The Umbelopsis ramanniana Sensu Lato Consists of Five Cryptic Species

Umbelopsis ramanniana is one of the most commonly reported species within the genus and an important oleaginous fungus. The morphology of the species varies remarkably in sporangiospores, columellae and chlamydospores. However, phylogenetic analyses based on ITS and nLSU rDNA had previously shown insufficiency in achieving species level identification in the genus Umbelopsis. In this study, by applying a polyphasic approach involving multi-gene (nSSU, ITS, nLSU, act1, MCM7 and cox1) phylogeny, morphology and maximum growth temperature, U. ramanniana sensu lato was revealed as a polyphyletic group and resolved with five novel taxa, namely U. curvata, U. dura, U. macrospora, U. microsporangia and U. oblongielliptica. Additionally, a key for all currently accepted species in Umbelopsis was also updated.


Introduction
Umbelopsis ramanniana, a widespread species in the genus Umbelopsis, is a promising oleaginous fungus in biochemistry and biotechnology. The species is well-known for accumulating large amounts of lipids, which makes the species useful in studying the mechanism of lipid biosynthesis [1][2][3] and for the biotransformation of oil [4,5]. Ecologically, U. ramanniana is a typical inhabitant of forest soils [6] and important in biological rehabilitation [7][8][9][10][11]; it is also frequently isolated from rhizospheres of forest plants [12], or as an endophyte of plants [13].
Umbelopsis ramanniana was first described as Mucor ramannianus by Möller [14]. Historically, there has been a long debate about the attribution of the species and position of the genus Umbelopsis in Mucorales [15]. For its similarity with Mortierella isabellina (basionym of U. isabellina), Linnermann [16] transferred the species to Mortierella sect. Pusilla (which was revised to sect. Isabellina by the author in 1969, nom. inval.). Mil'ko [17], however, retained the species in the genus Mucor and introduced a new section Ramannianus. Gams [18] maintained it as a species of Mortierella and proposed the subgenus Micromucor. Von Arx [19] elevated the subgenus to a genus rank as Micromucor in Mucoraceae and treated the species as Micromucor ramannianus. Meyer and Gams [20] combined the genera Umbelopsis and Micromucor based on the results of restriction fragment length polymorphism data of the whole nuclear ribosomal internal transcribed spacer (ITS) region and phylogenetic reconstruction of ITS1; and, therefore, the species was recombined as its current name U. ramanniana.
The species was first reported from pine mycorrhizas collected in Bavaria and Mark Brandenburg of Germany and was proposed with a poorly informative description, i.e., roseate colony, roundish to elongated sporangiospores and two types of chlamydospores [14]. Because of the lack of type information or illustrations, remarkable variations in morphology

Media, Cultivation and Morphological Observation
For DNA extraction, strains were cultivated in malt extract (ME: malt extract 2%, peptone 0.1%, and dextrose 2%) for 4-8 days at 20 • C. Isolates were cultivated at 18 • C for 7-14 days on malt extract agar (MEA: malt extract 2% and agar 2%) and cornmeal agar (CMA: cornmeal 2% and agar 2% agar) under natural light for morphophysiological studies [23]. To determine the maximum growth temperature, each strain was tested three times on PDA for 5 days between 25 and 45 • C.
Microscopic observations were conducted with a Zeiss AX10 Imager A2 light microscope using differential interference contrast illumination. Water or Shear's mounting medium was used for microscopic observation. Description of the sporangial state was based on an integrative observation of all strains within a certain taxon. Capitalized color designations in the descriptions were from Ridgway [31].

