Eduardo Leite Borba

Fly‐pollinated Pleurothallis (Orchidaceae) species have high genetic variability: evidence from isozyme markers

We conducted an isozyme study in 22 populations of five Pleurothallis (Orchidaceae) species (12 loci in nine enzymatic systems). The genetic variability in all populations is surprisingly high (P = 58-83%, A = 2.1-3.8, H(e) = 0.25-0.43) in spite of the fact that the five species are pollinated by small flies whose behavior enables self-pollination. We suggest that self-incompatibility, inbreeding depression, and mechanical barriers that prevent self-pollination in these species are responsible for the maintainance of the high genetic variability. These traits are uncommon in Orchidaceae, but have been observed in these and some other species pollinated by flies or other pollinators with behavior that facilitates self-pollination. The genetic similarity among conspecific populations is also high for species with very short-range flying pollinators. Only one population of P. teres presented values of genetic similarity lower than usually observed in allopatric conspecific populations. Morphology, however, does not support its segregation as a new taxon. All species can be recognized by their enzymatic patterns, and the results agree with recently proposed taxonomic realignments. Conversely, the supposed affinities among these species based on floral morphology are not supported, and we hypothesize that it may be due to convergence in species with similar pollinators.

Temporal variation in pollinarium size after its removal in species of Bulbophyllum: A different mechanism preventing self-pollination in Orchidaceae

InBulbophyllum involutum andB. ipanemense (Orchidaceae), two closely related species, shortly after removal the pollinarium has a diameter of approximately twice that of the entrance of the stigmatic cavity, requiring a mean time of 105 to 135 minutes to shrink in width and allow pollination. Because the pollinators of these species remain for some minutes in the same flower after removing the pollinia this mechanism, previously unknown inOrchidaceae, may be very important in preventing self-pollination. This mechanism does not occur inB. weddellii, and the pollinator does not remain in the flower after removing the pollinia. The smaller diameter of the stigmatic cavity inB. involutum reduces by 50% the chances of interspecific pollination withB. weddellii, and interspecific crossing is strictly unidirectional. This is important in maintaining isolation between these sympatric species, which share the same pollinators and have synchronized flowering.

A simple solid injection device for the analyses of Bulbophyllum (Orchidaceae) volatiles

Abstract The volatile components of three orchid species ( Bulbophyllum weddellii , B. ipanemense and B. involutum ) were detected using GC/MS. Due to the minute dimension and amount of flowers, special traps and extraction methods were applied and the analyses were finally successful using a special solid sample injector which was constructed in our own laboratory. The chemical composition comparison revealed the high similarity between the volatiles present in B. weddellii and B. involutum , which is coherent with the pollinator sharing. On the other hand, the volatiles of B. ipanemense , a species pollinated by other pollinator species, are different from the two previous species.

Morphological and histological characterization of the osmophores and nectaries of four species of Acianthera (Orchidaceae: Pleurothallidinae)

Nectar and floral odor are frequently associated with the presence and maintenance of specialized pollination systems in Orchidaceae. We studied flowers of four Acianthera species, a genus of myophilous orchids belonging to the largest fly-pollinated orchid group Pleurothallidinae, in order to characterize the secretory structures related to their pollination mechanism. Flowers at anthesis were sampled to detect volatile compounds and nectar; samples were fixed for light microscopy and scanning and transmission electron microscopy. The labellum presents epidermal cells and the first mesophyll layer involved with secretory processes. Cellular characteristics of these regions associated with the occurrence of sugars allowed us to recognize them as nectaries. Some portions of the sepals also shown to be involved with secretory processes and the presence of nitrogenated volatile compounds characterize them as osmophores. The production of nectar in these species makes the occurrence of sapromyophily questionable, even though these flowers present characteristics of this floral syndrome. The presence of osmophores on the sepals reinforces that this localization is common among the Pleurothallidinae, whilst they occur in a different region (labelum) in the other major fly-pollinated orchid group (Bulbophyllum).

