Identification and Isolation Pattern of Globisporangium spp. from a Sanionia Moss Colony in Ny-Ålesund, Spitsbergen Is., Norway from 2006 to 2018

Globisporangium spp. are soil-inhabiting oomycetes distributed worldwide, including in polar regions. Some species of the genus are known as important plant pathogens. This study aimed to clarify the species construction of Globisporangium spp. and their long-term isolation pattern in Sanionia moss in Ny-Ålesund, Spitsbergen Is., Norway. Globisporangium spp. were isolated at two-year intervals between 2006 and 2018 at a Sanionia moss colony, Ny-Ålesund, Spitsbergen Is., Norway. The isolates were obtained by using three agar media and were identified based on sequences of the rDNA-ITS region and cultural characteristics. Most of the Globisporangium isolates obtained during the survey were identified into six species. All six species were grown at 0 °C on an agar plate and used to infect Sanionia moss at 4 and/or 10 °C under an in vitro inoculation test. The total isolation frequency of Globisporangium gradually decreased throughout the survey period. The isolation frequency varied among the six species, and four of the species that showed a high frequency in 2006 were rarely isolated after 2016. The results suggested that Globisporangium inhabiting Sanionia moss in Ny-Ålesund has a unique composition of species and that most of the species reduced their population over the recent decade.


Introduction
Plant pathogenic fungi and oomycetes can affect individual growth and community structure in many wild plants [1]. Warmer temperatures can increase the relative abundance of phytopathogenic fungi and oomycetes [2,3]. Plant pathogens can also occur on mosses and vascular plant species in the polar regions [4]. Svalbard, a High Arctic archipelago, has been investigated for plant pathogenic fungi and oomycetes since the late 19th century [5,6]. Many plant inhabitants have been found, including at least 173 species of fungi and 3 of oomycetes [7]. Fourteen fungal species were also recorded in soil of the archipelago [8].

RDNA-ITS Analysis
All isolates obtained were compared with known species based on entire rDNA-ITS sequences. Genomic DNA of the obtained Globisporangium isolates was extracted from mycelium grown on V8 broth prepared according to Miller [32]. Mycelia were frozen in liquid nitrogen and ground using pestle and mortar. DNA extraction was performed using the DNeasy Plant kit (Qiagen, Hilden, Germany) following the manufacturer's instructions, and the DNA was then stored at −20 °C until used.
Sequences of the ITS region containing ITS1 and ITS2 were determined as follows. In the polymerase chain reaction (PCR), primer pairs ITS5 (5′ GGAAGTAAAAGTCG-TAACAAGG 3′) and ITS4 (5′ TCCTCCGCTTATTGATATGC 3′) described by White et al. [33] were used. Fifty microliters of PCR reaction mixture contained 25 µL 2×MightyAmp buffer ver.2, 0.5 µM of each primer, 0.25 µL MightyAmp DNA polymerase (Takara Bio, Shiga, Japan), and 1 µL template DNA. Amplification was carried out in a PerkinElmer 9700 thermal cycler (PerkinElmer Inc., Waltham, MA, USA). The amplification program consisted of a predenaturation at 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; and a final incubation at 72 °C for 7 min to complete the last extension. PCR products were used for sequence analysis.
The sequence reaction was performed using the primers ITS4 and ITS5. Products of the sequence reaction were analyzed with an ABI 3730 DNA Sequencer (Applied Biosystems). The sequences were aligned with relevant Globisporangium sequences obtained from the GenBank database using BLAST (http://www.ncbi.nlm.nih.gov/blast, accessed on 30 July 2021).
The BLAST search showed that the Globisporangium isolates obtained in this study were divided into six major taxonomic groups. A phylogenetic tree was therefore made based on randomly selected isolates from each of the major taxonomic groups (Figure 2, Table S1). The tree was constructed by MEGA version 5.2.2 [34] based on neighbor-joining (NJ) analysis [35]. To determine the support for each clade, a bootstrap analysis was

