A handheld NASBA analyzer for the field detection and quantification of Karenia brevis

Blooms of Karenia brevis, the red tide forming dinoflagellate in the Gulf of Mexico, cause a myriad of ecological and economic problems for coastal communities, including massive fish and mammal mortalities, and damage to tourism and fisheries/shellfish harvesting industries. There is a need for accurate detection and prediction of K. brevis blooms, including rapid and inexpensive monitoring of both water and shellfish meats to ensure the safety of shellfish harvested for human consumption. To address this issue, we have developed a protocol for easy field extraction of cellular RNA from water samples and coupled it with a handheld nucleic acid sequence-based amplification (NASBA) sensor that amplifies and detects target mRNA specific to the rbcL gene of K. brevis. This extraction protocol is a modified version of the Qiagen RNeasy Mini Kit spin protocol and requires no specialized equipment or training. Once extracted, the RNA is amplified and detected by NASBA in an in-house designed and produced handheld sensor that provides a real-time fluorescence plotting of the amplification. Both the field RNA extraction protocol and the handheld NASBA analyzer compared favorably to laboratory-based technologies. In duplicate reactions, the amplification curves generated with the handheld detector closely mirrored the curves generated with the bench top Nuclisens EasyQ NASBA analyzer and there was no difference in the sensitivity obtained using the handheld device versus the bench top models. This extraction protocol and detection sensor will be a valuable tool for rapidly monitoring K. brevis in field environments.     

Monitoring Karenia brevis blooms in the Gulf of Mexico using satellite ocean color imagery and other data

Harmful algal blooms (HABs) of Karenia brevis are a recurrent problem in the Gulf of Mexico, with nearly annual occurrences on the Florida southwest coast, and fewer occurrences on the northwest Florida and Texas coasts. Beginning in 1999, the National Oceanic and Atmospheric Administration has issued the Gulf of Mexico HAB Bulletins to support state monitoring and management efforts. These bulletins involve analysis of satellite imagery with field and meteorological station data. The effort involves several components or models: (a) monitoring the movement of an algal bloom that has previously been identified as a HAB (type 1 forecast); (b) detecting new blooms as HAB or non-HAB (type 2); (c) predicting the movement of an identified HAB (type 3); (d) predicting conditions favorable for a HAB to occur where blooms have not yet been observed (type 4). The types 1 and 2 involve methods of bloom detection requiring routine remote sensing, especially satellite ocean color imagery and in situ data. Prediction (types 3 and 4) builds on the monitoring capability by using interpretative and numerical modeling. Successful forecasts cover more than 1000 km of coast and require routine input of remotely sensed and in situ data.The data sources used in this effort include ocean color imagery from the Sea-Viewing Wide Field-of-View Sensor/OrbView-2 satellite and processed using coastal-specific algorithms, wind data from coastal and offshore buoys, field observations of bloom location and intensity provided by state agencies, and forecasts from the National Weather Service. The HAB Bulletins began in coordination with the state of Florida in autumn of 1999 and included K. brevis bloom monitoring (type 1), with limited advisories on transport (type 3) and the detection of blooms in new areas (type 2). In autumn 2000, we improved both the transport forecasts and detection capabilities and began prediction of conditions favorable for bloom development (type 4). The HAB Bulletins have had several successes. The state of Florida was advised of the potential for a bloom to occur at the end of September 2000 (type 4), and the state was alerted to the position of blooms in January 2000 and October 2001 in areas that had not been previously sampled (type 3). These successful communications of HAB activity allowed Florida agencies responsible for shellfish management and public health to respond to a rapidly developing event in a timely, efficient manner.     

