Beatriz Guzmán

Adaptive Radiation in Mediterranean Cistus (Cistaceae)

Background Adaptive radiation in Mediterranean plants is poorly understood. The white-flowered Cistus lineage consists of 12 species primarily distributed in Mediterranean habitats and is herein subject to analysis. Methodology/Principal Findings We conducted a “total evidence” analysis combining nuclear (ncpGS, ITS) and plastid (trnL-trnF, trnK-matK, trnS-trnG, rbcL) DNA sequences and using MP and BI to test the hypothesis of radiation as suggested by previous phylogenetic results. One of the five well-supported lineages of the Cistus-Halimium complex, the white-flowered Cistus lineage, comprises the higher number of species (12) and is monophyletic. Molecular dating estimates a Mid Pleistocene (1.04±0.25 Ma) diversification of the white-flowered lineage into two groups (C. clusii and C. salviifolius lineages), which display asymmetric characteristics: number of species (2 vs. 10), leaf morphologies (linear vs. linear to ovate), floral characteristics (small, three-sepalled vs. small to large, three- or five-sepalled flowers) and ecological attributes (low-land vs. low-land to mountain environments). A positive phenotype-environment correlation has been detected by historical reconstructions of morphological traits (leaf shape, leaf labdanum content and leaf pubescence). Ecological evidence indicates that modifications of leaf shape and size, coupled with differences in labdanum secretion and pubescence density, appear to be related to success of new species in different Mediterranean habitats. Conclusions/Significance The observation that radiation in the Cistus salviifolius lineage has been accompanied by the emergence of divergent leaf traits (such as shape, pubescence and labdanum secretion) in different environments suggets that radiation in the group has been adaptive. Here we argued that the diverse ecological conditions of Mediterranean habitats played a key role in directing the evolution of alternative leaf strategies in this plant group. Key innovation of morphological characteristics is supported by our dated phylogeny, in which a Mediterranean climate establishment (2.8 Ma) predated the adaptive radiation of the white-flowered Cistus.

Δ6-Desaturase sequence evidence for explosive Pliocene radiations within the adaptive radiation of Macaronesian Echium (Boraginaceae).

The oceanic islands of Macaronesia provide an ideal temporal and spatial context to test hypotheses of plant evolution using a novel set of phylogenetic markers, Delta(6)-desaturase sequences. In contrast to the limited resolution of standard molecular markers (nrDNA and plastid sequences), the Delta(6)-desaturase sequence phylogeny of Echium unequivocally reconstructs its active colonization across islands and archipelagos (Madeira, the Canary Islands, and Cape Verde), as well as its subsequent geographical and ecological speciation. Molecular-clock estimates using penalized likelihood and Bayesian inference reveal two radiation processes coincident with two dramatic climatic changes recorded in the region: the advent of the cold Canarian sea current (ca. 4 Ma) and the establishment of a strong seasonality in the Pleistocene (1.8 Ma). Though Echium had available all the diversity of present-day Macaronesian environments (xeric and mesic scrub, laurisilva, pine forest, and subalpine habitats) in the Miocene, evolutionary divergence appears to have been triggered by an extension of fluctuating xeric and mesic habitats with the advent of Pliocene conditions. These Echium radiations not only fulfill traditional predictions of adaptive radiation (i.e., common ancestry, rapid speciation, and phenotype-environment correlation), but also, uniquely among Macaronesian species, trait utility of woodiness. A Pliocene transition from annuality to a bush or tree-like condition occurred in early Echium lineages. Maintenance of woodiness in major lineages, and reversal to an herbaceous condition by three independent events, is reported for the first time in plants of oceanic islands.

Phylogenetic analysis of the angiosperm-floricolous insect–yeast association: Have yeast and angiosperm lineages co-diversified?

