Michael D. Pirie

A broader model for C4 photosynthesis evolution in plants inferred from the goosefoot family (Chenopodiaceae s.s.)

C4 photosynthesis is a fascinating example of parallel evolution of a complex trait involving multiple genetic, biochemical and anatomical changes. It is seen as an adaptation to deleteriously high levels of photorespiration. The current scenario for C4 evolution inferred from grasses is that it originated subsequent to the Oligocene decline in CO2 levels, is promoted in open habitats, acts as a pre-adaptation to drought resistance, and, once gained, is not subsequently lost. We test the generality of these hypotheses using a dated phylogeny of Amaranthaceae s.l. (including Chenopodiaceae), which includes the largest number of C4 lineages in eudicots. The oldest chenopod C4 lineage dates back to the Eocene/Oligocene boundary, representing one of the first origins of C4 in plants, but still corresponding with the Oligocene decline of atmospheric CO2. In contrast to grasses, the rate of transitions from C3 to C4 is highest in ancestrally drought resistant (salt-tolerant and succulent) lineages, implying that adaptation to dry or saline habitats promoted the evolution of C4; and possible reversions from C4 to C3 are apparent. We conclude that the paradigm established in grasses must be regarded as just one aspect of a more complex system of C4 evolution in plants in general.

Identifying clades in Asian Annonaceae: monophyletic genera in the polyphyletic Miliuseae

The tribe Miliuseae (Annonaceae) comprises six genera distributed in Asia: Alphonsea, Mezzettia, Miliusa, Orophea, Platymitra, and Phoenicanthus. A phylogenetic study to investigate the putative monophyly of the tribe and the intergeneric relationships is presented here. Nucleotide sequences of the plastid gene rbcL, trnL intron, and trnL-trnF intergenic spacer were analyzed from 114 Annonaceae taxa, including 24 Miliuseae species and two outgroups using maximum parsimony and Bayesian inference. The two data sets (rbcL and the trnL-trnF regions) were analyzed separately and in combination. Miliuseae were found to be polyphyletic due to the position of Mezzettia and are part of a large, predominantly Asian and Central-American clade (miliusoid clade). Although intergeneric relationships were poorly resolved, all genera, except Polyalthia, were monophyletic, supporting previous generic delimitation based on morphology. A group of three Polyalthia species seems the most likely sister group of Miliusa. Several infrageneric groups of Miliusa, Orophea, and Polyalthia are supported by both molecular and morphological data. No morphological synapomorphies have yet been found for the miliusoid clade. Molecular clades within the miliusoid clade, however, can be characterized by size and the shape of the outer petals, number of ovules per carpel, and the size of the fruits.

Do pollinator distributions underlie the evolution of pollination ecotypes in the Cape shrub Erica plukenetii

BACKGROUND AND AIMS According to the Grant-Stebbins model of pollinator-driven divergence, plants that disperse beyond the range of their specialized pollinator may adapt to a new pollination system. Although this model provides a compelling explanation for pollination ecotype formation, few studies have directly tested its validity in nature. Here we investigate the distribution and pollination biology of several subspecies of the shrub Erica plukenetii from the Cape Floristic Region in South Africa. We analyse these data in a phylogenetic context and combine these results with information on pollinator ranges to test whether the evolution of pollination ecotypes is consistent with the Grant-Stebbins model. METHODS AND KEY RESULTS Pollinator observations showed that the most common form of E. plukenetii with intermediate corolla length is pollinated by short-billed Orange-breasted sunbirds. Populations at the northern fringe of the distribution are characterized by long corollas, and are mainly pollinated by long-billed Malachite sunbirds. A population with short corollas in the centre of the range was mainly pollinated by insects, particularly short-tongued noctuid moths. Bird exclusion in this population did not have an effect on fruit set, while insect exclusion reduced fruit set. An analysis of floral scent across the range, using coupled gas chromatography-mass spectrometry, showed that the scent bouquets of flowers from moth-pollinated populations are characterized by a larger number of scent compounds and higher emission rates than those in bird-pollinated populations. This was also reflected in clear separation of moth- and bird-pollinated populations in a two-dimensional phenotype space based on non-metric multidimensional scaling analysis of scent data. Phylogenetic analyses of chloroplast and nuclear DNA sequences strongly supported monophyly of E. plukenetii, but not of all the subspecies. Reconstruction of ancestral character states suggests two shifts from traits associated with short-billed Orange-breasted sunbird pollination: one towards traits associated with moth pollination, and one towards traits associated with pollination by long-billed Malachite sunbirds. The latter shift coincided with the colonization of Namaqualand in which Orange-breasted sunbirds are absent. CONCLUSIONS Erica plukenetii is characterized by three pollination ecotypes, but only the evolutionary transition from short- to long-billed sunbird pollination can be clearly explained by the Grant-Stebbins model. Corolla length is a key character for both ecotype transitions, while floral scent emission was important for the transition from bird to moth pollination.

