Phylogeography above the species level for perennial species in a composite genus.

In phylogeography, DNA sequence and fingerprint data at the population level are used to infer evolutionary histories of species. Phylogeography above the species level is concerned with the genealogical aspects of divergent lineages. Here, we present a phylogeographic study to examine the evolutionary history of a western Mediterranean composite, focusing on the perennial species of Helminthotheca (Asteraceae, Cichorieae). We used molecular markers (amplified fragment length polymorphism (AFLP), internal transcribed spacer and plastid DNA sequences) to infer relationships among populations throughout the distributional range of the group. Interpretation is aided by biogeographic and molecular clock analyses. Four coherent entities are revealed by Bayesian mixture clustering of AFLP data, which correspond to taxa previously recognized at the rank of subspecies. The origin of the group was in western North Africa, from where it expanded across the Strait of Gibraltar to the Iberian Peninsula and across the Strait of Sicily to Sicily. Pleistocene lineage divergence is inferred within western North Africa as well as within the western Iberian region. The existence of the four entities as discrete evolutionary lineages suggests that they should be elevated to the rank of species, yielding H. aculeata, H. comosa, H. maroccana and H. spinosa, whereby the latter two necessitate new combinations.

Big thistle eats the little thistle: does unidirectional introgressive hybridization endanger the conservation of Onopordum hinojense?

Hybridization is known to have a creative role in plant evolution. However, it can also have negative effects on parental species. Onopordum is a large genus whose species frequently hybridize. In the Southwest Iberian Peninsula, the rare O. hinojense co-occurs with the widely distributed O. nervosum, and hybrids between these two taxa have been described as O. × onubense. In this study we determine the extinction risk in a hybrid zone, both for hybrids and parentals, using analyses of morphological and cytogenetic traits as well as genetic markers and demographic models. To investigate the introgression process we used amplified fragment length polymorphism (AFLP) markers, Bayesian analyses and genome scan methods. Morphology, genome size and molecular markers confirmed homoploid hybridization and also indicated unidirectional backcrossing of F1 hybrids with O. nervosum, which is likely to swamp O. hinojense, the parental with lower pollen size and a very low fruit set (8%). Genome scan methods revealed several loci significantly deviating from neutrality. Finally, our demographic modeling indicated that the higher fitness of O. nervosum threats the survival of O. hinojense by demographic swamping. Our study provides strong new evidence for a scenario of rapid extinction by unidirectional introgression and demographic swamping. The multifaceted approach used here sheds new light on the role of introgression in plant extinctions.

Effects of tree architecture on pollen dispersal and mating patterns in Abies pinsapo Boiss. (Pinaceae).

Plant architecture is crucial to pollination and mating in wind-pollinated species. We investigated the effect of crown architecture on pollen dispersal, mating system and offspring quality, combining phenotypic and genotypic analyses in a low-density population of the endangered species Abies pinsapo. A total of 598 embryos from three relative crown height levels (bottom, middle and top) in five mother plants were genotyped using eleven nuclear microsatellite markers (nSSRs). Paternity analysis and mating system models were used to infer mating and pollen dispersal parameters. In addition, seeds were weighed (N = 16 110) and germinated (N = 736), and seedling vigour was measured to assess inbreeding depression. Overall, A. pinsapo shows a fat-tailed dispersal kernel, with an average pollen dispersal distance of 113-227 m, an immigration rate of 0.84-26.92%, and a number of effective pollen donors (Nep ) ranging between 3.5 and 11.9. We found an effect of tree height and relative crown height levels on mating parameters. A higher proportion of seeds with embryo (about 50%) and a higher rate of self-fertilization (about 60%) were found at the bottom level in comparison with the top level. Seed weight and seedling vigour are positively related. Nevertheless, no differences were found in seed weight or in seedling-related variables such as weight and length of aerial and subterranean parts among the different relative crown height levels, suggesting that seeds from the more strongly inbred bottom level are not affected by inbreeding depression. Our results point to vertical isotropy for outcross-pollen and they suggest that self-pollen may ensure fertilization when outcross-pollen is not available in low-density population.

