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Kokia (Malvaceae) is a tree genus consisting only of three endangered and one extinct species endemic to the Hawaiian Islands. Morphological and molecular population analyses suggested conflicting hypotheses as to colonization and dispersal among the Islands. To resolve this, 12 nuclear and four chloroplast gene regions were sequenced and compared to establish phylogenetic hypotheses and to make biogeographic inferences. Seven gene regions yielded phylogenies consistent with the progression rule hypothesis suggesting dispersal from older to younger islands, seven other gene regions were equivocal, and two weakly supported an alternative phylogeny. Combined analyses of all genes strongly supported the progression rule hypothesis, with initial colonization to Kaua‘i, or possibly an older and now submerged island, with subsequent dispersal to O‘ahu, Moloka‘i, and Hawai‘i Islands. Morphological characters plotted onto the molecular phylogeny suggested that many morphological traits are plastic or nonhomologous. Our study illustrates the perils of evaluating relationships among widely divergent taxa using morphological characters.

Acacia koa (koa) is a valuable leguminous timber-wood tree endemic to the Hawaiian Islands. Over the past century, conversion of some native forests for agriculture and ranching, uncontrolled spread of invasive plant species and outbreak of a deadly fungal wilt disease severely reduced A. koa forests. However, recently there has been a growing interest in re-establishing A. koa as an agroforestry tree because the tree has significant importance in economy, ecology and culture of Hawai‘i. This review presents the current knowledge on A. koa, including its ecological roles, various morphological forms, genetics, evolution and development of methods for seedling selection and propagation. A better understanding and awareness of the nature of A. koa will help successful development of wilt-resistant A. koa trees with high wood quality that can support Hawaii’s ecology, economy and culture.

Moranopteris inaccessa, here described and illustrated, is a new species from southern Peru. Plants were collected in the Abra Malaga private conservation area from steep ledges accessible only with mountaineering equipment. Moranopteris inaccessa differs from its congeners by the combination of elongate membranaceous laminae that are 1-pinnate-pinnatifid. Found at 4,427 m, its distribution is over 500 m higher in elevation than any other species of Moranopteris. Phylogenetic analyses of atps, rbcL, and trnL-trnF molecular sequences, using maximum parsimony, maximum likelihood, and Bayesian analysis support M. inaccessa as sister to M. longisetosa. The two are similar in their elongate and highly divided leaves compared to other species of Moranopteris. The discovery of this new species highlights the value of undertaking botanical surveys of undisturbed vegetation patches in the high Andes that potentially host unrecognized plant diversity.

Microsatellite markers are valuable tools for determining amount and distribution of genetic diversity and differentiation within and between populations. In this study we examined the level of microsatellite variability within and among five populations of Lobelia villosa, a rare Hawaiian endemic lobeliad on the island of Kaua‘i. Populations of L. villosa were sampled from two regions on Kaua‘i: two populations at the Kilohana Lookout area and three populations from Alaka‘i Swamp. Nineteen microsatellite DNA primers were developed for L. villosa, 12 of which demonstrated polymorphism and were subsequently multiplexed and labeled for genotyping. An overall moderate degree of genetic differentiation was found within and between populations. Pairwise FST data showed population structure, and analysis with Structure software indicated two genetic clusters (K = 2) corresponding to the two sampled geographic regions. Although L. villosa exhibits moderate diversity, which exceeds that of other Hawaiia...

Proper taxonomic classification of species is essential to the conservation of rare species to ensure that they garner needed protection. This study was undertaken to determine relationships among the Hawaiian representatives of Cryptocarya. This genus is represented in Hawai‘i on the islands of Kaua‘i and O‘ahu, where it is currently classified as a single species (C. mannii) and not considered endangered. It had previously been recognized as two species, C. mannii on Kaua‘i where it is relatively abundant and C. oahuensis on O‘ahu. O‘ahu plants have been in decline since their discovery over 100 yr ago and now only one naturally occurring plant is known. RAPD analysis demonstrates that populations on Kaua‘i are genetically distinct from those on O‘ahu. Sequence analysis of nuclear (ITS) and plastid (trnK intron) gene regions shows distinctions among plants from different islands (ITS: four or five base substitutions plus one repeat length difference; trnK intron: one base substitution plus one...

Woody perennial plants on islands have repeatedly evolved from herbaceous mainland ancestors. Although the majority of species in

Genetic diversity of all three extant species of the Hawaiian endemic tree genus Kokia was examined using analyses of RAPD markers. All three species are federally endangered, two (K. cookei and K. drynarioides) critically so, and one (K. kauaiensis) is rare but with several populations. Samples extracted were PCR amplified with 10 primers to yield 115 genetic markers. Analyses indicate that genetic variation is present in all three species; a surprising result given that K. cookei survives only from self-fertilized propagules of the single surviving individual that was grafted on to the root stock of a related species, K. drynarioides. Principal coordinates and cluster analyses based on RAPD markers indicate that although K. kauaiensis has substantial genetic variation, samples are clearly separate from the other two species of the genus. In contrast, K. cookei and K. drynarioides collections are more similar but still distinguishable when analyzed separately. Presence of substantial genetic variation lends optimism to the eventual recovery of K. kauaiensis through conservation efforts. Combinations of RAPD primers are suggested that reveal diagnostic alleles for each of the three extant species and can thus be used for species identification.

