Donata Cafasso

Photosynthetic Mediterranean meadow orchids feature partial mycoheterotrophy and specific mycorrhizal associations

PREMISE OF THE STUDY We investigated whether four widespread, photosynthetic Mediterranean meadow orchids (Ophrys fuciflora, Anacamptis laxiflora, Orchis purpurea, and Serapias vomeracea) had either nutritional dependency on mycobionts or mycorrhizal fungal specificity. Nonphotosynthetic orchids generally engage in highly specific interactions with fungal symbionts that provide them with organic carbon. By contrast, fully photosynthetic orchids in sunny, meadow habitats have been considered to lack mycorrhizal specificity. METHODS We performed both culture-dependent and culture-independent ITS sequence analysis to identify fungi from orchid roots. By analyzing stable isotope ((13)C and (15)N) natural abundances, we also determined the degree of autotrophy and mycoheterotrophy in the four orchid species. KEY RESULTS Phylogenetic and multivariate comparisons indicated that Or. purpurea and Oph. fuciflora featured lower fungal diversity and more specific mycobiont spectra than A. laxiflora and S. vomeracea. All orchid species were significantly enriched in (15)N compared with neighboring non-orchid plants. Orchis purpurea had the most pronounced N gain from fungi and differed from the other orchids in also obtaining C from fungi. CONCLUSIONS These results indicated that even in sunny Mediterranean meadows, orchids may be mycoheterotrophic, with correlated mycorrhizal fungal specificity.

Fine-scale phylogeographical analysis of Mediterranean Anacamptis palustris (Orchidaceae) populations based on chloroplast minisatellite and microsatellite variation

The phylogeographical history of the rare marsh orchid Anacamptis palustris (Orchidaceae) was reconstructed using highly polymorphic chloroplast minisatellite and microsatellite loci. Allelic variation at chloroplast microsatellite loci was due to length variation in poly(A/T) repeats and was informative on a regional scale, but was not sufficient to unravel relationships among populations on a local geographical scale. The minisatellite locus, however, was found to be highly variable. Nine distinct repeat types were found and variation in repeat number occurred in five repeat types. The distribution of chloroplast haplotypes, combining microsatellite and minisatellite repeat type variation, provided a clear phylogeographical picture on a large geographical scale, whereas length variation in one highly polymorphic minisatellite repeat type provided fine‐scale phylogeographical information. Mediterranean populations could be divided into four main lineages, a western European lineage, a northern and central Italian lineage, a well‐isolated southern Italian (Apulian) lineage, and an eastern European lineage. Variation at the most variable minisatellite repeat type N revealed 19 alleles and allowed the study of seed‐mediated gene flow and an estimation of the ratio of pollen to seed flow among neighbouring populations.

Molecular evolution of a plastid tandem repeat locus in an orchid lineage.

The molecular evolution of a chloroplast minisatellite locus in the Anacamptis palustris (Orchidaceae) lineage and haplotype variation in two Italian A. palustris populations were investigated. A phylogenetic analyses of the chloroplast tRNALEU intron, where the minisatellite locus is located, revealed that a deletion in the ancestor of the A. palustris lineage led to the formation of two noncontiguous, complementary sequence motifs. We propose a model to explain the initial formation of the minisatellite repeat motif, starting with the two noncontiguous, complementary sequence motifs. A survey of minisatellite variation in four species of the A. palustris lineage revealed several haplotypes that differed not only in repeat number, but also in repeat organization. A haplotype network suggests that three different minisatellite loci evolved independently at the same position in the tRNALEU intron. A secondary structure model revealed that the A. palustris minisatellite repeat forms a stem region of the tRNALEU intron, which allows its notable expansion without negatively affecting splicing. Minisatellite variation was high in the two examined A. palustris populations where 20 haplotypes were detected, whereas no length variation was detected in a neighboring poly (A) microsatellite locus. We estimated a chloroplast minisatellite mutation rate of 3.2 × 10−3 mutations per generation. Southern blot analyses did not find evidence for chloroplast heteroplasmy. Based on the analysis of the largest known, extant A.palustris population, a stepwise mutation model (SMM) was inferred.

