Taxonomic significance of the epicuticular wax composition in species of the genus Clusia from Panama

We attempted to separate species of the genus Clusia according to the concentration of linear alkanes (C25 and C35), and the presence and diversity of terpenes in epicuticular wax extracts. We collected leaves of 15 Clusia species growing in mountain forests of Panama (Cerro Jefe 1007 m and Altos de Campana 800 m a.s.l.) and from cultivated plants at two lowland sites. Leaf surfaces were washed gently with hexane to extract epicuticular waxes, which were analyzed using gas chromatography and mass spectrometry. The predominant alkanes were C29, C31, and C33. In the extract the ratio C31/C29 was ≤1 in 6 of the 15 species analyzed: Clusia multiflora, Clusia peninsulae (Hammel ined.), Clusia stenophylla, Clusia liesneri, Clusia coclensis, and Clusia triflora. The concentrations of C29 and C33 were inversely related, the latter being above 10% in Clusia divaricata, Clusia pratensis, Clusia rosea, Clusia uvitana, and Clusia valerioi. Proportion of triterpenes was less than 5% in the species C. minor, C. pratensis, C. uvitana, C. rosea, Clusia cylindrica, C. divaricata, and C. valerioi. The rest contained squalene, and specific triterpenes such as β-amyrine in C. liesneri, betuline in Clusia osseocarpa, taraxerol in C. stenophylla, and lupeol in C. multiflora. The variety of triterpenes was higher in Clusia liesneri (5) and C. multiflora (3). The results suggest that groups of species can be distinguished within the genus according to the presence of terpenes and ratios of linear alkanes. These groups overlap with those generated by other classifications using morphology and nuclear ribosomal DNA.     

Floral Morphology and Relationships of Clusia gundlachii with a Discussion of Floral Organ Identity and Diversity in the Genus Clusia

The genus Clusia L. is highly variable in many floral features. Several Clusia species have floral organs of mixed or uncertain identity, such as organs that are transitional between bracteoles and sepals, petaloid sepals, and partly petaloid stamen rings. Unique in Clusia is the "corona" of Clusia gundlachii Stahl, a thick, urn-shaped structure that is initiated as a ring primordium. In male flowers it surrounds a synandrium, and in female flowers it surrounds the ovary and a row of staminodes. The corona combines features typical of both petals and stamens of other Clusia species. It is hypothesized that this corona may be the result of the altered expression patterns of the genes that determine floral organ identity. Clusia gundlachii has many floral features in common with two small genera that are sometimes included in Clusia: Havetiopsis and Oedematopus. These genera have four thick connivent petals. Their apparent close relationship makes it seem likely that the corona of C. gundlachii evolved via congenital fusion of such petals. The corona is also somewhat similar to the staminodial rings present in many Clusia species, but taxa in which such organs occur show little similarity to C. gundlachii in terms of other floral characters.     

Isolation of polymorphic microsatellite loci in the genus Clusia (Clusiaceae)

Microsatellite flanking region sequences may provide phylogenetically useful information. We isolated 13 polymorphic microsatellite loci from two species, Clusia minor (five loci) and Clusia nemorosa (eight loci), to aid in the determination of phylogenetic relationships within the genus Clusia. Eleven loci amplified across all 17 Clusia species tested, while two loci amplified in 10 out of 17 species. The extensive cross‐species amplification suggests that these loci may be useful for an examination of phylogenetic relationships in this genus.     

Oxygen isotope composition of CAM and C3 Clusia species: non-steady-state dynamics control leaf water 18O enrichment in succulent leaves

Leaf gas exchange and leaf water (18)O enrichment (Delta(18)O(L)) were measured in three Clusia species under field conditions during dry and wet seasons and in Miconia argentea during the dry season in the Republic of Panama. During the dry season, all three Clusia species used crassulacean acid metabolism (CAM); during the wet season Clusia pratensis operated in the C(3) mode, while Clusia uvitana and Clusia rosea used CAM. Large departures from isotopic steady state were observed in daytime Delta(18)O(L) of the Clusia species, especially during the dry season. In contrast, daytime Delta(18)O(L) was near isotopic steady state in the C(3) tree M. argentea. Across the full data set, non-steady-state predictions explained 49% of variation in observed Delta(18)O(L), whereas steady-state predictions explained only 14%. During the wet season, when Delta(18)O(L) could be compared with Clusia individuals operating in both C(3) and CAM modes, steady-state and non-steady-state models gave contrasting predictions with respect to interspecific variation in daytime Delta(18)O(L). The observed Delta(18)O(L) pattern matched that predicted for the non-steady state. The results provided a clear example of how non-steady-state control of leaf water (18)O dynamics can shift the slope of the relationship between transpiration rate and daytime Delta(18)O(L) from negative to positive.     