Metschnikowia (Saccharomycetales, Metschnikowiaceae/Metschnikowia clade) is an ascomycetous yeast genus whose species are associated mostly with angiosperms and their insect pollinators over all continents. The wide distribution of the genus, its association with angiosperm flowers, and the fact that it includes some of the best-studied yeasts in terms of biogeography and ecology make Metschnikowia an excellent group to investigate a possible co-radiation with angiosperm lineages. We performed phylogenetic analyses implementing Bayesian inference and likelihood methods, using a concatenated matrix (≈2.6 Kbp) of nuclear DNA (ACT1, 1st and 2nd codon positions of EF2, Mcm7, and RPB2) sequences. We included 77 species representing approximately 90% of the species in the family. Bayesian and parsimony methods were used to perform ancestral character reconstructions within Metschnikowia in three key morphological characters. Patterns of evolution of yeast habitats and divergence times were explored in the Metschnikowia clade lineages with the purpose of inferring the time of origin of angiosperm-associated habitats within Metschnikowiaceae. This paper presents the first phylogenetic hypothesis to include nearly all known species in the family. The polyphyletic nature of Clavispora was confirmed and Metschnikowia species (and their anamorphs) were shown to form two groups: one that includes mostly floricolous, insect-associated species distributed in mostly tropical areas (the large-spored Metschnikowia clade and relatives) and another that comprises more heterogeneous species in terms of habitat and geographical distribution. Reconstruction of character evolution suggests that sexual characters (ascospore length, number of ascospores, and ascus formation) evolved multiple times within Metschnikowia. Complex and dynamic habitat transitions seem to have punctuated the course of evolution of the Metschnikowiaceae with repeated and independent origins of angiosperm-associated habitats. The origin of the family is placed in the Late Cretaceous (71.7 Ma) with most extant species arising from the Early Eocene. Therefore, the Metschnikowiaceae likely radiated long after the Mid-Cretaceous radiations of angiosperms and their diversification seems to be driven by repeated radiation on a pre-existing diverse resource.

Investigating reproductive incompatibility barriers in a Mediterranean rockrose (Cistus ladanifer)

Cistus ladanifer L. (Cistaceae) is a shrubby species, primarily found as a dominant species of the Mediterranean evergreen scrub. Intervarietal (var. ladanifer and var. maculatus) cross compatibility and self-incompatibility (SI) were studied in six Iberian populations of C. ladanifer. In addition, temporal variation in SI across 2 years and variability in the strength of SI throughout the flowering season were analysed in a subset of two populations. The two coloured floral morphs of C. ladanifer were compatible, and pollination had statistically similar ability to fruit set when crossing individuals of the two varieties (440 flowers). Results from hand self-pollination indicate negative self-compatibility, regardless of individuals being sampled from different populations, varieties and years (160 flowers in 2003 and 1620 flowers in 2006). In addition, SI strength did not vary along the flowering season. All these results lead us to argue that, in contrast to the results of its sister species (Cistus salviifolius), the highly polymorphic C. ladanifer possesses an extremely stable SI system but a weak reproductive barrier between varieties.

Extensive Pollen Flow but Few Pollen Donors and High Reproductive Variance in an Extremely Fragmented Landscape

Analysing pollen movement is a key to understanding the reproductive system of plant species and how it is influenced by the spatial distribution of potential mating partners in fragmented populations. Here we infer parameters related to levels of pollen movement and diversity of the effective pollen cloud for the wind-pollinated shrub Pistacia lentiscus across a highly disturbed landscape using microsatellite loci. Paternity analysis and the indirect KinDist and Mixed Effect Mating models were used to assess mating patterns, the pollen dispersal kernel, the effective number of males (Nep) and their relative individual fertility, as well as the existence of fine-scale spatial genetic structure in adult plants. All methods showed extensive pollen movement, with high rates of pollen flow from outside the study site (up to 73–93%), fat-tailed dispersal kernels and large average pollination distances (δ = 229–412 m). However, they also agreed in detecting very few pollen donors (Nep = 4.3–10.2) and a large variance in their reproductive success: 70% of males did not sire any offspring among the studied female plants and 5.5% of males were responsible for 50% of pollinations. Although we did not find reduced levels of genetic diversity, the adult population showed high levels of biparental inbreeding (14%) and strong spatial genetic structure (Sp = 0.012), probably due to restricted seed dispersal and scarce safe sites for recruitment. Overall, limited seed dispersal and the scarcity of successful pollen donors can be contributing to generate local pedigrees and to increase inbreeding, the prelude of genetic impoverishment.