rDNA-ITS Analysis
All isolates obtained were compared with known species based on entire rDNA-ITS sequences. Genomic DNA of the obtained Globisporangium isolates was extracted from mycelium grown on V8 broth prepared according to Miller [32]. Mycelia were frozen in liquid nitrogen and ground using pestle and mortar. DNA extraction was performed using the DNeasy Plant kit (Qiagen, Hilden, Germany) following the manufacturer's instructions, and the DNA was then stored at −20 • C until used.
Sequences of the ITS region containing ITS1 and ITS2 were determined as follows. In the polymerase chain reaction (PCR), primer pairs ITS5 (5 GGAAGTAAAAGTCGTAA-CAAGG 3 ) and ITS4 (5 TCCTCCGCTTATTGATATGC 3 ) described by White et al. [33] were used. Fifty microliters of PCR reaction mixture contained 25 µL 2×MightyAmp buffer ver. 2, 0.5 µM of each primer, 0.25 µL MightyAmp DNA polymerase (Takara Bio, Shiga, Japan), and 1 µL template DNA. Amplification was carried out in a PerkinElmer 9700 thermal cycler (PerkinElmer Inc., Waltham, MA, USA). The amplification program consisted of a predenaturation at 95 • C for 5 min; 35 cycles of 95 • C for 30 s, 55 • C for 30 s, and 72 • C for 1 min; and a final incubation at 72 • C for 7 min to complete the last extension. PCR products were used for sequence analysis.
The sequence reaction was performed using the primers ITS4 and ITS5. Products of the sequence reaction were analyzed with an ABI 3730 DNA Sequencer (Applied Biosystems). The sequences were aligned with relevant Globisporangium sequences obtained from the GenBank database using BLAST (http://www.ncbi.nlm.nih.gov/blast, accessed on 30 July 2021).
The BLAST search showed that the Globisporangium isolates obtained in this study were divided into six major taxonomic groups. A phylogenetic tree was therefore made based on randomly selected isolates from each of the major taxonomic groups ( Figure 2, Table S1). The tree was constructed by MEGA version 5.2.2 [34] based on neighbor-joining (NJ) analysis [35]. To determine the support for each clade, a bootstrap analysis was performed with 1000 replications. Pythium aphanidermatum strain CBS118.80 was used as an outgroup.

Characterizations of Morphology and Hyphal Growth Speed
One Globisporangium strain from each of the six major taxonomic groups were used for characterization of morphology and hyphal growth speed. The strains used were 10G16V2, 10G15W, 10C17N1, 10G34N1, 10C12N1, and 10G26N1 ( Figure 2, Table S1).
Morphology of the strains was examined in grass-leaf water culture [36]. All strains were grown on CMA, potato dextrose agar (PDA; Becton Dickinson and Company), or V8 juice agar at 4-17 • C. A piece of agar medium was placed in a Petri dish containing a shallow layer of sterilized water, to which some 1-2 cm leaf pieces of gramineous weeds sterilized by autoclave were added. After incubation at 4-17 • C until Globisporangium strains colonized the leaves, sterilized pond water was added. Spore formation and the shape of said spores were examined by optical microscope (Olympus BX 43, Tokyo, Japan).

Isolation Pattern
Isolation frequency was determined as the number of the moss shoots that isolated Globisporangium spp. divided by the total number of the moss shoots examined. The isolation frequency was compared yearly by least significant differences based on a Tukey-Kramer Honestly Significant Difference test (p < 0.05) by JMP 13 (SAS Institute, Cary, NC, USA).

Infectivity to Sanionia Moss
Eleven Globisporangium strains from the six major taxonomic groups were used to test infectivity to Sanionia moss (S. uncinata). The strains used were 10G16V2, 10G15W, 10C17N1, 10G34N1, 10C12N1, 10G26N1, 18G11V1, 18C29N1, 18C32N1, 18C17N2, and 18C14N1 ( Figure 2, Table S1). Stem-leaf sections (15 mm long, 0.5 mm wide) of the Sanionia obtained in Ny-Ålesund were placed on plates containing a KNOP agar medium [38] amended with 1.5% agar and were grown in a growth chamber at 10 • C for 3-4 months with continuous light (80 mmol m −2 s −1 measured at the level of the plants). A CMA plug (8 mm diameter) from each strain of Globisporangium, grown at 15 • C for 1 week, was placed in the center of the plate containing the Sanionia moss sections. Uninfected CMA was used as a control. The plates were kept at 0 • C, 4 • C, and 15 • C in darkness for approximately one month in a growth chamber. Infectivity was confirmed by optical microscopic observation. Recovery of the inoculated Globisporangium strains from the infected stem leaves was done using NARM medium. There were 16 replicates for each strain, using one moss segment for each replicate.

