Harvey E. Ballard

EVOLUTION AND BIOGEOGRAPHY OF THE WOODY HAWAIIAN VIOLETS (VIOLA, VIOLACEAE): ARCTIC ORIGINS, HERBACEOUS ANCESTRY AND BIRD DISPERSAL

Abstract Specialists studying the genus Viola have consistently allied the Hawaiian violets comprising section Nosphinium—most of which are subshrubs or treelets—with putatively primitive subshrubs in certain South American violet groups. Hawaiian violets also possess inflorescences, a floral disposition otherwise found only in other genera of the Violaceae, thus strengthening the hypothesis of a very ancient origin for the Hawaiian species. A survey of phylogenetic relationships among infrageneric groups of Viola worldwide using nuclear rDNA internal transcribed spacer (ITS) sequences revealed a dramatically different biogeographic origin for the Hawaiian violets: A monophyletic Hawaiian clade was placed in a close sister relationship with the amphi‐Beringian tundra violet, V. langsdorffii s. l., in a highly derived position. This remarkable and unforeseen relationship received strong clade support values across analyses, and monophyly of the Hawaiian lineage was further indicated by a unique 26‐base‐pair deletion in section Nosphinium. The high polyploid base chromosome number (n⋍ 40) in the Hawaiian violets relates them to Alaskan and eastern Siberian populations in the polyploid V. langsdorffii complex. More than 50 species of the 260 allochthonous birds wintering in the Hawaiian Islands are found to breed in the Arctic, occupying habitats in which individual birds might have encountered ancestral V. langsdorffii populations and served as dispersers to the central Pacific region. Acquisition of derived morphological traits (e.g., arborescence and inflorescences), significance of a confirmed Arctic origin for a component of the Hawaiian flora, and the likelihood of other “cryptic” Arctic elements in the Hawaiian flora deserving independent molecular phylogenetic corroboration are discussed.

Shrinking the Violets: Phylogenetic Relationships of Infrageneric Groups in Viola (Violaceae) Based on Internal Transcribed Spacer DNA Sequences

A phylogenetic study of the genus Viola used internal transcribed spacer (ITS) DNA sequences for 44 taxa representing many infrageneric groups in Viola plus outgroups Hybanthus concolor and Noisettia orchidiflora. Parsimony and maximum likelihood approaches place Latin American sections basal in Viola, sup- porting an Andean origin for the genus. Groups of sect. Chamaemelanium, mostly stemmed and yellow-flow- ered with x = 6 chromosomes, intermingle with groups of sect. Nomimium that are stemless and white- or blue- flowered with x = 12 or an aneuploid number. Neither section is monophyletic, and the assemblage forms a weak clade or grade, depending on the analysis. The remaining sect. Nomimium groups with primarily blue flowers and x = 10 and aneuploid or polyploid numbers form a clade including Hawaiian sect. Nosphinium, with pansies of sect. Melanium (typically stemmed with multicolored flowers and x = 5 to 17) at the base. Phy- logenetic relationships from ITS data herald the need for drastic remodeling of Viola. Proposed changes include splitting sect. Chamaemelanium and some of sect. Nomimium into several sections, transferring the remainder of Nomimium to the segregate sect. Viola, and merging Hawaiian sect. Nosphinium with the amphi- Beringian V. langsdorffii complex under the Langsdorffianae in sect. Viola.

Inferring Species Networks from Gene Trees in High-Polyploid North American and Hawaiian Violets (Viola, Violaceae)

Abstract The phylogenies of allopolyploids take the shape of networks and cannot be adequately represented as bifurcating trees. Especially for high polyploids (i.e., organisms with more than six sets of nuclear chromosomes), the signatures of gene homoeolog loss, deep coalescence, and polyploidy may become confounded, with the result that gene trees may be congruent with more than one species network. Herein, we obtained the most parsimonious species network by objective comparison of competing scenarios involving polyploidization and homoeolog loss in a high-polyploid lineage of violets (Viola, Violaceae) mostly or entirely restricted to North America, Central America, or Hawaii. We amplified homoeologs of the low-copy nuclear gene, glucose-6-phosphate isomerase (GPI), by single-molecule polymerase chain reaction (PCR) and the chloroplast trnL-F region by conventional PCR for 51 species and subspecies. Topological incongruence among GPI homoeolog subclades, owing to deep coalescence and two instances of putative loss (or lack of detection) of homoeologs, were reconciled by applying the maximum tree topology for each subclade. The most parsimonious species network and the fossil-based calibration of the homoeolog tree favored monophyly of the high polyploids, which has resulted from allodecaploidization 9–14 Ma, involving sympatric ancestors from the extant Viola sections Chamaemelanium (diploid), Plagiostigma (paleotetraploid), and Viola (paleotetraploid). Although two of the high-polyploid lineages (Boreali-Americanae, Pedatae) remained decaploid, recurrent polyploidization with tetraploids of section Plagiostigma within the last 5 Ma has resulted in two 14-ploid lineages (Mexicanae, Nosphinium) and one 18-ploid lineage (Langsdorffianae). This implies a more complex phylogenetic and biogeographic origin of the Hawaiian violets (Nosphinium) than that previously inferred from rDNA data and illustrates the necessity of considering polyploidy in phylogenetic and biogeographic reconstruction.