DNA Extraction, Amplification and Sequence Analyses
Total genomic DNA extraction, amplification and sequencing of partial nuclear small subunit (nSSU) rDNA, ITS and D1-D3 region of nLSU rDNA, and the partial γ-actin gene (act1) were conducted according to the protocols described by Wang et al. [23,32]. The partial regions of DNA replication licensing factor (MCM7) and mitochondrial cytochrome c oxidase subunit 1 (cox1) were amplified using the primer pairs Mcm7-8af (or MCM-709f)/MCM7-16r [33,34] and cox1/cox4 [35], respectively. Polymerase chain reaction (PCR) program of the above two loci included an initial denaturation at 94 • C for 5 min, 39 cycles of 94 • C for 1 min, 53 • C for 1 min and 72 • C for 50 s and a final extension of 72 • C for 10 min. DNA sequencing was performed at Majorbio Bio-technology Company Ltd. (Beijing, China) with the PCR primers. Generated sequences were assembled for consensus in Sequencher 4.1.4 (Gene Codes Corp., Ann Arbor, Michigan); then, they were aligned with MAFFT 6.952 [36,37] and optimized manually in BioEdit 7.1.3.0 [38]. Sequence data generated in this study are deposited in GenBank (Table S1).
Phylogenetic analysis based on the nLSU rDNA sequences was performed by using the neighbor-joining (NJ) method executed in MEGA7 [39] with Kimura 2-parameter model. The topology of the trees was assessed by 1000 bootstrap replications. For multi-gene phylogenetic analyses, optimized sequence alignments of nSSU, ITS, nLSU, act1, MCM7 and cox1 were combined with SequenceMatrix1.7.8 [40]. Phylogenetic analyses using the maximum likelihood (ML), maximum parsimony (MP) and strict clock Bayesian inference (BI) algorithms were performed with RAxML8.0.23 [41], MEGA7 [39] and MrBayes v. 3.0b4 [42,43], respectively. The parameters for ML, MP and BI analyses were set following the methods described by Wang et al. [23,32]. Trees were visualized with Figtree [44] and edited in Adobe Illustrator CS4. The node reliability was assessed by no less than 70% of maximum likelihood bootstrap proportion (MLBP) and maximum parsimony bootstrap support value (MPBS) and no less than 95% of Bayesian posterior probability values (BPP) [45].

Molecular Phylogenetic Analyses
In order to investigate the phylogenetic relationships of the strains sequenced in this study with the three subclades recognized by Ogawa et al. [28], the nLSU rDNA sequences of the U. ramanniana and related species determined by Ogawa et al. [28] were retrieved from GenBank and integrated with nLSU sequences of the strains determined in this study for a phylogenetic analysis. The phylogenetic tree ( Figure S1) shows that the strains identified as U. ramanniana are polyphyletic and grouped together with U. angularis, U. gibberispora, U. heterosporus, U. swartii, and U. westeae and U. wiegerinckiae, with high bootstrap values. When more strains were added, the three subclades designated by Ogawa et al. [28] were not resolved as three well-supported linages ( Figure S1). Six U. ramanniana strains sequenced in this study (CGMCC 3.15777-3.15781 and NRRL 1296) were grouped in a clade together with the strains of subclade I. This clade also includes U. swartii, and U. westeae, but with low bootstrap support (<50%). The strains of the subclade II and subclade III of Ogawa et al. [28] and 13 new isolates employed in this study formed a well-supported (96%) clade together with U. angularis, U. gibberispora, U. heterosporus and U. wiegerinckiae. As mentioned by Ogawa et al. [28], the results indicate that strains of the U. ramanniana complex represent an assemblage of several genetically distinct species, but nLSU rDNA alone is insufficient in resolving the relationships of the strains in this species complex. Therefore, a phylogenetic construction based on multiple genes was performed in this study to clarify cryptic species in the U. ramanniana complex.
