Four New Species of Dictyostelids from Soil Systems in Northern Thailand

Dictyostelid cellular slime molds (dictyostelids) are ubiquitous microorganisms found in the uppermost layers of most soils. Reports on the species diversity of dictyostelids in Southeast Asia, particularly Thailand, are few in number. A survey for dictyostelids performed in northern Thailand in 2008 recovered 15 distinctive forms, including several common species and a number of forms morphologically different from anything already described. Five of the latter were formally described as new to science in a previous paper. An additional five isolates appeared to be morphologically distinct, and this was supported by DNA sequence data and phylogenetic analysis. These isolates representing four species are described herein as species new to science. Detailed descriptions and illustrations of these new species are provided.


Introduction
Dictyostelid cellular slime molds (dictyostelids) are a ubiquitous component of soils, where they feed upon bacteria and other microbes and thus play a major role in maintaining the balance between these organisms in the soil microhabitat [1,2]. Dictyostelids are amoebozoans, a distinct branch of eukaryotes, separate from plants, fungi, and animals. During the past 20 years, as a result of ongoing surveys in areas of the world where dictyostelids remain an understudied group, the number of species has essentially doubled. Molecular studies performed over the past decade have revised the traditional system of classification used for dictyostelids [3], and a SSU-based phylogeny has established that there are four major clades [4].
In 2008, a total of 40 samples for isolation of dictyostelids were collected from four localities in northern Thailand to obtain data on the occurrence and distribution of dictyostelids in this region of the world. One of the localities sampled was located within a tropical cloud forest in Doi Inthanon National Park, Chom Thong District, Chiang Mai Province (18 • 35 32" N, 98 • 29 12" E). In addition to several common species of dictyostelids, nine isolates that could not be assigned to any described species were also recovered from the sampling. All of these isolates were subjected to a detailed morphological study of subcultures in addition to DNA sequence analyses, and five were described as new to science in a previous publication [5]. Five additional isolates, all obtained from samples collected from the tropical cloud forest (elevation 2500 m) in Doi Inthanon National Park,

Isolation and Cultivation
The samples were processed using the methods described by Cavender and Raper [6]. Each sample was weighed, and enough sterile distilled water was added to obtain an initial soil/water dilution of 1:10. This mixture was shaken to disperse the material and to suspend the cells of the dictyostelids present. A 5.0 mL volume of this initial dilution was added to 7.5 mL of sterile, distilled water to create a 1:25 dilution of sample material. Aliquots (each 0.5 mL) of this suspension were added to each of two or three 95 × 15 mm Petri dishes prepared with hay (leached and dried, mostly Poa sp.) infusion agar [2]. This produced a final dilution of 0.02 g of soil per plate. Approximately 0.4 mL of a heavy suspension of 12-24 h Escherichia coli was added to each culture plate, and plates were incubated under diffused light at 20-25 • C. Each plate was examined at least once daily for several days following the appearance of initial aggregations, and the location of each aggregate clone was marked. Aggregations, pseudoplasmodia, and sorocarps appeared in the plates over a period that ranged from 2 d to 3 w. Isolates of interest were subcultured from spores on low-nutrient agar with E. coli. Spores were also conserved in tubes of silica gel granules at 4 • C, as described by Raper [2]. Isolates considered to be of interest were studied in more detail, which involved an initial characterization of morphological features and obtaining a first set of images of morphological structures. Later, subcultures of these isolates were sent to Vadell and Liu. All isolates considered in the present study were deposited in the Dicty Stock Center at Northwestern University in the United States and then at Jilin Agricultural University (HMJAU) in China.

Morphological Observations
The characteristic stages in the life cycle, including cell aggregation and the formation of pseudoplasmodia and sorocarps, were photographed in the Liu laboratory under a dissecting microscope (Axio Zoom V16, Carl Zeiss Microscopy GmbH, Göttingen, Germany) with a 1.5× objective and a 10× ocular. Slides with sorocarps were prepared with water as the mounting medium. Features of spores, sorophores, and sorocarps were observed and measured on the slides by using a light microscope (Axio Imager A2, Carl Zeiss Microscopy GmbH, Göttingen, Germany), with a 10× ocular and 10, 40, and 100× (oil) objectives. Photographs were taken with a Axiocam 506 color microscope camera (Carl Zeiss Microscopy GmbH, Göttingen, Germany).
Detailed examinations of the development and overall morphology of subcultured clones of the five isolates from Doi Inthanon National Park were performed. These isolates were observed at magnifications from 50 to 200×, either in hydric conditions or in xeric media conditions to observe the effects of dehydration and conservation of the hydric contents of the sori over time, migration, stolon formation, and sorogen development. Observations of early aggregations and fruiting bodies were made after 2-30 d incubation under diffuse illumination at 18-26 • C. Optimal temperatures for growth and fruiting body formation, when determined, were measured in an incubator at 18, 20, 24, and 26 (±0.5 • C), under low-diffuse illumination. All major stages of development and behavior (in young and old cultures) of each isolate were observed, described, measured, and drawn by hand in India ink, carefully noting the patterns and shapes of early and late sorogens and behavior [7,8]. The morphological behavior was compared and contrasted with independent observations. Structures were studied through the top, side, and bottom of the Petri dish, making use of variations in light intensity to discriminate the various special perspectives and differences (e.g., basal zones, type and arrangement of cells or the accumulations of slime supporting the base of the sporophore, presence of granulated slime and cushions, relative degree of hydric conditions, and mound shapes) [8], using magnifications provided by the 4 and 12× lenses of a dissecting microscope along with observations made with a compound phase contrast microscope. The general criteria used for the features observed were based on Raper [2], and the drawings of the four species described herein are presented.