The global evolutionary pattern among the dracaenoid plant genera Dracaena, Pleomele, and Sansevieria has remained ambiguous. Their classification has been disputed due to different taxonomic interpretation and the difficulty in morphological characterization of the species and genera. This study explores the phylogenetic analysis of 95 species representing all three genera using four combined chloroplast intergenic spacer DNA regions (trnL-trnF, ndhF-rpl32, trnQ-rps16, and rpl32-trnL). The combined dataset was analyzed using parsimony, maximum likelihood, and Bayesian analysis. Results show that 1) dracaenoids are monophyletic; 2) the Hawaiian Pleomele species are the sister group to the remainder of the dracaenoid clade; 3) species of Dracaena and the remaining Pleomele are intermixed; 4) Sansevieria is monophyletic, but nested within Dracaena; 5) the Central American species D. americana and D. cubensis are the sister group to the remainder of the non-Hawaiian dracaenoid species. The Hawaiian Pleomele are morphologically and phylogenetically distinct from the remaining dracaenoids and are recognized as a distinct genus, Chrysodracon, and new species combinations are made. Dracaena is paraphyletic in regards to Sansevieria and those species should be recognized as species of Dracaena. The polyphyletic status of Pleomele species nested within Dracaena confirms previous morphology based studies that combined these genera.

 Premise of the study: Floral nectar sugar compositions have, for several decades, been used to predict a plant species’ pollinator guild. Plants possessing a generalist ornithophilous pollination syndrome produce nectar that is dilute (8–12% w/v sugars) with a low sucrose to hexose (glucose and fructose) ratio. The Hawaiian lobeliad genus Clermontia contains 22 endemic species of shrubs and small trees that are believed to have evolved fl owers adapted for pollination by now mostly extinct or endangered endemic passerines in the Drepanidinae and Mohoidae.  Methods: We analyzed the nectar sugar compositions, concentration, and nectar standing crop of 23 taxa to test the assumption that Clermontia taxa have evolved fl oral traits in response to selection pressures from these avian pollinators.  Key results: All Clermontia taxa produced nectar with sugar concentrations (mean: 9.2% w/v ± 1.8 SD) comparable to the nectar of other plant species with a generalized bird pollination system. Nectar sugars were overwhelmingly composed of hexoses in all taxa (mean sucrose/hexose ratio: 0.02 ± 0.02). Nectar standing crop volumes varied widely among taxa, ranging from 9.7 µL ± 7.1 to 430.5 µL ± 401.8 (mean volume: 177.8 ± 112.0).  Conclusions: Collectively, the nectar traits indicate that Clermontia species possess a generalist passerine pollination syndrome.

The Hawaiian Euphorbia is an assemblage of 17 species, seven of which are endangered and four others rare. Euphorbia kuwaleana is an endangered species of small shrubs restricted to three small populations in West O'ahu, Hawai'i. The species has declined to fewer than 1,000 individuals largely due to habitat encroachment by alien plant species and the periodic fires that occur in the vicinity. Genetic variation was assessed among individuals in two populations to determine what impact small population size has had on genetic diversity within the species using RAPD markers. Results demonstrate that polymorphism within these populations is high (mean = 82.5%), equal to or exceeding that of many other nonendangered Hawaiian species. Genetic similarities within (0.741) and among (0.716) populations, FST (0.072), and PCO analysis all indicate differentiation among the populations although in close geographical proximity (

Hybridization between alien plant species and their native congeners represents a major global threat to native floras. In Hawai'i, two introduced cotton species, Pima cotton (G. barbadense) and Upland cotton (G. hirsutum), have naturalized on the main Hawaiian Islands. Both species are capable of producing fertile hybrids with the native Hawaiian cotton (G. tomentosum). Hybrid populations between Pima and Hawaiian cotton were documented at Nanakuli on the Wai'anae coast of O'ahu in 1964 by the late Stanley G. Stephens. Extant populations of Pima and Hawaiian cotton in the vicinity of those documented by Stephens (1964) were screened using both morphological and molecular (microsatellite markers) techniques to assess persistence of the original hybrid population and potential gene flow that may have occurred between the two cotton species. We did not relocate the original hybrid populations described in Stephens (1964), although a herbarium voucher that was verified as a hybrid indicates that at...

Understanding the reproductive biology of rare plant species is fundamental to managing their restoration. Brighamia insignis is a critically endangered Hawaiian lobeliad endemic to the islands of Kauai and Niihau. The flowers appear to be adapted for moth pollination although its putative pollinator is believed to be extinct or very rare. To confirm the pollination syndrome, document the breeding system, and identify potential pollinators of B. insignis: 1) a suite of floral characters were examined, 2) pollination treatments were performed, and 3) diurnal and nocturnal floral visitor observations were conducted at an ex situ site on Kaua‘i. Brighamia insignis flowers contain sucrose-rich nectar and emit a strong floral scent containing benzyl alcohol, linalool, and methyl salicylate. Pollination treatments revealed that the species is primarily outcrossing with several of the study plants also capable of low levels of selfing. However, most of the plants had low pollen production and viability. No moths and only occasional non-native insect species visited the flowers of B. insignis. None of these insect visitors appeared to be serving as effective pollinators. In spite of this lack of flower visitation by moths, analysis of nectar and floral scent support a moth pollination syndrome in B. insignis. The potential loss of pollinators suggests that restoring populations of B. insignis may not be feasible; human assisted cross-pollination would be necessary for fruit and seed to set, as only 1% of the control and self treatment flowers formed fruit. Therefore, this species appears dependent upon intense human management to prevent its extinction. (VLID)4393823

Thirty-one microsatellite markers, developed from Acacia koa, were evaluated for cross-amplification of seven nonnative species of Acacia in the Hawaiian Islands. The number of alleles per locus ranged from one to seven, with 16 of the 31 markers showing polymorphism in at least two of the nonnative Acacia species. Of the Acacia species examined, A. melanoxylon showed the highest percentage of amplified loci.