Phylogeography of SW Mediterranean firs: different European origins for the North African Abies species.

The current distribution of Western Mediterranean Abies species is a result of complex geodynamic processes and climatic oscillations that occurred in the past. Abies sect. Piceaster offers a good study model to explore how geo-climatic oscillations might have influenced its expansion and diversification on both sides of the W Mediterranean basin. We investigated the genetic variation within and among nine populations from five Abies species by molecular markers with high and low mutation rates and contrasting inheritance (AFLP and cpSSR). Analyses revealed the opening of the Strait of Gibraltar as an effective barrier against gene flow between the Southern Iberian (A. pinsapo) and North African (A. marocana and A. tazaotana) firs. The A. pinsapo populations in Spain and likewise those of the A. marocana - A. tazaotana population complex were not differentiated, and no evidence was found to distinguish A. tazaotana at the species level. Diversification of Abies across North Africa could occur by way of at least two vicariant events from Europe, in the west, giving rise to the A. marocana - A. tazaotana complex, and in the east, giving A. numidica. Secondary contacts among species from Abies sect. Piceaster (A. pinsapo and A. numidica), and with A. alba (Abies sect. Abies) are also indicated. However, there is a closer relationship between the Algerian fir (A. numidica) and the North Mediterranean widespread A. alba, than with the Moroccan firs (A. marocana and A. tazaotana) or the Southern Iberian (A. pinsapo). We also discuss the distribution range of these taxa in its paleogeological and paleoclimatic context, and propose that part of the modern geography of the South-Western Mediterranean firs might be traced back to the Tertiary.

Nuclear microsatellite primers for the endangered relict fir, Abies pinsapo (Pinaceae) and cross-amplification in related Mediterranean Species.

Twelve nuclear microsatellite primers (nSSR) were developed for the endangered species Abies pinsapo Boiss. to enable the study of gene flow and genetic structure in the remaining distribution areas. Microsatellite primers were developed using next-generation sequencing (454) data from a single Abies pinsapo individual. Primers were applied to thirty individuals from the three extant localities. The number of alleles per locus ranged from one to four. Cross-amplification was tested for other Abies species from the Mediterranean Basin, and most of the loci showed higher polymorphisms in the Mediterranean species than in A. pinsapo. These microsatellite markers provide tools for conservation genetic studies in Abies pinsapo as well other Abies species from the Mediterranean Basin.

Phenotypic consequences of polyploidy and genome size at the microevolutionary scale: a multivariate morphological approach.

• Chromosomal duplications and increases in DNA amount have the potential to alter quantitative plant traits like flower number, plant stature or stomata size. This has been documented often across species, but information on whether such effects also occur within species (i.e. at the microevolutionary or population scale) is scarce. • We studied trait covariation associated with polyploidy and genome size (both monoploid and total) in 22 populations of Dianthus broteri s.l., a perennial herb with several cytotypes (2x, 4x, 6x and 12x) that do not coexist spatially. Principal component scores of organ size/number variations were assessed as correlates of polyploidy, and phylogenetic relatedness among populations was controlled using phylogenetic generalized least squares. • Polyploidy covaried with organ dimensions, causing multivariate characters to increase, remain unchanged, or decrease with DNA amount. Variations in monoploid DNA amount had detectable consequences on some phenotypic traits. According to the analyses, some traits would experience phenotypic selection, while others would not. • We show that polyploidy contributes to decouple variation among traits in D. broteri, and hypothesize that polyploids may experience an evolutionary advantage in this plant lineage, for example, if it helps to overcome the constraints imposed by trait integration.

Mycorrhizal fungi and parasitic plants: Reply.