First records of Clusia Haliday and Clusiodes Coquillett (Diptera: Clusiidae) from Korea

This study reports the first Korean records of the genera Clusia Haliday and Clusiodes Coquillett (Diptera: Clusiidae), with four newly recorded species, Clusia intermedialis (Mamaev), Clusia nigromaculata (Mamaev), Clusia unita Mamaev, and Clusiodes flaveolus Mamaev. Morphological diagnosis is provided with a key to Korean Clusiidae. The mitochondrial cytochrome c oxidase I (COI) gene sequences of Clusia and Clusiodes are analyzed for DNA barcoding. Molecular analyses of both genera result in well-defined species clusters with significant barcode gaps, and also identify problematic data in the public database.     

High degree of conservation of nuclear microsatellite loci in the genus Clusia.

In plants, microsatellites and their flanking DNA are rarely conserved across a whole genus, let alone other genera in the same family. Therefore, the possibility of using microsatellite primers developed for a species across a large number of plant species in the same genus is often limited. Remarkably, dinucleotide nuclear microsatellites developed for Clusia minor and for Clusia nemorosa amplified homologous microsatellites in species across the whole genus Clusia. In this present study, we report on the DNA sequence variation across the genus of 3 microsatellite loci with varying levels of variation. Compared over the species, there was a correlation between the lengths of the microsatellite loci. Interrupts occurred multiple times and did not seem to lead to the death of the microsatellite. These highly conserved markers will be useful for studying the variable reproductive systems in the genus Clusia.     

Phosphoenolpyruvate carboxylase genes in C3, crassulacean acid metabolism (CAM) and C3/CAM intermediate species of the genus Clusia: rapid reversible C3/CAM switches are based on the C3 housekeeping gene

The genus Clusia includes species that exhibit either the C3 or crassulacean acid metabolism (CAM) mode of photosynthesis, or those that are able to switch between both modes according to water availability. In order to screen for species-specific genetic variability, we investigated the key carboxylase for CAM, phosphoenolpyruvate carboxylase (PEPC). Sequence analysis of DNA isolated from the obligate CAM species, Clusia hilariana, the obligate C3 species, Clusia multiflora, and an intermediate species that can switch between C3 and CAM photosynthesis, Clusia minor, revealed three different isoforms for C. hilariana and one each for the other two species. Sequence alignments indicated that PEPC from the intermediate species had high homology with the C3 protein and with one of CAM plant proteins. These were assumed to constitute 'housekeeping' proteins, which can also support CAM in intermediate species. The other two isoforms of the CAM plant C. hilariana were either CAM-specific or showed homologies with PEPC from roots. Phylogenetic trees derived from neighbour-joining analysis of amino acid sequences from 13 different Clusia species resulted in two distinct groups of plants with either 'housekeeping' PEPC only, or additionally CAM-related isoforms. Only C. hilariana showed the third, probably root-specific isoform. The high homology of the PEPC from the intermediate species with the C3 protein indicates that for the reversible transition from the C3 to CAM mode of photosynthesis, the C3 type of PEPC is sufficient. Its expression, however, is strongly increased under CAM-inducing conditions. The use of the C3 isoform could have facilitated the evolution of CAM within the genus, which occurred independently for several times.     

Habitat use by Oryzomys subflavus (Rodentia) in an open shrubland formation in Restinga de Jurubatiba National Park, RJ, Brazil.

