Ice nucleation and its e ff ect on the atmospheric transport of fungal spores from the classes Agaricomycetes , Ustilaginomycetes , and Eurotiomycetes

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Tables Figures these spores is Ustilaginomycetes > Agaricomycetes Eurotiomycetes.We show that at temperatures below −20 • C, all of the fungal spores studied here are less efficient ice nuclei compared to Asian mineral dust on a per surface area basis.We used our new freezing results together with data in the literature to compare the freezing temperatures of spores from the phyla Basidiomycota and Ascomycota, which together make up 98 % of known fungal species found on Earth.The data show that within both phyla (Ascomycota and Basidiomycota) there is a wide range of freezing properties, and also that the variation within a phylum is greater than the variation between the average freezing properties of the phyla.Using a global chemistry-climate transport model, we investigated whether ice nucleation on the studied spores, followed by precipitation, can influence the atmospheric transport and global distributions of these spores in the atmosphere.Simulations show that inclusion of ice nucleation scavenging of these fungal spores in mixed-phase clouds can decrease the annual mean concen-
Ice nucleation on fungal spores may be important because ice nucleation on these spores could influence the frequency and properties of ice and mixed-phase clouds in the atmosphere.On a global annual scale, ice nucleation on fungal spores may be less important than ice nucleation on mineral dust (Hoose et al., 2010;Sesartic et al., 2013), but ice nucleation on fungal spores could still influence the frequency and properties of ice and mixed-phase clouds regionally and seasonally (Delort et al., 2010;DeMott and Prenni, 2010;Möhler et al., 2007;Morris et al., 2004Morris et al., , 2011;;Pöschl et al., 2010;Sands et al., 1982;Phillips et al., 2009;Creamean et al., 2013;Spracklen and Heald, 2013).As examples, numerous field studies have illustrated that at certain times, locations and conditions, a fraction of the total ice nuclei population may contain biological material (Pratt et al., 2009;Prenni et al., 2009;Garcia et al., 2012;Huffman et al., 2013;Tobo et al., 2013;Hader et al., 2013).Ice nucleation on fungal spores may also be important because ice nucleation on fungal spores, followed by precipitation, may be an impor-Figures

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Full tant removal mechanism of these spores from the atmosphere (Morris et al., 2013a).
The main mechanism for the spread of fungi is by wind-dissemination of spores (Webster and Weber, 2007), and accurate models to predict the spread of fungal spores are needed.In the past researchers have predicted the spread of fungal spores using various models (Andrade et al., 2009;Aylor, 1986Aylor, , 2003;;Fröhlich-Nowoisky et al., 2012;Isard et al., 2005;Kim and Beresford, 2008;Magarey et al., 2007;Pan et al., 2006;Pfender et al., 2006;Skelsey et al., 2008;Wang et al., 2010;Wilkinson et al., 2012), but ice nucleation followed by precipitation was not included in these models as a sink of the spores from the atmosphere.Even though ice nucleation on fungal spores may be important, very little is known about this topic.
Fungi can be classified into 35 different classes (Hibbett et al., 2007).So far, the ice nucleation properties of spores from the following classes have been investigated: Dothideomycetes, Exobasidiomycetes, Eurotiomycetes, Pucciniomycetes, Sordariomycetes (Haga et al., 2013;Morris et al., 2013b;Iannone et al., 2011;Jayaweera and Flanagan, 1982;Pouleur et al., 1992;Pummer et al., 2013).To add to the limited amount of data on the ice nucleation properties of fungal spores, we studied the ice nucleation properties of 12 different species of fungal spores chosen from three classes: Agaricomycetes, Ustilaginomycetes, and Eurotiomycetes.The species and classes of fungal spores investigated are listed in Table 1.We focused on these classes since they are thought to be abundant in the atmosphere.Recently, Pummer et al. (2013) studied a few of the same species as were studied here (Agaricus bisporus and Aspergillus niger ).Our studies are complimentary to the studies by Pummer et al. ( 2013) since we measured the cumulative number of IN per spore as a function of temperature, as well as the ice nucleation active surface site density as a function of temperature, while Pummer et al. (2013) reported the temperature at which 50 % of all of the droplets froze using an average spore mass concentration of about 20 mg mL −1 .
Agaricomycetes is a class of fungi that includes many types of mushroom species and are cosmopolitan (Webster and Weber, 2007).This class includes Agaricus bisporus, the common or button mushroom.Spores from this class have been found to Introduction