Multi-gene phylogenetic analyses were carried out based on the sequence data of six loci generated in this study or retrieved from GenBank (Table S1). A total of 86 isolates representing all currently accepted taxa of Umbelopsis were analyzed with Mortierella minutissima and M. verticillata as outgroups. Six sequence alignments (including gaps) were obtained with 1667 characters in nSSU, 723 in ITS, 1013 in nLSU, 820 in act1, 1032 in MCM7 and 1742 in cox1, respectively. The final combined sequence matrix consists of 6996 characters, including 4915 constant, 777 parsimony uninformative and 1304 parsimony informative characters. The most parsimonious tree resulted in a tree length of 4454 steps, consistency index (CI) of 0.655, retention index (RI) of 0.929 and rescaled consistency index (RC) of 0.608. For the Bayesian inference, the GTR + I + G model was selected for nSSU, act1 and MCM7, GTR + G model for ITS and nLSU and HKY + G model for cox1, respectively. The ML, MP and BI analyses produced similar trees with nodes supported by high bootstrap values. The BI phylogeny tree ( Figure 1) is presented with BI, ML and MP bootstrap values indicated along branches.

Maximum Growth Temperature
A total of 25 strains in the Umbelopsis ramanniana species complex were tested three times for their maximum growth temperature. Detailed results for each culture are presented in Table S2. The maximum growth temperature of those strains varies from 31 • C to 38 • C, which indicates that there may be cryptic species in the species complex. According to above mentioned phylogenetic clades, the maximum growth temperature ranges for the clades C1 to C6 ( Figure 1

Morphology and Taxonomy
Like in other species of the Umbelopisis, morphology is an important basis for the taxonomy of the U. ramanniana species complex. The following characteristics are of prime importance to the classification of Umbelopsis: colony color, the pattern and length of branches, the type and shape of sporangia, the shape and size of columellae and sporangiospores, and the formation of chlamydospores. In this study, more criteria, such as colony diameter, the deliquescence of sporangial walls and the possession of collars, have been adopted to investigate differences in the U. ramanniana complex. Combined with the results of phylogenetic analyses, the mainly morphological comparison is summarized in Table 1.
Based on the results of the multi-gene phylogeny (Figure 1), morphological comparison (Table 1) and maximum growth temperature test (Table S2), five new species are introduced for the isolates formerly identified as U. ramanniana. These five novel taxa are described here. Meanwhile, U. ramannianus is re-described based on our isolated speci-mens. Additionally, accompanied by these new members, a diagnostic key for all taxa of Umbelopsis is updated herein. Notes: The ex-type culture CBS 219.47 of U. curvata was originally identified as U. ramanniana by Domsch et al. [6] and then illustrated by Meyer and Gams [20]. According to the nLSU phylogeny by Ogawa et al. [28], this culture belonged to subclades III of the U. ramanniana complex. In the present study, multi-gene phylogenetic analyses revealed that U. curvata was distant from the cluster consisting of U. angularis and U. ramannina ( Figure 1) and formed a sister cluster to a group that contained U. heterosporus and U. wiegerinckiae. Morphologically, U. curvata differs from U. ramanniana by forming sporangiospores that are larger (3.2-4.5 × 2.0-2.4 µm) and curved; and can be distinguished from U. angularis by not exhibiting angular sporangiospores. Compared to irregular sporangiospores and columellae in U. heterosporus, U. curvata possesses distinct and depressed globose columella and smooth and ellipsoidal sporangiospores. Umbelopsis curvata can be distinguished from U. wiegerinckiae by its pinkish colony and sympodial branching. Additionally, U. curvata is somewhat similar to U. macrospora and U. oblongielliptica in the shape of sporangiospores (ellipsoidal and curving to one side; Table 1), but they are distantly related in molecular phylogeny ( Figure 1). U. angularis by not exhibiting angular sporangiospores. Compared to irregular sporangiospores and columellae in U. heterosporus, U. curvata possesses distinct and depressed globose columella and smooth and ellipsoidal sporangiospores. Umbelopsis curvata can be distinguished from U. wiegerinckiae by its pinkish colony and sympodial branching. Additionally, U. curvata is somewhat similar to U. macrospora and U. oblongielliptica in the shape of sporangiospores (ellipsoidal and curving to one side; Table 1), but they are distantly related in molecular phylogeny ( Figure 1).  Etymology: dura referring to the permanent nature of sporangial walls, which dissolve slowly and appear stronger than the other species in the U. ramanniana complex.
