The evolution of the duckweed ionome mirrors losses in structural complexity.

BACKGROUND AND AIMS: The duckweeds (Lemnaceae) consist of 36 species exhibiting impressive phenotypic variation, including the progressive evolutionary loss of a fundamental plant organ, the root. Loss of roots and reduction of vascular tissues in recently derived taxa occur in concert with genome expansions of ≤14-fold. Given the paired loss of roots and reduction in structural complexity in derived taxa, we focus on the evolution of the ionome (whole-plant elemental contents) in the context of these fundamental changes in body plan. We expect that progressive vestigiality and eventual loss of roots might have both adaptive and maladaptive consequences that are hitherto unknown. METHODS: We quantified the ionomes of 34 accessions in 21 species across all duckweed genera, spanning 70 Myr in this rapidly cycling plant (doubling times are as rapid as ~24 h). We related both micro- and macroevolutionary ionome contrasts to body plan remodelling and showed nimble microevolutionary shifts in elemental accumulation and exclusion in novel accessions. KEY RESULTS: We observed a robust directional trend in calcium and magnesium levels, decreasing from the ancestral representative Spirodela genus towards the derived rootless Wolffia, with the latter also accumulating cadmium. We also identified abundant within-species variation and hyperaccumulators of specific elements, with this extensive variation at the fine (as opposed to broad) scale. CONCLUSIONS: These data underscore the impact of root loss and reveal the very fine scale of microevolutionary variation in hyperaccumulation and exclusion of a wide range of elements. Broadly, they might point to trade-offs not well recognized in ionomes.

Two lineages of Lemna aequinoctialis (Araceae, Lemnoideae) based on physiology, morphology, and phylogeny.

Lemna aequinoctialis Welw. is a widely spread species that has diverse physiological and molecular properties. Flower characteristics are important factors in deducing taxonomical status; however, owing to the rarity of flowering observations in Lemna, studying them has been a prolonged challenge. In this study, physiological and morphological analyses were conducted by inducing flowering, and molecular analysis was done based on the two chloroplast DNA loci (matK, atpF-atpH intergeneric spacer) of L. aequinoctialis sensu Landolt (1986) from 70 strains found in 70 localities in Japan, Korea, Thailand, and the US. In total, 752 flowering fronds from 13 strains were observed based on axenic conditions. Two different trends in flower organ development-protogyny and adichogamy-were detected in these strains. Their physiological traits were divided into two groups, showing different morphological features based on frond thickness, root cap, and anther sizes. Molecular analysis showed two lineages corresponding to two physiological groups. These were identified as L. aequinoctialis sensu Beppu et al. (1985) and L. aoukikusa Beppu et Murata based on the description of the nomenclature of L. aoukikusa. These were concluded as independent taxa and can be treated as different species. Furthermore, the distribution of L. aoukikusa is not only limited to Japan.

Plant evolution and environmental adaptation unveiled by long-read whole-genome sequencing of Spirodela.

Aquatic plants have to adapt to the environments distinct from where land plants grow. A critical aspect of adaptation is the dynamics of sequence repeats, not resolved in older sequencing platforms due to incomplete and fragmented genome assemblies from short reads. Therefore, we used PacBio long-read sequencing of the Spirodela polyrhiza genome, reaching a 44-fold increase of contiguity with an N50 (a median of contig lengths) of 831 kb and filling 95.4% of gaps left from the previous version. Reconstruction of repeat regions indicates that sequentially nested long terminal repeat (LTR) retrotranspositions occur early in monocot evolution, featured with both prokaryote-like gene-rich regions and eukaryotic repeat islands. Protein-coding genes are reduced to 18,708 gene models supported by 492,435 high-quality full-length PacBio complementary DNA (cDNA) sequences. Different from land plants, the primitive architecture of Spirodela's adventitious roots and lack of lateral roots and root hairs are consistent with dispensable functions of nutrient absorption. Disease-resistant genes encoding antimicrobial peptides and dirigent proteins are expanded by tandem duplications. Remarkably, disease-resistant genes are not only amplified, but also highly expressed, consistent with low levels of 24-nucleotide (nt) small interfering RNA (siRNA) that silence the immune system of land plants, thereby protecting Spirodela against a wide spectrum of pathogens and pests. The long-read sequence information not only sheds light on plant evolution and adaptation to the environment, but also facilitates applications in bioenergy and phytoremediation.