The degradation of Karenia brevis toxins utilizing ozonated seawater

Florida red tides impose both an economic and health impact on the state. The purpose of this research was to examine the effectiveness of ozone to reduce the numbers of Florida red tide organism (Karenia brevis Davis) and its associated toxins in an artificial seawater environment. The results obtained in this experiment showed an approximate 1.25 log₁₀ unit reduction in the major toxin groups recovered after 10 min of ozone exposure (approximately 135 mg). In initial trials, K. brevis toxins were extracted and reintroduced into an artificial seawater (ASW) media. Subsequent experiments exposed whole cell K. brevis culture to ozone treatment. Samples from both experiments displayed approximately 1.10 log₁₀ unit reduction in total toxin and an approximate 1.25 log₁₀ unit reduction in three of the six major toxins associated with K. brevis (PxTx-1, -2, -9). The reduction in toxin concentration, as measured by high performance liquid chromatography (HPLC) analysis, displayed a positive correlation with the reduction of toxicity as determined by a fish (Cyprinodon variegatus) bioassay. Despite large total doses of ozone applied, as compared to levels that might be found at a commercial ozonation facility, some toxins were still recoverable by HPLC after ozone treatment.     

Diel vertical migration thresholds of Karenia brevis (Dinophyceae)

Light and nutrient availability change throughout dinoflagellate diel vertical migration (DVM) and/or with sub-population location in the water column along the west Florida shelf. Typically, the vertical depth of the shelf is greater than the distance a sub-population can vertically migrate during a diel cycle, limiting the ability of a sub-population to photosynthetically fix carbon toward the surface and access nutrients sub-surface. This project investigated changes of Karenia brevis (C.C. Davis) G. Hansen et Moestrup intracellular carbon, nitrogen, internal nitrate (iNO₃), free amino acid (FAA), and total lipid concentrations in high-light, nitrate-replete (960 μmol quanta m⁻² s⁻¹, 80 μM NO₃), and high-light, nitrate-reduced (960 μmol quanta m⁻² s⁻¹, <5 μM NO₃) mesocosms. The nitrate-reduced mesocosm had a slowed cell division rate when compared to the nitrate-replete mesocosm. Minimum intracellular carbon, nitrogen, iNO₃, FAA, and total lipid concentrations during the largest surface sub-population aggregations led to the conclusion that daughter cells resulting from cell division received unequal shares of the parental resources and that this inequality influenced migration behavior. Nutrient reduced daughter cells were more strongly influenced by light and phototaxis for carbon production than their replete same cell division sister cells during vertical migration thus rapidly increasing the fulfillment of constituents through photosynthesis. Vertical migration was consistent with an optimization scheme based on threshold limits through utilization or formation of photosynthate. We propose a simplified conceptual model describing how K. brevis is transported along the benthos of the west Florida shelf from off-shore to on-shore. Dynamic carbon thresholds are also suggested for future DVM modeling efforts on K. brevis populations transported between nitrogen replete and nitrogen reduced environmental conditions.     

Growth, uptake, and assimilation of ammonium, nitrate, and urea, by three strains of Karenia brevis grown under low light

Observations of near-bottom populations of Karenia brevis suggest that these cells may derive nutrients from the sediment–water interface. Cells undergoing a metabolic-mediated migration may be in close proximity to enhanced concentrations of nutrients associated with the sediment during at least a fraction of their diel cycle. In this study, the growth, uptake and assimilation rates of ammonium, nitrate, and urea by K. brevis were examined on a diel basis to better understand the potential role of these nutrients in the near-bottom ecology of this species. Three strains of K. brevis, C6, C3, and CCMP 2229, were grown under 12:12 light dark cycle under 30 μmol photons m⁻² s⁻¹ delivered to the surface plain of batch cultures. Nitrogen uptake was evaluated using ¹⁵N tracer techniques and trichloroacetic acid extraction was used to evaluate the quantity of nitrogen (N) assimilated into cell protein. Growth rates ranged from a low of 0.12 divisions day⁻¹ for C6 and C3 grown on nitrate to a high of 0.18 divisions day⁻¹ for C3 grown on urea. Diurnal maximum uptake rates, ρ_max, varied from 0.41 pmol-N cell⁻¹ h⁻¹ for CCMP 2229 grown on nitrate, to 1.29 pmol-N cell⁻¹ h⁻¹ for CCMP 2229 grown on urea. Average nocturnal uptake rates were 29% of diurnal rates for nitrate, 103% of diurnal uptake rates for ammonium and 56% of diurnal uptake rates for urea. Uptake kinetic parameters varied between substrates, between strains and between day and night measurements. Highest maximum uptake rates were found for urea for strains CCMP2229 and C3 and for ammonium for strain C6. Rates of asmilation into protein also varied day and night, but overall were highest for urea. The comparison of maximal uptake rates as well as assimilation efficiencies indicate that ammonium and urea are utilized (taken up and assimilated) more than twice was fast as nitrate on a diel basis.     