Isolation and Identification
In total, 434 isolates of Globisporangium spp. were obtained from the Sanionia moss during the 2006-2018 survey. All the isolates obtained were compared with known species based on the entire rDNA-ITS sequences through the GenBank database. The phylogenetic analysis of the sequences revealed that all isolates obtained were divided into six taxonomic groups of Globisporangium spp., which formed each monophyletic clade based on neighborjoining (NJ) analyses ( Figure 2). There was one exception: strain 12G14W1 did not belong to any of the six taxonomic groups. Since the maximum identities of these taxonomic groups against known species [22,[39][40][41] were low (86.7 to 96.7%, Table S1), with the exception of one group with 99.8-100% similarity to G. polare (Table S1), they were named as Globisporangium sp. 1, sp. 2 (=G. polare), sp. 3, sp. 4, sp. 5, and sp. 6 ( Figure 2). Globisporangium strain 12G14W1 was isolated only once, in 2012. The phylogenetic position of the strain 12G14W1 was the closest to G. kandovanense [41], but further analysis was not conducted because of loss of the strain. Characteristics of morphology and hyphal growth speed of Globisporangium spp. 1-6 are described below.
Globisporangium sp. 1 strain 10G16V2: Main hyphae were up to 5 µm in diameter. Sporangia were not observed. Hyphal swellings were observed in single culture. Oogonia did not develop in single culture, but developed in dual culture with OPU1276. Strain OPU1276 was isolated from the present study site in July 2003 and showed identical rDNA-ITS sequence with strain 10G16V2 (Figure 2, Table S1). Oogonia were globose (Figure 3a), smooth, terminal sometimes intercalary, and 20.0-24.5 (mean 21.7) µm in diameter. Antheridia were monoclinous, with 1-4 per oogonium. Oospores were aplerotic, globose, smooth, and 16.5-21.5 (mean 18.7) µm in diameter, with one per oogonium. The thickness of the oospore wall was 0.5-1.5 (mean 1.0) µm. The minimum, optimum, and maximum temperatures for growth on PCA were 0 • C, 25 • C, and 28 • C, with daily growth rates at 2.7 mm, 18.3 mm, and 15.7 mm, respectively ( Figure 4). The strain did not grow at 31 • C but showed regrowth when the dishes were placed at 22 • C. Globisporangium sp. 1 was closely phylogenetically related to G. spinosum, G. sylvaticum, and P. macrosporum ( Figure 2) but was distinguished from these known species by the size and shape of its oogonia.
Microorganisms 2021, 9, x FOR PEER REVIEW 6 of 13 position of the strain 12G14W1 was the closest to G. kandovanense [41], but further analysis was not conducted because of loss of the strain. Characteristics of morphology and hyphal growth speed of Globisporangium spp. 1-6 are described below.
Globisporangium sp. 1 strain 10G16V2: Main hyphae were up to 5 µm in diameter. Sporangia were not observed. Hyphal swellings were observed in single culture. Oogonia did not develop in single culture, but developed in dual culture with OPU1276. Strain OPU1276 was isolated from the present study site in July 2003 and showed identical rDNA-ITS sequence with strain 10G16V2 (Figure 2, Table S1). Oogonia were globose (Figure 3a), smooth, terminal sometimes intercalary, and 20.0-24.5 (mean 21.7) µm in diameter. Antheridia were monoclinous, with 1-4 per oogonium. Oospores were aplerotic, globose, smooth, and 16.5-21.5 (mean 18.7) µm in diameter, with one per oogonium. The thickness of the oospore wall was 0.5-1.5 (mean 1.0) µm. The minimum, optimum, and maximum temperatures for growth on PCA were 0 °C, 25 °C, and 28 °C, with daily growth rates at 2.7 mm, 18.3 mm, and 15.7 mm, respectively ( Figure 4). The strain did not grow at 31 °C but showed regrowth when the dishes were placed at 22 °C. Globisporangium sp. 1 was closely phylogenetically related to G. spinosum, G. sylvaticum, and P. macrosporum ( Figure 2) but was distinguished from these known species by the size and shape of its oogonia. Globisporangium sp. 2 strain 10G15W2: Main hyphae were up to 6 µm in diameter. Sporangia were terminal and globose or sometimes subglobose. Zoospores were formed at 4-15 °C. Oogonia did not develop in single culture but developed in dual culture with G. polare CBS118202 [22]. Oogonia were globose, smooth, terminal or sometimes intercalary, and 17.3-26.9 (mean 23.1) µm in diameter (Figure 3b). Antheridia were diclinous, with 1-3 per oogonium. Oospores were aplerotic, globose, smooth, and 14.4-24.5 (mean 19.7) µm in diameter, with one per oogonium. The thickness of the oospore wall was 0.7-1.5 (mean 1.0) µm. The minimum, optimum, and maximum temperatures for growth on PCA were 0 °C, 22 °C, and 28 °C, with daily growth rates at 1.7 mm, 12.1 mm, and 9.4 mm, respectively ( Figure 4). The growth rate at 25 °C was 11.2 mm per day. Since these taxonomic features matched those of G. polare, Globisporangium sp. 2 was identified as G. polare Globisporangium sp. 2 strain 10G15W2: Main hyphae were up to 6 µm in diameter. Sporangia were terminal and globose or sometimes subglobose. Zoospores were formed at 4-15 • C. Oogonia did not develop in single culture but developed in dual culture with G. polare CBS118202 [22]. Oogonia were globose, smooth, terminal or sometimes intercalary, and 17.3-26.9 (mean 23.1) µm in diameter (Figure 3b). Antheridia were diclinous, with 1-3 per oogonium. Oospores were aplerotic, globose, smooth, and 14.4-24.5 (mean 19.7) µm in diameter, with one per oogonium. The thickness of the oospore wall was 0.7-1.5 (mean 1.0) µm. The minimum, optimum, and maximum temperatures for growth on PCA were 0 • C, 22 • C, and 28 • C, with daily growth rates at 1.7 mm, 12.1 mm, and 9.4 mm, respectively ( Figure 4). The growth rate at 25 • C was 11.2 mm per day. Since these taxonomic features matched those of G. polare, Globisporangium sp. 2 was identified as G. polare [22]. The result of the morphological study is in concordance with the result of the phylogenetic study. genetic study.