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Ustilagomycycetes are agricultural pathogens that belong to the group of smut fungi, causing widespread damage to crops and cereals (Webster and Weber, 2007).Hence, understanding the transport of these spores in the atmosphere (which may involve ice nucleation) is of interest from an economic perspective.
Eurotiomycetes are found on all types of decaying material and are one of the most ubiquitous types of fungi (Webster and Weber, 2007).Eurotiomycetes have also been identified as important human allergens (see, for example: Horner et al. (1995) and references therein).The specific types of spores from the class Eurotiomycetes that were studied here are from the Penicillium and Aspergillus genera.These spores are frequently present in surface air (Pyrri andKapsanaki-Gotsi, 2012, 2007;Goncalves et al., 2010;Mallo et al., 2011;Shelton et al., 2002;Fröhlich-Nowoisky et al., 2009), Introduction

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In addition to studying the ice nucleation properties of spores from the classes Agaricomycetes, Ustilaginomycetes, and Eurotiomycetes, we also investigated whether ice nucleation on these spores, followed by precipitation, can influence the transport and removal of these spores in the atmosphere.We did this using a global chemistryclimate transport model and by comparing simulations with and without ice nucleation included in the model.These simulations suggest that ice nucleation on the spores studied may be important for the transport of these spores to remote regions (such as the marine boundary layer, polar regions, and the upper troposphere).
Fungal spores from the phyla Basidiomycota and Ascomycota make up approximately 98 % of known fungal species found on Earth (James et al., 2006).Recent field measurements observed higher ratios of Ascomycota to Basidiomycota in remote marine regions compared to continental regions (Fröhlich-Nowoisky et al., 2012).One possible explanation for this was that Basidiomycota spores are more efficient ice nuclei compared to Ascomycota spores, resulting in Basidiomycota spores being more efficiently removed from the atmosphere by precipitation from ice and mixed-phase clouds (Fröhlich-Nowoisky et al., 2012).We also use our new freezing data, together Introduction

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Full with freezing data from the literature to assess if fungal spores from the phylum Basidiomycota are better ice nuclei than fungal spores from the phylum Ascomycota.

Spore samples and slide preparation
Agaricus bisporus, the common button mushroom, was purchased from a local grocery store in Vancouver, British Columbia (B.C.), Canada.The remaining Agaricomycetes fungi (Amanita muscaria, Boletus zelleri, Lepista nuda, Trichaptum abietinum) were harvested from the Pacific Spirit Regional Park surrounding the University of British Columbia (UBC) campus in Vancouver, B.C. To prepare slides containing Agaricomycetes spores, sections of the fruiting body were placed on a wire mesh in a sealed and humidified chamber.Hydrophobic glass slides were placed underneath the wire mesh, exposed to the part of the fungus that releases spores under humidified conditions.Over time, spores were naturally released and deposited onto the glass slides.
Ustilaginomycetes spores (Ustilago nuda, Ustilago nigra, Ustilago avenae) were acquired from the Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, Manitoba, Canada.Slides containing Ustilaginomycetes spores were prepared analogously to the method used by Haga et al. (2013) for rust (Pucciniomycetes) spores.Briefly, a sealed glass vessel containing spores and a small fan were immersed in a sonicator bath.The combination of the fan and the vibrations from the sonicator bath generated a spore cloud within the vessel and resulted in spores being deposited onto glass slides that were suspended on a wire mesh within the vessel.

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Full In short, the fungi were grown in petri dishes and approximately 10 3 glass beads (0.5 mm in diameter) (BioSpec Products Inc.) were rolled over the surface of the fungal culture, which resulted in the spores being dislodged from the culture and attached to the beads.The beads and spores were then transferred to a sample vial containing ultrapure water.This resulted in a suspension of spores, while the glass beads settled to the bottom of the sample vial.The resulting spore suspension was sprayed onto hydrophobic glass slides using a Meinhard nebulizer (model TR-30-A1).Pencillium spores were aerosolized from fungal cultures and deposited on a hydrophobic glass slide using a custom-built flow cell recently developed in our laboratory and previously used for depositing Cladosporium spores on hydrophobic surfaces (Iannone et al., 2011).

