Evolution of the genus Aristolochia - Systematics, Molecular Evolution and Ecology

Evolution of Piperales – matK gene and trnK intron sequence data reveal lineage specific resolution contrast. Piperales are one of the largest basal angiosperm orders with a nearly worldwide distribution. The order includes three species rich genera, Piper (ca. 1,000 species), Peperomia (ca. 1,500-1,700 species), and Aristolochia s. l. (ca. 500 species). Sequences of the matK gene and the non-coding trnK group II intron are analysed for a dense set of 105 taxa representing all families (except Hydnoraceae) and all generic segregates (except Euglypha within Aristolochiaceae) of Piperales. A large number of highly informative indels are found in the Piperales trnK/matK dataset. Within a narrow region approximately 500 nt downstream in the matK coding region (CDS), a length variable simple sequence repeat (SSR) expansion segment occurs, in which insertions and deletions have led to short frame-shifts. These are corrected shortly afterwards, resulting in a maximum of 6 amino acids being affected. Furthermore, additional non-functional matK copies were found in Zippelia begoniifolia, which can easily be discriminated from the functional open reading frame (ORF). The trnK/matK sequence data fully resolve relationships within Peperomia, whereas they are not effective within Piper. The resolution contrast is correlated with the rate heterogenity between those lineages. Parsimony, Bayesian and likelihood analyses result in virtually the same topology, and converge on the monophyly of Piperaceae and Saururaceae. Lactoris gains high support as sister to Aristolochiaceae subf. Aristolochioideae, but the different tree inference methods yield conflicting results with respect to the relationships of subfam. Asaroideae. In Piperaceae, a clade formed by the monotypic genus Zippelia and the small genus Manekia (=Sarcorhachis) is sister to the two large genera Piper and Peperomia. Systematics of pipevines – Combining morphological and fast-evolving molecular characters to investigate the relationships within subfamily Aristolochioideae (Aristolochiaceae) A combined phylogenetic analysis of the Aristolochioideae was conducted based on 72 morphological characters and molecular datasets (matK gene, trnK intron, trnL intron, trnL-trnF spacer). The analysis sampled 33 species as the ingroup, including two species of Thottea and 30 species of Aristolochia and the monotypic genus Euglypha, which represent all the infrageneric taxa formally described; Saruma henryi and Asarum caudatum were used as the outgroup. The results corroborate a sister-group relationship between Thottea and Aristolochia, and the paraphyly of Aristolochia with respect to Euglypha that consequently should be included into Aristolochia. Two of the three subgenera within Aristolochia (Isotrema and Pararistolochia) are shown to be monophyletic, whereas the signal obtained from the different datasets about the relationships within subg. Aristolochia is low and conflicting, resulting in collapsed or unsupported branches. The relationship between the New World and the Old World species of subgenus Aristolochia is conflictive because morphological data support these two groups as monophyletic, whereas molecular data show the monophyletic Old World species of Aristolochia nested within the New World species. A sister group relationship is proposed between A. lindneri and pentandrous species, which suggests that a group of five species from central and southern South America (including A. lindneri) could be monophyletic and sister to Aristolochia subsection Pentandrae, a monophyletic taxon consisting of about 35 species from southern USA, Mesoamerica, and the West Indies. Colonisation, phylogeography and evolution of endemism in Mediterranean Aristolochia (Aristolochiaceae). This study provides evidence for a multiple colonisation of the western Old World from Asian ancestors within Aristolochia section Diplolobus (subsection Aristolochia and Podanthemum). Within subsection Podanthemum it is assumed, that the colonisation of the African continent happened at least two times independently. In contrast, for subsection Aristolochia, a rapid morphological radiation in the Near East (or close to this area) with subsequent star like colonisation of the different current distribution areas, which is not paralleled on the molecular level, appears to be more likely. Phylogenetic tree reconstruction is unsupported for these clades, but most clades are highly supported as monophyletic. Interestingly the Mediterranean and temperate Eurasian species, which are morphologically distinct (A. pistolochia, A. clematitis) are not clustering within the main clades, but are independent lineages. Analogue, A. rigida a species from Somalia is well-supported sister to the subsection Aristolochia. Within subsection Podanthemum the colonisation event from an Asian ancestor is clearly traceable, whereas in subsection Aristolochia the path is not traceable, since the ancestors are extinct or not present in the connecting areas. Within the Mediterranean, Near East and Caucasian species of subsection Aristolochia two morphologically and biogeographically well supported groups can be identified: the Near East/Caucasian species and the West Mediterranean species. The previous groupings for the latter, based on morphological characters, could be substantiated only partly by our results. This study provides the first phylogeny of all West Mediterranean species. In addition an independent complex is established including some micro endemic species. The phylogenetic results are discussed with respect to biogeography, and morphology, to give a first insight into the radiation and colonisation of the genus Aristolochia in the Mediterranean region. Universal primers for a large cryptically simple cpDNA microsatellite region in Aristolochia. We provide a new and valuable marker to study species relationships and population genetics in order to trace evolutionary, ecological, and conservational aspects in the genus Aristolochia. Universal primers for amplification and subsequent sequencing of a chloroplast microsatellite locus inside the trnK intron are presented. Utility of the primers has been tested in 32 species representing all clades of Aristolochia, including population studies within the A. pallida complex, A. clusii and A. rotunda. The microsatellite region is characterized as a (AnTm)k repeat of 22–438 bp containing a combination of different repeats arranged as ‘cryptically simple’. Trapped! Pollination of Aristolochia pallida Willd. in the Mediterranean A first study of the pollination biology of a Mediterranean Aristolochia species in its natural habitat is presented. 183 flowers of Aristolochia pallida were investigated, which in total contained 73 arthropods, dominated by two groups of Diptera, Sciaridae (37%) and Phoridae (19%). However, only Phoridae are regarded as potential pollinators, since pollen has been found exclusively on the body surfaces of these insects. All Phoridae belong to the genus Megaselia and are recognised as four undescribed species. The measurements of flower and insect dimensions suggest that size is an important constrain for successful pollination: 1) the insects must have a definitive size for being able to enter the flower and 2) must be able to get in touch with the pollen. Only very few insect groups found in Aristolochia pallida fulfil these size requirements. However, size alone is not a sufficient constrain as too many fly species of the same size might be trapped but not function as pollinators. Instead, specific attraction is required as otherwise pollen is lost. Since all trapped Phoridae are males, a chemical attraction (pheromones) is proposed as an additional constrain. Since A. pallida flowers are protogynous, the record of Megaselia loaded with pollen found in a flower during its female stage proves that this insect must have been visited at least one different flower during its male stage before. Further on, this observation provides strong evidence that the flowers are cross-pollinated. All these factors indicate a highly specialised pollination of Aristolochia pallida by Megaselia species

