Sterling C. Keeley

Chloroplast DNA Variation in the Asteraceae: Phylogenetic and Evolutionary Implications

The sunflower family (Asteraceae) is one of the largest families of flowering plants, consisting of approximately 1,100 genera and 25,000 species (Jeffrey, 1978). The family has been variously divided into two or three subfamilies and 10 to 17 tribes (reviewed in Jansen et al., 1991a; Bremer, 1987). In addition to the disagreement concerning intrafamilial relationships, the phylogenetic affinities of the Asteraceae to other angiosperm families have been the topic of considerable debate (Cronquist, 1955,Cronquist, 1977, 1981; Carlquist, 1976; Wagenitz, 1976; Stebbins, 1977; Turner, 1977a; Jeffrey, 1977; Bremer, 1987). Until recently, systematic studies in the family relied on more traditional taxonomic characters and less rigorous phylogenetic methods. During the past five years we have been examining chloroplast DNA (cpDNA) variation in the Asteraceae to evaluate phylogenetic relationships at a wide range of taxonomic levels using several cladistic methods. We have accumulated restriction site data for approximately 250 genera in the family from all currently recognized tribes. In addition, we have sequenced the gene encoding the large subunit of ribulose-l,5-bisphosphate carboxylase (rbcL) from representative species in the Asteraceae and putatively related families. Our studies, which have produced the largest molecular data set for any plant family, have allowed us to perform phylogenetic comparisons from the intraspecific to the interfamilial levels.

Insights into the evolution of the tribe Arctoteae (Compositae: subfamily Cichorioideae s.s.) using trnL-F, ndhF, and ITS

Compositae (Asteraceae) are the largest flowering plant family (23,000 to 30,000 species) and its members are found throughout the world in both temperate and tropical habitats. The subfamilies and tribes of Compositae remained relatively constant for many years; recent molecular studies, however, have identified new subfamilial groups and identified previously unknown relationships. Currently there are 35 tribes and 10 subfamilies (Baldwin & al., 2002; Panero & Funk, 2002). Some of the tribes and subfamilies have not been tested for monophyly and without a clear understanding of the major genera that form each tribe and subfamily, an accurate phylogeny for the family cannot be reconstructed. The tribe Arctoteae (African daisies) is a diverse and interesting group with a primarily southern African distribution (ca. 17 genera, 220 species). They are especially important in that most of the species are found in the Cape Floral Kingdom, the smallest floral kingdom and the subject of intense conservation interest. Arctoteae are part of the monophyletic subfamily Cichorioideae s.s. Other tribes in the subfamily include Eremothamneae, Gundelieae, Lactuceae, Liabeae, Moquineae, and Vernonieae, and these were all evaluated as potential outgroups. Ultimately 29 ingroup taxa and 16 outgroup taxa with a total of 130 sequences (125 newly reported), from three genetic regions, two from chloroplast DNA (trnL-F and ndhF) and one from the nuclear genome (ITS), were used to evaluate the tribe and its proposed outgroups. Each molecular region is examined separately, the chloroplast markers are examined together, and the data are combined. The data were analyzed with and without outgroups and problem taxa using parsimony and maximum likelihood methods. The analyses showed robust support for two outgroup clades, Liabeae-Vernonieae and Gundelieae-Lactuceae and two main subtribes within Arctoteae: Arctotineae and Gorteriinae. Support for monophyly of Arctoteae is weak. Within Arctoteae, some taxa of interest are easily placed: Didelta, Cuspidia and Heterorhachis are consistently part of subtribe Gorteriinae, Cymbonotus, the Australian genus, is nested within subtribe Arctotineae, and Haplocarpha is at the base of Arctotineae. Berkheya, Haplocarpha, and Hirpicium are probably paraphyletic. Furthermore, Platycarpha most likely does not belong in Arctoteae, and Heterolepis and the tribe Eremothamneae are within Arctoteae but not within either of the two main subtribes. After some rearrangements, the two main subtribes, Arctotineae and Gorteriinae, are monophyletic and the latter has three clades. The study shows that the unusual taxa are of critical importance, and they should be included in any molecular analysis. Adequate representation of the ingroup is also important as all previous studies of Arctoteae had involved only a few taxa from the core subtribes, and so did not reveal the problems. Multiple outgroups evaluated in an iterative manner had pronounced effects on the relationships within the ingroup, not only on the position of the root. Finally, unrooted consensus trees and unrooted phylograms were found to be very useful in analyzing the data, allowing for examination of placement of taxa without the bias of a rooted tree.