Freezing experiments
The instrument used to study the immersion freezing properties of the fungal spores consisted of an optical microscope (Zeiss Axiolab A with a 10 × or 50 × objective) coupled with a flow cell that had temperature and relative humidity control.This apparatus or a similar apparatus has been used in many previous ice nucleation studies (Iannone et al., 2011;Dymarska et al., 2006;Koop et al., 1998;Chernoff and Bertram, 2010;Wheeler and Bertram, 2012), and will only be described briefly here.A hydrophobic glass cover slide is used to support the fungal spores inside the flow cell.

Spore properties and number of spores per drop
Using microscope images, we calculated the average projected 2-D surface area per spore and the spore dimensions (length and width) for each species.From this information and the 2-D surface area of spores per drop (Sect.2.2), we determined the number of spores per drop.
Scanning electron microscopy (SEM) images, acquired at the UBC Bioimaging Facility, were used to determine the surface features of the spores.Images for A. muscaria, B. zelleri, L. nuda, T. abietinum, A. brasiliensis, A. niger, U. nuda, U. nigra, and U. avenae were acquired using variable pressure SEM (Hitachi S2600 VP-SEM). A. bisporus, Penicillium sp., and P. brevicompactum spores were imaged by field emission SEM (Hitachi S4700 FESEM).Glass slides containing Agaricomycetes spores (A. bisporus, A. muscaria, B. zelleri, L. nuda, T. abietinum) and Aspergillus spores (A.brasiliensis and A. bisporus) were affixed onto aluminum SEM stubs using double-sided glue tabs, sputter coated with 12 nm gold (Au) palladium (Pd) and mounted in the SEM instrument.Similarly, loose Ustilaginomycetes (U. nuda, U. avenae, U. nigra) spores were deposited directly onto double-sided glue tabs, affixed onto aluminum SEM stubs, sputter coated with 8 nm Au and mounted in the SEM instrument.To prepare Penicillium sp. and P. brevicompactum samples for imaging, cultures of each fungal species were first fixed using osmium vapor (4 % OsO 4 (aq)), and then a portion of the culture was excised and mounted onto an aluminum SEM stub using Aquadag (a graphite based adhesive), and finally the samples were allowed to air dry prior to imaging.For the SEM experiments, images were captured using secondary electrons, a working distance between 5 and 15 mm, and an acceleration voltage between 3 and 25 kV.

Global chemistry-climate transport model, EMAC
We simulated the transport and removal of atmospheric aerosol particles in a global chemistry-climate model, ECHAM/MESSy Atmospheric Chemistry (EMAC).The EMAC model is a numerical chemistry and climate simulation system that includes Introduction

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Full sub-models describing tropospheric and middle atmosphere processes and their interaction with oceans, land and human influences (Jöckel et al., 2006(Jöckel et al., , 2005;;Kerkweg et al., 2006;Tost et al., 2006;Lawrence and Rasch, 2005).It uses the Modular Earth Submodel System (MESSy) to link multi-institutional computer codes.The core atmospheric model is the 5th generation European Centre Hamburg general circulation model (ECHAM5 GCM) (Roeckner et al., 2003).For the present study, we applied EMAC (ECHAM5 version 5.3.01,MESSy version 1.9) in the T63 L31 resolution, i.e. with a spherical truncation of T63 (corresponding to a quadratic Gaussian grid of approximately 1.9 by 1.9 • in latitude and longitude, or approximately 140 km × 210 km at mid-latitudes) with 31 vertical hybrid pressure levels up to 10 hPa.This model resolution provides a reasonable balance between accuracy and computational expense: tracer transport in the ECHAM5 GCM has been shown to be comparatively insensitive to further increases in model resolution (Aghedo et al., 2010).Simulations were performed for five years and an additional year of spin-up time, with the model setup described in detail by Burrows et al. (2009), and with modifications to the scavenging scheme (Tost et al., 2010), including the slow downward transport of crystal-borne species due to the sedimentation of crystals, and the release and repartitioning of tracers associated with evaporation of droplets and melting of ice crystals.Briefly, aerosols are treated as monodisperse passive aerosol tracers, emitted at the Earth's surface.Atmospheric transport is simulated (including advection and parameterized convective mass flux), and tracers are removed by dry and wet deposition to the surface.The EMAC model, used in similar configurations, has been shown to be capable of realistic simulations of aerosol transport and deposition for the transport of African dust to Europe (Gläser et al., 2012) and of radioactive aerosol particles from the Chernobyl accident (Lelieveld et al., 2012).
Removal processes of particles simulated in the model included sedimentation, dry deposition, impaction scavenging, and nucleation scavenging by liquid, mixed-phase, and ice clouds.We use the methods outlined by Burrows et al. (2009) to describe sedimentation, dry deposition, and impaction scavenging.Nucleation scavenging was Introduction