Diagnosis: Umbelopsis dura ( Figure 3) differs from other species due to slowly deliquescent sporangial walls and Indian red to dark vinaceous-brown colonial color.
Etymology: dura referring to the permanent nature of sporangial walls, which dissolve slowly and appear stronger than the other species in the U. ramanniana complex.
Diagnosis: Umbelopsis dura ( Figure 3) differs from other species due to slowly deliquescent sporangial walls and Indian red to dark vinaceous-brown colonial color.  Notes: Umbelopsis dura can be separated from all other Umbelopsis species in the cox1 gene by a significant insertion. Phylogenetic inferences show that this species is closely related to U. oblongielliptica. However, they can be easily distinguished by the characteristics of the growth rate (41-45 mm vs. 60-62 mm in diam. on CMA after 10 days at 18 • C; Table 1), the deliquescence of sporangial walls (slowly vs. quickly) and the shape and size of sporangiospores (ovoid and 2.8-3.3 × 1.9-2.4 µm vs. oblong-ellipsoidal and 4.0-5.0 × 1.6-2.0 µm; Table 1). Although the colony colors of U. dura and U. angularis are somewhat similar, the two species differ in their sporangiospores shape and are distantly related in molecular phylogeny (Figure 1). Etymology: macrospora referring to the size of its sporangiospores, bigger than other species in the U. ramanniana complex.
Maximum growth temperature: 38 °C. Notes: The only strain NRRL 5844 of U. macrospora was received as U. ramanniana. Previously, the strains NRRL 5884 and NRRL 1296 (the ex-hotype of U. oblongielliptica) Maximum growth temperature: 38 • C. Notes: The only strain NRRL 5844 of U. macrospora was received as U. ramanniana. Previously, the strains NRRL 5884 and NRRL 1296 (the ex-hotype of U. oblongielliptica) were widely used as U. ramanniana in the phylogenetic analyses. However, Nagy et al. [26] reported that the chromosomal banding patterns in those two cultures are significantly diverse in orthogonal field alternation gel electrophoreses (OFAGE) and contour clamped homogeneous electric field gel electrophoreses (CHEF). In the present study, multi-gene phylogenetic inferences show that U. macrospora is basal to the U. oblongielliptica/ U. dura cluster (BPP: 1.00; MLBP: 94; MPBS: 100). However, it was separated from this cluster in the nLSU tree ( Figure S1). This species is morphologically similar to U. oblongielliptica but differs in the growth rate (46 mm vs. 60-62 mm in diam. after 10 days at 18 • C), sporangiospore size (4.0-4.9 × 2.4-2.8 µm vs. 4.0-5.0 × 1.6-2.0 µm) and macro-chlamydospores (undiscovered vs. abundant). Considering the significant divergences in the nLSU tree ( Figure S1), morphological characteristics and chromosomal banding patterns demonstrated by Nagy et al. [26], those two isolates were treated as different species.
Etymology: oblongielliptica referring to the shape of sporangiospores. Diagnosis: Umbelopsis oblongielliptica ( Figure 6) differs from other species by forming oblong-ellipsoidal sporangiospores (3.2-) 4.0-5.0 (-5.5) × 1.6-2.0 (-2.5) µm. This species is also characterized by the rapid colony extension (up to 64-70 mm in diameter after 10 d at 18 °C on MEA) and the production of abundant macro-chlamydospores on the substrate of both CMA and MEA.    Notes: The strains of clade C2 in the multi-gene phylogenetic tree ( Figure 1) were characterized by producing ellipsoidal spores, small but distinct columellae and two types of chlamydospores, which is compatible with Möller's description for U. ramanniana [14].