Evidence from Chloroplast DNA for the Recognition of a New Tribe, the Tarchonantheae, and the Tribal Placement of Pluchea (Asteraceae)

The genera Pluchea, Brachylaena, and Tarchonanthus were originally placed in the Vernonieae by Cassini, but have since been assigned to other tribes. Pluchea has most often been assigned to the Inuleae (subfam. Asteroideae). Brachylaena and Tarchonanthus have been assigned to six different tribes, including the Inuleae, in both the subfam. Lactucoideae and Asteroideae. The primary reason for this diversity of tribal assignments has been the anomalous morphology of these latter two genera. Both are unisexual, dioecious, woody shrubs or trees with filiform florets. In addition, distribution is restricted to Africa, Madagascar and associated islands. Chloroplast DNA variation was examined for these three problematical genera and compared to 64 genera in 15 tribes. Complete restriction site maps were constructed for 11 enzymes; a total of 944 mutations were found, 339 of which were phylogenetically informative. The data were used to construct phylo- genetic trees using both Wagner and Dollo parsimony and the resulting monophyletic groups were evaluated using the bootstrap method. Pluchea placed within the Inuleae s.l. (subfam. Asteroideae) in all equally parsimonious trees. Brachylaena and Tarchonanthus formed a distinct monophyletic lineage at or near the base of the subfam. Lactucoideae. These two genera did not group consistently with any currently accepted tribe and are thus recognized as a new tribe, the Tarchonantheae (Cassini) Keeley & Jansen.

Forest age correlates with fine-scale spatial structure of Matsutake mycorrhizas.

Examining the fine-scale spatial structure of fungal populations can tell us much about how individual species reproduce and disperse throughout natural landscapes. Here we study the fine-scale genetic structure of Tricholoma matsutake, a prized edible and medicinal mushroom, by systematic sampling of mycorrhizas within fairy rings in 50-y-old and old-growth forests in two villages. Using single nucleotide polymorphism DNA markers we show that mycorrhizas in both forest age classes in both villages showed high levels of genotypic diversity, consistent with a reproductive life history predominated by outcrossing via basidiospore dispersal. Both the percentage of polymorphic loci within fairy rings, as well as genotype diversity were higher in old-growth compared to 50-y-old forests. Fifty-year-old forests showed significant spatial autocorrelation between pairs of mycorrhizas up to 42m, and a pattern consistent isolation-by-distance structure. Spatial patterns in old-growth forests were random. Furthermore, AMOVA analysis indicates that 11% of molecular variance in 50-y-old forests is partitioned between villages, whereas no significant variance is partitioned between villages in old-growth forests. We conclude that populations of T. matsutake in 50-y-old forests are the result of a founder effect maintained by local inoculation sources. This pattern attenuates as forests age and accumulate inocula from more distance sources. We speculate on how genetic mosaicism within T. matsutake fairy rings may structure populations within a chronosequence. Finally, we discuss how population spatial dynamics and dispersal strategy in T. matsutake contrast with other ectomycorrhizal species.

Cladistics of the bryopsidales: A preliminary analysis

The Bryopsidales contains some of the most species rich and ecologically dominant algae in tropical ecosystems. However, the evolutionary relationships among the 29 genera and several hundred species of this order remain poorly resolved. Because of a lack of known reproductive characters for many taxa, evolutionary hypotheses grouped genera by similarities in morphological characters. To apply standard cladistical analyses to further our understanding of this group, this study presents the first comprehensive compilation of reported morphological, reproductive, and subcellular characteristics for genera in the Bryopsidales. Computer‐assisted cladistical analyses ultimately identified phylogenetically informative and uninformative characters. Although the topology of the trees generated in this study is expected to change as additional data are added to this matrix, many traditional groupings and recent groupings based on molecular data were supported.

Molecular phylogeny and dating of Asteliaceae (Asparagales): Astelia s.l. evolution provides insight into the Oligocene history of New Zealand