Inferring nativity and biogeographic affinities of central and marginal populations of Froelichia floridana (Amaranthaceae) from Inter-Simple Sequence Repeat (ISSR) markers1

and southeastern United States. We analyzed the biogeographic patterns of this species to answer a series of questions regarding its historical dispersal and recent range expansion, and the relationships of a disjunct population in southern Ohio using a combination of genetic analyses based on Inter-Simple Sequence Repeat markers (ISSR) and herbarium studies. Amounts of genetic diversity showed a marked reduction in marginal populations in the far north and northeast of the species range, corresponding to areas where herbarium records show the species has become naturalized during the past 100 years. Our analysis suggests the recognition of three biogeographic lineages within this polymorphic species complex: a Great Plains lineage, an associated Midwestern Mississippian lineage, and an Atlantic Coastal Plain lineage. These lineages have been loosely recognized as taxonomic varieties: the western lineages as F. floridana var. campestris (Small) Fern., and the eastern lineage as F. floridana var. floridana (Nutt.) Moq.; however, our data suggest that the continued recognition of these varieties is not warranted. The disjunct population in Ohio, formerly believed to be a remnant of a once larger distribution and presently state-listed as a rare species, shows patterns of genetic diversity and differentiation comparable to recently established marginal populations in other states, and was most likely derived from the Great Plains or Midwestern lineages.

A Phylogeny of the Violaceae (Malpighiales) Inferred from Plastid DNA Sequences: Implications for Generic Diversity and Intrafamilial Classification

Abstract The Violaceae consist of 1,000–1,100 species of herbs, shrubs, lianas, and trees that are placed in 22 recognized genera. In this study we tested the monophyly of genera with a particular focus on the morphologically heterogeneous Rinorea and Hybanthus, the second and third most species-rich genera in the family, respectively. We also investigated intrafamilial relationships in the Violaceae with taxon sampling which included all described genera and several unnamed generic segregates. Phylogenetic inference was based on maximum parsimony, maximum likelihood, and Bayesian analyses of DNA sequences from the trnL/trnL-F and rbcL plastid regions for 102 ingroup accessions. Results from phylogenetic analyses showed Rinorea and Hybanthus to be polyphyletic, with each genus represented by three and nine clades, respectively. Results also showed that most intrafamilial taxa from previous classifications of the Violaceae were not supported. The phylogenetic inferences presented in this study illustrate the need to describe new generic segregates and to reinstate other genera, as well as to revise the traditionally accepted intrafamilial classification, which is artificial and principally based on the continuous and homoplasious character state of floral symmetry.

A Phylogeny of Rinorea (Violaceae) Inferred from Plastid DNA Sequences with an Emphasis on the African and Malagasy Species

Abstract Rinorea is the second most species-rich genus in the Violaceae and has a pantropical distribution. In this study we investigated relationships among species of Rinorea and tested the monophyly of newly revised infrageneric groupings of African and Malagasy species. Phylogenetic inference was based on maximum parsimony, maximum likelihood, and Bayesian analyses of plastid trnL/trnL—F and trnD—trnE regions. Results showed the divergent Neotropical Rinorea ‘Apiculata’ group to be a strongly supported clade which was consistently resolved outside of the Rinorea s.S. clade. The Rinorea s.S. clade was strongly supported and was comprised of Neotropical and Old World clades. The Old World clade was strongly supported and contained 11 clades that corresponded to newly revised infrageneric groupings of African and Malagasy species. An additional Old World clade was recovered which contained all Asian species sampled in the study and was resolved as sister to the Malagasy endemic Rinorea subsect. Verticillatae. Maximum parsimony analysis recovered two Neotropical clades, while maximum likelihood and Bayesian analyses recovered a weakly supported monophyletic Neotropical clade. This study represents the first estimation of the phylogeny of Rinorea with sampling across its distribution and breadth of morphological diversity. The results presented here have implications for generic limits in Rinorea, infrageneric taxonomy of the African and Malagasy species, and biogeography of Rinorea in Madagascar.