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Full prescribed with a scavenging parameter, R, where R nuc, liq and R nuc, ice are the fraction of particles embedded in cloud droplets or ice particles within a cloud, respectively.For warm liquid clouds (i.e.clouds at temperatures > 0 • C) we assume that R nuc, liq is unity, as done previously (Heald and Spracklen, 2009).A value of unity assumes that the particles are efficient cloud condensation nuclei (CCN).Here, we are using the immersion freezing results to assess implications for fungal spore global transport.In order for the spores to act as immersion-freezing nuclei, they would first have to be incorporated in liquid cloud droplets during the droplet nucleation and growth process, which is ensured by the assumption R nuc, liq = 1.For mixed-phase clouds (i.e.clouds at temperatures between 0 we assume that R nuc, ice is 0.05 for both simulations mentioned above (IN-Inactive and IN-Active).This value is consistent with measurements of cloud scavenging at low temperatures (Henning et al., 2004;Verheggen et al., 2007).Although scavenging at temperatures below −35 • C may also differ between IN-Active and IN-Inactive, the sensitivity of modeled aerosol transport to scavenging at these temperatures is extremely low (Burrows et al., 2013).A summary of the nucleation scavenging coefficients used in the different simulations is included in Table 2.
The in-cloud local rate of change in the mixing ratio of particles, i.e. the number of particles per number of air molecules (X i ), due to nucleation scavenging and subse-Introduction

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Full where ∆t is the length of the model time step; f cl is the grid-box mean cloud fraction; F snow and F rain are the fluxes of snow and rain, respectively; iwc and lwc are the ice water content and liquid water content of the cloud, respectively; and R nuc, ice and R nuc, liq are the nucleation scavenging efficiencies of cloud ice and cloud liquid water, respectively (and as described in Table 2).
For nucleation scavenging the same scavenging parameterizations were applied to both stratiform clouds and to parameterized convective clouds.Simulations were run for 3 µm and 8 µm diameter spherical particles, which overlaps with the sizes (volume equivalent diameters) of the spores studied here (see Table 1).In the simulations fungal spores were emitted globally and continuously at a single constant rate from all land surfaces, excluding land ice.Gilbertson and Ryvarden (1987), Lincoff (1981), Mathre (1982), Mitchell and Walter (1999), Pasanen et al. (1991), Raper et al. (1949), andVanky (1987).SEM images of the fungal spores studied are shown in Fig. 1.In many of the images, the spores appear deflated (A.bisporus, U. nuda, U. nigra, U. avenae, A. brasiliensis, A. niger, Penicillium sp., P. brevicompactum) or as partial spheroids (T.abietinum).These features are likely due to the sample processing prior to imaging Introduction

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Full The median number of spores per droplet is given for each spore species in Table 1.Also included in Fig. 2 (as x's), are previous results from Iannone et al. (2011) for homogeneous nucleation using the same experimental method as used here.Figure 2 shows that the fungal spores studied here initiate freezing at warmer freezing temperatures compared to the homogeneous results of Iannone et al. (2011).Since the number of spores per droplet varies for the different fungal species studied, conclusions about the relative freezing properties of these spores should not be formulated from Fig. 2.