Reproductive systems and crossing potential in three species of Bulbophyllum (Orchidaceae) occurring in Brazilian 'campo rupestre' vegetation

Experimental self-, cross- and interspecific pollinations were carried out inBulbophyllum weddellii, B. involutum andB. ipanemense (Orchidaceae). The last two species are closely related, butB. weddellii is somewhat more distant. The three species are self-compatible, with pollination by a vector necessary for fruit formation. In all crossing types, high rates of fruit abortion and formation of seeds without embryos were observed, particularly in interspecific crosses that involvedB. weddellii. Crosses between the more closely relatedB. involutum andB. ipanemense produced fruit formation rates and seed viability similar to those obtained in intraspecific crosses. These results agree with current hypotheses that suggest that interspecific crossing rates reflect phylogenetic proximity. Examination of pollen tube growth and aborted fruits suggests that a series of factors may be involved in the high abortion rate for fruits and large proportion of seeds without embryos. The low interspecific fertility noted forB. weddellii andB. involutum is important in the maintenance of isolation between these species that are sympatric, flower in the same period and share the same pollinators.

Self-incompatibility and myophily in Octomeria (Orchidaceae, Pleurothallidinae) species

Pleurothallidinae (ca. 4,000 spp.) is the largest myophilous group in Orchidaceae. Recently three self-compatible bird-pollinated genera were included at the base of the group, while Octomeria (plus Brachionidium) is the sister group to the remaining subtribe in the former circumscription. Studies of reproductive biology of three Octomeria species were carried out to determine the occurrence of myophily and self-incompatibility in the genus, to help trace the evolution of these characteristics in the subtribe. The three species showed a strong self-incompatibility and are intercompatible. Octomeria crassifolia and O. grandiflora are pollinated by Sciaridae (Diptera) species, presenting high specificity. These results indicate that both self-incompatibility and myophily are basal characteristics in the former delimitation of the Pleurothallidinae and possibly have been kept in the derived lineages, representing biological synapomorphies of the group.

Demography, and genetic and morphological variability of the endangered Sophronitis sincorana (Orchidaceae) in the Chapada Diamantina, Brazil

We carried out a demographic study and evaluated the genetic and morphological variability in five populations of the endangered Sophronitis sincorana (Orchidaceae) endemic to Northeastern Brazil, based on allozyme and morphometric analyses. Plant density was approximately 0.5 plants/m2, and the projected total number of plants was approximately 50,000 individuals. However, fruit set and recruitment of individuals are rare. The genetic variability was very high in all populations (P = 100, A = 3.0−3.5, He = 0.33−0.48), and all populations presented similar values of morphological variability. Low genetic and morphological structuring were found in the species (FST = 0.053, AMRPP = 0.018). The elevated coefficient of endogamy encountered in populations of S. sincorana indicates the occurrence of structuring within the populations. The lack of correlation between morphological and genetic variation in this species indicates that none of the markers examined should be used separately for either conservation purposes.

Biosystematics of Chamaecrista sect. Absus subsect. Baseophyllum (Leguminosae-Caesalpinioideae) based on allozyme and morphometric analyses

We carried out genetic and morphometric analyses in 33 populations belonging to all nine described and one putative taxa of Chamaecrista subsect. Baseophyllum. Genetic variability was low in all taxa. Morphological and genetic data are consistent with the status of C. depauperata and C. coriacea. However, divergence between C. cytisoides var. cytisoides and all the other six conspecific varieties is higher than the divergence between it and the other two species. Our data support the recognition of eight species in the subsection Baseophyllum. Chamaecrista coriacea and C. depauperata are supported in their original circumscription. However, the varieties ascribed to C. cytisoides are best treated as six different species. We propose five new combinations in order to raise varieties of C. cytisoides to specific rank: C. blanchetii, C. brachystachya, C. confertiformis, C. decora, C. unijuga. We also propose synonimization of one variety, C. cytisoides var. micrantha, under C. brachystachya.

Geographical genetic structuring and phenotypic variation in the Vellozia hirsuta (Velloziaceae) ochlospecies complex