Multiple shifts to different pollinators fuelled rapid diversification in sexually deceptive Ophrys orchids

Episodes of rapid speciation provide unique insights into evolutionary processes underlying species radiations and patterns of biodiversity. Here we investigated the radiation of sexually deceptive bee orchids (Ophrys). Based on a time-calibrated phylogeny and by means of ancestral character reconstruction and divergence time estimation, we estimated the tempo and mode of this radiation within a state-dependent evolutionary framework. It appears that, in the Pleistocene, the evolution of Ophrys was marked by episodes of rapid diversification coinciding with shifts to different pollinator types: from wasps to Eucera bees to Andrena and other bees. An abrupt increase in net diversification rate was detected in three clades. Among these, two phylogenetically distant lineages switched from Eucera to Andrena and other bees in a parallel fashion and at about the same time in their evolutionary history. Lack of early radiation associated with the evolution of the key innovation of sexual deception suggests that Ophrys diversification was mainly driven by subsequent ecological opportunities provided by the exploitation of novel pollinator groups, encompassing many bee species slightly differing in their sex pheromone communication systems, and by spatiotemporal fluctuations in the pollinator mosaic.

Characterization of a minisatellite repeat locus in the chloroplast genome of Orchis palustris (Orchidaceae)

Abstract. We describe the occurrence of a tandem repeat in the chloroplast genome of the marsh orchid, Orchis palustris. The repeat unit is an AT-rich, 16-bp sequence located in the chloroplast tRNALEU intron. Southern blot analysis confirmed that the O. palustris tRNALEU intron including the minisatellite locus has not been transferred to the nucleus, but is indeed located on the chloroplast genome. The 16-bp repeat unit was found to be present in all O. palustris accessions studied, as well as in the closely related O. laxiflora. Variation in repeat numbers among individuals was found in O. palustris from central and northern Italy; and this was consistently associated with a 13-bp sequence motif preceding the repeat. This motif was absent from O. palustris from southern Italy, Greece, and from O. laxiflora. In these accessions, no variation in repeat numbers was found. Our results suggest that the O. palustris chloroplast minisatellite locus evolved relatively recently, presumably in central Italy, and may represent a valuable marker for population genetic studies.

Transitions between self-compatibility and self-incompatibility and the evolution of reproductive isolation in the large and diverse tropical genus Dendrobium (Orchidaceae)

BACKGROUND AND AIMS The evolution of interspecific reproductive barriers is crucial to understanding species evolution. This study examines the contribution of transitions between self-compatibility (SC) and self-incompatibility (SI) and genetic divergence in the evolution of reproductive barriers in Dendrobium, one of the largest orchid genera. Specifically, it investigates the evolution of pre- and postzygotic isolation and the effects of transitions between compatibility states on interspecific reproductive isolation within the genus. METHODS The role of SC and SI changes in reproductive compatibility among species was examined using fruit set and seed viability data available in the literature from 86 species and ∼2500 hand pollinations. The evolution of SC and SI in Dendrobium species was investigated within a phylogenetic framework using internal transcribed spacer sequences available in GenBank. KEY RESULTS Based on data from crossing experiments, estimations of genetic distance and the results of a literature survey, it was found that changes in SC and SI significantly influenced the compatibility between species in interspecific crosses. The number of fruits produced was significantly higher in crosses in which self-incompatible species acted as pollen donor for self-compatible species, following the SI × SC rule. Maximum likelihood and Bayesian tests did not reject transitions from SI to SC and from SC to SI across the Dendrobium phylogeny. In addition, postzygotic isolation (embryo mortality) was found to evolve gradually with genetic divergence, in agreement with previous results observed for other plant species, including orchids. CONCLUSIONS Transitions between SC and SI and the gradual accumulation of genetic incompatibilities affecting postzygotic isolation are important mechanisms preventing gene flow among Dendrobium species, and may constitute important evolutionary processes contributing to the high levels of species diversity in this tropical orchid group.

Phylogeographic patterns, genetic affinities and morphological differentiation between Epipactis helleborine and related lineages in a Mediterranean glacial refugium