DNA Isolation, PCR Amplification and Sequencing
The spores of all five isolates being studied were collected with a sterile tip and mixed with the lysis buffer of the MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (Takara Bio Inc., Kusatsu, Japan) following the manufacturer's protocol. The genomic DNA solution was used directly for the small subunit (SSU) PCR amplification using the primers 18SF-A (AACCTGGTTGATCCTGCCAG) and 18SR-B (TGATCCTTCTGCAGGTTCAC) [9] along with D542F (ACAATTGGAGGGCAAGTCTG3) and D1340R (TCGAGGTCTCGTC-CGTTATC) [4]. PCR products were sent to Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing. Sequences obtained were deposited in the GenBank database. The isolates and the NCBI GenBank accession numbers of SSU DNA sequences considered in present study are listed in Table 1.

Phylogenetic Analysis
The five newly generated sequences were checked and then submitted to GenBank, as noted above. The SSU sequences were aligned and compared using the program ClustalW Multiple alignment version 2.1 (Institut Pasteur, Paris, France) [10] and then manually adjusted in BioEdit version 7.0.9.0 (Manchester, UK) [11]. Maximum likelihood (ML) analyses were performed using IQ-TREE v.1.6.12 (Insitut Pasteur, Paris, France) [12] with 1000 replicates of ultrafast-likelihood bootstrapping to obtain node support values by the "-bb 1000" option, and further optimized using a hill-climbing nearest-neighbor interchange (NNI) by the "-bnni" option [13]. The "-nt AUTO" option was used to automatically determine the best number of cores given the current data. In the ML analyses of SSU sequences, TVMe+R5 (IQTree, Vienna, Austria) model was chosen as the best-fit model according to BIC by IQ-TREE with the "-m TVMe+R5" option.

Data Availability
Sequence data are available in GenBank (www.ncbi.nlm.nih.gov/genbank/, accessed on 14 February 2022, Accession Numbers OM677255, OM677256, OM677257, OM677258, and OM677259). The nomenclature of the new species in the present study is available in MycoBank (www.mycobank.org, accessed on 13 February 2022 for MB842981, MB842982 and MB842984, accessed on 14 February 2022 for MB842989). Sequence alignment was uploaded as supplementary material (File S1).