Globisporangium sp. 3 strain 10C17N1: Main hyphae were up to 5 µm in diameter. Globose sporangia were observed in single culture (Figure 3c). Sexual reproductive organs did not produce in single or dual culture. The minimum, optimum, and maximum temperatures for growth on PCA were 0 °C, 22 °C, and 28 °C, with daily growth rates at 1.1 mm, 11.9 mm, and 7.8 mm, respectively (Figure 4). The growth rate at 25 °C was 11.2 mm per day. Globisporangium sp. 3 did not grow at 31 °C but showed regrowth at 22 °C. Globisporangium sp. 4 strain 10G34N1: Main hyphae were up to 5 µm in diameter. Globose sporangia were observed in single culture (Figure 3d). Sexual reproductive organs were not produced in single or dual culture. The minimum, optimum, and maximum temperatures for growth on PCA were 0 °C, 19 °C, and 28 °C, with daily growth rates at 2.1 mm, 11.0 mm, and 6.9 mm, respectively ( Figure 4). The growth rate at 25 °C was 9.4 mm per day. The strain did not grow at 31 °C but showed regrowth at 22 °C. Globisporangium spp. 3 and 4 were closely related to G. nagaii based on rDNA-ITS sequences ( Figure 2). Since asexual stages of Globisporangium spp. 3 and 4 were not formed in this study, additional taxonomic study is needed to distinguish Globisporangium spp. 3 and 4 from G. nagaii.
Globisporangium sp. 5 strain 10C12N1: Main hyphae were up to 6 µm in diameter. Globose sporangia, hyphal swellings, and sexual reproductive organs were observed in single culture. Oogonia were globose, smooth, terminal, and 20.0-26.0 (mean 22.9) µm in diameter (Figure 3e). Antheridia were monoclinous or occasionally diclinous, with 1-2 per oogonium. Oospores were aplerotic or occasionally plerotic, globose, smooth, and 19.0-26.0 (mean 22.2) µm in diameter, with one per oogonium. The thickness of the oospore wall was 0.2-2.0 (mean 1.1) µm. The minimum, optimum, and maximum temperatures for growth on PCA were 0 °C, 22 °C, and 25 °C, with daily growth rates at 1.0 mm,  (Figure 3c). Sexual reproductive organs did not produce in single or dual culture. The minimum, optimum, and maximum temperatures for growth on PCA were 0 • C, 22 • C, and 28 • C, with daily growth rates at 1.1 mm, 11.9 mm, and 7.8 mm, respectively ( Figure 4). The growth rate at 25 • C was 11.2 mm per day. Globisporangium sp. 3 did not grow at 31 • C but showed regrowth at 22 • C.
Globisporangium sp. 4 strain 10G34N1: Main hyphae were up to 5 µm in diameter. Globose sporangia were observed in single culture (Figure 3d). Sexual reproductive organs were not produced in single or dual culture. The minimum, optimum, and maximum temperatures for growth on PCA were 0 • C, 19 • C, and 28 • C, with daily growth rates at 2.1 mm, 11.0 mm, and 6.9 mm, respectively ( Figure 4). The growth rate at 25 • C was 9.4 mm per day. The strain did not grow at 31 • C but showed regrowth at 22 • C.
Globisporangium spp. 3 and 4 were closely related to G. nagaii based on rDNA-ITS sequences ( Figure 2). Since asexual stages of Globisporangium spp. 3 and 4 were not formed in this study, additional taxonomic study is needed to distinguish Globisporangium spp. 3 and 4 from G. nagaii.
Globisporangium sp. 5 was also distinguished from G. rostratifingens and G. rostratum by growing at 0 • C.
Globisporangium sp. 6 strain 10G26N1: Main hyphae were up to 5 µm in diameter. Sporangia were not observed. Hyphal swellings were observed in single culture (Figure 3f). Sexual reproductive organs and sporangia were formed neither in single nor dual culture. The minimum, optimum, and maximum temperatures for growth on PCA were 0 • C, 22 • C, and 28 • C, with daily growth rates at 0.9 mm, 7.1 mm, and 5.0 mm, respectively ( Figure 4). The growth rate at 25 • C was 7.0 mm per day. Like Globisporangium sp. 5, sp. 6 is phylogenetically closely related with G. kandovanense, G. rostratifingens, and G. rostratum. Although the species identity was unclear for Globisporangium sp. 6, this species could be distinguished from G. rostratifingens and G. rostratum by growing at 0 • C. The species also differed from G. kandovanense by not forming sporangia.
All the Globisporangium strains obtained were identified as one of six species, i.e., Globisporangium sp. 1, ibid sp. 2 (=G. polare), ibid sp. 3, ibid sp. 4, ibid sp. 5, and ibid sp. 6, except for the strain 12G14W1. Strains of Globisporangium spp. 1 to 6 grew at 0 • C on agar plates and infected the Sanionia moss at 4 to 10 • C. Among the six species, only Globisporangium sp. 2 was a known species and was G. polare [22]. The other five remained unknown species. G. polare was first described from Sanionia moss with brown discoloration under snow cover in Longyearbyen, Spitsbergen Is., and has been found only in polar regions [22]. The phylogenetic position of the strain 12G14W1 was closest to G. kandovanense which was isolated from Lolium perenne with snow rot symptoms in a natural grassland in East Azerbaijan province, Iran [41]. The present results, together with previous reports, suggest that Globisporangium in Sanionia moss colonies in Ny-Ålesund not only has a unique species composition, but also shows adaptation to cold environments. Further study is needed to describe the new species for the unknown Globisporangium spp.