Toxic effects of Pb on Spirodela polyrhiza (L.): Subcellular distribution, chemical forms, morphological and physiological disorders.

The impact of lead (Pb) on Spirodela polyrhiza was studied to determine the subcellular distribution, chemical forms, and resulting morphophysiological modifications after treatments with 20 or 80 µM Pb(NO3)2 for 10 days. At the subcellular level, the Pb uptake by S. polyrhiza was mainly compartmentalized in the cell walls (70%), and the majority of Pb (approximately 70%) was extracted using 1 M NaCl and 2% acetic acid (HAc). Visual symptoms of phytotoxcity, surface roughness and closure of stomata, were observed in Pb-treated fronds. Electron-dense precipitates were present in cell walls, and changes to the ultrastructure were most noticeably exhibited in organelle shape, internal organization, and size of the plastoglobules of chloroplasts. Toxic concentrations of Pb induced oxidative stress in fronds, characterized by an accumulation of malondialdehyde (MDA) and decreased chlorophyll and unsaturated fatty acid contents. Pb exposure increased ABS/RC, TRo/RC, DIo/RC, Vj, and φDo (Fv/Fm), indicating that reaction centers were transformed to dissipation sinks, leading to a decrease in the efficiency of photosystem II, which was evident from the decreased values of Fv/Fo, Fv/Fm, ψEo, φEo, RC/ABS, and PIabs. These results indicated that decreased photosynthesis in Pb-treated fronds was partially ascribed to the lower pigment content, inhibition of electron transport, inactivation of the reaction centers, damage to the chloroplast ultrastructure, and stomatal closure. The physiological implications of subcellular distribution and chemical forms are discussed in relation to Pb accumulation and detoxification. However, Pb accumulation significantly impaired photosynthesis and membrane integrity in the fronds of S. polyrhiza.

Morphological and structural characterization of the attachment system in aerial roots of Syngonium podophyllum.

MAIN CONCLUSION: The attachment of aerial roots of Syngonium podophyllum involves a multi-step process adjusted by multi-scale structures. Helical-crack root hairs are first found in the attachment system, representing specialized structures for surface anchorage. The morphological variability of attachment organs reflects diverse climbing strategies. One such anchoring mode in clinging-climbers involves the time-dependent interaction between roots and the support: By naturally occurring adhesive roots with root hairs, the plant can ascend on supports of any shape and size. As a typical root-climber, Syngonium podophyllum develops elongate aerial roots at nodes. Here, we studied its attachment behavior from the external morphology to the internal structure in detail. Through SEM and LM observation on several root-substrate interfaces, we suggested that the attachment of aerial roots was mediated by a multi-step process, in which root hairs played significant roles in releasing mucilaginous substance and securing the durable anchorage. We summarized all the types of shape changes of root hairs with particular focus on the abnormal transition from a tube to a helical-crack ribbon. We demonstrated our understanding with respect to the formation of the helical-crack root hairs, based on the structural evidence of cellulose microfibrils orientation on the cell wall lamellae. The helical-crack root hairs serving as energy-dissipating units retard the failure of adhesion under high winds and loads.

A multigenerational effect of parental age on offspring size but not fitness in common duckweed (Lemna minor).

Classic theories on the evolution of senescence make the simplifying assumption that all offspring are of equal quality, so that demographic senescence only manifests through declining rates of survival or fecundity. However, there is now evidence that, in addition to declining rates of survival and fecundity, many organisms are subject to age-related declines in the quality of offspring produced (i.e. parental age effects). Recent modelling approaches allow for the incorporation of parental age effects into classic demographic analyses, assuming that such effects are limited to a single generation. Does this 'single-generation' assumption hold? To find out, we conducted a laboratory study with the aquatic plant Lemna minor, a species for which parental age effects have been demonstrated previously. We compared the size and fitness of 423 laboratory-cultured plants (asexually derived ramets) representing various birth orders, and ancestral 'birth-order genealogies'. We found that offspring size and fitness both declined with increasing 'immediate' birth order (i.e. birth order with respect to the immediate parent), but only offspring size was affected by ancestral birth order. Thus, the assumption that parental age effects on offspring fitness are limited to a single generation does in fact hold for L. minor. This result will guide theorists aiming to refine and generalize modelling approaches that incorporate parental age effects into evolutionary theory on senescence.

