analysis of mycorrhizal fungi based on nuclear ribosomal internal transcribed spacer (ITS) sequences, and symbiotic seed germination experiments. Most of the mycorrhizal isolates from T. variegata fell into four different clades of Ceratobasidium, while most of those from I. utricularioides were restricted to a single clade of the same genus. Seeds of T. variegata germinated equally well with fungi from both T. variegata and I. utricularioides, but seeds of I. utricularioides germinated significantly better with its own isolates. Seeds of I. utricularioides germinated and developed faster than those of T. variegata. Both the molecular phylogeny and the seed germination experiments showed that T. variegata is a generalist in its association with fungal symbionts. In contrast, I. utricularioides is more specialized and more effective at exploiting a specific fungal clade. Our data are consistent with the theoretical trade-offs between specialized and generalized interactions.

Does flower color variation matter in deception pollinated Psychilis monensis (Orchidaceae)?

Negative frequency dependent selection (FDS) had been proposed as a mechanism for the maintenance of the high levels of variability in floral traits of rewardless plants. Thus far the evidence has been equivocal for discontinuous traits. We experimentally tested the FDS hypothesis for continuous variation in flower color of Psychilis monensis, a rewardless, epiphytic orchid of Mona Island, Puerto Rico. P. monensis flowers all year long with a peak in June and July. Plants are self-incompatible. Individual flowers last 9-15 days if unpollinated. Over a 9-month observation of an unmanipulated population, a high percentage of plants showed pollinarium removals (79%) and fruit initiation (63%), but the actual percentage of flowers visited was very low (12%) and final fruit set was as low as the lowest recorded for orchids (2.4%). In a FDS experiment using a Latin Square design, we manipulated flower color in three populations. Over 50% of the variation in either male or female reproductive success was explained by time and site with no significant effect of treatment except as part of a three-way interaction of time x site x treatment. Paired comparisons with the controls gave equivocal results for both male and female measures of reproductive success. Major community changes had occurred during the FDS experiment with flower activity falling dramatically and by the third run of the experiment, only P. monensis was in flower. Coincidentally, the numbers of effective visits increased with time, presumably as pollinators became less discriminating in search of new food resources. Thus, negative frequency dependent selection is either sporadic or non-existent. Reproductive success was statistically related to locality and date, which was reflected in the flowering phenology of the local communities. High natural levels of color variation may be more influenced by drift than selection.

Diversity and host specificity of endophytic Rhizoctonia-like fungi from tropical orchids.

All orchids have an obligate relationship with mycorrhizal symbionts. Most orchid mycorrhizal fungi are classified in the form-genus Rhizoctonia. This group includes anamorphs of Tulasnella, Ceratobasidium, and Thanatephorus. Rhizoctonia can be classified according to the number of nuclei in young cells (multi-, bi-, and uninucleate). From nine Puerto Rican orchids we isolated 108 Rhizoctonia-like fungi. Our isolates were either bi- or uninucleate, the first report of uninucleate Rhizoctonia-like fungi as orchid endophytes. We sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal DNA from 26 isolates and identified four fungal lineages, all related to Ceratobasidium spp. from temperate regions. Most orchid species hosted more than one lineage, demonstrating considerable variation in mycorrhizal associations even among related orchid species. The uninucleate condition was not a good phylogenetic character in mycorrhizal fungi from Puerto Rico. All four lineages were represented by fungi from Tolumnia variegata, but only one lineage included fungi from Ionopsis utricularioides. Tropical epiphytic orchids appear to vary in degree of specificity in their mycorrhizal interactions more than previously thought.