In a recent study (American Journal of Botany 97: 730-737), we described the first case of a tripartite association in natural conditions among a holoparasitic plant (Cytinus), its host Cistaceae species, and mycorrhizal fungi at an anatomical level. In a letter to the editor, Brundrett (American Journal of Botany 98: 595-596) commented on our manuscript and questioned our conclusions, arguing that they are not adequately supported by the data. We reject this point of view and believe that the controversy has arisen because of the parasitic way of life of Cytinus. We maintain and demonstrate that there is enough evidence in the data that we presented to confirm the existence of mycorrhizal associations in the Cytinus-Cistaceae complex, supporting the functionality of the tripartite association. Most holoparasitic plants have been considered as nonmycorrhizal. However, it is not advisable to be categorical in drawing conclusions about the mycorrhizal status of a group of plants that has not been fully studied.

Spatial and temporal patterns of floral scent emission in Dianthus inoxianus and electroantennographic responses of its hawkmoth pollinator.

Scent emission is important in nocturnal pollination systems, and plant species pollinated by nocturnal insects often present characteristic odor compositions and temporal patterns of emission. We investigated the temporal (day/night; flower lifetime) and spatial (different flower parts, nectar) pattern of flower scent emission in nocturnally pollinated Dianthusinoxianus, and determined which compounds elicit physiological responses on the antennae of the sphingid pollinator Hyles livornica. The scent of D.inoxianus comprises 68 volatile compounds, but is dominated by aliphatic 2-ketones and sesquiterpenoids, which altogether make up 82% of collected volatiles. Several major and minor compounds elicit electrophysiological responses in the antennae of H. livornica. Total odor emission does not vary along day and night hours, and neither does along the life of the flower. However, the proportion of compounds eliciting physiological responses varies between day and night. All flower parts as well as nectar release volatiles. The scent of isolated flower parts is dominated by fatty acid derivatives, whereas nectar is dominated by benzenoids. Dissection (= damage) of flowers induced a ca. 20-fold increase in the rate of emission of EAD-active volatiles, especially aliphatic 2-ketones. We suggest that aliphatic 2-ketones might contribute to pollinator attraction in D. inoxianus, even though they have been attributed an insect repellent function in other plant species. We also hypothesize that the benzenoids in nectar may act as an honest signal ('nectar guide') for pollinators.

Endozoochory by beetles: a novel seed dispersal mechanism.

BACKGROUND AND AIMS: Due in part to biophysical sized-related constraints, insects unlike vertebrates are seldom expected to act as primary seed dispersers via ingestion of fruits and seeds (endozoochory). The Mediterranean parasitic plant Cytinus hypocistis, however, possesses some characteristics that may facilitate endozoochory by beetles. By combining a long-term field study with experimental manipulation, we tested whether C. hypocistis seeds are endozoochorously dispersed by beetles. METHODS: Field studies were carried out over 4 years on six populations in southern Spain. We recorded the rate of natural fruit consumption by beetles, the extent of beetle movement, beetle behaviour and the relative importance of C. hypocistis fruits in beetle diet. KEY RESULTS: The tenebrionid beetle Pimelia costata was an important disperser of C. hypocistis seeds, consuming up to 17·5 % of fruits per population. Forty-six per cent of beetles captured in the field consumed C. hypocistis fruits, with up to 31 seeds found in individual beetle frass. An assessment of seeds following passage through the gut of beetles indicated that seeds remained intact and viable and that the proportion of viable seeds from beetle frass was not significantly different from that of seeds collected directly from fruits. CONCLUSIONS: A novel plant-animal interaction is revealed; endozoochory by beetles may facilitate the dispersal of viable seeds after passage through the gut away from the parent plant to potentially favourable underground sites offering a high probability of germination and establishment success. Such an ecological role has until now been attributed only to vertebrates. Future studies should consider more widely the putative role of fruit and seed ingestion by invertebrates as a dispersal mechanism, particularly for those plant species that possess small seeds.

Radiative evolution of polyploid races of the Iberian carnation Dianthus broteri (Caryophyllaceae).