Asteliaceae (4 genera, 36 species) are found on both continents and island archipelagos in the southern hemisphere and across the Pacific. The circumscription of Asteliaceae and intrageneric relationships are poorly understood. We generated a phylogeny including all genera and 99% of the species using DNA sequence data from chloroplast (trnL, psbA-trnH, rps16, and petL-psbE) and nuclear (NIA-i3) regions. Relaxed clock methods were applied to infer the age of the family and the timing of cladogenic events. Generic delimitations change as a result of this study. Collospermum is nested within Astelia and is recognized here only at the subgeneric level. Further, Astelia subgenera Astelia, Asteliopsis, and Tricella are paraphyletic and to achieve monophyly their recircumscriptions are proposed. Despite the presence of Asteliaceae taxa on multiple Gondwanan landmasses and proposed Cretaceous origins for the family, radiation of genera was during the Tertiary. The largest and oldest genus, Astelia s.l. (including Collospermum), radiated around the Eocene/Oligocene boundary (ca. 34.2 million years ago (Ma)). Astelia s.l. subgenera diverged from the Oligocene/Miocene boundary onwards (<24.0 Ma). These dates suggest that current distributions are most likely to be the result of long-distance dispersal. Alpine taxa in New Zealand and Australia radiated during the Late Miocene/Pliocene. These results are congruent with Astelia micro- and macro-fossil data and suggest that Astelia s.l. either persisted in New Zealand during the proposed Oligocene marine transgression or dispersed from Australia after the subsequent expansion of terrestrial habitat.

Studies on the Gymnantheminae (Vernonieae: Asteraceae) III: restoration of the genus Strobocalyx and the new genus Tarlmounia

ABSTRACT The genus Strobocalyx is resurrected from the synonymy of Gymnanthemum and Vernonia, and the genus Tarlmounia is described. Both of these Asian and Malaysian genera have distinct stylar nodes, blunt stylar hairs and specialized tricolporate, echinate pollen. Strobocalyx is typified by the Asian and Indonesian S. arborea, and combinations are provided for the 6 other East Asian and Malaysian species: Vernonia bockiana, V. chunii, V. esculenta, V. solanifolia, V. sylvatica and V. vidalii. Tarlmounia contains only Vernonia elliptica.

Leaky dioecy in Diospyros (Ebenaceae) endemic to the Island of Mauritius

Dioecy, a rather rare phenomenon in the plant kingdom seems to be more prevalent on oceanic islands. The high incidence of dioecy on these islands could result from dioecious colonists among which a small percentage show leaky dioecy, which is an ability to self-fertilise. In this study, we report the occurrence of leaky dioecy in one of the 11 extant Diospyros species endemic to Mauritius. Female flowers on the leaky dioecious plants were artificially pollinated and bagged. Populations of D. egrettarum, D. leucomelas, D. melanida, D. revaughanii, D. tessellaria were all male-biased with a ratio of at least 2:1. Leaky dioecy occurred only in one Diospyros species, D. egrettarum where hermaphrodite plants represented 2% of the populations studied. Seeds collected from them had the same germination rate (approximately 40%) as the ones obtained from strictly unisexual female plants of D. egrettarum. The fact that leaky dioecy led to the production of fertile seeds opens the possibility that a single pioneer Diospyros plant could have played a major role in the establishment of reproductively viable populations in Mauritius.

Pollen morphology and classification of the Vernonieae (Compositae)

Summary Pollen morphology and chloroplast DNA data are very useful in defining the tribes of the Compositae. Within the tribe Vernonieae, pollen is a good character for delimiting sections, subsections, and series in the genus Vernonia. The prevalence of the unspecialized type A pollen in the genera segregated from Vernonia limits the utility of this character for placing these taxa. Both the pollen and chloroplast DNA data indicate that the tribe needs much research to define its constituent monophyletic groups.

A long distance dispersal hypothesis for the Pandanaceae and the origins of the Pandanus tectorius complex.

Pandanaceae (screwpines) is a monocot family composed of c. 750 species widely distributed in the Paleotropics. It has been proposed that the family may have a Gondwanan origin with an extant Paleotropical distribution resulting from the breakup of that supercontinent. However, fossils supporting that hypothesis have been recently reassigned to other families while new fossil discoveries suggest an alternate hypothesis. In the present study, nuclear and chloroplast sequences were used to resolve relationships among Pandanaceae genera. Two well-supported fossils were used to produce a chronogram to infer whether the age of major intra-familial lineages corresponds with the breakup of Gondwana. The Pandanaceae has a Late Cretaceous origin, and genera on former Gondwanan landmasses began to diverge in the Late Eocene, well after many of the southern hemisphere continents became isolated. The results suggest an extant distribution influenced by long-distance-dispersal. The most widespread group within the family, the Pandanus tectorius species complex, originated in Eastern Queensland within the past six million years and has spread to encompass nearly the entire geographic extent of the family from Africa through Polynesia. The spread of that group is likely due to dispersal via hydrochory as well as a combination of traits such as agamospermy, anemophily, and multi-seeded propagules which can facilitate the establishment of new populations in remote locations.