PREMISE OF THE STUDY Vellozia hirsuta forms a complex presenting wide morphological and anatomical variation, resulting in five specific names and 14 morpho-anatomical patterns occurring in disjunct populations. We carried out a phylogeographical study to investigate the existence of correlation among the genetic and morphological patterns within this complex, and to determine whether it is composed of various species or should be treated as an ochlospecies, a species having widely polymorphic and weakly polytypic complex variation, with morphological characteristics varying independently. METHODS We carried out phylogeographical analyses using cpDNA rpl32F-trnL intergenic region. KEY RESULTS We found 20 haplotypes in 23 populations sampled. The populations are genetically structured (Φ(ST) = 0.818) into four phylogeographical groups demonstrating geographical structuring but with no correlation with morpho-anatomical patterns. Our analyses do not support recognizing any of the species now synonymized under Vellozia hirsuta. The northern populations were the most genetically differentiated and could be considered a distinct taxon, as they are also morphologically different. CONCLUSIONS It is recommended that Vellozia hirsuta be considered a single enormously variable species. The patterns of variation within V. hirsuta probably are related to climatic changes that occurred during the Pleistocene Epoch in tropical Brazil when reductions in forest cover favored the expansion of V. hirsuta populations into extensive lowland areas. The expansion of forest cover at the end of the glaciations would have again restricted the occurrence of campos rupestres vegetation to high elevations, which constitute the current centers of diversity of this species.

Evidence of natural hybridization and introgression in Bulbophyllum involutum Borba, Semir & F. Barros and B. weddellii (Lindl.) Rchb. f. (Orchidaceae) in the Chapada Diamantina, Brazil, by using allozyme markers

Evidence of natural hybridization and introgression in Bulbophyllum involutum Borba, Semir & F. Barros and B. weddellii (Lindl.) Rchb. f. (Orchidaceae) in the Chapada Diamantina, Brazil, by using allozyme markers). Hybridization between B. involutum and B. weddellii (Orchidaceae) has been first observed in the Serra do Cipo, Minas Gerais State, Brazil, the hybrid being described as B. ×cipoense Borba & Semir. In this study, allozime electrophoresis was used to test the hypothesis of occurrence of hybridization between these two species, as suggested by morphological characters, in the Chapada Diamantina, Bahia State, Brazil. The lack of a diagnostic locus does not allow definite confirmation of the natural hybridization, although this hypotheses is reinforced by the absence of exclusive alleles in the putative hybrid individuals. The existence of several different genotypes points out to either population derived from multiple hybridization events or the hybrids produced offspring. Homozigosity in some morphologically intermediate individuals of alelles which are exclusive to B. involutum and high genetic similarity between them reinforce the hypotheses of introgression in B. involutum, but not in B. weddellii. Genetic variability observed in B. weddellii (He = 0.21) and B. involutum (He = 0.35) is high. Bulbophyllum weddellii and B. involutum presented very high genetic similarity values (0.94). These species, although vegetatively similar, have been placed in different sections based on floral morphology. The results suggest that these species may be more related than previously supposed. RESUMO - (Evidencia de hibridacao e introgressao em Bulbophyllum involutum Borba, Semir & F. Barros e B. weddellii (Lindl.) Rchb. f. (Orchidaceae) na Chapada Diamantina, Brasil, a partir de marcadores alozimaticos). Hibridacao entre B. involutum e B. weddellii (Orchidaceae) foi observada pela primeira vez na Serra do Cipo, Estado de Minas Gerais, Brasil, o hibrido sendo descrito como B. ×cipoense Borba & Semir. Neste estudo, foi utilizada eletroforese de alozimas para testar a hipotese de ocorrencia de hibridacao entre estas especies, como sugerido por caracteres morfologicos, na Chapada Diamantina, Estado da Bahia, Brasil. A falta de um locus diagnostico nao permite confirmacao definitiva da hibridacao natural, embora esta hipotese seja reforcada pela ausencia de alelos exclusivos nos individuos supostamente hibridos. A existencia de varios genotipos diferentes indica que ou a populacao e derivada de multiplos eventos de hibridacao, ou que os hibridos estao se reproduzindo. Homozigose em alguns individuos morfologicamente intermediarios de alelos exclusivos a especie B. involutum e a elevada similaridade genetica entre eles reforcam a hipotese de introgressao em B. involutum, mas nao em B. weddellii. A variabilidade genetica observada em B. weddellii (He = 0,21) e B. involutum (He = 0,35) e elevada. Bulbophyllum weddellii e B. involutum apresentaram valor de similaridade genetica muito elevado (0,94). Estas especies, embora vegetativamente muito similares, tem sido colocadas em diferentes secoes baseado na morfologia floral. Os resultados encontrados sugerem que estas especies sao mais relacionadas do que previamente suposto. Palavras-chave - Bulbophyllum, hibridacao, introgressao, Orchidaceae, variabilidade genetica