A comparison of leaf crystal macropatterns in the two sister genera Piper and Peperomia (Piperaceae)

Premise of the study: This is the first large-scale study comparing leaf crystal macropatterns of the species-rich sister genera Piper and Peperomia. It focuses on identifying types of calcium oxalate crystals and their macropatterns in leaves of both genera. The Piper results are placed in a phylogenetic context to show evolutionary patterns. This information will expand knowledge about crystals and provide specific examples to help study their form and function. One example is the first-time observation of Piper crystal sand tumbling in chlorenchyma vacuoles. Methods: Herbarium and fresh leaves were cleared of cytoplasmic content and examined with polarizing microscopy to identify types of crystals and their macropatterns. Selected hydrated herbarium and fresh leaf punches were processed for scanning electron microscopy and x-ray elemental analysis. Vibratome sections of living Piper and Peperomia leaves were observed for anatomical features and crystal movement. Key results: Both genera have different leaf anatomies. Piper displays four crystal types in chlorenchyma-crystal sand, raphides, styloids, and druses, whereas Peperomia displays three types-druses, raphides, and prisms. Because of different leaf anatomies and crystal types between the genera, macropatterns are completely different. Crystal macropattern evolution in both is characterized by increasing complexity, and both may use their crystals for light gathering and reflection for efficient photosynthesis under low-intensity light environments. Conclusions: Both genera have different leaf anatomies, types of crystals and crystal macropatterns. Based on Piper crystals associated with photosynthetic tissues and low-intensity light, further study of their function and association with surrounding chloroplasts is warranted, especially active crystal movement