AimThe taxon cycle hypothesis describes the cyclic movement of taxa during range expansion and contraction, accompanied by an evolutionary shift from open and often coastal vegetation to closed, and often inland forest vegetation in island systems. The Hawaiian Archipelago is an ideal system to test this hypothesis given the linear fashion of island formation and a relatively well-understood geological history.LocationHawaiian Islands.MethodsWe sampled 153 individuals in 15 of the 16 native species of HawaiianEuphorbiasectionAnisophyllumon six major Hawaiian Islands, plus 11 New World close relatives, to elucidate the biogeographic movement of this lineage along the Hawaiian island chain. We used a concatenated chloroplast DNA data set of more than eight kilobases in aligned length and applied maximum likelihood and Bayesian inference for phylogenetic reconstruction. Connectivity among islands and habitat types was estimated using BayesTraits. Age and phylogeographic patterns were co-estimated using BEAST. In addition, we used nuclear ribosomal ITS and the low-copy genesLEAFYandG3pdhCto investigate the reticulate relationships within this radiation.ResultsWe estimate that HawaiianEuphorbiafirst arrived on Kauai or Niihau ca. 5 million years ago and subsequently diverged into 16 species on all major Hawaiian Islands. During this processEuphorbiadispersed from older to younger islands in a stepping-stone fashion through open, dispersal-prone habitats. Taxa that occupy closed vegetation on Kauai and Oahu evolvedin situfrom open vegetation taxa of the same island. Consequently, widespread species tend to occupy habitats with open vegetation, whereas single island endemic species predominantly occur in habitats with closed canopy and are only found on the two oldest islands of Kauai and Oahu.Main conclusionsThe spatial and temporal patterns of dispersal and range shifts in HawaiianEuphorbiasupport an intra-volcanic-archipelago version of the taxon cycle hypothesis.

Aim To estimate the rate of adaptive radiation of endemic Hawaiian Bidens and to compare their diversification rates with those of other plants in Hawaii and elsewhere with rapid rates of radiation. Location Hawaii. Methods Fifty-nine samples representing all 19 Hawaiian species, six Hawaiian subspecies, two Hawaiian hybrids and an additional two Central American and two African Bidens species had their DNA extracted, amplified by polymerase chain reaction and sequenced for four chloroplast and two nuclear loci, resulting in a total of approximately 5400 base pairs per individual. Internal transcribed spacer sequences for additional outgroup taxa, including 13 non-Hawaiian Bidens, were obtained from GenBank. Phylogenetic relationships were assessed by maximum likelihood and Bayesian inference. The age of the most recent common ancestor and diversification rates of Hawaiian Bidens were estimated using the methods of previously published studies to allow for direct comparison with other studies. Calculations were made on a per-unit-area basis. Results We estimate the age of the Hawaiian clade to be 1.3–3.1 million years old, with an estimated diversification rate of 0.3–2.3 species/million years and 4.8 × 10−5 to 1.3 × 10−4 species Myr−1 km−2. Bidens species are found in Europe, Africa, Asia and North and South America, but the Hawaiian species have greater diversity of growth form, floral morphology, dispersal mode and habitat type than observed in the rest of the genus world-wide. Despite this diversity, we found little genetic differentiation among the Hawaiian species. This is similar to the results from other molecular studies on Hawaiian plant taxa, including others with great morphological variability (e.g. silverswords, lobeliads and mints). Main conclusions On a per-unit-area basis, Hawaiian Bidens have among the highest rates of speciation for plant radiations documented to date. The rapid diversification within such a small area was probably facilitated by the habitat diversity of the Hawaiian Islands and the adaptive loss of dispersal potential. Our findings point to the need to consider the spatial context of diversification – specifically, the relative scale of habitable area, environmental heterogeneity and dispersal ability – to understand the rate and extent of adaptive radiation.

Acacia koa A. Gray (koa) is a leguminous tree endemic to the Hawaiian Islands and can be divided into morphologically distinguishable groups of A. koaia Hillebrand, A. koa and populations that are intermediate between these extremes. The objectives of this investigation were to distinguish among divergent groups of koa at molecular levels, and to determine genetic diversity within and among the groups. Phylogenetic analyses using the ITS/5.8S rDNA and trnK intron sequences did not separate the representative koa types into distinct clusters. An unweighted pair group method with arithmetic mean cluster analysis and principal coordinate analysis, based on allele profiles of 12 microsatellite loci for 215 individual koa samples, separated the population into three distinct clusters consistent with their morphology, A. koaia, A. koa and intermediate forms. There was an average of 8.8 alleles per polymorphic locus (AP) among all koa and koaia individuals. The intermediate populations had the highest genetic diversity (H′ = 1.599), AP (7.9) and total number of unique alleles (21), whereas A. koaia and A. koa showed similar levels of genetic diversity (H′ = 0.965 and 0.943, respectively). No correlation was observed between geographic distance and genetic distance as determined by a Mantel test (r = 0.027, P = 0.91). The data presented here support previous recommendations that morphological variation within koa should be recognized at the subspecific level rather than as distinct species.