SUMMARY: *The micro-evolutionary mechanisms that drive large-scale radiations are not completely understood, partly because of a shortage of population-level studies aimed at identifying putative causes of rapid evolutionary change. The Dianthus broteri complex, representing the largest polyploid series known to date for any species in the genus (2x, 4x, 6x and 12x cytotypes), belongs to a lineage that was recently found to have diversified at unusually rapid rates. *We used a combination of genome sequencing (internal transcribed spacer (ITS), plus chloroplast DNA (cpDNA) regions trnH-psbA, psbA-trnK and trnK-matK) and amplified fragment length polymorphism (AFLP) fingerprinting in 25 populations to infer the evolutionary history of extant polyploid races. *The haplotype, ribotype and AFLP reconstructions showed a star-shaped arrangement suggesting a pattern of radiative evolution. The major, widespread haplotype occurred at all ploidy levels, whereas 20 minor haplotypes were restricted to single populations and cytotypes. In addition, AFLP analyses retrieved well-supported cytogeographic groups: six clades were clearly differentiated in terms of ploidy level and geography. Molecular data indicate that gene flow among different cytotypes is rare or nonexistent. *Our study supports a scenario of rapid diversification in carnations in which autopolyploidy and allopolyploidy, in interaction with geography and/or isolation, have played prominent roles.

Phylogeny and genetic structure of Erophaca (Leguminosae), a East-West Mediterranean disjunct genus from the Tertiary.

The genus Erophaca comprises a single herbaceous perennial species with two subspecies distributed at opposite ends of the Mediterranean region. We used nrDNA ITS to investigate the phylogeny of the genus, and AFLP markers (9 primers, 20 populations) to establish the genetic relationship between subspecies, and among populations at each side of the Gibraltar Strait. According to nrDNA ITS, Erophaca is monophyletic, old (Miocene), and sister to the Astragalean clade. Life form attributes and molecular clock estimates suggest that Erophaca is one of the many Tertiary relicts that form part of the present Mediterranean flora. Within the occidental subspecies, European plants are clearly derived from North-African populations (Morocco) which, despite being rare on a regional scale, present the highest genetic diversity (as estimated by private and rare fragment numbers). In general, genetic diversity decreased with increasing distance from Morocco. AFLP and nrDNA ITS markers evidenced that the Eastern and the Western subspecies are genetically distinct. Possible causes for their disjunct distribution are discussed.

Anatomical relations among endophytic holoparasitic angiosperms, autotrophic host plants and mycorrhizal fungi: A novel tripartite interaction.

Mycorrhizae are widespread mutualistic symbioses crucial for the functioning of terrestrial ecosystems. Not all plants associate with mycorrhizae; most parasitic plants have been suggested to be nonmycorrhizal because they have developed alternative strategies to obtain nutrients. In endophytic parasitic plants, whose vegetative bodies grow completely inside their mycorrhizal host roots, the opportunity for establishing a tripartite association seems evident, but information on these systems is lacking. In studying natural associations among the endophytic holoparasite Cytinus hypocistis, their Cistaceae host species, and associated mycorrhizal fungi, we found that mycorrhizae were associated with the hosts and the parasites, reaching high frequencies of colonization. In parasitic and host root tissues, mycorrhizal fungi spread in the parenchymatic cells by intracellular growth and formed hyphal coils and vesicles, while the cambium and the vascular tissues were never colonized. This report is the first on a tripartite association of an endophytic parasitic plant, its host, and mycorrhizae in natural conditions, representing a novel trophic interaction not previously reported within the angiosperms. Additional studies on the interactions occurring among these three players are needed because they may be crucial to our understanding of how this mutualistic-antagonistic system is functioning and evolving.

Pleistocene refugia and polytopic replacement of diploids by tetraploids in the Patagonian and Subantarctic plant Hypochaeris incana (Asteraceae, Cichorieae).