Verhuellia is a segregate lineage in Piperaceae: more evidence from flower, fruit and pollen morphology, anatomy and development

Background and Aims The perianthless Piperales, i.e. Saururaceae and Piperaceae, have simple reduced flowers strikingly different from the other families of the order (e.g. Aristolochiaceae). Recent molecular phylogenies proved Verhuellia to be the first branch in Piperaceae, making it a promising object to study the detailed structure and development of the flowers. Based on recently collected material, the first detailed study since 1872 was conducted with respect to morphology, anatomy and development of the inflorescence, pollen ultrastructure and fruit anatomy. Methods Original Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Light Microscopy (LM) observations on Verhuellia lunaria were compared with Piperaceae, Saururaceae and fossils. Key results The inflorescence is an indeterminate spike with sessile flowers, each in the axil of a bract, developing in acropetal, helical succession. Flowers consist of two (occasionally three) stamens with basifixed tetrasporangiate anthers and latrorse dehiscence by a longitudinal slit. The gynoecium lacks a style but has three to four stigma branches and a single, basal orthotropous, and unitegmic ovule. The fruit is a drupe with large multicellular epidermal protuberances. The pollen is very small, inaperturate, and areolate with hemispherical microechinate exine elements. Conclusions Despite the superficial similarities with different genera of Piperaceae and Saururaceae, the segregate position of Verhuellia revealed by molecular phylogenetics is supported by morphological, developmental and anatomical data presented here. Unitegmic ovules and inaperturate pollen, which are synapomorphies for the genus Peperomia, are also present in Verhuellia

The first plastid genome of the Holoparasitic Genus Prosopanche (Hydnoraceae)

Plastomes of parasitic and mycoheterotrophic plants show different degrees of reduction depending on the plants' level of heterotrophy and host dependence in comparison to photoautotrophic sister species, and the amount of time since heterotrophic dependence was established. In all but the most recent heterotrophic lineages, this reduction involves substantial decrease in genome size and gene content and sometimes alterations of genome structure. Here, we present the first plastid genome of the holoparasitic genus Prosopanche, which shows clear signs of functionality. The plastome of Prosopanche americana has a length of 28,191 bp and contains only 24 unique genes, i.e., 14 ribosomal protein genes, four ribosomal RNA genes, five genes coding for tRNAs and three genes with other or unknown function (accD, ycf1, ycf2). The inverted repeat has been lost. Despite the split of Prosopanche and Hydnora about 54 MYA ago, the level of genome reduction is strikingly congruent between the two holoparasites although highly dissimilar nucleotide sequences are observed. Our results lead to two possible evolutionary scenarios that will be tested in the future with a larger sampling: 1) a Hydnoraceae plastome, similar to those ofHydnora and Prosopanche today, existed already in the most recent common ancestor and has not changed much with respect to gene content and structure, or 2) the genome similarities we observe today are the result of two independent evolutionary trajectories leading to almost the same end point. The first hypothesis would be most parsimonious whereas the second would point totaxon dependent essential gene sets for plants released from photosynthetic constraints.Fil: Jost, Matthias. Technische Universität Dresden.; AlemaniaFil: Naumann, Julia. Technische Universität Dresden.; AlemaniaFil: Rocamundi, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Cocucci, Andrea Aristides. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Wanke, Stefan. Technische Universität Dresden.; Alemani

Discordant phylogenomic placement of Hydnoraceae and Lactoridaceae within Piperales using data from all three genomes