Reticulation, data combination, and inferring evolutionary history: an example from Danthonioideae (Poaceae).

We explore the potential impact of conflicting gene trees on inferences of evolutionary history above the species level. When conflict between gene trees is discovered, it is common practice either to analyze the data separately or to combine the data having excluded the conflicting taxa or data partitions for those taxa (which are then recoded as missing). We demonstrate an alternative approach, which involves duplicating conflicting taxa in the matrix, such that each duplicate is represented by one partition only. This allows the combination of all available data in standard phylogenetic analyses, despite reticulations. We show how interpretation of contradictory gene trees can lead to conflicting inferences of both morphological evolution and biogeographic history, using the example of the pampas grasses, Cortaderia. The characteristic morphological syndrome of Cortaderia can be inferred as having arisen multiple times (chloroplast DNA [cpDNA]) or just once (nuclear ribosomal DNA [nrDNA]). The distributions of species of Cortaderia and related genera in Australia/New Guinea, New Zealand, and South America can be explained by few (nrDNA) or several (cpDNA) dispersals between the southern continents. These contradictions can be explained by past hybridization events, which have linked gains of complex morphologies with unrelated chloroplast lineages and have erased evidence of dispersals from the nuclear genome. Given the discrepancies between inferences based on the gene trees individually, we urge the use of approaches such as ours that take multiple gene trees into account.

Ancient paralogy in the cpDNA trnL-F region in Annonaceae: implications for plant molecular systematics

The plastid trnL-F region has proved useful in molecular phylogenetic studies addressing diverse evolutionary questions from biogeographic history to character evolution in a broad range of plant groups. An important assumption for phylogenetic reconstruction is that data used in combined analyses contain the same phylogenetic signal. The trnL-F region is often used in combined analyses of multiple chloroplast markers. These markers are assumed to contain congruent phylogenetic signal due to lack of recombination. Here we show that trnL-F sequences display a phylogenetic signal conflicting with that of other chloroplast markers in Annonaceae, and we demonstrate that this conflict results from ancient paralogy. TrnL-F copy 2 diverged from trnL-F copy 1 (as used in family-wide phylogenetic analyses) in a direct ancestor of the Annonaceae. Although this divergence dates back 88 million years or more, the exons of both copies appear to be intact. In this case, assuming that (putative) chloroplast markers contain the same phylogenetic signal results in an incorrect topology and an incorrect estimate of ages. Our study demonstrates that researchers should be cautious when interpreting gene phylogenies, irrespective of the genome from which they are presumed to have been sampled.