Results
Five isolates representing four new species [Cavenderia helicoidea (Figures 1 and 2), C. parvibrachiata (Figures 3 and 4), C. protumula (Figures 5 and 6) and C. ungulata (Figures 7  and 8)] of dictyostelids were recovered from samples collected from a tropical cloud forest in northern Thailand. Morphological characteristics and phylogenetic studies of the SSU sequences both support the taxonomic placement in Cavenderia of all four of the species (Table 2, Figure 9).              Table 1. The tree was derived using IQ-TREE with ultrafast bootstrap approximation (UFBoot) by TVMe+R5 model. The phylogeny is rooted according to Sheikh et al. [3]. Sorocarps were mostly prone, decumbent, helical, solitary, or clustered to gregarious, 0.23-2.85 mm long, zero to five branches (2C). Sorophores were hyaline to white or slightly yellow, with one to several tiers of cells, sometimes with a few branches. Branches 0.4-0.7 mm long. Tips acuminate or piliform with one or two tiers of cells, 4.8-5.6 µm wide (2D). Bases were clavate to round, consisting of one to six cells, with one or more terminal cells protruding, 13.3-23.3 µm wide (2E). Sori were hyaline-white to cream, globose, with a 39-241 µm diam. Spores of fallen sori germinated immediately. Spores were elliptical-oblong, 5.6-10.5 × 2.9-4.4 µm, with irregular consolidated polar granules (PG), occasionally slightly subpolar, germinating rapidly (2F). Typical myxamoeba (2G). Aggregations radiated or took the shape of irregular mounds, as psedoplasmodia with stalks (2A). Sorogens were in tight clusters. Early and late sorogens may migrate, leaving traces of slime and sections of immature sorophores (2B).
Comments: This species appears morphologically most similar to C. protodigitata, but the latter is smaller in length. Both share the fast fading of the yellow pigment very early during morphogenesis. This fading of the yellow pigment is a common feature among the smaller members of the Thailand species in group 1 (sensu Sheikh et al. [3]) that have been studied, as a relict feature tending to disappear. This isolate is also larger than C. ungulata and C. aureostabilis [5], having similarly elongated, thin fruiting bodies also common in both species, and its sorocarps are more regular, sigmoid, and broken, and the bases are more variable. Spores are relatively large, and do not germinate immediately. cells (8C). Smaller solitary sorocarps were unbranched and varying considerably in height, sometimes surround the bases of larger sorophores (8B). Branches were 0.5-1.0 mm long. Tips were variable, obtuse to acuminate or clavate, with one to two tiers of cells (mostly one), 6.1-10.0 µm wide (8E). Bases were clavate with one or two tiers of cells, 8.9-17.2 µm wide (8F). Sori were pale white, globose, and 29-182 µm wide. Spores were elliptical-oblong, 5.0-9.8 × 2.3-4.6 µm, with prominent, large, consolidated PG, sticky, and often surrounded by a clear narrow halo (8G). Aggregations radiated, consisting of irregular mounds with short irregular streams (8A). Pseudoplasmodia migrated. Myxamoebae had many median vacuoles (8H).
Comments: Cavenderia ungulata is separated from any other known species of dictyostelids by its very irregular sorophores with claw-like clavate bases, sticky regular spores with large regular PG (2 µm), with halos and dispersed small vacuoles in the spore body, a homogeneous content, continuous development and growth, and migration of early sorogens. This species adapts well to different conditions, and aggregations are either those with small irregular radiate streams or tenuous aggregations that develop dendroid ample streams partitioning into small, thick, round, or slightly elongated separate pseudoplasmodia. Some features resemble those of C. aureostipes, although this species is very small, crowded, and delicate. This species is much smaller than C. aureostabilis [5] and even smaller than C. helicoidea with a different spore morphology and aggregation.

Discussion
Historically, species of dictyostelids have been described solely on the basis of morphological characters [2] and only recently has it been possible to complement such morphological information with DNA sequence analysis. In general, DNA sequence data have supported morphology-based species determination of dictyostelids. However, DNA sequence information recently has led to an extensive revision of the higher levels of classification within the dictyostelids [3].
In little more than 50 years, the number of known species of dictyostelids has increased from about 40 to more than 160. Surveys for these organisms performed in regions of the world where previous investigations were either limited or completely lacking [14][15][16][17] largely account for this dramatic increase. However, more intensive studies in areas where previous records of dictyostelids already existed have yielded unexpected results. For example, Cavender et al. [18] reported 10 new species of dictyostelids from the Great Smoky Mountains National Park in the eastern United States.
In an earlier survey of dictyostelids in Southeast Asia performed in 1970 by Cavender, samples were collected from three localities in Thailand [19]. These were Kao Yai National Park in southern Thailand along with collecting sites near Chiang Dao and Chiang Mai in northern Thailand. All three sites were characterized by tropical semi-deciduous forests, and only Chiang Dao is located at an elevation comparable to that of Doi Inthanon, where the samples considered herein were collected. The data from this survey were summarized in Cavender [19]. A total of nine species were isolated, including a new group 1 species (Cavenderia bifurcata) from Chiang Dao.
The sequences obtained for the four species described in the present paper appear to indicate that they are closely related and probably evolved in situ. A similar situation was reported by Cavender et al. [18] for several morphologically similar species collected in a single small collecting site located at higher elevations the Great Smoky Mountains National Park in the United States. As a general observation, the species composition and diversity of the assemblage of dictyostelids in northern Thailand are relatively similar to what has been reported in previous studies of both the dictyostelids of Southeast Asia [19] and the American tropics [15]. In both regions, those species that appear to be endemic are rare. The four new and apparently rare species isolated from the tropical cloud forest at Doi Inthanon appear to be surviving as organisms adapted to a cool environment characterized by high levels of organic matter.
The major contribution of the present study is that it adds four new species of dictyostelids to the known biodiversity of these organisms in Thailand, all of Southeast Asia, and the world as a whole. The sampling effort involved certainly was not adequate to characterize the entire assemblage of dictyostelids present in the general study areas investigated. As such, it represents a potential starting point for additional future studies.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The datasets generated for this study can be found in the GenBank and MycoBank.