Original ResearchPhylogenetic relationships within the magnoliid order Piperales have been studied extensively, yet the relationships of the monotypic family Lactoridaceae and the holoparasitic Hydnoraceae to the remainder of the order remain a matter of debate. Since the first confident molecular phylogenetic placement of Hydnoraceae among Piperales, different studies have recovered various contradictory topologies. Most phylogenetic hypotheses were inferred using only a few loci and have had incomplete taxon sampling at the genus level. Based on these results and an online survey of taxonomic opinion, the Angiosperm Phylogeny Group lumped both Hydnoraceae and Lactoridaceae in Aristolochiaceae; however, the latter family continues to have unclear relationships to the aforementioned taxa. Here we present extensive phylogenomic tree reconstructions based on up to 137 loci from all three subcellular genomes for all genera of Piperales. We infer relationships based on a variety of phylogenetic methods, explore instances of phylogenomic discordance between the subcellular genomes, and test alternative topologies. Consistent with these phylogenomic results and a consideration of the principles of phylogenetic classification, we propose to exclude Hydnoraceae and Lactoridaceae from the broad circumscription of Aristolochiaceae, and instead favor recognition of four monophyletic and morphologically well circumscribed families in the perianth-bearing Piperales: Aristolochiaceae, Asaraceae, Hydnoraceae, and Lactoridaceae, with a total of six families in the orderinfo:eu-repo/semantics/publishedVersio

Molecular phylogenetics and new (infra)generic classification to alleviate polyphyly in tribe Hydrangeeae (Cornales : Hydrangeaceae)

Tribe Hydrangeeae of Hydrangeaceae currently contains nine morphologically diverse genera, many of which are well-known garden ornamentals. Previous studies have shown eight of these genera to be phylogenetically nested within Hydrangea, rendering the latter polyphyletic. To clarify the phylogeny of tribe Hydrangeeae, the present study sequenced four chloroplast regions and ITS for an extensive set of taxa, including the type for all nine genera involved. The resulting phylogenetic hypotheses corroborate the polyphyly of Hydrangea. Since polyphyletic taxa are deemed unacceptable by both sides in the ongoing debate concerning the adherence to strict monophyly in biological classifications, a new (infra)generic classification for tribe Hydrangeeae is proposed. In order to create a stable, evolutionary informative classification a broader circumscription of the genus Hydrangea is proposed, to include all eight satellite genera of the tribe. Such treatment is considered highly preferable to an alternative where Hydrangea is to be split into several morphologically potentially unidentifiable genera. To facilitate the acceptance of the new classification proposed here, and in order to create a classification with high information content, the familiar generic names were maintained as section names where possible

A genome-scale mining strategy for recovering novel rapidly-evolving nuclear single-copy genes for addressing shallow-scale phylogenetics in Hydrangea

Background: Identifying orthologous molecular markers that potentially resolve relationships at and below species level has been a major challenge in molecular phylogenetics over the past decade. Non-coding regions of nuclear low-or single-copy markers are a vast and promising source of data providing information for shallow-scale phylogenetics. Taking advantage of public transcriptome data from the One Thousand Plant Project (1KP), we developed a genome-scale mining strategy for recovering potentially orthologous single-copy markers to address low-scale phylogenetics. Our marker design targeted the amplification of intron-rich nuclear single-copy regions from genomic DNA. As a case study we used Hydrangea section Cornidia, one of the most recently diverged lineages within Hydrangeaceae (Cornales), for comparing the performance of three of these nuclear markers to other "fast" evolving plastid markers. Results: Our data mining and filtering process retrieved 73 putative nuclear single-copy genes which are potentially useful for resolving phylogenetic relationships at a range of divergence depths within Cornales. The three assessed nuclear markers showed considerably more phylogenetic signal for shallow evolutionary depths than conventional plastid markers. Phylogenetic signal in plastid markers increased less markedly towards deeper evolutionary divergences. Potential phylogenetic noise introduced by nuclear markers was lower than their respective phylogenetic signal across all evolutionary depths. In contrast, plastid markers showed higher probabilities for introducing phylogenetic noise than signal at the deepest evolutionary divergences within the tribe Hydrangeeae (Hydrangeaceae). Conclusions: While nuclear single-copy markers are highly informative for shallow evolutionary depths without introducing phylogenetic noise, plastid markers might be more appropriate for resolving deeper-level divergences such as the backbone relationships of the Hydrangeaceae family and deeper, at which non-coding parts of nuclear markers could potentially introduce noise due to elevated rates of evolution. The herein developed and demonstrated transcriptome based mining strategy has a great potential for the design of novel and highly informative nuclear markers for a range of plant groups and evolutionary scales