INperSpore(T)
Using the method presented by Vali (1971), the cumulative number of ice nuclei per spore as a function of temperature (INperSpore(T)) was determined from the number of spores per drop and the fraction of droplets frozen as a function of temperature (Fig. 2).For details, see Haga et al. (2013).Shown in Fig. 3 Jayaweera and Flanagan (1982) and Pummer et al. (2013) are the only other studies to measure ice nucleation ability of spores from the three classes investigated here.The results from Jayaweera and Flanagan (1982) are included in Fig. 3 for comparison (filled symbols).The results from Pummer et al. ( 2013) are not included in Fig. 3 since they did not report the cumulative number of IN per spore and it was not possible to calculate this value from their experimental description.
From Fig. 3, when comparing the temperatures at which the cumulative number of ice nuclei per spore is equal to 0.01, all of the Ustilaginomycetes, Agaricomycetes and Eurotiomycetes spores studied here are poorer IN than those studied by Jayaweera and Flanagan (1982).Comparing just the Penicillium results from the current study with those from Jayaweera and Flanagan (1982) suggests that: (1) Penicillium sp. and P. brevicompactum are poor IN compared to P. digitatum, P. notatum, and P. frequentens, and (2) even within the same genus, the INperSpore values can vary drastically (e.g. the freezing temperature at an INperSpore value of 0.01 varies by approximately 20 • C in Fig. 3 and looking at the Penicillium results).
It is important to consider the possibility that the fungal spores studied here may contain bacterial or fungal contaminants on their surface, and to discuss the likelihood that these contaminants would change the ice nucleating properties of the spores (Morris et al., 2013b).From the SEM images presented in Fig. 1, there is no indication of any bacterial or fungal surface contamination on the fungal spores (D.Horne, UBC Bioimaging Facility, personal communication, 2013).Since the Eurotiomycetes spores studied here (A.brasiliensis, A. niger, Penicillium sp., P. brevicompactum) were grown in culture, any bacterial contamination of the spores would have been apparent in the Introduction

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Full culture stage of these experiments.The remaining spores (with the exception of A. bisporus) were qualitatively tested for surface contamination by bacteria and/or fungi using the same method from Haga et al. (2013) for rust and bunt spores: the spores to be tested were cultured in media (nutrient agar and tryptic soy agar) that would support contaminant growth and the cultures were monitored for two weeks.The test indicated low levels of surface contamination for four of the fungal spores studied here.Specifically, U. avenae, U. nigra, U. nuda cultures indicated surface contamination on the spores after 4 days, and colonies were observed in the T. abietinum culture after 13 days.For the remaining spores, B. zelleri, L. nuda, and A. muscaria, there was no indication of surface contamination.From the discussion above, it seems unlikely that the ice nucleation properties of the spores studied here are appreciably affected by surface contamination by fungi or bacteria.However, we cannot rule out this possibility and, so, the INperSpore spectra presented in the current study should be taken as upper limits.
Mineral dust particles are considered to be important atmospheric ice nuclei.For comparison in Fig. 3

Ice nucleation active surface site density, n s
Since heterogeneous ice nucleation is a surface process, larger particles may be more efficient IN on a per particle basis because they have more surface area available for 5028 Introduction

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Full nucleation.To take into account the difference in surface area per particle, we calculated the ice nucleation active surface site density, n s , for the fungal spores in the current study.The ice nucleation active surface site density corresponds to the number of ice nucleation sites per unit surface area of a particular type of IN (Connolly et al., 2009), and was calculated according to: To calculate the surface area for each fungal spore (A aer ), we approximated all spores other than the Aspergillus spores to be prolate spheroids having length and width shown in Table 1.Aspergillus spores were approximated as spherical with diameters given in Table 1.
The temperature dependent n s values for the fungal spores studied here are shown in Fig. 4. For comparison, included in Fig. 4 are n s values for Asian mineral dust (Niemand et al., 2012) (experiments ACI04_16 and ACI04_19), and also results from Jayaweera and Flanagan (1982) for Eurotiomycetes spores.The n s values for the Eurotiomycetes spores studied by Jayaweera and Flanagan (1982) were calculated using spore dimensions and morphologies from the literature (Raper et al., 1949).Specifically, P. digitatum, was approximated to be a prolate spheroid having dimensions 7.75 µm × 5.5 µm; and both P. notatum and P. frequentens spores were assumed to be spheres with a diameter of 3.25 µm.
After accounting for surface area, Fig. 4 shows that the n s spectra for all of the fungal species studied here are similar.Figure 4 also shows that a difference in surface area cannot explain the difference in freezing temperatures within the same genus observed in Fig. 3 for species of Penicillium.Finally, Fig. 4 shows that, at temperatures below approximately −20 • C, all of the fungal spores studied here are less efficient ice nuclei compared to Asian mineral dust on a per surface area basis.Introduction