Evidence for the recognition of Viola appalachiensis

Because most taxonomists merge the Appalachian endemic Viola appalachiensis with the southeastern V. walteri, distinctions between the two taxa and the morphologically related V. conspersa were reexamined. Herbarium studies, morphometric analyses, and scanning electron mi- croscopy of leaf vestiture and style morphology clearly distinguish V. appalachiensis from V. walteri. Most features of V. appalachiensis occur in V. conspersa or V. walteri, but its combination of character states is unique and not intermediate. Flowering specimens of V. appalachiensis produce variably stainable pollen, but herbarium specimens in fruit usually bear well-formed cleistogamous and chasmogamous seeds. Several locations for putative hybrids with V. conspersa and V. striata provide further, indirect evidence of the fertility of V. appalachiensis for outcrossing and sexual reproduction. Ecological and distributional information, coupled with morphological patterns, suggest that V. appalachiensis may have arisen from hybridization of V. conspersa and V. walteri during or after the Pleistocene glacial epoch, with subsequent genetic segregation of a population with a non-inter- mediate morphology and comparatively high fertility. Because it behaves like other well-defined sexual species in section Viola and is isolated ecologically or geographically from them, V. appala- chiensis warrants recognition as a species distinct from V. walteri.

Gibberellins are involved but not sufficient to trigger a shift between chasmogamous-cleistogamous flower types in Viola pubescens1

Abstract At least 50 angiosperm families have plants that produce both chasmogamous flowers and cleistogamous flowers. Various environmental and physiological factors, including the plant growth regulators gibberellins (GAs), have been reported to influence the flower types. Here, the relationship between GAs and flower production was studied for the first time in Viola, a genus famous for the large number of species with the mixed breeding system. Orthologs of genes for GA20 oxidase (VGA20ox) and GA3 oxidase (VGA3ox) were identified by reverse transcription polymerase chain reaction (RT-PCR) from a widespread North American species, Viola pubescens. Semi-quantitative RT-PCR indicated that both genes had increased expression in chasmogamous flowers as compared to cleistogamous flowers, supporting a role for GA in the differential production of flower type. However, the application of exogenous GA3 (the most common commercially available GA) to V. pubescens failed to induce a conversion of production of cleistogamous flowers to chasmogamous ones. Thus, increased levels of GAs in the floral buds appeared be related to flower type in the chasmogamous-cleistogamous mixed breeding system in V. pubescens, but exogenous application was not sufficient to induce an alteration in the type of flower produced.

Ixchelia, a new genus of Violaceae from Mexico and Mesoamerica

Ixchelia, a new genus of Violaceae, is described and illustrated, and new combinations are made for its two species, Ixchelia mexicana and Ixchelia uxpanapana, which are transferred from Hybanthus and Rinorea, respectively. In previous molecular phylogenetic studies, both Hybanthus and Rinorea have been shown to be polyphyletic, necessitating the erection of several genera for the constituent clades. Our recognition of Ixchelia as a distinct genus is based on morphological data and molecular phylogenetic evidence, both of which support its monophyly and its significant divergence from related genera. The new genus is most closely related to the Hawaiian endemic genus Isodendrion and the New World genus Pombalia, but it differs from them by having one to several flowers per inflorescence, a weakly zygomorphic corolla, the basal portion of the petals not fused, the stamen filaments fused into a tube, and the fruits containing three subspherical-ovoid seeds per capsule.ResumenIxchelia, un nuevo género de Violaceae, se describe e ilustra, y nuevas combinaciones se hacen para sus dos especies, Ixchelia mexicana e Ixchelia uxpanapana, que son transferidos desde Hybanthus y Rinorea, respectivamente. Tanto Hybanthus y Rinorea han demostrado ser polifiléticos, lo que exige la descripción de varios géneros para los clados constituyentes. Nuestro reconocimiento de Ixchelia como un género distinto se basa en datos morfológicos y una evidencia filogenética molecular, los cuales apoyan su monofilia y su divergencia significativa de géneros afines. El nuevo género está más estrechamente relacionado con el género Isodendrion endémico de Hawai y el género Pombalia del Nuevo Mundo, pero se diferencia por las inflorescencias con una a varias flores, corolas débilmente zigomórficas, porción basal de pétalos no fusionada, filamentos estaminales fusionados en un tubo, y los frutos que contienen tres semillas subesférica-ovoides por cápsula.

Phenetic analysis shows conspecificity between Hispaniolan Viola domingensis Urban and North American Viola macloskeyi sensu lato (Violaceae)

Phenetic investigations were undertaken to evaluate differences between the cloud forest endemicViola domingensis of the Dominican Republic and the morphologically similarV. macloskeyi of continental North America. Cluster, principal components (PCA), and canonical variates analyses (CVA) of leaf characters and of ratios derived from them consistently separateV. macloskeyi subsp.macloskeyi of the Pacific states fromV. domingensis and transcontinentalV. macloskeyi subsp.pallens. However, all analyses portray the latter two as extensively intergradient extremes of a continuum, withV. domingensis plants tending to be smaller. Phenetic analyses of capsule and seed features, which are often divergent among other species of subsect.Stolonosae, showV. domingensis to be nested within a broaderV. macloskeyi subsp.pallens. Similarly, floral morphology does not provide observable differences between the pair. Results support the synonymy ofV. domingensis underV. macloskeyi subsp.pallens and reveal that it represents a disjunct outlier of an otherwise continental North American violet.