Comparative analysis of two algicidal bacteria active against the red tide dinoflagellate Karenia brevis

The red tide dinoflagellate Karenia brevis blooms annually along the eastern Gulf of Mexico, USA, and is often linked to significant economic losses through massive fish kills, shellfish harvest closures, and the potential threat to humans of neurotoxic shellfish poisonings as well as exposure to aerosolized toxin. As part of an effort to enhance the strategies employed to manage and mitigate these events and their adverse effects, several approaches are being investigated for controlling blooms. Previous studies have established the presence of algicidal bacteria lethal to K. brevis in these waters, and we aim to characterize bacterial–algal interactions, evaluate their role as natural regulators of K. brevis blooms, and ultimately assess possible management applications. Herein, the algicidal activity of a newly isolated Cytophaga/Flavobacterium/Bacteroidetes (CFB)-bacterium, strain S03, and a previously described CFB-bacterium, strain 41-DBG2, was evaluated against various harmful algal bloom (HAB) and non-HAB species (23 total), including multiple clones of K. brevis, to evaluate algal target specificity. Strains S03 and 41-DBG2, which employ direct and indirect modes of algicidal lysis, respectively, killed 20% and 40% of the bacteria-containing isolates tested. Interestingly, no bacteria-free algal cultures were resistant to algicidal attack, whereas susceptibility varied occasionally among bacteria-containing isolates of a single algal taxon originating from either the same or different geographic location. The dynamics of K. brevis culture death appeared to differ according to whether the algicidal bacterium did or did not require direct contact with algal cells, with the former most rapidly affecting K. brevis morphology and causing cell lysis. Both bacterial strains promoted the formation of a small number of cyst-like structures in the K. brevis cultures, possibly analogous to temporary cysts formed by other dinoflagellates exposed to certain types of stress. Results were also consistent with earlier work demonstrating that bacterial assemblages from certain cultures can confer resistance to attack by algicidal bacteria, again indicating the complexity and importance of microbial interactions, and the need to consider carefully the potential for using such bacteria in management activities.     

Microbial community interactions and population dynamics of an algicidal bacterium active against Karenia brevis (Dinophyceae)

The population dynamics of Cytophaga strain 41-DBG2, a bacterium algicidal to the harmful algal bloom (HAB) dinoflagellate Karenia brevis, were investigated in laboratory experiments using fluorescent in-situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Following its introduction into non-axenic K. brevis cultures at concentrations of 10³ or 10⁵ bacterial cells per milliliter, 41-DBG2 increased to 10⁶ cells per milliliter before initiation of its algicidal activity. Such threshold concentrations were not achieved when starting algal cell numbers were relatively low (10³ cells per milliliter), suggesting that the growth of this bacterium may require high levels of dissolved organic matter (DOM) excreted by the algae. It remains to be determined whether this threshold concentration is required to trigger an algicidal response by 41-DBG2 or, alternatively, is the point at which the bacterium accumulates to an effective killing concentration. The ambient microbial community associated with these algal cultures, as determined by DGGE profiles, did not change until after K. brevis cells were in the process of lysing, indicating a response to the rapid input of algal-derived organic matter. Resistance to algicidal attack exhibited by several K. brevis clones was found to result from the inhibition of 41-DBG2 growth in the presence of currently unculturable bacteria associated with those clones. These bacteria apparently prevented 41-DBG2 from reaching the threshold concentration required for initiation of algicidal activity. Remarkably, resistance and susceptibility to the algicidal activity of 41-DBG2 could be transferred between K. brevis clones with the exchange of their respective unattached bacterial communities, which included several dominant phylotypes belonging to the α-proteobacteria, γ-proteobacteria, and Cytophaga–Flavobacterium–Bacteroides (CFB) groups. We hypothesize that CFB bacteria may be successfully competing with 41-DBG2 (also a member of the CFB) for nutrients, thereby inhibiting growth of the latter and indirectly providing resistance against algicidal attack. We conclude that if algicidal bacteria play a significant role in regulating HAB dynamics, as some authors have inferred, bacterial community interactions are crucial factors that must be taken into consideration in future studies.     