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Full Figure 5 illustrates that the inclusion of ice nucleation scavenging of fungal spores in mixed-phase clouds can decrease the surface annual mean mixing ratios of fungal spores over the oceans and in polar regions.Since the source of fungal spores in the simulations was land surfaces, excluding land ice, the decrease in the surface concentrations of fungal spores over these regions can be explained by a decrease in the long-distance transport of fungal spores from land surfaces to these remote regions due to ice nucleation followed by precipitation.In stark contrast, scavenging by ice nucleation in mixed-phase clouds increased surface concentrations of fungal spores in the region between roughly 50-80 • S.This can be explained by increased downward transport of ice-borne spores from mixed-phase clouds at higher altitudes to lower altitudes, where ice evaporates and releases spores in the region between 50-80 • S.This explanation is supported by results shown in Fig. 6, which shows the percent Introduction

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Full change in zonally averaged vertical profile of annual mean mixing ratio.This figure illustrates that the inclusion of ice nucleation scavenging in mixed-phase clouds leads to a decrease in mean mixing ratios in the mid-to upper troposphere, particularly in the mid-latitudes, where precipitation scavenging in mixed-phase clouds exerts a strong control on aerosol transport to polar regions (Bourgeois and Bey, 2011;Zhang et al., 2012).Below 5 km and in the region between 50 and 80 • S, there is an increase in concentrations of fungal spores when ice nucleation in mixed-phase clouds is included.An explanation for this observation is that ice nucleation and precipitation at high altitudes, followed by evaporation of the ice crystals at heights between 0 and 5 km, leads to increased fungal spore concentrations at these altitudes.

Ascomycota vs. Basidiomycota
The phyla Basidiomycota and Ascomycota together make up 98 % of known fungal species found on Earth (James et al., 2006).Recent field measurements found increased concentrations of Ascomycota spores relative to Basidiomycota spores in remote marine regions and it was suggested (based on preliminary ice nucleation results) that these findings may be explained by Basidiomycota spores being better IN compared to Ascomycota spores (Fröhlich-Nowoisky et al., 2012).To investigate further whether there is a systematic difference in the ice nucleating ability of the Ascomycota and the Basidiomycota fungi, we have compared the cumulative number of ice nuclei per spore and the ice nucleation active surface site density of Basidiomycota  2011) using the same procedure as described above (i.e.procedures described in Sects.2.2 and 2.3).All of the other IN-perSpore values shown in Fig. 7 were provided in the original studies (Haga et al., 2013;Morris et al., 2013b;Jayaweera and Flanagan, 1982).Based on Fig. 7, some 5031 Introduction

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Full of the Ascomycota spores (Penicillium sp. and P. brevicompactum) are the poorest IN, but on the other hand, some of the spores from this phylum are also the best IN (P.digitatum, P. notatum, P. frequentens, A. brasiliensis, C. herbarum).Within both phyla (Ascomycota and Basidiomycota) there is a wide range of freezing properties.Also, the variation within each phylum is greater than the variation between the average freezing properties of each phylum.Overall, we conclude that Basidiomycota spores are not all better IN compared to Ascomycota spores.Figure 7 also shows that the ice nucleating ability of Asian mineral dust particles studied by Niemand et al. (2012) is approximately in the middle of the range observed for Basidiomycota and Ascomycota fungal spores.
Figure 8 shows previous results of n s for fungal spores from the classes Basidiomycota and Ascomycota.To calculate n s for the spores studied by Morris et al. (2013b), we approximated the spores as prolate spheroids based on literature values for spore morphology and size ranges (Cummins and Husain, 1966;Bruckart et al., 2007;Anikster et al., 2005;Anikster et al., 2004;Laundon and Waterston, 1964).Specifically, we used dimensions of length and width as follows: 29.0 µm × 25.0 µm for P.
Figure 8 shows that, once normalized to surface area, Basidiomycota spores are not all better IN compared to Ascomycota spores.Figure 8 also shows that the variability in freezing properties within both phyla (Basidiomycota and Ascomycota) cannot be accounted for by a difference in surface area between different spore species.Finally, Introduction

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Full Fig. 8 shows that, at temperatures below −20 • C, all fungal spores for which data are available are poorer immersion IN than Asian mineral dust on a per surface area basis.