Infectivity to Sanionia Moss
Globisporangium spp. 1, 2, 3, 4, and 6 infected the moss cells by penetration and colonization of mycelia at 4 • C and/or 10 • C (Table 1). Only one of the three strains tested of Globisporangium sp. 1 managed to colonize the moss cells, because the other two strains were lost when the test was done. Among the six species, Globisporangium spp. 1-4 consistently formed hyphae, oospores, and sporangia into the stem leaves of the moss cells (Table 1). At least one strain of all six groups produced sporangia or hyphal swellings inside the moss cells ( Figure 5). All the strains infected the moss without showing any symptoms such as blight or discoloration of shoots and leaves until about 2 months after inoculation. The Globisporangium spp. were reisolated from the nonsymptomatic moss (Table 1).
Lévesque and de Cock [42] characterized phylogenetic clades of Pythium involving Globisporangium. Based on their clades, the Globisporangium spp. found in this study belong to clades E, F, and G [42]. Globisporangium sp. 1 belonged to clade F. This clade includes important crop pathogens such as G. spinosum, G. irregulare, G. sylvaticum, and G. debaryanum. Globisporangium spp. 2 (=G. polare), 3, and 4 belonged to clade G. This clade also includes important plant pathogens such as G. iwayamai, G. paddicum, and G. okanoganense, which cause snow rot of wheat and barley in Asia and the USA [25]. Globisporangium spp. 5 and 6 belonged to clade E, which includes weak pathogens of many plants [37]. This suggests that Globisporangium spp. 1 to 6 could be potential crop pathogens.  10 Infection was found more than 50% of the plant part examined, +: infection was found less than 50% of the plant part examined, −: no infection. Recovery of Globisporangium spp. from stem-leaves was calculated from the number of stem leaves from which Globisporangium was recovered after 4 weeks of incubation at 4 °C.