Chamaesyce skottsbergii var. skottsbergii is federally listed as an endangered taxon, and is found in small and isolated populations restricted to calcareous soils in dry shrubland habitats on the Hawaiian islands of Oahu and Molokai. Concern over the genetic relationship among these disjunct populations arose as a result of threats to the habitat of the Oahu population. The populations were examined using random amplified polymorphic DNA (RAPD) markers and sequence analysis of the internal transcribed spacer (ITS) region of the rDNA cistron. Chamaesyce skottsbergii var. vaccinioides, a closely related variety found in several small populations on Molokai, was used for baseline comparison of the genetic divergence among populations. RAPD analysis demonstrated that variation within and among populations is the highest for any Hawaiian species examined. Polymorphism was greater than 95% within populations and was 99.4% at the species level. Similarly, measures of genetic similarity indicate that differentiation among these populations is higher than is known for some species. Both RAPD and ITS sequence analysis indicate that populations of C. skottsbergii var. skottsbergii on Oahu and Molokai are genetically distinct, and the extent of this genetic differentiation supports the recognition of these populations as distinct varieties. The Molokai population is in fact much more closely related to var. vaccinioides than to var. skottsbergii on Oahu, and thus should be recognized by the previously used variety name, C. skottsbergii var. audens. Further conservation measures for each of the varieties are addressed.

SUMMARY The taxonomic and phylogenetic concepts within the Sargassum C. Agardh (Sargassaceae) species complex were evaluated through molecular phylogenetic analyses using portions of the chloroplast encoded rbcLS Operon. According to more conservative sequences (rbcL), Turbinaria (Turner) J. Agardh is a close and well-supported sister lineage to the Sargassum species complex and an appropriate external outgroup for analyses of subgenera and subsections within Sargassum. Both rbcL and more rapidly evolving rbcLS spacer sequences indicated that the East Asiatic genus Myagropsis (Mertens et Turner) Fensholt, along with Sargassum sinicola Setchell et Gardner, represent the closest lineage to Sargassum and form appropriate internal outgroups. The rbcLS spacer region supported three of four subgeneric designations by J. Agardh and sectional levels within the subgenus Sargassum. However, some aspects of Agardh's system were not supported: many of the subsectional ranks or the phyletic concepts; Phyllotrichia was not monophyletic as a subgenus, and its species were also not the most ancestral of Sargassum; and subgenus Sargassum was not the most derived subgenus within the genus. This modern phylogeny suggests a deep evolutionary history for subgenus Sargassum with rapid speciation in closely related subsections and series, and a sister relationship between subgenera Arthrophycus and Bactrophycus.

A population of putative hybrids between the endemic Rubus hawaiensis and naturalized R. rosifolius was discovered in KValley, on the island of Maui in the Hawaiian archipelago. The goal of this study was to molecularly characterize this natural hybridization event, investigate the mode of hybridization, and determine the male fertility of the hybrid individuals. Both morphological and RAPD marker data indicate that the putative hybrid individuals are the progeny of R. rosifolius and R. hawaiensis. All 39 hybrid individuals sampled had the chloroplast DNA haplotype of R. rosifolius. Thus hybridization appears to be asymmetric, with R. rosifolius acting as the maternal parent. All hybrid individuals assessed for pollen stainability were sterile, and there was no evidence of backcrossing to either parent. This result suggests that hybrids are of the first filial generation and that variation among hybrids reflects differences within the parental populations. Sympatric populations of R. hawaiensis and R. rosifolius occur on four islands and six additional alien species of Rubus are naturalized and sympatric with R. hawaiensis in Hawai'i. Further investigation is merited to assess whether hybridization may pose a threat to the long term viability of R. hawaiensis. This study highlights the increasing frequency and negative consequences of native-alien hybridization and the importance of maintaining active alien species control programs in the Hawaiian Islands.

Touchardia latifolia (Urticaceae) is an endemic species to the Hawaiian Islands. Although widespread and extensively cultivated by early Hawaiians for cordage, populations are now greatly diminished and increasingly rare. Reproductively dioecious, T . latifolia is expected to have high genetic diversity. However, it is thought that diversity may be reduced because of the reduction in the number of populations and their size. Examination of random amplified polymorphic DNA (RAPD) markers from seven populations indicates that genetic diversity (based on individual similarities) is in fact low relative to other dioecious and outcrossing Hawaiian species. Patterns within islands are not correlated with geographic distance; therefore, loss of diversity is likely associated with genetic drift. In contrast, variation among islands is associated with island biogeography and therefore colonization from one island to another was probably progressive down the island chain.

The endemic Hawaiian raspberries Rubus hawaiensis and R. macraei (both subgenus Idaeobatus) had been thought to be closely related species until recent molecular studies demonstrated otherwise. These studies suggest that they are the products of separate colonizations to the Hawaiian Islands. Affinities of R. hawaiensis to R. spectabilis of western North America were clearly confirmed. However, no clear relation to R. macraei has been published. This study was initiated to examine species of subg. Idaeobatus from the surrounding Pacific region as well as species from other subgenera to better evaluate biogeographic and phylogenetic affinities of R. macraei by means of chromosome analysis and molecular data using the chloroplast gene ndhF. Results show that R. macraei clusters in a clade with species of blackberries, subg. Rubus, and of these it is most closely linked to R. ursinus. Chromosomally, R. macraei is 2n = 6x = 42, a number that would be a new report for subg. Idaeobatus. However, polyploidy is common in subg. Rubus. Analyses indicate that R. macraei and R. hawaiensis are derived from separate colonizations from North America and that similarities between them are due to convergent evolution in the Hawaiian environment.