Metschnikowia proteae sp. nov., a nectarivorous insect-associated yeast species from Africa

A collection of yeasts isolated from nectar of flowers of Protea caffra (Proteaceae) and associated scarab beetles (Atrichelaphinis tigrina, Cyrtothyrea marginalis, Trichostetha fascicularis and Heterochelus sp.) and drosophilid flies in South Africa, contained 28 isolates that could not be assigned to known species. Comparisons of the D1/D2 domains of the large subunit rRNA gene demonstrated the existence of three separate phylotypes with an affinity to the genus Metschnikowia and more specifically to the beetle-associated large-spored Metschnikowia clade. Twenty-six strains that had similar D1/D2 sequences were mixed in all pairwise combinations. They were found to mate and give rise to large asci typical of those in the clade. The name Metschnikowia proteae sp. nov. (type strain EBDT1Y1(T) = CBS 12522(T) = NRRL Y-48784(T); allotype strain EBDC2Y2 = CBS 12521 = NRRL Y-48785) is proposed to accommodate this novel species. The ecology of this novel yeast species is discussed in relation to its potential plant and insect host species. The additional two single strains isolated from Heterochelus sp. represent two novel undescribed species (Candida sp. 1 EBDM2Y3 and Candida sp. 2 EBDM8Y1). As these single strains are probably haploid mating types of Metschnikowia species, their description is deferred until the species are sufficiently well sampled to permit meaningful descriptions.

Similar reproductive success of the two petal colour polymorphisms of Cistus ladanifer (Cistaceae)

Abstract Variation in flower colour can reflect adaptive responses to selective pressures generated by pollinators. Cistus ladanifer is a Mediterranean shrub that displays self-incompatibility, entomophyly and polymorphism for the presence of a dark coloured blotch in the base of their petals (var. maculatus with blotched petals and var. ladanifer with unblotched petals). In order to investigate the effect of the petal blotch and geography in C. ladanifer reproductive success, variation in female reproductive output (flower production, fruit set, seed set) was analysed in four and three Iberian populations of var. ladanifer and var. maculatus, respectively. Additionally, we tested whether co-existence of these two C. ladanifer phenotypes yielded different reproductive output (ovule production, seed set) in a subset of two populations. Significant differences in flower production (56.4–229.4%), fruit set (64.9–93.8%) and seed set (82.8–98.0%) among populations were reported. Geographical and taxonomic variables did not significantly explain this variation. Significant results between floral morphs within a single population were found neither for ovule nor for seed production. Our findings suggest that the petal blotch in C. ladanifer should be considered to be neutral in its effect on fruit and seed production and its occurrence may simply reflect a relaxed selection under current conditions.

Metschnikowia drakensbergensis sp. nov. and Metschnikowia caudata sp. nov., endemic yeasts associated with Protea flowers in South Africa

In a taxonomic study of yeasts recovered from nectar of flowers and associated insects in South Africa, 11 strains were found to represent two novel species. Morphological and physiological characteristics and sequence analyses of the large-subunit rRNA gene D1/D2 region, as well as the actin, RNA polymerase II and elongation factor 2 genes, showed that the two novel species belonged to the genus Metschnikowia. Metschnikowia drakensbergensis sp. nov. (type strain EBD-CdVSA09-2(T) =CBS 13649(T) =NRRL Y-63721(T); MycoBank no. MB809688; allotype EBD-CdVSA10-2(A) =CBS13650(A) =NRRL Y-63720(A)) was recovered from nectar of Protea roupelliae and the beetle Heterochelus sp. This species belongs to the large-spored Metschnikowia clade and is closely related to Metschnikowia proteae, with which mating reactions and single-spored asci were observed. Metschnikowia caudata sp. nov. (type strain EBD-CdVSA08-1(T) =CBS 13651(T) =NRRL Y-63722(T); MycoBank no. MB809689; allotype EBD-CdVSA57-2(A) =CBS 13729(A) =NRRL Y-63723(A)) was isolated from nectar of Protea dracomontana, P. roupelliae and P. subvestita and a honeybee, and is a sister species to Candida hainanensis and Metschnikowia lopburiensis. Analyses of the four sequences demonstrated the existence of three separate phylotypes. Intraspecies matings led to the production of mature asci of unprecedented morphology, with a long, flexuous tail. A single ascospore was produced in all compatible crosses, regardless of sequence phylotype. The two species appear to be endemic to South Africa. The ecology and habitat specificity of these novel species are discussed in terms of host plant and insect host species.