Molecular characterization of Anthurium genotypes by using DNA fingerprinting and SPAR markers.

We characterized single primer amplification reaction (SPAR) molecular markers from 20 genotypes of Anthurium andraeanum Lind., including 3 from commercial varieties and 17 from 2 communities in the State of Espírito Santo, Brazil. Twenty-four SPAR, consisting of 7 random amplified polymorphic DNA and 17 inter-simple sequence repeat markers were used to estimate the genetic diversity of 20 Anthurium accessions. The set of SPAR markers generated 288 bands and showed an average polymorphism percentage of 93.39%, ranging from 71.43 to 100%. The polymorphism information content (PIC) of the random amplified polymorphic DNA primers averaged 0.364 and ranged from 0.258 to 0.490. Primer OPF 06 showed the lowest PIC, while OPAM 14 was the highest. The average PIC of the inter-simple sequence repeat primers was 0.299, with values ranging from 0.196 to 0.401. Primer UBC 845 had the lowest PIC (0.196), while primer UCB 810 had the highest (0.401). By using the complement of Jaccard's similarity index and unweighted pair group method with arithmetic mean clustering, 5 clusters were formed with a cophenetic correlation coefficient of 0.8093, indicating an acceptable clustering consistency. However, no genotype clustering patterns agreed with the morphological data. The Anthurium genotypes investigated in this study are a germplasm source for conservational research and may be used in improvement programs for this species.

Morphological diversity of the velamen radicum in the genus Anthurium (Araceae).

Epiphytes develop anatomical features to improve efficiency of the uptake of water and nutrients, such as absorptive foliar scales or a velamen radicum. Despite substantial studies on the occurrence, morphology, development and phylogeny of the velamen, most of the available literature is focused on Orchidaceae, making current knowledge on velamen clearly biased. A recent publication firmly established that velamina are common in Anthurium species. Thus, this study provides further insights by describing velamen morphological characteristics of Anthurium species and classifying them into different velamen types. Furthermore, we investigate if the different velamen morphological traits are clade-specific and phylogenetically conserved within the genus. Using SEM, we performed a morphological study on 89 Anthurium species, describing six micromorphological traits of velamen and exodermis, following traits used to classify Orchidaceae velamen by Porembski & Barthlott (1988). We distinguished nine velamen types, including two that are unique to Anthurium and not similar to any type found in Orchidaceae. Comparing velamen morphology within the phylogenetic tree of Anthurium revealed clear phylogenetic signals. This study provides detailed morphological descriptions among 89 species of Anthurium from the Araceae, and substantially broadens our knowledge of this tissue. However, velamen function has been even less studied, with hardly anything known about functional significance of having secondary cell wall thickening and perforations on velamen cell walls. Therefore, a logical next step would be to connect these anatomical features to their functions.

How did the swiss cheese plant get its holes?

Adult leaf fenestration in "Swiss cheese" plants (Monstera Adans.) is an unusual leaf shape trait lacking a convincing evolutionary explanation. Monstera are secondary hemiepiphytes that inhabit the understory of tropical rainforests, where photosynthesis from sunflecks often makes up a large proportion of daily carbon assimilation. Here I present a simple model of leaf-level photosynthesis and whole-plant canopy dynamics in a stochastic light environment. The model demonstrates that leaf fenestration can reduce the variance in plant growth and thereby increase geometric mean fitness. This growth-variance hypothesis also suggests explanations for conspicuous ontogenetic changes in leaf morphology (heteroblasty) in Monstera, as well as the absence of leaf fenestration in co-occurring juvenile tree species. The model provides a testable hypothesis of the adaptive significance of a unique leaf shape and illustrates how variance in growth rate could be an important factor shaping plant morphology and physiology.

Reconstructing the origin and elaboration of insect-trapping inflorescences in the Araceae.