Throughout the world DNA banks are used as storage repositories for genetic diversity of organisms ranging from plants to insects to mammals. Designed to preserve the genetic information for organisms of interest, these banks also indirectly preserve organisms’ associated microbiomes, including fungi associated with plant tissues. Studies of fungal biodiversity lag far behind those of macroorganisms, such as plants, and estimates of global fungal richness are still widely debated. Utilizing previously collected specimens to study patterns of fungal diversity could significantly increase our understanding of overall patterns of biodiversity from snapshots in time. Here, we investigated the fungi inhabiting the phylloplane among species of the endemic Hawaiian plant genus, Clermontia (Campanulaceae). Utilizing next generation DNA amplicon sequencing, we uncovered approximately 1,780 fungal operational taxonomic units from just 20 DNA bank samples collected throughout the main Hawaiian Islands. Using these historical samples, we tested the macroecological pattern of decreasing community similarity with decreasing geographic proximity. We found a significant distance decay pattern among Clermontia associated fungal communities. This study provides the first insights into elucidating patterns of microbial diversity through the use of DNA bank repository samples.

We examined the global historical biogeography of grammitid ferns (Polypodiaceae) within a phylogenetic context. We inferred phylogenetic relationships of 190 species representing 31 of the 33 currently recognized genera of grammitid ferns by analyzing DNA sequence variation of five plastid DNA regions. We estimated the ages of cladogenetic events on an inferred phylogeny using secondary fossil calibration points. Historical biogeographical patterns were inferred via ancestral area reconstruction. Our results supported four large-scale phylogenetic and biogeographic patterns: (1) a monophyletic grammitid clade that arose among Neotropical polypod ancestors about 31.4 Ma; (2) a paraphyletic assemblage of clades distributed in the Neotropics and the Afro-Malagasy region; (3) a large clade distributed throughout the Asia–Malesia–Pacific region that originated about 23.4 Ma; and, (4) an Australian or New Zealand origin of the circumaustral genus Notogrammitis. Most genera were supported as monophyletic except for Grammitis, Oreogrammitis, Radiogrammitis, and Zygophlebia. Grammitid ferns are a well-supported monophyletic group with two biogeographically distinct lineages: a primarily Neotropical grade exhibiting several independent successful colonizations to the Afro-Malagasy region and a primarily Paleotropical clade exhibiting multiple independent dispersals to remote Pacific islands and temperate, austral regions.

Summary Despite the normally strong link between geographic proximity and relatedness of recently diverged taxa, truly puzzling biogeographic anomalies to this expectation exist in nature. Using a dated phylogeny, population genetic structure and estimates of ecological niche overlap, we tested the hypothesis that two geographically very disjunct, but morphologically very similar, island endemics (Acacia heterophylla from Reunion Island and A. koa from the Hawaiian archipelago) are the result of dispersal between these two island groups, rather than independent colonization events from Australia followed by convergent evolution. Our genetic results indicated that A. heterophylla renders A. koa paraphyletic and that the former colonized the Mascarene archipelago directly from the Hawaiian Islands ≤ 1.4 million yr ago. This colonization sequence was corroborated by similar ecological niches between the two island taxa, but not between A. melanoxylon from Australia (a sister, and presumed ancestral, taxon to A. koa and A. heterophylla) and Hawaiian A. koa. It is widely accepted that the long-distance dispersal of plants occurs more frequently than previously thought. Here, however, we document one of the most exceptional examples of such dispersal. Despite c. 18 000 km separating A. heterophylla and A. koa, these two island endemics from two different oceans probably represent a single taxon as a result of recent extreme long-distance dispersal.

Hybridization between Dubautia ciliolata and D. scabra occurring on a mosaic of lava flows of 1855 and 1935 on the island of Hawai'i was examined using random amplified polymorphic DNA (RAPD) markers. The RAPD data indicate that D. ciliolata plants, nearly restricted to the 1855 lava flow, contain higher levels of genetic variation than do D. scabra plants occurring on the 1935 lava flow. Seventy-one markers were specific to D. ciliolata and 60 to D. scabra; 40 of these were "constant" (found in all individuals) in one or the other species. Hybrids sampled were determined to represent F(1), filial hybrids beyond the F(1), and backcross progeny. All backcrosses were unidirectional with D. ciliolata acting as the recurrent parent. No hybrid, including an artificially produced F(1), had all 40 constant markers, suggesting that at least some loci for these markers were heterozygous in the parents. However, several hybrids exhibited a loss of many of the species markers, suggesting that they were later filial hybrid generation plants. The apparent occurrence of unidirectional introgression at the study site may be providing D. ciliolata plants with genetic plasticity to colonize the new lava flow previously occupied only by D. scabra.

Randomly amplified polymorphic DNA (RAPD) markers provide data consistent with the conclusion based on morphological characters that the recently named taxon Cibotium x heleniae is indeed of hybrid origin. This assertion is supported by (1) placement of C. x heleniae intermediate to the parent taxa, as determined by genetic similarity data; (2) location of C. x heleniae individuals on a clade intermediate to the parent species in the cladistic analysis; and (3) clustering of the C. x heleniae individuals between clusters of parental individuals in principal components analysis. Additivity of parental genetic markers in the putative hybrid ranged from 54 to 64%, providing additional though modest support for the hypothesized origin of C. x heleniae. Our results indicate that RAPD data can be of considerable value in assessing potential hybridity of individuals in naturally occurring populations.