A Generic Classification of the Danthonioideae (Poaceae)1

Abstract We present a new generic classification of the largely Southern Hemisphere grass subfamily Danthonioideae. This classification is based on an almost completely sampled and well-resolved molecular phylogeny and on a complete morphological data set. We have attempted to delimit monophyletic genera (complicated by the presence of apparent intergeneric hybridization), which are diagnosable, as well as morphologically and ecogeographically coherent. We recognize 17 genera, including five new genera (Austroderia N. P. Barker & H. P. Linder, Capeochloa H. P. Linder & N. P. Barker, Chimaerochloa H. P. Linder, Geochloa H. P. Linder & N. P. Barker, and Tenaxia N. P. Barker & H. P. Linder), and two sections newly designated for Pentameris P. Beauv. (section Dracomontanum H. P. Linder & Galley and section Pentaschistis (Nees) H. P. Linder & Galley). Of the remaining 12 genera, the delimitations of seven are changed: Merxmuellera Conert is much reduced by the segregation of Geochloa, Capeochloa, and Tenaxia; Pentameris is expanded to include Prionanthium Desv. and Pentaschistis (Nees) Spach; Cortaderia Stapf is expanded by the inclusion of Lamprothyrsus Pilg., but reduced by the segregation of its New Zealand species into the new genus Austroderia; a large Rytidosperma Steud. is assembled out of Joycea H. P. Linder, Austrodanthonia H. P. Linder, Notodanthonia Zotov, Erythranthera Zotov, Pyrrhanthera Zotov, and Monostachya Merr.; and the species previously assigned to Karroochloa Conert & Türpe, Schismus P. Beauv., Urochlaena Nees, and Tribolium Desv. have been reassigned to only two genera. Finally, the Himalayan species of Danthonia DC. are transferred to Tenaxia and the remaining African species of Danthonia to Merxmuellera. The 281 species that we recognize in the subfamily are listed under their new genera, which are arranged in the phylogenetic sequence evident from the molecular phylogeny. The 100 necessary new combinations include: Merxmuellera grandiflora (Hochst. ex A. Rich.) H. P. Linder, Geochloa decora (Nees) N. P. Barker & H. P. Linder, G. lupulina (L. f.) N. P. Barker & H. P. Linder, G. rufa (Nees) N. P. Barker & H. P. Linder, Capeochloa arundinacea (P. J. Bergius) N. P. Barker & H. P. Linder, C. cincta (Nees) N. P. Barker & H. P. Linder, C. cincta subsp. sericea (N. P. Barker) N. P. Barker & H. P. Linder, C. setacea (N. P. Barker) N. P. Barker & H. P. Linder, Pentameris praecox (H. P. Linder) Galley & H. P. Linder, P. tysonii (Stapf) Galley & H. P. Linder, P. acinosa (Stapf) Galley & H. P. Linder, P. airoides Nees subsp. jugorum (Stapf) Galley & H. P. Linder, P. alticola (H. P. Linder) Galley & H. P. Linder, P. ampla (Nees) Galley & H. P. Linder, P. andringitrensis (A. Camus) Galley & H. P. Linder, P. argentea (Stapf) Galley & H. P. Linder, P. aristidoides (Thunb.) Galley & H. P. Linder, P. aristifolia (Schweick.) Galley & H. P. Linder, P. aspera (Thunb.) Galley & H. P. Linder, P. aurea (Steud.) Galley & H. P. Linder, P. aurea subsp. pilosogluma (McClean) Galley & H. P. Linder, P. bachmannii (McClean) Galley & H. P. Linder, P. barbata (Nees) Steud. subsp. orientalis (H. P. Linder) Galley & H. P. Linder, P. basutorum (Stapf) Galley & H. P. Linder, P. borussica (K. Schum.) Galley & H. P. Linder, P. calcicola (H. P. Linder) Galley & H. P. Linder, P. calcicola var. hirsuta (H. P. Linder) Galley & H. P. Linder, P. capensis (Nees) Galley & H. P. Linder, P. capillaris (Thunb.) Galley & H. P. Linder, P. caulescens (H. P. Linder) Galley & H. P. Linder, P. chippindalliae (H. P. Linder) Galley & H. P. Linder, P. chrysurus (K. Schum.) Galley & H. P. Linder, P. clavata (Galley) Galley & H. P. Linder, P. colorata (Steud.) Galley & H. P. Linder, P. dentata (L. f.) Galley & H. P. Linder, P. dolichochaeta (S. M. Phillips) Galley & H. P. Linder, P. ecklonii (Nees) Galley & H. P. Linder, P. exserta (H. P. Linder) Galley & H. P. Linder, P. galpinii (Stapf) Galley & H. P. Linder, P. holciformis (Nees) Galley & H. P. Linder, P. horrida (Galley) Galley & H. P. Linder, P. humbertii (A. Camus) Galley & H. P. Linder, P. insularis (Hemsl.) Galley & H. P. Linder, P. juncifolia (Stapf) Galley & H. P. Linder, P. longipes (Stapf) Galley & H. P. Linder, P. malouinensis (Steud.) Galley & H. P. Linder, P. microphylla (Nees) Galley & H. P. Linder, P. minor (Ballard & C. E. Hubb.) Galley & H. P. Linder, P. montana (H. P. Linder) Galley & H. P. Linder, P. natalensis (Stapf) Galley & H. P. Linder, P. oreodoxa (Schweick.) Galley & H. P. Linder, P. pallida (Thunb.) Galley & H. P. Linder, P. pholiuroides (Stapf) Galley & H. P. Linder, P. pictigluma (Steud.) Galley & H. P. Linder, P. pictigluma var. gracilis (S. M. Phillips) Galley & H. P. Linder, P. pictigluma var. mannii (Stapf ex C. E. Hubb.) Galley & H. P. Linder, P. pseudopallescens (H. P. Linder) Galley & H. P. Linder, P. pungens (H. P. Linder) Galley & H. P. Linder, P. pusilla (Nees) Galley & H. P. Linder, P. pyrophila (H. P. Linder) Galley & H. P. Linder, P. reflexa (H. P. Linder) Galley & H. P. Linder, P. rigidissima (Pilg. ex H. P. Linder) Galley & H. P. Linder, P. rosea (H. P. Linder) Galley & H. P. Linder, P. rosea subsp. purpurascens (H. P. Linder) Galley & H. P. Linder, P. scandens (H. P. Linder) Galley & H. P. Linder, P. setifolia (Thunb.) Galley & H. P. Linder, P. tomentella (Stapf) Galley & H. P. Linder, P. trifida (Galley) Galley & H. P. Linder, P. triseta (Thunb.) Galley & H. P. Linder, P. trisetoides (Hochst. ex Steud.) Galley & H. P. Linder, P. velutina (H. P. Linder) Galley & H. P. Linder, P. veneta (H. P. Linder) Galley & H. P. Linder, Cortaderia hieronymi (Kuntze) N. P. Barker & H. P. Linder, C. peruviana (Hitchc.) N. P. Barker & H. P. Linder, Austroderia fulvida (Buchanan) N. P. Barker & H. P. Linder, A. richardii (Endl.) N. P. Barker & H. P. Linder, A. splendens (Connor) N. P. Barker & H. P. Linder, A. toetoe (Zotov) N. P. Barker & H. P. Linder, A. turbaria (Connor) N. P. Barker & H. P. Linder, Chimaerochloa archboldii (Hitchc.) Pirie & H. P. Linder, Tenaxia aureocephala (J. G. Anderson) N. P. Barker & H. P. Linder, T. cachemyriana (Jaub. & Spach) N. P. Barker & H. P. Linder, T. cumminsii (Hook. f.) N. P. Barker & H. P. Linder, T. disticha (Nees) N. P. Barker & H. P. Linder, T. dura (Stapf) N. P. Barker & H. P. Linder, T. guillarmodiae (Conert) N. P. Barker & H. P. Linder, T. stricta (Schrad.) N. P. Barker & H. P. Linder, T. subulata (A. Rich.) N. P. Barker & H. P. Linder, Schismus schismoides (Stapf ex Conert) Verboom & H. P. Linder, Tribolium curvum (Nees) Verboom & H. P. Linder, T. pleuropogon (Stapf) Verboom & H. P. Linder, T. purpureum (L. f.) Verboom & H. P. Linder, T. tenellum (Nees) Verboom & H. P. Linder, Rytidosperma bipartitum (Kunth) A. M. Humphreys & H. P. Linder, R. diemenicum (D. I. Morris) A. M. Humphreys & H. P. Linder, R. fulvum (Vickery) A. M. Humphreys & H. P. Linder, R. lepidopodum (N. G. Walsh) A. M. Humphreys & H. P. Linder, R. pallidum (R. Br.) A. M. Humphreys & H. P. Linder, R. popinensis (D. I. Morris) A. M. Humphreys & H. P. Linder, R. remotum (D. I. Morris) A. M. Humphreys & H. P. Linder. Typifications are designated for the following names: Achneria Munro ex Benth. & Hook. f., Avena aristidoides Thunb., A. elephantina Thunb., Danthonia crispa Nees var. trunculata Nees, Danthonia sect. Himantochaete Nees, D. zeyheriana Steud. var. trichostachya Stapf, Geochloa lupulina, Pentameris aristidoides, and P. holciformis.