PREMISE OF THE STUDY: Floral traps are among the most sophisticated devices that have evolved in angiosperms in the context of pollination, but the evolution of trap pollination has not yet been studied in a phylogenetic context. We aim to determine the evolutionary history of morphological traits that facilitate trap pollination and to elucidate the impact of pollinators on the evolution of inflorescence traps in the family Araceae. • METHODS: Inflorescence morphology was investigated to determine the presence of trapping devices and to classify functional types of traps. We inferred phylogenetic relationships in the family using maximum likelihood and Bayesian methods. Character evolution of trapping devices, trap types, and pollinator types was then assessed with maximum parsimony and Bayesian methods. We also tested for an association of trap pollination with specific pollinator types. • KEY RESULTS: Inflorescence traps have evolved independently at least 10 times within the Araceae. Trapping devices were found in 27 genera. On the basis of different combinations of trapping devices, six functional types of traps were identified. Trap pollination in Araceae is correlated with pollination by flies. • CONCLUSIONS: Trap pollination in the Araceae is more common than was previously thought. Preadaptations such as papillate cells or elongated sterile flowers facilitated the evolution of inflorescence traps. In some clades, imperfect traps served as a precursor for the evolution of more elaborate traps. Traps that evolved in association with fly pollination were most probably derived from mutualistic ancestors, offering a brood-site to their pollinators.

Relationships within the Araceae: comparison of morphological patterns with molecular phylogenies.

PREMISE OF THE STUDY: The first family-wide molecular phylogeny of the Araceae, a family of about 3800 published species in 120 genera, became available in 1995, followed by a cladistic analysis of morpho-anatomical data in 1997. The most recent and comprehensive family-wide molecular phylogeny was published in 2008 and included species from 102 genera. We reanalyzed the molecular data with a more complete genus sampling and compared the resulting phylogeny with morphological and anatomical data, with a view to contributing to a new formal classification of the Araceae. METHODS: We analyzed 113 aroid genera and 4494 aligned nucleotides that resulted from adding 11 genera to the 2008 molecular matrix. We also analyzed 81 morphological characters in the context of the molecular phylogeny, using an extended version of the 1997 morpho-anatomical data set. KEY RESULTS: The resulting maximum-likelihood phylogeny is well resolved and supported, and most of the 44 larger clades also have morphological or anatomical synapomorphies as well as ecological or geographic cohesion. Of the 44 clades, 16 are here newly circumscribed and informally named. However, some relationships remain poorly supported within the Aroideae subfamily. The most problematic placement is Calla within Aroideae, which conflicts with the distribution of morphological, anatomical, and palynological character states. CONCLUSIONS: The comparison of the molecular analysis with morphological and anatomical data presented here represents an important basis for a new formal classification for the Araceae and for the understanding of the evolution of this ancient family, a monocot group known in the fossil record from the early Cretaceous.

Genome of the world's smallest flowering plant, Wolffia australiana, helps explain its specialized physiology and unique morphology.

Watermeal, Wolffia australiana, is the smallest known flowering monocot and is rich in protein. Despite its great potential as a biotech crop, basic research on Wolffia is in its infancy. Here, we generated the reference genome of a species of watermeal, W. australiana, and identified the genome-wide features that may contribute to its atypical anatomy and physiology, including the absence of roots, adaxial stomata development, and anaerobic life as a turion. In addition, we found evidence of extensive genome rearrangements that may underpin the specialized aquatic lifestyle of watermeal. Analysis of the gene inventory of this intriguing species helps explain the distinct characteristics of W. australiana and its unique evolutionary trajectory.

Distinct responses of frond and root to increasing nutrient availability in a floating clonal plant.

Current knowledge on responses of aquatic clonal plants to resource availability is largely based on studies manipulating limited resource levels, which may have failed to capture the "big picture" for aquatic clonal plants in response to resource availability. In a greenhouse experiment, we grew the floating clonal plant Spirodela polyrhiza under ten nutrient levels (i.e., 1/64×, 1/32×, 1/16×, 1/8×, 1/4×, 1/2×, 1×, 2×, 4× and 8×full-strength Hoagland solution) and examined their responses in terms of clonal growth, morphology and biomass allocations. The responses of total biomass and number of ramets to nutrient availability were unimodal. A similar pattern was found for frond mass, frond length and frond width, even though area per frond and specific frond area fluctuated greatly in response to nutrient availability. In contrast, the responses of root mass and root length to nutrient availability were U-shaped. Moreover, S. polyrhiza invested more to roots under lower nutrient concentrations. These results suggest that nutrient availability may have distinct influences on roots and fronds of the aquatic clonal plant S. polyrhiza, resulting in a great influence on the whole S. polyrhiza population.