Extant chromophytic algae have been suggested to have originated via the engulfment of a photo synthetic alga by a colorless protist. The dinoflagellate Peridinium foliaceum (Stein) Biecheler contains a reduced chlorophyll c–containing endosymbiont and, thus, represents an evolutionary intermediate stage in the establishment of chloroplasts. Although the exact phylogenetic relationship of the symbiont to extant algal species is unknown, it had been suggested that the P. foliaceum symbiont was either a diatom or a chrysophyte. Identification of the closest living relative of the P. foliaceum symbiont would provide a free-living model system with which the photosynthetic symbiont could be compared. Nucleotide sequence analysis of rbcL and rbcS (encoding the large and small subunits ofribulose-1,5-bisphosphate carboxylase/oxygenase) by the P. foliaceum symbiont was performed to provide insights into its identity. Cloned restriction fragments from a chloroplast DNA library were screened, and clones encoding the rbcLS operon were sequenced. Parsimony phylogenetic analysis was performed for each gene. Our data strongly suggest that the symbiont originated from a photosynthetic diatom.

The endpoints of the large inverted repeat (IR) of chloroplast DNA in flowering plants differ by small amounts between species. To quantify the extent of this movement and define a possible mechanism for IR expansion, DNA sequences across the IR-large single-copy (IR-LSC) junctions were compared among 13 Nicotiana species and other dicots. In most Nicotiana species the IR terminates just upstream of, or somewhere within, the 5' portion of the rps19 gene. The truncated copy of this gene, rps19', varies in length even between closely related species but is of constant size within a single species. In Nicotiana, six different rps19' structures were found. A phylogenetic tree of Nicotiana species based on restriction site data shows that the IR has both expanded and contracted during the evolution of this genus. Gene conversion is proposed to account for these small and apparently random IR expansions. A large IR expansion of over 12 kb has occurred in Nicotiana acuminata. The new IR-LSC junction in this species lies within intron 1 of the clpP gene. This rearrangement occurred via a double-strand DNA break and recombination between poly (A) tracts in clpP intron 1 and upstream of rps19. Nicotiana acuminata chloroplast DNA contains a "molecular fossil' of the IR-LSC junction that existed prior to this dramatic rearrangement.

Expression of the vestigial plastid genome of the nonphotosynthetic, parasitic flowering plant Epifagus virginiana was examined by northern analysis and by characterization of cDNAs. Probes for each of 12 plastid genes tested hybridized to all lanes of northern blots containing total RNA prepared from stems and fruits of Epifagus and from leaves of tobacco. Certain transcript patterns in Epifagus plastids are highly complex and similar to those of tobacco operons. In contrast, genes such as rps2, which have become orphaned in Epifagus as a result of evolutionary loss of formerly cotranscribed genes, show simpler transcript patterns in Epifagus than in tobacco. Sizing and sequencing of cDNAs generated by reverse transcriptase-PCR for three genes, rps12, rpl2, and clpP, show that their transcripts are properly cis- and/or trans-spliced at the same five group II intron insertion sites used in photosynthetic plants. A single, conventional C--U edit in rps12 was found among the total of 1401 nucleotides of cDNA sequence that was determined for the three genes. An octanucleotide sequence identical to a putative guide RNA of plant organelles and perfectly complementary to the rps12 edit site itself was identified just 200 bp upstream of the edit site. These data, together with previous results from the complete sequencing of the Epifagus plastid genome, provide compelling evidence that this degenerate genome is nonetheless expressed and functional. Analysis of the putative maturase MatK, encoded by the group II intron of trnK in photosynthetic land plants but by a freestanding gene in Epifagus, leads us to hypothesize that it acts 'in trans' to assist the splicing of group II introns other than the one in which it is normally encoded.

The genus Chrysodracon has six endemic species in the Hawaii Islands. Chrysodracon hawaiiensis is endemic to Hawaii Island and was described as a distinct species in 1980. It was listed as an endangered species on the International Union for the Conservation of Nature and Natural Resources (IUCN) Red List in 1997. This woody plant species was, at one time, common in exposed dry forests, but it became very rare due to grazing pressure and human development. The tree species Chrysodracon auwahiensis (C. auwahiensis), endemic to Maui and Molokai, still has large adult populations in dry lands of the islands, but unfortunately no regeneration from seed has been reported in those areas for many years. The two endemic species were examined using the molecular technique of random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) to determine the genetic structure of the populations and the amount of variation. Both species possess similar genetic structure. Larger and smaller populations of both species contain similar levels of genetic diversity as determined by the number of polymorphic loci, estimated heterozygosity, and Shannon’s index of genetic diversity. Although population diversity of Chrysodracon hawaiiensis (C. hawaiiensis) is thought to have remained near pre-disturbance levels, population size continues to decline as recruitment is either absent or does not keep pace with senescence of mature plants. Conservation recommendations for both species are suggested.