The Restinga de Jurubatiba has at least 10 plant formations, including open Clusia shrubland. This formation is composed of dense shrubs of many shapes and sizes, where Clusia hilariana is one of the most important plant species. Shrublands with Clusia (CC) are poorer in plant species and less dense than shrublands without Clusia (SC). Oryzomys subflavus (Rodentia) is the most abundant small mammal species in the open Clusia shrubland. We tested the hypothesis that the abundance of rodents would increase with the size of the patch and would be higher in SC shrublands. Rodents were captured, marked and released in three 780-m-long transects. At each capture site, we evaluated the shape of the shrubland patch, calculated the area and noted the category of the shrubland. Using ANCOVA, we ascertained whether the abundance of Oryzomys subflavus increased with the sampled area and used CC and SC shrublands differently. We also verified if the size of patches used by rodents varies in the same frequency as the size of available shrublands. Rodent abundance was found to increase significantly with the area. There were no differences in the size of the patches used by rodents and the frequency of the size of available patches. This finding indicates that O. subflavus, in the study area, is a generalist species that uses its habitat according to availability.     

Superoxide dismutase activity in C<Subscript>3</Subscript> and C<Subscript>3</Subscript>/CAM intermediate species of <Emphasis Type="Italic">Clusia</Emphasis>

The C₃-CAM intermediate Clusia minor L. and the C₃ obligate Clusia multiflora H.B.K. plants were exposed for 7 d to a combination of drought stress and high irradiance of about 1200 μmol m⁻² s⁻¹ for 12 h per day. In both species under these conditions a strong decrease in stomatal conductance was observed at dawn and dusk. Changes in stomatal behaviour of C. minor were accompanied by only a low nocturnal accumulation of malate and citrate. Thus, in C. minor drought stress applied in combination with high irradiance limited CAM expression, and possibly this is the main reason why C. minor prefers semi-shaded sites in the field. The mitochondrial MnSOD, in both well watered and stressed plants of two species showed strong diurnal oscillations with maximum activity at dusk. These oscillations can be explained by the engagement of mitochondria in dissipation of an excess of reducing equivalents. In plants which are able to carry out CAM metabolism tricarboxylic acid cycle is expected to be down regulated in the dark period to prevent breakdown of the entire malate and citrate.     

Evolution of morphological diversity and resin secretion in flowers of Clusia (Clusiaceae): insights from ITS sequence variation

The taxonomically poorly known, neotropical genus Clusia (Clusiaceae) comprises over 300 species of trees, shrubs and hemiepiphytes. The flowers are morphologically highly diverse and offer either nectar, pollen or resin as a reward for pollinators. Resin production in flowers is a poorly studied phenomenon, known from only five angiosperm genera. Variation in sequences of the nuclear ribosomal ITS region was analysed cladistically in order to trace the evolution of floral resin production in Clusia , and as a first step towards a phylogenetically based reclassification of the genus. The most parsimonious trees show that the genera Havetia, Havetiopsis, Oedematopus and Quapoya are nested inside Clusia. Traditionally, Clusia has been divided into sections based on androecial variation, and most of these groups are supported as monophyletic based on the ITS data. Sections Retinostemon, Havetia and Cochlanthera together form one of very few well-supported suprasectional groups. Character optimisation experiments suggest that resiniferous flowers have evolved at least three times independently in Clusia , but a character-evolution model with a single origin for floral resin is only slightly less parsimonious. Clades of resiniferous species are morphologically the most diverse, and it is concluded that resin production has been a key innovation triggering floral morphological diversification. Secondary losses of resin and switches to apomixis seem to be correlated with colonisation of habitats and areas where resin-collecting bees are less frequent.     

On the nature of facultative and constitutive CAM: environmental and developmental control of CAM expression during early growth of Clusia, Kalanchöe, and Opuntia

The capacity to induce crassulacean acid metabolism developmentally (constitutive CAM) and to up-regulate CAM expression in response to drought stress (facultative CAM) was studied in whole shoots of seven species by measuring net CO(2) gas exchange for up to 120 day-night cycles during early growth. In Clusia rosea, CAM was largely induced developmentally. Well-watered seedlings began their life cycle as C(3) plants and developed net dark CO(2) fixation indicative of CAM after the initiation of the fourth leaf pair following the cotyledons. Thereafter, CAM activity increased progressively and drought stress led to only small additional, reversible increases in dark CO(2) fixation. In contrast, CAM expression was overwhelmingly under environmental control in seedlings and mature plants of Clusia pratensis. C(3)-type CO(2) exchange was maintained under well-watered conditions, but upon drought stress, CO(2) exchange shifted, in a fully reversible manner, to a CAM-type pattern. Clusia minor showed CO(2) exchange reponses intermediate to those of C. rosea and C. pratensis. Clusia cretosa operated in the C(3) mode at all times. Notably, reversible stress-induced increases of dark CO(2) fixation were also observed during the developmental progression to pronounced CAM in young Kalancho? daigremontiana and Kalancho? pinnata, two species considered constitutive CAM species. Drought-induced up-regulation of CAM was even detected in young cladodes of a cactus, Opuntia ficus-indica, an archetypal constitutive CAM species. Evidently, the defining characteristics of constitutive and facultative CAM are shared, to variable degrees, by all CAM species.     