Response of Pistia stratiotes to heavy metals (Cr, Ni, and Zn) and phosphorous.

The effects of Cr, Ni, Zn, and P exposure on the root anatomic structure, growth, and chlorophyll a concentration of Pistia stratiotes L. were studied. Plastic aquaria containing 50 g of wet plants and 5 L of pond water added with the contaminant(s) were disposed. The treatments were: (1) Cr, (2) Ni, (3) Zn, (4) P, (5) Cr + Ni + Zn, (6) Cr + Ni + Zn + P, and (7) control. Contaminant additions were done seven times. In each addition, concentrations of 1 mg of metals or 5 mg of P per liter of water were added. Chlorophyll a was an indicator more sensitive to Zn and Cr toxicity than the relative growth rate. Ni and Cr + Ni + Zn treatments were the most toxic ones, in which biomass and the root anatomical parameters (root length, cross-sectional areas [CSAs] of root, stele, and metaxylem vessels) decreased significantly. The addition of P to the treatment with combined metals attenuated the decrease in plant growth and root length, and caused a significant increase in CSAs of total metaxylem vessels, suggesting that P increased the tolerance of P. stratiotes to metals. This fact has important implications for the use of this macrophyte in constructed wetlands for industrial wastewater treatment.

[Comparative identification on Dai medicine "Pokou" and its processing product].

OBJECTIVE: To research identification methods of the Dai Medicine "Pokou" (the rhizome of Homalomena gigantea) and its processing product, and provide basis for identification of the drug in further research and application. METHODS: Macroscopic, microscopic observation and TLC and FTIR techniques were used to authenticate this raw medicine and its processing product. RESULTS: There were certain differences in the macroscopic features. The TLC result and infrared spectra of the samples had also obvious differences. The methods for identification of this raw medicine and its processing product were established, The detailed tissue and powder of this medicine were drawn. CONCLUSION: The results provided the basis for identification of the medicine and establishment of its quality standard.

Influence of nitrogen and phosphorus concentrations and ratios on Lemna gibba growth responses to triclosan in laboratory and stream mesocosm experiments.

The effects of co-occurring nutrient and contaminant stressors are very likely to interact in aquatic systems, particularly at the level of primary producers. Site-specific nitrogen (N) and phosphorus (P) concentrations are often much lower and differ in relative availability than those used in nutrient-saturated laboratory assays for aquatic plants, which can introduce uncertainty in prospective ecological hazard and risk assessments. Because triclosan, an antimicrobial agent included in personal care products, potentially presents high relative risk among antimicrobial agents to aquatic plants and algae, we performed laboratory experiments with the model aquatic macrophyte Lemna gibba across a gradient of environmentally relevant N:P levels with and without triclosan co-exposure. Frond numbers (7 d) were significantly higher in N:P treatments of 16 and 23 but were lower in N:P of 937 and 2,500 treatments relative to standardized control media (N:P=3). When triclosan co-exposure occurred at high nutrient concentrations, frond number median effective concentration values at N:P 0.75, 3, and 16 were more than twofold lower than triclosan median effective concentration values in low nutrient media N:P ratios. However, a triclosan median effective concentration for frond number was twofold lower at N:P of 2,500 than at other N:P ratios in low concentration media. Influences of P enrichment on triclosan toxicity to L. gibba were further explored during a 14-d outdoor experimental stream mesocosm study. Effects of 2.6 and 20.8 microg L(-1) triclosan on L. gibba growth rates were more pronounced with increasing P treatment levels, which was generally consistent with our laboratory observations. Findings from these laboratory and field studies indicate that site-specific nutrient concentrations and ratios should be considered during assessments of primary producer responses to chemical stressors.

Palyno-anatomical studies of monocot taxa and its taxonomic implications using light and scanning electron microscopy.