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 (

The most widely distributed dinoflagellate plastid contains chlorophyll c(2) and peridinin as the major carotenoid. A second plastid type, found in taxa such as Karlodinium micrum and Karenia spp., contains chlorophylls c(1) + c(2) and 19'-hexanoyloxy-fucoxanthin and/or 19'-butanoyloxy-fucoxanthin but lacks peridinin. Because the presence of chlorophylls c(1) + c(2) and fucoxanthin is typical of haptophyte algae, the second plastid type is believed to have originated from a haptophyte tertiary endosymbiosis in an ancestral peridinin-containing dinoflagellate. This hypothesis has, however, never been thoroughly tested in plastid trees that contain genes from both peridinin- and fucoxanthin-containing dinoflagellates. To address this issue, we sequenced the plastid-encoded psaA (photosystem I P700 chlorophyll a apoprotein A1), psbA (photosystem II reaction center protein D1), and "Form I" rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase) genes from various red and dinoflagellate algae. The combined psaA + psbA tree shows significant support for the monophyly of peridinin- and fucoxanthin-containing dinoflagellates as sister to the haptophytes. The monophyly with haptophytes is robustly recovered in the psbA phylogeny in which we increased the sampling of dinoflagellates to 14 species. As expected from previous analyses, the fucoxanthin-containing dinoflagellates formed a well-supported sister group with haptophytes in the rbcL tree. Based on these analyses, we postulate that the plastid of peridinin- and fucoxanthin-containing dinoflagellates originated from a haptophyte tertiary endosymbiosis that occurred before the split of these lineages. Our findings imply that the presence of chlorophylls c(1) + c(2) and fucoxanthin, and the Form I rbcL gene are in fact the primitive (not derived, as widely believed) condition in dinoflagellates.

Insular oceanic islands provide excellent opportunities for the study of evolutionary processes and adaptive radiation. The Hawaiian Plantago radiation comprises six endemic taxa showing considerable inter- and intraspecifi c morphological and ecological diversity. The rDNA internal (ITS) and external (ETS) transcribed spacers and two recently described chloroplast spacers, ndhF - rpl 32 and rpl 32- trnL , were sequenced to study phylogenetic relationships within this morphologically complex group. Phylogenetic analysis provided strong evidence for the monophyly of Hawaiian Plantago , suggesting that the lineage arose from a single long-distance dispersal event. Inconsistencies between nuclear and chloroplast phylogenies suggest a history of hybridization. The basal, unresolved dichotomy of the combined phylogeny is consistent with rapid phenotypic diversifi cation of the major lineages early in the history of this group. Speciation has largely occurred allopatrically, with divergence a result of intraisland ecological shifts between bog and woodland habitats and interisland dispersal events. Most interisland colonizations were from older to younger islands with initial colonization of Kaua ‘ i. In our analysis, P. pachyphylla is paraphyletic and taxonomic separation of the distinct morphotypes of this species appears justifi ed. Furthermore, the apparent hybrid ancestry and unique morphology and habitat of the endangered P. princeps var. longibracteata support its recognition at the specifi c rank.

Complete nucleotide sequencing shows that the plastid genome of Epifagus virginiana, a nonphotosynthetic parasitic flowering plant, lacks all genes for photosynthesis and chlororespiration found in chloroplast genomes of green plants. The 70,028-base-pair genome contains only 42 genes, at least 38 of which specify components of the gene-expression apparatus of the plastid. Moreover, all chloroplast-encoded RNA polymerase genes and many tRNA and ribosomal protein genes have been lost. Since the genome is functional, nuclear gene products must compensate for some gene losses by means of previously unsuspected import mechanisms that may operate in all plastids. At least one of the four unassigned protein genes in Epifagus plastid DNA must have a nongenetic and nonbioenergetic function and, thereby, serve as the reason for the maintenance of an active genome. Many small insertions in the Epifagus plastid genome create tandem duplications and presumably arose by slippage mispairing during DNA replication. The extensive reduction in genome size in Epifagus reflects an intensification of the same processes of length mutation that govern the amount of noncoding DNA in chloroplast genomes. Remarkably, this massive pruning occurred with a virtual absence of gene order change.

We have determined the nucleotide sequence of a 7 kb (1 kb = 10 3 base-pairs) region that includes the entire small single-copy region (SSC) of the plastid genome of Epifagus virginiana , a non-photosynthetic, parasitic flowering plant. The SSC (4.8 kb) is considerably smaller than those of photosynthetic plants due to the complete deletion of all photosynthetic, chlororespiratory and ribosomal protein genes. This leaves only two genes: a protein gene of 1738 codons whose product is unlikely to be involved in bioenergetic processes and a leucine tRNA gene ( trn L UAG ). Both genes span junctions between the inverted repeat and the SSC, with the consequence that the terminal 20 base-pairs of the repeat is transcribed in both directions and functions both as the 3′ end of the tRNA gene and as an internal segment of orf1738 . We find that the region of tobacco plastid DNA homologous to Epifagus orf1738 contains a single open reading frame (ORF) of 1901 codons rather than the three ORFs of 1244, 273 and 228 codons originally reported. However, we confirm that the equivalent region of the bryophyte Marchantia contains two genes (1068 and 464 codons) corresponding to the N and C-terminal portions of the dicot protein. In contrast, rice plastid DNA contains a severely truncated pseudogene at this locus.

Recent findings have established cracks in the straight-laced image of the plastid genome as a molecule whose sole function is photosynthesis and whose gene content is highly conserved. Genes for numerous non-photosynthetic functions have been identified. Algal plastid genomes contain many genes with no homologs in angiosperms, and the recent transfer of genes from the plastid to the nuclear genome has been described. Wholesale abandonment of genes encoding photosynthetic and gene-expression functions has occurred in the plastid genomes of a non-green plant and alga. The origins of plastid DNA, its use in phylogenetic studies, and the origins of plastid introns are also reviewed.