Ice nucleation on fungal spores
To add to the limited amount of data on the ice nucleation properties of fungal spores, we studied the ice nucleation properties of 12 different species of fungal spores chosen from three classes: Agaricomycetes, Ustilaginomycetes, and Eurotiomycetes.We focused on these classes since they are thought to be abundant in the atmosphere, and also because there is currently little quantitative data on the ice nucleation ability of spores from these classes.
All of the spores studied caused freezing of water droplets above homogeneous freezing temperatures.The cumulative number of ice nuclei per spore was 0.001 at temperatures between −19 • C and −29 • C, 0.01 between −25.5 • C and −31 • C, and 0.1 between −26 • C and −36 • C (Fig. 3).On average, the order of ice nucleating ability for these spores was Ustilaginomycetes > Agaricomycetes Eurotiomycetes.On a per surface area basis, all of the fungal spores studied had similar freezing properties (Fig. 4).In addition, at temperatures below −20 • C, all of the fungal spores studied here were less efficient ice nuclei compared to Asian mineral dust on a per surface area basis.
Comparing our results with previous results, we show that within the same genus, the freezing temperatures can vary drastically (e.g. the freezing temperature at an IN-perSpore value of 0.01 can vary by approximately 20 • C).We used our new freezing results together with data from the literature to compare the freezing temperatures of spores from the phyla Basidiomycota and Ascomycota, which together make up 98 % of known fungal species found on Earth (James et al., 2006).The data show that within Introduction

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Full both phyla (Ascomycota and Basidiomycota) there is a wide range of freezing properties and that not all Basidiomycota spores are better IN than Ascomycota spores.

Global transport of fungal spores
Using a global chemistry-climate transport model, we show that ice nucleation on the fungal spores studied can modify their atmospheric transport and global distributions by providing an additional removal mechanism of the spores from the atmosphere.Specifically, we show that the inclusion of ice nucleation scavenging of fungal spores in mixed-phase clouds can (1) decrease the surface annual mean mixing ratios of fungal spores over the oceans and polar regions and (2) decrease the annual mean mixing ratios in the upper troposphere.
To our knowledge, this study is the first to model globally the effect of IN activity on the distribution of fungal spores in the atmosphere and their long-distance transport.While the treatment of ice nucleation scavenging is simplified, this study gives an initial indication of the regions that are most sensitive to removal of spores due to scavenging by ice nucleation, as well as the potential magnitude of these effects.
In the simulations we assumed that the fungal spores were efficient cloud condensation nuclei, as done previously (Heald and Spracklen, 2009).Experiments to verify this assumption are needed.In addition, the freezing results shown here were from immersion freezing only.Experiments that explore ice nucleation by these fungal spores in other freezing modes would be useful, and they would complement the current results.Also, experiments that explore the effect of acidic coatings on the ice nucleation ability of these spores would be useful as these coatings could potentially change the ice nucleation ability of the spores, similar to mineral dust (Eastwood et al., 2009;Archuleta et al., 2005;Attard et al., 2012;Chernoff and Bertram, 2010;Cziczo et al., 2009;Gallavardin et al., 2008;Kanji et al., 2008;Knopf and Koop, 2006;Koehler et al., 2010;Koop and Zobrist, 2009;Möhler et al., 2008;Niedermeier et al., 2011Niedermeier et al., , 2010;;Reitz et al., 2011;Salam et al., 2007;Sullivan et al., 2010;Yang et al., 2011;Zobrist et al., 2008).