Detection of Karenia brevis blooms on the west Florida shelf using in situ backscattering and fluorescence data

Using shipboard data collected from the central west Florida shelf (WFS) between 2000 and 2001, an optical classification algorithm was developed to differentiate toxic Karenia brevis blooms (>10⁴ cells l⁻¹) from other waters (including non-blooms and blooms of other phytoplankton species). The identification of K. brevis blooms is based on two criteria: (1) chlorophyll a concentration ≥1.5 mg m⁻³ and (2) chlorophyll-specific particulate backscattering at 550 nm ≤ 0.0045 m² mg⁻¹. The classification criteria yielded an overall accuracy of 99% in identifying both K. brevis blooms and other waters from 194 cruise stations. The algorithm was validated using an independent dataset collected from both the central and south WFS between 2005 and 2006. After excluding data from estuarine and post-hurricane turbid waters, an overall accuracy of 94% was achieved with 86% of all K. brevis bloom data points identified successfully. Satisfactory algorithm performance (88% overall accuracy) was also achieved when using underway chlorophyll fluorescence and backscattering data collected during a repeated alongshore transect between Tampa Bay and Florida Bay in 2005 and 2006. These results suggest that it may be possible to use presently available, commercial optical backscattering instrumentation on autonomous platforms (e.g. moorings, gliders, and AUVs) for rapid and timely detection and monitoring of K. brevis blooms on the WFS.     

A modified assay to determine hemolytic toxin variability among Karenia clones isolated from the Gulf of Mexico

Karenia brevis (formerly Gymnodinium breve) is a toxic marine dinoflagellate generally restricted to the Gulf of Mexico and is the main causative organism in fish kills, shellfish intoxications and respiratory distress in humans following bloom events. K. mikimotoi is a morphologically similar co-occurring species which is toxic in other parts of the world oceans, but has not been recognized as a major contributor in toxicity of blooms within the Gulf of Mexico. Recently there has been increasing evidence of the simultaneous production of a variety of bioactive compounds in addition to potent neurotoxins (brevetoxin) in Karenia brevis isolates. These compounds are potentially ichthyotoxic and have been shown to cause hemolysis in several bioassays [Eshbach, E., Scharsack, J., John, U., Medlin, L., 2001. Improved erythrocyte lysis assay in microtitre plates for the sensitive detection and efficient measurement of haemolytic compounds from ichthyotoxic algae. J. Appl. Toxicol. 21, 513–519; Kirkpatrick, B., Fleming, L.E., Squicciarini, D., Backer, L.C., Clark, R., Abraham, W., Benson, J., Cheng, Y.S., Johnson, D., Pierce, R., Zaias, J., Bossart, G.D., Baden, D.G., 2004. Literature review of Florida red tide: implications for human health effects. Harmful Algae 3, 99–115]. Presence of hemolytic compounds may therefore add to the overall toxicity levels of bloom events. Current monitoring methods include assays which are highly sensitive in brevetoxin detection and yet may not target other harmful compounds.By adapting protocols developed by Eshbach et al. [Eshbach, E., Scharsack, J., John, U., Medlin, L., 2001. Improved erythrocyte lysis assay in microtitre plates for the sensitive detection and efficient measurement of haemolytic compounds from ichthyotoxic algae. J. Appl. Toxicol. 21, 513–519], Red drum (Sciaenops ocellatus) erythrocytes were used to create a modified bioassay to detect hemolytic activity of crude algal extracts. Red drum was selected because it is endemic to coastal areas throughout the Gulf of Mexico and is sensitive to Karenia blooms, and thus makes this species a valid ecological target. Preliminary data has shown this method is sensitive for use in assessing hemolysis induced by laboratory cultures down to levels of 1 × 10³ cells mL⁻¹. Results showed an unexpectedly high level of hemolytic activity among K. mikimotoi clones, with one Texas strain inducing significantly higher hemolysis compared to Florida K. brevis isolates. Using this approach, future research efforts will examine the difference in production of hemolytic compounds among various Karenia clones.     