Flowers as a reservoir of yeast diversity: description of Wickerhamiella nectarea f.a. sp. nov., and Wickerhamiella natalensis f.a. sp. nov. from South African flowers and pollinators, and transfer of related Candida species to the genus Wickerhamiella as new combinations

Abstract Flowers offer favourable microenvironments for yeast growth, and are increasingly recognised as a rich source of novel yeast species. Independent surveys of yeasts associated with flowers and pollinators in South Africa led to the discovery of 38 strains of two new species. Physiological profiles and analysis of the internal transcribed spacer and the D1/D2 domains of the large subunit rRNA gene showed that they represent two novel species that belong to the Wickerhamiella clade. We describe the species as Wickerhamiella nectarea f.a. sp. nov. (type strain EBDCdVSA11‐1T, CBS 14162T, NRRL Y‐63791T) and W. natalensis f.a. sp. nov. (type strain EBDCdVSA7‐1T, CBS 14161T, NRRL Y‐63790T). We extend the known range of flower‐associated Wickerhamiella species to South Africa and discuss the ecology and phylogenetic relationships of the clade in relation to its host species and biogeography. Examination of growth characteristics supports that the Wickerhamiella clade exhibits a high degree of evolutionary lability, and that specialisation to different niches may occur rapidly. We review the current status of floral yeast biodiversity and nectar as a reservoir of species diversity, and the importance of pollinators and biogeography. In addition, 18 species formerly assigned to the genus Candida are reassigned formally to the genus Wickerhamiella.

Phylogenetic and paleobotanical evidence for late Miocene diversification of the Tertiary subtropical lineage of ivies (Hedera L., Araliaceae)

BackgroundHedera (ivies) is one of the few temperate genera of the primarily tropical Asian Palmate group of the Araliaceae, which extends its range out of Asia to Europe and the Mediterranean basin. Phylogenetic and phylogeographic results suggested Asia as the center of origin and the western Mediterranean region as one of the secondary centers of diversification. The bird-dispersed fleshy fruits of ivies suggest frequent dispersal over long distances (e.g. Macaronesian archipelagos), although reducing the impact of geographic barriers to gene flow in mainland species. Genetic isolation associated with geographic barriers and independent polyploidization events have been postulated as the main driving forces of diversification. In this study we aim to evaluate past and present diversification patterns in Hedera within a geographic and temporal framework to clarify the biogeographic history of the genus.ResultsPhylogenetic (biogeographic, time divergence and diversification) and phylogeographic (coalescence) analyses using four DNA regions (nrITS, trnH-psbA, trnT-trnL, rpl32) revealed a complex spatial pattern of lineage divergence. Scarce geographic limitation to gene flow and limited diversification are observed during the early-mid Miocene, followed by a diversification rate increase related to geographic divergence from the Tortonian/Messinian. Genetic and palaeobotanical evidence points the origin of the Hedera clade in Asia, followed by a gradual E-W Asian extinction and the progressive E-W Mediterranean colonization. The temporal framework for the E Asia - W Mediterranean westward colonization herein reported is congruent with the fossil record. Subsequent range expansion in Europe and back colonization to Asia is also inferred. Uneven diversification among geographic areas occurred from the Tortonian/Messinian onwards with limited diversification in the newly colonized European and Asian regions. Eastern and western Mediterranean regions acted as refugia for Miocene and post-Miocene lineages, with a similar role as consecutive centers of centrifugal dispersal (including islands) and speciation.ConclusionsThe Miocene Asian extinction and European survival of Hedera question the general pattern of Tertiary regional extinction of temperate angiosperms in Europe while they survived in Asia. The Tortonian/Messinian diversification increase of ivies in the Mediterranean challenges the idea that this aridity period was responsible for the extinction of the Mediterranean subtropical Tertiary flora. Differential responses of Hedera to geographic barriers throughout its evolutionary history, linked to spatial isolation related to historical geologic and climatic constraints may have shaped diversification of ivies in concert with recurrent polyploidy.