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Asian mineral dust [Niemand et al., 2012] Fig. 8.Comparison between ice nucleation active surface site density spectra (n s vs. T ) for Ascomycota and Basidiomycota fungal spores calculated using the data in Fig. 7 and using approximations for spore size and shape detailed in the text.Included are results from the current study as well as results from the following studies: Haga et al. (2013), Iannone et al. (2011), Jayaweera and Flanagan (1982), and Morris et al. (2013b).Also shown in the figure are previous results for submicron Asian mineral dust (Niemand et al., 2012).
Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | First, water vapor is condensed on the spores to grow droplets approximately 30-150 µm in size by adjusting the dew point of the carrier gas to 2 • C and the temperature of the flow cell to 0 • C. Next, the flow cell was isolated from the humidified flow and the temperature was lowered at a rate of 5-10 • C min −1 down to −40 • C. Finally, the temperature was raised to room temperature to evaporate the droplets, leaving only the fungal spores on the glass slide.Digital video and temperature were recorded during the freezing experiments.For each droplet, the freezing temperature and the 2-dimensional (2-D) surface area of the fungal spores contained within each droplet were determined.Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | quent precipitation is: Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |(see Sect.2.3) or from a loss in turgidity of the fungal spores in the vacuum of the SEM instrument.3.2Fraction of droplets frozen vs. temperatureShown in Fig.2is the fraction of frozen droplets as a function of temperature, including all freezing events that were observed during these experiments.Results for droplets containing Agaricomycetes spores are shown in blue (open symbols), Ustilaginomycetes spores are shown in green (open symbols with horizontal line), and Eurotiomycetes spores are in red (open symbols with vertical line).
Fig. 3, the cumulative number of ice nuclei per spore was 0.001 between −19 • C Discussion Paper | Discussion Paper | Discussion Paper | (half-filled diamonds) we have included recent immersion freezing results by Niemand et al. (2012) for submicron Asian mineral dust (experiments ACI04_16 and ACI04_19).INperParticle(T) for the Asian mineral dust was calculated from the ice nucleation active surface site density, n s , values from Niemand et al. (2012) and as described by Haga et al. (2013).With the exception of U. nigra and A. brasiliensis, all of the spores studied here are poorer IN than the Asian mineral dust studied by Niemand et al. (2012) on a per particle basis.The INperSpore(T) values for U. nigra are similar to the INperParticle(T) values for Asian mineral dusts over the entire temperature range studied by Niemand et al. (2012); for A. brasiliensis, the INperSpore(T) values are comparable to the INperParticle(T) values for the dust only at temperatures below approximately −25 • C.
Discussion Paper | Discussion Paper | Discussion Paper | We used the global chemistry-climate transport model EMAC to study the effects of ice nucleation on the transport and distribution of fungal spores in the atmosphere.Two scavenging scenarios were compared: in the first scenario (IN-Inactive), ice nucleation scavenging by fungal spores was set to zero for mixed-phase cloud temperatures (i.e.below 0 to −35 • C); in the second scenario (IN-Active), ice nucleation scavenging for fungal spores was set to zero for T > −25 • C, but unity for −25 ≥ T > −35 • C (see Table 2).Results from the simulations are presented in terms of percentage difference in the fungal spore mixing ratio between the two scavenging scenarios (i.e.(IN-Active -IN-Inactive)/IN-Inactive × 100 %).Positive non-zero values indicate increased concentrations due to scavenging from heterogeneous ice nucleation at temperatures between −25 • C and −35 • C, whereas negative non-zero values indicate decreased concentrations due to scavenging from heterogeneous ice nucleation over this temperature range.The results from these simulations are given in Figs. 5 and 6.In both figures, the top and bottom panels correspond to results for 3 µm and 8 µm particles, respectively.Differences between simulations with 3 µm and 8 µm particles are minor.
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Table 1 are the average dimensions of the spores studied here, determined by optical microscopy.Values reported in Table 1 are consistent with the literature values: Bockus et al. (2010), • C and −36 • C. Also, on average, the order of ice nucleating ability for these spores is Ustilaginomycetes > Agaricomycetes Eurotiomycetes.To illustrate this point, as an example, the cumulative number of ice nuclei per spore was 0.01 at approximately −27 • C for the Ustilaginomycetes spores, 0.01 at approximately −29 • C for the Agaricomycetes spores, and 0.01 at approximately −28.5 • C for the Eurotiomycetes spores.

Table 1 .
Description of the fungal spores investigated in the current study.