Palyno-anatomical study of monocots taxa using Light and Scanning Electron Microscopy (SEM) was first time conducted with a view to evaluating their taxonomic significance. Studied plants were collected from different eco-climatic zones of Pakistan ranges from tropical, sub-tropical, and moist habitats. The aim of this study is to use palyno-anatomical features for the correct identification, systematic comparison, and investigation to elucidate the taxonomic significance of these features, which are useful to taxonomists for identifying monocot taxa. A signification variation was observed in quantitative and qualitative characters by using the standard protocol of light microscopy (LM) and SEM. Epidermal cell length varied from maximum in Allium griffthianum (480 ± 35.9) µm at the adaxial surface to minimum in Canna indica (33.6 ± 8.53) µm on abaxial surface. Maximum exine thickness was observed in Canna indica (4.46) µm and minimum in Allium grifthianum (0.8) µm. Variation was observed in shape and exine ornamentation of the pollen, shape of the epidermal cell, number, size, and type of stomata, guard cell shape, and anticlinal wall pattern. Based on these palyno-anatomical features a taxonomic key was developed, which help in the discrimination of studied taxa. In conclusion, LM and SEM pollen and epidermal morphology is explanatory, significant, and can be of special interest for the plant taxonomist in the correct identification of monocots taxa.

Near-infrared fluorescence imaging in the largely unexplored window of 900-1,000 nm.

Near-infrared (NIR) fluorescence imaging has relied on fluorophores that emit in the 700-900 nm NIR-Ia or 1,000-1,700 nm NIR-II window for generating deep-tissue images. Up until now, there have been few fluorophores developed for the 900-1,000 nm NIR-Ib window. This is largely because NIR-Ib light is thought to be strongly absorbed by water. Methods: Here we found that six heptamethine dyes had distinct emission peaks in both the NIR-Ia and NIR-Ib window. We tested the performance of these contrast agents by introducing them into the leaves of the common house plant Epipremnum aureum with early stage anthracnose leaf infections from Khaya senegalensis, as well as injecting them into the hind feet of nude mice and tails of tumour-bearing mice in vivo. Results: Heptamethine dyes yielded superior images of leaf venation, anthracnose infection locations, sentinel lymph nodes, brain tumours and subcutaneous tumours in the NIR-Ib window. We found that NIR-Ib images had markedly enhanced signal-to-background ratio because autofluorescence, scattering and light absorption by biological tissues and water were weaker at longer wavelengths. Conclusion: NIR-Ib fluorescence imaging was a powerful method for studying sentinel lymph nodes, tumours, leaf veins and early anthracnose infection locations in plant leaves. The findings challenge our current view of NIR fluorescence imaging and may have important implications for biomedical research and image-guided cancer surgery.

Comparative profiling of microRNAs and their effects on abiotic stress in wild-type and dark green leaf color mutant plants of Anthurium andraeanum 'Sonate'.

MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in the regulation of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data, including Anthurium andraeanum. To identify miRNAs and their target genes in A. andraeanum and study their responses to abiotic stresses, we conducted deep-sequencing of two small RNA (sRNA) libraries prepared from young leaves of wild-type (WT) and dark green (dg) leaf color mutant plants of A. andraeanum 'Sonate'. A total of 53 novel miRNAs were identified, 32 of which have been annotated to 18 miRNA families. 10 putative miRNAs were found to be differentially expressed in WT and dg, among which two miRNAs were significantly up-regulated and eight down-regulated in dg relative to WT. One differentially expressed miRNA, Aa-miR408, was dramatically up-regulated in dg. qRT-PCR analysis and heterologous expression of Aa-miR408 in Arabidopsis under different stress treatments suggest that Aa-miR408 is involved in abiotic stress responses in A. andraeanum. Our results provide a foundation for further dissecting the roles of miRNAs and their targets in regulating abiotic stress tolerance in A. andraeanum.

Floral diversity and pollination strategies of three rheophytic Schismatoglottideae (Araceae).

Homoplastic evolution of 'unique' morphological characteristics in the Schismatoglottideae - many previously used to define genera - prompted this study to compare morphology and function in connection with pollination biology for Aridarum nicolsonii, Phymatarum borneense and Schottarum sarikeense. Aridarum nicolsonii and P. borneense extrude pollen through a pair of horned thecae while S. sarikeense sheds pollen through a pair of pores on the thecae. Floral traits of spathe constriction, presence and movement of sterile structures on the spadix, the comparable role of horned thecae and thecae pores, the presence of stamen-associated calcium oxalate packages, and the timing of odour emission are discussed in the context of their roles in pollinator management. Pollinators for all investigated species were determined to be species of Colocasiomyia (Diptera: Drosophilidae).