Phylogenetic relationships in the harmful dinoflagellate Cochlodinium polykrikoides (Gymnodiniales, Dinophyceae) inferred from LSU rDNA sequences

Phylogenetic relationships among chain-forming Cochlodinium species, including the harmful red tide forming dinoflagellate Cochlodinium polykrikoides, were investigated using specimens collected from coastal waters of Canada, Hong Kong, Japan, Korea, Malaysia, México, Philippines, Puerto Rico, and USA. The phylogenetic tree inferred from partial (D1–D6 regions) large subunit ribosomal RNA gene (LSU rDNA) sequences clearly differentiated between C. polykrikoides and a recently described species, Cochlodinium fulvescens. Two samples collected from the Pacific coasts of North America (British Columbia, Canada and California, USA) having typical morphological characters of C. fulvescens such as the sulcus located in the intermediate region of the cingulum, were closely related to C. fulvescens from western Japan in the phylogenetic tree. Cochlodinium polykrikoides formed a monophyletic group positioned as a sister group of the C. fulvescens clade with three well-supported sub-clades. These three clades were composed of (1) East Asian, including specimens collected from Hong Kong, western Japan, and southern Korea, (2) Philippines, from Manila Bay, Philippines and Omura Bay, Japan, and (3) American/Malaysian, from the Atlantic coasts of USA, the Pacific coast of México, Puerto Rico, and Borneo Island, Malaysia. Each of these clades is considered to be a so-called “ribotype” representing the population inhabiting each region, which is distinguished based on ribosomal RNA gene sequences in the species despite similarities in their morphological characters.     

Sensitive and rapid detection of Karenia mikimotoi (Dinophyceae) by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay was developed to detect the genomic DNA of Karenia mikimotoi using a set of four specific primers based on a ribosomal DNA internal transcribed spacer (ITS). The sensitivity of this LAMP assay was 100-fold higher than regular PCR, and its specificity was validated using other algae as a comparison. Two visual detection approaches were feasible to interpret the positive or negative results. This technology may have the potential to aid in forecasting red-tides on the scene because of its high sensitivity, specificity and rapid detection.     

Development and field application of rRNA-targeted probes for the detection of Cochlodinium polykrikoides Margalef in Korean coastal waters using whole cell and sandwich hybridization formats

The dinoflagellate, Cochlodinium polykrikoides Margalef, has been responsible for mass mortalities of both wild and farmed fish along the Korean coast on virtually an annual basis since 1982. Economic impacts to the fishing and aquaculture industries are extensive, with a loss of USD $95 million reported in 1995 alone. The use of taxon-specific molecular probes for harmful algal species is recognized as a promising approach for the early detection of bloom formation and as part of an effective mitigation strategy. We have developed and successfully applied large subunit ribosomal RNA (LSU rRNA)-targeted probes in both whole cell and sandwich hybridization assay (SHA) formats for the species-specific detection of C. polykrikoides in Korean coastal waters. Sequences of the D1–D3 variable regions used to design probes were identical between five Korean and one Hong Kong C. polykrikoides isolates, while sequences for several N. American Cochlodinium isolates differed to varying degrees from the former. The automated SHA detected C. polykrikoides at levels as low as 1–3 cells/L in the field, demonstrating its suitability for detecting the target species at pre-bloom concentrations. This method should thus prove valuable to existing monitoring programs aimed at providing aquaculture interests with an early warning of frequently devastating bloom events.     

Morphology, toxin composition and LSU rDNA phylogeny of Alexandrium minutum (Dinophyceae) from Denmark, with some morphological observations on other European strains

The morphology of Alexandrium minutum Halim from Denmark was studied and compared to the morphology of material from Portugal, Spain, France and Ireland. Strains from Denmark and the French coast of the English Channel differed from the typical minutum morphotype by the absence of a ventral pore. Cells without a pore also dominated field material from Ireland but a small fraction (6%) did have a pore. Many cells had a heavily areolated theca. In the exponential growth phase, the PSP-toxin profile of the Danish strain of A. minutum was dominated by C1 and C2 (up to 70%), whereas GTX2 and 3 made up more than 17%, and STX almost 13%. Cells entering the stationary phase contained 30% STX with a concomitant decrease of the other toxins. Partial large subunit rDNA sequences (664 bp) confirmed that the Danish A. minutum strain clusters together with other European strains of this species, and a strain from Australia. However, sequencing of this part of the gene did not resolve intraspecific relationships and could not differentiate populations with or without pore and/ or different toxin signatures. A strain from New Zealand had a remarkably high sequence divergence (up to 6%) compared to the other strains of A. minutum and its identity should be further investigated. A distribution map of A. minutum has been compiled and it is suggested that A. minutum and A. angustitabulatum Taylor are conspecific.     