We report the phylogeographic pattern of the Patagonian and Subantarctic plant Hypochaeris incana endemic to southeastern South America. We applied amplified fragment length polymorphism (AFLP) and chloroplast DNA (cpDNA) analysis to 28 and 32 populations, respectively, throughout its distributional range and assessed ploidy levels using flow cytometry. While cpDNA data suggest repeated or simultaneous parallel colonization of Patagonia and Tierra del Fuego by several haplotypes and/or hybridization, AFLPs reveal three clusters corresponding to geographic regions. The central and northern Patagonian clusters (approximately 38-51 degrees S), which are closer to the outgroup, contain mainly tetraploid, isolated and highly differentiated populations with low genetic diversity. To the contrary, the southern Patagonian and Fuegian cluster (approximately 51-55 degrees S) contains mainly diploid populations with high genetic diversity and connected by high levels of gene flow. The data suggest that H. incana originated at the diploid level in central or northern Patagonia, from where it migrated south. All three areas, northern, central and southern, have similar levels of rare and private AFLP bands, suggesting that all three served as refugia for H. incana during glacial times. In southern Patagonia and Tierra del Fuego, the species seems to have expanded its populational system in postglacial times, when the climate became warmer and more humid. In central and northern Patagonia, the populations seem to have become restricted to favourable sites with increasing temperature and decreasing moisture and there was a parallel replacement of diploids by tetraploids in local populations.

Distribution and diversity of cytotypes in Dianthus broteri as evidenced by genome size variations.

BACKGROUND AND AIMS: Studying the spatial distribution of cytotypes and genome size in plants can provide valuable information about the evolution of polyploid complexes. Here, the spatial distribution of cytological races and the amount of DNA in Dianthus broteri, an Iberian carnation with several ploidy levels, is investigated. METHODS: Sample chromosome counts and flow cytometry (using propidium iodide) were used to determine overall genome size (2C value) and ploidy level in 244 individuals of 25 populations. Both fresh and dried samples were investigated. Differences in 2C and 1Cx values among ploidy levels within biogeographical provinces were tested using ANOVA. Geographical correlations of genome size were also explored. KEY RESULTS: Extensive variation in chromosomes numbers (2n = 2x = 30, 2n = 4x = 60, 2n = 6x = 90 and 2n = 12x =180) was detected, and the dodecaploid cytotype is reported for the first time in this genus. As regards cytotype distribution, six populations were diploid, 11 were tetraploid, three were hexaploid and five were dodecaploid. Except for one diploid population containing some triploid plants (2n = 45), the remaining populations showed a single cytotype. Diploids appeared in two disjunct areas (south-east and south-west), and so did tetraploids (although with a considerably wider geographic range). Dehydrated leaf samples provided reliable measurements of DNA content. Genome size varied significantly among some cytotypes, and also extensively within diploid (up to 1.17-fold) and tetraploid (1.22-fold) populations. Nevertheless, variations were not straightforwardly congruent with ecology and geographical distribution. CONCLUSIONS: Dianthus broteri shows the highest diversity of cytotypes known to date in the genus Dianthus. Moreover, some cytotypes present remarkable internal genome size variation. The evolution of the complex is discussed in terms of autopolyploidy, with primary and secondary contact zones.

AFLP and breeding system studies indicate vicariance origin for scattered populations and enigmatic low fecundity in the Moroccan endemic Hypochaeris angustifolia (Asteraceae), sister taxon to all of the South American Hypochaeris species.

We used Amplified Fragment Length Polymorphism markers (AFLP) and breeding system studies to investigate the population structure and reproductive biology of Hypochaeris angustifolia (Asteraceae: Cichorieae). This species is endemic to altiplanos of the Atlas Mountains (Morocco) where it occurs in scattered populations, and it is the sister species to c. 40 species of this genus in South America. PCoA, NJ, and Bayesian clustering, revealed that the populations are very isolated whilst AFLP parameters show that almost all populations have marked genetic divergence. We contend that these features are more in accord with a vicariance origin for the scattered populations of H. angustifolia, rather than establishment by long-distance dispersal. The breeding system studies revealed that H. angustifolia is a self-incompatible species, with low fecundity in natural and in experimental crosses, probably due to a low frequency of compatible phenotypes within and between the populations.