Isolation Pattern
Isolation frequency of the total population of Globisporangium spp. was maintained between 2006 and 2010, and significantly (p < 0.05) decreased from 2012 to 2018 ( Figure 6). The total population was lowest in 2018 during the twelve-year period. The changes in the isolation pattern were different for the six Globisporangium spp. (Figure 7).

Isolation Pattern
Isolation frequency of the total population of Globisporangium spp. was maintained between 2006 and 2010, and significantly (P < 0.05) decreased from 2012 to 2018 ( Figure  6). The total population was lowest in 2018 during the twelve-year period. The changes in the isolation pattern were different for the six Globisporangium spp. (Figure 7). Globisporangium spp. 1, 3, 4, and 6 consistently decreased from 2012 on. Globisporangium sp. 1 was not recorded in 2012, 2016, and 2018. Globisporangium spp. 2 (=G. polare) and 5 maintained their population, although the population differed from year to year.

Isolation Pattern
Isolation frequency of the total population of Globisporangium spp. was maintained between 2006 and 2010, and significantly (P < 0.05) decreased from 2012 to 2018 ( Figure  6). The total population was lowest in 2018 during the twelve-year period. The changes in the isolation pattern were different for the six Globisporangium spp. (Figure 7). Globisporangium spp. 1, 3, 4, and 6 consistently decreased from 2012 on. Globisporangium sp. 1 was not recorded in 2012, 2016, and 2018. Globisporangium spp. 2 (=G. polare) and 5 maintained their population, although the population differed from year to year.   Quantitative isolation from 2006 to 2018 demonstrated that total population of Globisporangium significantly decreased during the twelve-year period. Most of the Globisporangium spp. decreased their population. Only Globisporangium spp. 2 (=G. polare) and 5 showed little decreasing. The reason for the population decreasing is difficult to explain, but it may be influenced by climate changes in Arctic regions [43,44]. The influence of climate changes has already been recognized in the species composition and distribution of the Arctic vegetation [45,46]. Globisporangium spp. inhabiting Arctic regions are cold-adapted mesophiles rather than true psychrophiles (cold-loving), because they can grow at 20-25 • C. Mycelia of Globisporangium spp. are less freeze-resistant than those of fungi, even though a few isolates of G. polare are tolerant [28]. However, Globisporangium spp. can be highly tolerant to freezing when they have infected plant tissues [28]. The present in vitro study confirmed consistent infection of wet living moss by all six Globisporangium spp. under cold conditions. Previous and current results suggest that Globisporangium spp. found in the study site mainly increase their population during the summer period by infecting Sanionia moss, although they can grow at 0 • C under snow cover. Since Globisporangium requires wet conditions to produce hyphae, sporangia, and oospores [37], a consistent moist condition during the summer period is necessary to maintain its population. Romero et al. [3] reported that humidity is a primary driving factor for outbreaks of plant diseases caused by fungi and oomycetes. The recent continuous warming in the Arctic regions will decrease the diversity of mosses [11,46], which can be host plants of Globisporangium in the region. Better understanding of taxonomic and ecological features of the Arctic Globisporangium is needed, because they have unique species constructions and are probably vulnerable to climate changes.

Conclusions
At least six species of Globisporangium were found in single colony of the Sanionia moss in Ny-Ålesund, Spitsbergen Is., Norway. Among them, G. polare was the only known species, which has only been found in polar regions. The other five were unknown species and remain to be described as new species. All six species grew at 0 • C on an agar plate. All of them infected Sanionia moss under an in vitro inoculation test. Quantitative isolations of Globisporangium spp. from 2006 to 2018 showed that most of the species reduced their population over the recent decade at the study site. Much like other plantparasitic oomycetes, the present Globisporangium spp. require a consistent moist condition to maintain their population. Recent climate change is influencing humidity in the Arctic region and could become a factor in the population reduction of the Globisporangium spp. Considering the unique species construction of Globisporangium found in this study, further evaluations are needed to provide better understanding of the taxonomic and ecological features of these species.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/microorganisms9091912/s1, Table S1: Information for Globisporangium strains used in the phylogenetic tree of Figure 2.

Data Availability Statement:
The data presented in present paper are available in this article.