Clusia blattophila sp. nov. (Clusiaceae) from an inselberg in French Guiana

Clusia blattophila (Clusiaceae) is described as a new species. It is distinguished from related species in C. sect. Oedematopus by e.g. the relatively large and reflexed petals and from other Clusia spp. by having fewer (6–16) stamens and only four petals. Unique traits are the almost sessile inflorescence and the presence of a secretory pit at the floral apex in staminate flowers. The species grows epilithically and is known only from the Nouragues inselberg, a massive granite outcrop in eastern French Guiana. The name refers to its cockroach pollinators.     

Diel leaf growth cycles in Clusia spp. are related to changes between C3 photosynthesis and crassulacean acid metabolism during development and during water stress

This study reports evidence that the timing of leaf growth responds to developmental and environmental constraints in Clusia spp. We monitored diel patterns of leaf growth in the facultative C₃-crassulacean acid metabolism (CAM) species Clusia minor and in the supposedly obligate CAM species Clusia alata using imaging methods and followed diel patterns of CO₂ exchange and acidification. Developing leaves of well-watered C. minor showed a C₃-like diel pattern of gas exchange and growth, with maximum relative growth rate (RGR) in the early night period. Growth slowed when water was withheld, accompanied by nocturnal CO₂ exchange and the diel acid change characteristic of CAM. Maximum leaf RGR shifted from early night to early in the day when water was withheld. In well-watered C. alata , similar changes in the diel pattern of leaf growth occurred with the development of CAM during leaf ontogeny. We hypothesize that the shift in leaf growth cycle that accompanies the switch from C₃ photosynthesis to CAM is mainly caused by the primary demand of CAM for substrates for nocturnal CO₂ fixation and acid synthesis, thus reducing the availability of carbohydrates for leaf growth at night. Although the shift to leaf growth early in the light is presumably associated with the availability of carbohydrates, source–sink relationships and sustained diurnal acid levels in young leaves of Clusia spp. need further evaluation in relation to growth processes.     

The Genus Clusia L.: Molecular Evidence for Independent Evolution of Photosynthetic Flexibility

Abstract: Members of the Clusiaceae genus Clusia (tropical trees and shrubs) belong to the small group of dicotyledonous trees which are able to perform crassulacean acid metabolism (CAM). Most of the species are able to switch between C₃ and CAM modes of photosynthesis and only a few are restricted to either C₃ or CAM. In order to discover possible phylogenetic relationships with regard to the mode of photosynthesis, we investigated 17 species of the genus Clusia, and one species each of the Clusiaceae genera Oedematopus and Hypericum on the basis of internal transcribed spacer (ITS) sequences between the 18S and 26S coding regions of nuclear ribosomal DNA. Little length variation was detected in the ITS region of Clusia species. ITS1 sequences ranged from 255 to 260 bp and ITS2 sequences from 208 to 210 bp. Neighbour-joining and parsimony analyses of these sequences resulted in considerable differences in cluster formation when compared to a classification based on morphological characteristics. The molecular data also give no indication of a group-specific evolution of modes of photosynthesis, i.e., C₃ and CAM. We thus conclude that CAM has evolved independently several times within the genus Clusia.     

Floral biology and reproductive ecology ofClusia nemorosa (Clusiaceae) in northeastern Brazil

Between March 1993 and May 1995 the floral biology and the phenology ofClusia nemorosa G. Mey. (Clusiaceae) were studied in two dioecious populations in Pernambuco State, and some observations were made in gynodioecious populations, that were encountered only in Bahia State. The species flowers from June to January/February. The flowers are bowl shaped, and last only one day. The floral reward is resin which is produced by staminodia in all types of flowers. The sex ratio was 1:1 for the studied populations. The pollen viability was approximately 98%. Apomixis was not observed. The rate of fruit set, in hermaphrodite individuals, under natural conditions and the sex ratio of the gynodioecious populations studied suggest that the hermaphrodite individuals may behave as males.Clusia nemorosa is melittophilous and was visited by 16 species of bees. The main pollinators wereEuplusia mussitans, E. iopyhrra, Eulaema cingulata, E. nigrita, Euglossa cordata andEuglossa sp.     