A rapid and inexpensive method for the direct PCR amplification of DNA from plants

UNLABELLED PREMISE OF THE STUDY We present a rapid and inexpensive alternative to DNA isolation for polymerase chain reaction (PCR) amplification from plants. • METHODS AND RESULTS The method involves direct PCR amplification from material macerated in one buffer, followed by dilution and incubation in a second buffer. We describe the procedure and demonstrate its application for nuclear and plastid DNA amplification across a broad range of vascular plants. • CONCLUSIONS The method is fast, easy to perform, cost-effective, and consequently ideal for large sample numbers. It represents a considerable simplification of present approaches requiring DNA isolation prior to PCR amplification and will be useful in plant systematics and biotechnology, including applications such as DNA barcoding.

The Recent Recombinant Evolution of a Major Crop Pathogen, Potato virus Y

Potato virus Y (PVY) is a major agricultural disease that reduces crop yields worldwide. Different strains of PVY are associated with differing degrees of pathogenicity, of which the most common and economically important are known to be recombinant. We need to know the evolutionary origins of pathogens to prevent further escalations of diseases, but putatively reticulate genealogies are challenging to reconstruct with standard phylogenetic approaches. Currently available phylogenetic hypotheses for PVY are either limited to non-recombinant strains, represent only parts of the genome, and/or incorrectly assume a strictly bifurcating phylogenetic tree. Despite attempts to date potyviruses in general, no attempt has been made to date the origins of pathogenic PVY. We test whether diversification of the major strains of PVY and recombination between them occurred within the time frame of the domestication and modern cultivation of potatoes. In so doing, we demonstrate a novel extension of a phylogenetic approach for reconstructing reticulate evolutionary scenarios. We infer a well resolved phylogeny of 44 whole genome sequences of PVY viruses, representative of all known strains, using recombination detection and phylogenetic inference techniques. Using Bayesian molecular dating we show that the parental strains of PVY diverged around the time potatoes were first introduced to Europe, that recombination between them only occurred in the last century, and that the multiple recombination events that led to highly pathogenic PVY(NTN) occurred within the last 50 years. Disease causing agents are often transported across the globe by humans, with disastrous effects for us, our livestock and crops. Our analytical approach is particularly pertinent for the often small recombinant genomes involved (e.g. HIV/influenza A). In the case of PVY, increased transport of diseased material is likely to blame for uniting the parents of recombinant pathogenic strains: this process needs to be minimised to prevent further such occurrences.

Revision and Biogeography of Centrolobium (Leguminosae - Papilionoideae)

Abstract A taxonomic revision and biogeographic study of the genus Centrolobium (Leguminosae - Papilionoideae) is presented. Centrolobium includes important timber trees distributed disjunctly in seasonally dry tropical forests and rain forests in Central and South America, from Panama to south-eastern Brazil. It is characterized by large samaroid pods with a spiny seed case and an abundance of orange peltate glands covering the leaves and inflorescences. Taxonomic distinctions between some species of Centrolobium have been a source of confusion. Here, seven species are recognized: C. robustum, C. microchaete, C. tomentosum, C. ochroxylum, C. sclerophyllum, C. paraense, and C. yavizanum. Previously recognized varieties of C. paraense, C. paraense var. paraense and C. paraense var. orinocense, are not maintained. Phylogenetic analysis of DNA sequence data from the internal transcribed spacer region of nuclear ribosomal DNA and the plastid matK gene and trnL-trnF intron and spacer support the monophyly of the genus. Different molecular dating methods indicate that the Centrolobium crown group and lineages found to the west and east of the Andes diverged before the Pleistocene. Divergences between species occurring east of the Andes, particularly in Bolivia and south-eastern Brazil are more recent, but nevertheless unlikely to be explained by Pleistocene climatic changes.

When do different C4 leaf anatomies indicate independent C4 origins? Parallel evolution of C4 leaf types in Camphorosmeae (Chenopodiaceae)

Broad-scale phylogenetic studies give first insights in numbers, relationships, and ages of C4 lineages. They are, however, generally limited to a model that treats the evolution of the complex C4 syndrome in different lineages as a directly comparable process. Here, we use a resolved and well-sampled phylogenetic tree of Camphorosmeae, based on three chloroplast and one nuclear marker and on leaf anatomical traits to infer a more detailed picture of C4 leaf-type evolution in this lineage. Our ancestral character state reconstructions allowed two scenarios: (i) Sedobassia is a derived C3/C4 intermediate, implying two independent gains of C4 in Bassia and Camphorosma; or (ii) Sedobassia is a plesiomorphic C3/C4 intermediate, representing a syndrome ancestral to the Bassia/Camphorosma/Sedobassia lineage. In Bassia, a kochioid leaf type (Bassia muricata and/or Bassia prostrata type) is ancestral. At least three independent losses of water-storage tissue occurred, resulting in parallel shifts towards an atriplicoid leaf type. These changes in leaf anatomy are adaptations to different survival strategies in steppic or semi-desert habitats with seasonal rainfall. In contrast, Camphorosma shows a fixed C4 anatomy differing from Bassia types in its continuous Kranz layer, which indeed points to an independent origin of the full C4 syndrome in Camphorosma, either from an independent C3 or from a common C3/C4 intermediate ancestor, perhaps similar to its C3/C4 intermediate sister genus Sedobassia. The enlarged bundle sheath cells of Sedobassia might represent an important early step in C4 evolution in Camphorosmeae.