We used DNA sequencing and gel blot surveys to assess the integrity of the chloroplast gene infA, which codes for translation initiation factor 1, in300 diverse angiosperms. Whereas most angiosperms appear to contain an intact chloroplast infA gene, the gene has repeatedly become defunct in approximately 24 separate lineages of angiosperms, including almost all rosid species. In four species in which chloroplast infA is defunct, transferred and expressed copies of the gene were found in the nucleus, complete with putative chloroplast transit peptide sequences. The transit peptide sequences of the nuclear infA genes from soybean and Arabidopsis were shown to be functional by their ability to target green fluorescent protein to chloroplasts in vivo. Phylogenetic analysis of infA sequences and assessment of transit peptide homology indicate that the four nuclear infA genes are probably derived from four independent gene transfers from chloroplast to nuclear DNA during angiosperm evolution. Considering this and the many separate losses of infA from chloroplast DNA, the gene has probably been transferred many more times, making infA by far the most mobile chloroplast gene known in plants.

The nucleotide sequence of a 7.4 kb region containing the entire plastid ribosomal RNA operon of the nongreen parasitic plant Epifagus virginiana has been determined. Analysis of the sequence indicates that all four rRNA genes are intact and almost certainly functional. In contrast, the split genes for tRNA(Ile) and tRNA(Ala) present in the 16S-23S rRNA spacer region have become pseudogenes, and deletion upstream of the 16S rRNA gene has removed a tRNA(Val) gene and most of the promoter region for the rRNA operon. The rate of nucleotide substitution in 16S and 23S rRNAs is several times higher in Epifagus than in tobacco, a related photosynthetic plant. Possible reasons for this, including relaxed translational constraints, are discussed.

Prochlorophytes similar to Prochloron sp. and Prochlorothrix hollandica have been suggested as possible progenitors of the plastids of green algae and land plants because they are oxygenic photosynthetic prokaryotes that possess chlorophyll b. We have sequenced the P. hollandica genes encoding the photosystem II (PSII) reaction-center protein Dl (psbA) and the large and small subunits of Rubisco (rbcL and rbcS) for comparison with those of other taxa to assess the phylogenetic placement of this species. P. hollandica has two psbA genes that encode identical D1 proteins. The gene sequences predict a seven-amino-acid deletion at the carboxy terminus of the deduced D1 protein relative to all cyanobacterial sequences examined, a feature that previously had been observed only in plant psbA sequences. Phylogenetic analysis of deduced D1 protein sequences indicated that the phylogenetic position of P. hollandica is either intermediate between cyanobacteria and chloroplasts or on a branch associated with Synechococcus sp. strain Pasteur Culture Collection (PCC) 7942 (one of the strains hitherto known as Anacystis nidulans) adjacent to the chloroplast lineage. The P. hollandica rbcL and rbcS genes are arranged in an operon, as is the case in cyanobacterial and other photosynthetic bacterial genomes and in the cyanelle genome of the eukaryote Cyanophora paradoxa. Phylogenetic analysis using deduced amino-acid sequences of rbcL and rbcS indicated that P. hollandica is more closely related to cyanobacteria than to the green plastid lineage. We also examined a group of genes that are arranged differently in the genomes of chloroplasts and cyanobacteria. The psbB, psbH, petB, and petD genes, encoding components of PSII and the cytochrome b 6 /f complex, are arranged as an operon in chloroplast genomes. In cyanobacterial genomes, petB and petD form an operon, but psbB and psbH are not part of this cluster. In P. hollandica these genes are arranged as in cyanobacteria, with psbB and psbH not closely linked to each other or to the petBD operon. However, psbH and petBD are preceded by identical 93-bp elements near transcription start sites for each cistron, suggesting that the clustering of these genes in chloroplasts and the presence of putative regulatory elements in the prochlorophyte may represent two mechanisms for coordinating their expression. These studies of gene sequences and gene arrangement support previous analyses of 16S rRNA sequences which indicated that P. hollandica is a close relative of the cyanobacteria, and reveal few special similarities at the gene level among prochlorophytes and chloroplasts.

The endosymbiotic origin of chloroplasts from cyanobacteria has long been suspected and has been confirmed in recent years by many lines of evidence. Debate now is centered on whether plastids are derived from a single endosymbiotic event or from multiple events involving several photosynthetic prokaryotes and/or eukaryotes. Phylogenetic analysis was undertaken using the inferred amino acid sequences from the genes psbA, rbcL, rbcS, tufA and atpB and a published analysis (Douglas and Turner, 1991) of nucleotide sequences of small subunit (SSU) rRNA to examine the relationships among purple bacteria, cyanobacteria and the plastids of non-green algae (including rhodophytes, chromophytes, a cryptophyte and a glaucophyte), green algae, euglenoids and land plants. Relationships within and among groups are generally consistent among all the trees; for example, prochlorophytes cluster with cyanobacteria (and not with green plastids) in each of the trees and rhodophytes are ancestral to or the sister group of the chromophyte algae. One notable exception is that Euglenophytes are associated with the green plastid lineage in psbA, rbcL, rbcS and tufA trees and with the non-green plastid lineage in SSU rRNA trees. Analysis of psbA, tufA, atpB and SSU rRNA sequences suggests that only a single bacterial endosympbiotic event occurred leading to plastids in the various algal and plant lineages. In contrast, analysis of rbcL and rbcS sequences strongly suggests that plastids are polyphyletic in origin, with plastids being derived independently from both purple bacteria and cyanobacteria. A hypothesis consistent with these discordant trees is that a single bacterial endosymbiotic event occurred leading to all plastids, followed by the lateral transfer of the rbcLS operon from a purple bacterium to a rhodophyte.