Polyisoprenylated benzophenones from Clusia floral resins

From the floral resins of various Clusia species, seven polyisoprenylated benzophenones were isolated. HPLC allowed their quantification in all resins, revealing a distribution of benzophenone derivatives distinct from each other. In some species the staminal oils were collected and oleic, stearic and palmitic acids were the main constituents.     

Daily rhythm of MnSOD in the C<Subscript>3</Subscript>-CAM intermediate <Emphasis Type="Italic">Clusia fluminensis</Emphasis> Planch. et Triana.

The C₃-CAM intermediate plant Clusia fluminensis under well-watered at low light conditions opens stomata during the light period. In leaf extracts of this plant we have found two copper-zinc superoxide dismutases (CuZnSODs) and two manganese SODs: MnSOD-like protein (MnSOD II) and MnSOD I. Daily rhythm of the MnSOD I shows maximum activity during the afternoon hours and it is accompanied by only a very small tendency to increase in catalase (CAT) activity and lowering of citrate level.     

Comparison of Photosynthetic Responses of the Sympatric Tropical C3 Species Clusia multiflora H. B. K. and the C3-CAM Intermediate Species Clusia minor L. to Irradiance and Drought Stress in a Phytotron1

Abstract: Clusia multiflora H. B. K., an obligate C₃ species and Clusia minor L. a C₃/CAM intermediate species, are two physio-types of a similar morphotype. They can sympatrically occupy secondary savanna sites exposed to high insolation in the tropics. In C. multiflora severe stress, i.e., switching shade-grown plants to high light plus drought, resulted in leaves browning or yellowing and becoming necrotic. However, in long-term light stress C. multiflora was able to grow new leaves with their photosynthetic apparatus fit for high light conditions. Shade-grown C. minor readily overcame switching to high light conditions and drought, responding by a rapid change from C₃ photosynthesis to CAM. Decreasing _soil led to increased abscisic acid levels in the leaves of C. minor, however CAM induction was not directly related to this and was mainly determined by increased PPFD. Both species were capable of rapid accumulation of zea-xanthin for acute photoprotection following high PPFD exposure. The maximum capacity for zeaxanthin accumulation was larger in C. minor, but under steady high PPFD it only partially made use of this capacity, relying on high internal CO₂ concentrations of Phase Ill of CAM, in addition to zeaxanthin, for acute photo-protection. Thus, by different means the two species perform well under high light conditions. However, C. multiflora needs time for development of adapted leaves under such stress conditions while the more flexible C. minor can readily switch from low light to high light conditions.     

Contrasting response of seedlings of two tropical species Clusia minor and Clusia multiflora to mycorrhizal inoculation in two soils with different pH

Differences in mycotrophic growth and response to phosphorus (P) fertilization were studied in seedlings of two woody native species: Clusia minor L. and Clusia multiflora H.B.K. from a cloud montane forest of tropical America. Greenhouse investigation was undertaken to determine the relationships between mycorrhizal dependency of host species associated with P utilization and growth in two different soils contrasting in pH (acidic and neutral) and nutrient content. Four treatments were performed: sterilized soil; sterilized soil plus 375 mg/kg of triple superphosphate (TSP); sterilized soil inoculated with Scutellospora fulgida (20 g/pot); and sterilized soil plus S. fulgida and TSP, with 10 replications per treatment for the two species. Results showed that both Clusia species presented high growth response to increasing P availability, which indicates that the root morphology (magnolioid roots) of these species is not a limiting factor for the incorporation of P from soils. Plants inoculated with arbuscular mycorrhizal fungi (AMF) in acidic soil had significantly increased shoot and root biomass, leaf area and height, in comparison to the biomass of P-fertilized plants and nonmycorrhizal plants. In neutral soil, seedlings of C. minor and C. multiflora were negatively affected by inoculation with AMF. In contrast, a significant decrease in growth was observed when inoculated plants were compared with noninoculated plants on neutral soil. Results indicate that an increase in the availability of a limiting nutrient (P) can turn a balanced mutualistic relationship into a less balanced nonmutualistic one.