BACKGROUND AND AIMS In the Mediterranean basin, the Italian peninsula has been suggested to be one of the most important glacial refugia for temperate tree species. The orchid genus Epipactis is widely represented in the Italian peninsula by widespread species and several endemic, localized taxa, including selfing and outcrossing taxa. Here the phylogenetic and phylogeographic relationships in a group of closely related taxa in Epipactis are investigated with the aim of understanding the role of this refugial area for cladogenesis and speciation in herbaceous species, such as terrestrial orchids. METHODS Ribosomal DNA (rDNA) was employed to assess phylogenetic relationships, and plastid sequence variation in the rbcL-accD spacer was used to reveal phylogeographic patterns among plastid haplotypes using a parsimony network. KEY RESULTS Low genetic variation and shared ribotypes were detected in rDNA, whereas high levels of sequence variation and a strong phylogeographic structure were found in the examined plastid region. The parsimony plastid haplotype network identified two main haplotype groups, one including E. atrorubens/microphylla/muelleri/leptochila and the other including all accessions of E. helleborine and several localized and endemic taxa, with a combination of widespread and rare haplotypes detected across the Italian peninsula. A greater genetic divergence separated the Italian and other European accessions of E. helleborine. CONCLUSIONS Phylogenetic and phylogeographic patterns support a working hypothesis in which the Italian peninsula has only recently been colonized by Epipactis, probably during the most recent phase of the Quaternary age and, nevertheless, it acted as a remarkable centre of diversification for this orchid lineage. Changes in pollination strategy and recurrent shifts in mating system (from allogamy to autogamy) could have represented the mechanism promoting this rapid diversification and the observed high taxonomic complexity detected in the E. helleborine species complex.

Genetic variation in time and space: the use of herbarium specimens to reconstruct patterns of genetic variation in the endangered orchid Anacamptis palustris

Habitat alteration, fragmentation and destruction as a consequence of human impact are a global phenomenon. Here we document changes in genetic variation in the marsh orchid Anacamptis palustris as a consequence of such habitat changes. We examined historical specimens that were collected during the nineteenth and early twentieth centuries, prior to the most recent massive habitat changes affecting this species. Sequences of a hypervariable region in the plastid DNA, located in the tRNALEU intron, from herbarium vouchers were compared with those from a near-exhaustive survey of the extant A. palustris populations on the Italian peninsula. It was found that private haplotypes and alleles found in small, extant populations were once widespread and more common in historic populations and that alleles, once present in historic populations, have gone extinct. In addition, genetic differentiation among populations has increased over time and haplotype frequencies significantly differ among historic and extant populations. These results document that human induced habitat changes have reduced genetic diversity and increased the importance of random genetic drift in this species. It is concluded that the analysis of herbarium specimens may provide important insights into changes of genetic diversity over time and may be critical for correct inference of the evolutionary history of rare and endangered species.

Organization of a dispersed repeated DNA element in the Zamia genome

Occurrence and genomic organization of dispersed elements containing ZpS1 satellite repeats have been investigated in a wide representation of species of the old plant genus Zamia (Zamiaceae, Cycadales). In Z. paucijuga, the ZpS1 repeat is organized as long satellite DNA arrays and as short arrays inserted into AT-rich dispersed elements. A comparative study by Southern analysis shows that these unusual dispersed elements containing the ZpS1 repeat are present with different organizations in all investigated Zamia species. In some species these elements are present with a low copy number, while in other species secondary amplification events, involving specific sequence clusters, appear to have generated characteristic dispersed elements in a high copy number. Among Zamia species, several groups share similar restriction patterns, as the Zamia loddigesii complex and the Caribbean species suggesting a general correlation between organization and genomic representation of the dispersed repeated sequence and the pattern of phyletic relationships in the genus. However, the finding of different patterns also among closely related species suggests a complex history of amplifications and losses of these dispersed repetitive elements that cannot be always easily traced through the phylogenetic reconstruction of this ancient plant group.

Multiple and different genomic rearrangements of the rbcL gene are present in the parasitic orchid Neottia nidus-avis

In parasitic plants that have lost most, if not all, of their photosynthetic genes, the genome of their plastids has also undergone a dramatic reduction. For example, photosynthetic genes, such as rbcL, frequently become pseudogenes, in which large portions of the gene have been found to be deleted. Orchids are flowering plants with several parasitic lineages. This is consistent with the observation that parasitic orchids can invade pre-existing mutualistic associations between ectomycorrhizal trees and fungi to obtain fixed carbon and nutrients. In addition, some parasitic species are devoid of chlorophyll, and consequently, have lost their photosynthetic capacity. Here, the organization of the plastid genome of the parasitic orchid Neottia nidus-avis (L.) Rich. was investigated using sequencing and hybridization experiments. In particular, genomic rearrangements in the rbcL region of this parasitic orchid were analyzed. At least three distinct rbcL sequences were found to be present as pseudogenes and were likely located in the plastid genome. Based on these results, it is hypothesized that N. nidus-avis contains different plastomes, each with a different pseudogene, and these can exist within the same individual plant.