Development of microsatellite markers in the marine phytoplankton Karenia mikimotoi (Dinophyceae)

The marine phytoplankton, Karenia mikimotoi, causes severe red tides which are associated with mass mortality of marine fish, and have expanded their distributions in the coastal waters of western Japan. To assess the dispersal mechanism, a population genetic study using highly polymorphic genetic markers is one of the crucial approaches. Here we developed 12 polymorphic microsatellite markers from K. mikimotoi. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from 5 to 23, and the estimate of gene diversity was from 0.551 to 0.933 across the 12 microsatellites. We consider these loci potentially useful for detailing the genetic structure and gene flow among K. mikimotoi populations.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Effect of salinity on the distribution, growth, and toxicity of Karenia spp.

The first recorded bloom of Karenia spp., resulting in brevetoxin in oysters, in the low salinity waters of the Northern Gulf of Mexico (NGOMEX) occurred in November 1996. It raised questions about the salinity tolerance of Karenia spp., previously considered unlikely to occur at salinities <24 psu, and the likelihood that the bloom would reoccur in the NGOMEX. Salinity was investigated as a factor controlling Karenia spp. abundance in the field, using data from the NGOMEX 1996 bloom and Florida coastal waters from 1954 to 2004, and growth and toxin production in cultures of Karenia brevis (Davis) G. Hansen and Moestrup. During the NGOMEX bloom, Karenia spp. occurred much more frequently at low salinities than in Florida coastal waters over the last 50 years. The data suggest that the NGOMEX bloom started on the NW Florida Shelf, an area with a higher frequency of Karenia spp. at low salinities than the rest of Florida, and was transported by an unusual westward surface current caused by Tropical Storm Josephine. The minimum salinity at which growth occurred in culture ranged between 17.5 and 20 psu, but the optimal salinity ranged between low values of 20 or 25 and high values of 37.5–45 psu, depending on the clone. The effect of salinity on toxin production in one clone of K. brevis was complex, but at all salinities brevetoxin levels were highest during the stationary growth phase, suggesting that aging, high density blooms may pose the greatest public health threat. The results demonstrate that Karenia spp. can be a public health threat in low salinity areas, but the risk in the NGOMEX is relatively low. No bloom has occurred since the 1996 event, which was probably associated with a special set of conditions: a bloom along the Florida Panhandle and a tropical storm with a track that set up a westward current.     

Phylogenetic relationships among species and genera of Lycoperdaceae based on ITS and LSU sequence data from north European taxa

Phylogenetic relationships, limits of species, and genera within Lycoperdaceae, were inferred by use of ITS and LSU nu-rDNA sequence data. Lycoperdaceae was confirmed as monophyletic, and Mycenastrum corium as a sister taxon to the ingroup. Four major clades were identified and received weak to moderate support and correspond with the genera Lycoperdon, Bovista, Calvatia, and Disciseda. The Lycoperdon clade includes species from Lycoperdon, Vascellum, Morganella, Handkea, Bovistella, and Calvatia. The structure within the Lycoperdon clade is unresolved and several clades are more or less unsupported, which suggests treating the supported Lycoperdon clade as the genus Lycoperdon. L. nigrescens and L. caudatum occur on single branches and their phylogenetic positions could not be resolved. The phylogenetic analyses identified 31 species of Lycoperdon, 11 species of Bovista, six species of Calvatia, and two species of Disciseda. In Lycoperdon three new species were recognized. A new species closely related to B. limosa is identified and discussed. A classification of Lycoperdaceae is proposed based on the results of the phylogenetic analyses. Morphological characters of species within and among identified clades are discussed.