The ant-pollination system of Cytinus hypocistis (Cytinaceae), a Mediterranean root holoparasite.

BACKGROUND AND AIMS: The genus Cytinus is composed of rootless, stemless and leafless parasites whose flowers are only visible during the reproductive period when they arise from the host tissues. Most of the taxa occur in Madagascar and South Africa, where mammal pollination has been suggested for one species. There is only one species in the Mediterranean region, and its pollination system has been unknown. Here, a long-term field observation study is combined with experimental pollination treatments in order to assess the pollination biology and reproductive system in the Mediterranean species Cytinus hypocistis. METHODS: Field studies were carried out in six populations in southern Spain over 4 years. Temporal and spatial patterns of variation in the composition and behaviour of floral visitors were characterized. Pollen loads and pollen viability were observed, and exclusion and controlled-pollination treatments were also conducted. KEY RESULTS: Cytinus hypocistis is a self-compatible monoecious species that relies on insects for seed production. Ants were the main visitors, accounting for 97.4 % of total floral visits, and exclusion experiments showed that they act as true pollinators. They consistently touched reproductive organs, carried large pollen loads and transported viable pollen, although the different ant species observed in the flowers differed in their pollination effectiveness. The abundance of flying visitors was surprisingly low, and only the fly Oplisa aterrima contributed to fruit production and cross-pollination. CONCLUSIONS: Mutualistic services by ant are essential for the pollination of Cytinus hypocistis. Although this parasite does not exhibit typical features of the 'ant-pollination syndrome', many other characteristics indicate that it is evolving to a more specialized ant-pollination system. The striking interspecific differences in the pollination systems of Mediterranean Cytinus (ant-pollinated) and some South African Cytinus (mammal-pollinated) make this genus an excellent model to investigate the divergent evolution of pollination systems in broadly disjunct areas.

Genetic races associated with the genera and sections of host species in the holoparasitic plant Cytinus (Cytinaceae) in the Western Mediterranean basin.

* Speciation via race formation is an important evolutionary process in parasites, producing changes that favour their development on particular host species. Here, the holoparasitic plant Cytinus, which has diverse host species in the family Cistaceae, has been used to study the occurrence of such races. * Amplified fragment length polymorphism (AFLP) analyses were performed on 174 individuals of 22 populations parasitizing 10 Cistaceae species in the Western Mediterranean basin. * Neighbour-joining, multivariate ordination analyses, and individual-based Bayesian analyses, clustered Cytinus populations into five well-characterized genetic races that, overall, agreed with the taxonomic sections of their hosts. In the AMOVA, among-races differences accounted for almost 50% of the genetic variation. The isolation-by-distance model was not supported by a Mantel test among Cytinus populations (r = 0.012; P = 0.456). All races showed low within-population genetic diversity, probably as a result of restricted pollen flow aggravated by flowering asynchrony, restricted seed dispersion, or stochastic processes. * The genetic differentiation among the five races of Cytinus is congruent with the view that these races are well-characterized lineages that have evolved independently as a result of selective pressures imposed by their hosts. This pattern, with genetically distinctive groups associated with the infrageneric sections of the host species, has not been reported previously for parasitic angiosperms.

Range-wide phylogeography of Juniperus thurifera L., a presumptive keystone species of western Mediterranean vegetation during cold stages of the Pleistocene.

We investigate the range-wide population structure and phylogeography of thuriferous juniper (Juniperus thurifera L.), a species with a highly disjunct distribution in the western Mediterranean. We genotyped a total of 327 individuals from 20 populations using amplified fragment length polymorphisms (AFLP). Different analyses such as principal co-ordinate analysis (PCoA), nonmetric multidimensional scaling of F(ST) distances among populations, unweighted pair group method with arithmetic mean (UPGMA), and Bayesian clustering revealed that the Strait of Gibraltar acted as an efficient barrier against gene flow between the Moroccan and European populations for a very long time, and consequently support that the Moroccan populations should be recognised as a distinct subspecies (J. thurifera L. subsp. africana (Maire) Romo and Boratynsky). The Algerian population was genetically more closely related to the European than to the Moroccan ones, probably due to dispersal events from Europe to Algeria. With respect to the mainland European populations, our data are not conclusive to reject any of the two following hypotheses: (1) the Iberian Peninsula was subdivided into different gene pools, and was the source for the colonisation of the Pyrenees and the Alps; and (2) the pattern we see today is partly the result of immigration into the Iberian Peninsula, e.g. from the Alps. Finally, the Corsican population was closely related genetically to two northern Iberian populations most probably due to relatively recent long-distance dispersal.

Phylogeography of North African Atlas cedar (Cedrus atlantica, Pinaceae): Combined molecular and fossil data reveal a complex Quaternary history.

Northwest Africa is a major hotspot of plant biodiversity, but very little is known about the Quaternary range dynamics of plant species in this region. Here we investigate the range-wide population structure and phylogeography of Atlas cedar (Cedrus atlantica), an emblematic forest tree endemic to Morocco and Algeria. We genotyped 261 individuals from 11 populations using AFLP markers. Data were analyzed using both conventional F(ST)-based techniques and Bayesian clustering. Overall population differentiation was high (F(ST) = 0.25). Two major groups of populations were identified, one distributed through the Rif and Middle Atlas mountains in Morocco and the other through the Algerian Tell Atlas and Aurès mountains as well as the Middle Atlas. Combined molecular and fossil data indicate that C. atlantica survived the Last Glacial Maximum in at least three disjunct refugia along the coast of the Mediterranean Sea, whereas the Middle Atlas, today the core of the species range, has been colonized relatively recently (<10000 yr BP). The colonization history of individual populations has left clear imprints in their present-day diversity, which may vary greatly even between nearby stands. Our study illustrates how integrating different data sources and analytical approaches can help elucidate complex range dynamics that would otherwise remain undeciphered.

The endophytic system of mediterranean Cytinus (cytinaceae) developing on five host Cistaceae species.

BACKGROUND AND AIMS: One of the most extreme manifestations of parasitism is found in the genus Cytinus, a holoparasite whose vegetative body is reduced to an endophytic system living within its host root. There are two species of Cytinus in the Mediterranean, C. hypocistis and C. ruber, which parasitize various genera of Cistaceae, one of the most characteristic families of the Mediterranean scrublands. The aim of this work is to describe the endophytic systems of C. hypocistis and C. ruber, and their tissue relationships with their host. METHODS: Roots from five different hosts infected with C. hypocistis and C. ruber were harvested, and examined by anatomical techniques under light microscopy to elucidate the characteristics of the endophytic system of Cytinus, and to determine if differences in endophytic systems occur between the two species and in response to different hosts. KEY RESULTS: The endophyte structure is similar in both Cytinus species irrespective of the host species. In the initial stages of the endophyte, rows of parenchymal cells spread through the host pericyclic derivatives and phloem, and begin to generate small nodules in the outermost region of the host xylem. Later the nodules anastomose, and bands of parasitic tissue are formed. The host cambium continues to develop xylem tissue, and consequently the endophyte becomes enclosed within the xylem. The bands of parasitic tissue fuse to form a continuous sheath. This mature endophyte has well-developed vascular system with xylem and phloem, and forms sinkers with transfer cells that grow through the host xylem. CONCLUSIONS: The endophytic system of Cytinus develops in all host root tissues and reaches its most mature stages in the host xylem. It is more complex than previously reported, showing parenchyma, xylem and phloem tissues. This is the first report of well-developed phloem in a holoparasitic endophytic species.