Moreover, they formed a sister lineage to U. angularis, which was a variant of U. ramanniana in the sense of Linnermann [15,16]. As a result, the strains in clade C2 were regarded as representative of the U. ramanniana in this study.
Umbelopisis ramanniana can be distinguished from U. angularis by having ellipsoidal sporangiospores and a lighter colony color. Umbelopisis curvata (C1, Figure 1) can be distinguished from U. ramanniana (C2, Figure 1) by its columellae shape and the shape and size of sporangiospores (Table 1). In addition, slight morphological variations were observed in several isolates of this clade. The sporangiophores of isolates CGMCC 3.6646 and CGMCC 3.15774 are shorter than other isolates, which may be intraspecific variations caused by differences in geographical environment.
In this species complex, remarkable variations were found in the monophyletic cluster consisting of U. microsporangia, U. dura, U. oblongielliptica and U. macrospora (C3, C4, C5 and C6, respectively; Figure 1). Umbelopisis microsporangia and U. dura produce smaller ovoid sporangiospores; however, some of them are oblong-ellipsoidal to ellipsoidal in U. oblongielliptica and U. macrospora (Figure 1 and Table 1). Compared to U. microsporangia and other taxa in the U. ramanniana complex, the deliquescence of sporangia walls in U. dura is slower. Moreover, the colony diam. after 10 days at 18 • C in the clade U. microsporangia is 35-40 mm, which is 41-45 mm in U. dura. Regarding their morphology, two single strain clades C5 and C6 can be easily distinguished from each other by their colony diam., the shape and size of columellae, sporangiospores size and macro-chlamydospores (abundant vs. undiscovered). Therefore, we treated them as different taxa U. oblongielliptica and U. macrospora.

Discussion
The noticeable variations of Umbelopsis ramanniana have been described by the morphological characteristics of their sporangiospores shape, columella size and chlamydospore production, and by their ubiquitous ecological distribution by many studies [22,24,27,28]. However, it was difficult to make a clear delimitation as those morphological characteristics vary continuously among the isolates. Previous research indicated that ITS and nLSU rDNA were not sufficient in achieving species-level identification for the genus Umbelopsis [15,20,27,28,46]. In the present study, based on a combined analysis of multi-loci phylogeny (nSSU, ITS and nLSU rDNA, act1, MCM7 and cox1), morphology and maximum growth temperature, isolates previously identified as U. ramanniana were re-examined and divided into six clades, including five morphologically cryptic lineages. They are described herein as novel species, namely U. curvata, U. dura, U. macrospora, U. microsporangia and U. oblongielliptica.
Umbelopsis ramanniana was meagerly described by Möller [14] without illustration or typification. Based on the culture isolated from mycorrhiza of trees in Eberswalde (Brandenburg, Germany; habitation in protolog) by Möller, Lendner [47] provided a more detailed description and illustration of sporangia, columella and chlamydospores for the species. According to the two studies mentioned above, this species produces roseate colonies, ellipsoidal sporangiospores (2.5 × 1.7 µm), small but distinct columellae and two types of chlamydospores. In this study, the isolates in the clade C2 ( Figure 1) tend to fit the descriptions of Möller [14] and Lendner [47] best. Moreover, the clade C2 formed a sister linage to U. angularis, which is a closely related species to U. ramanniana in the sense of Linnemann [16]. Therefore, strains in the clade C2 were chosen as the deputies of U. ramanniana, although there may have been some arbitrariness.
Based on the sequences of the nLSU rDNA D1/D2 region, Ogawa et al. [28] examined intraspecific variations of U. ramanniana and pointed out that this species is an assemblage of several genetically distinct species. In the present study, U. ramanniana was polyphyletic in the nLSU tree ( Figure S1), which supports the conclusion of Ogawa et al. [28]. When 23 strains of U. ramanniana were added to the subclades identified by Ogawa et al. [28], more divergent clades were recognized in this species complex. However, due to the low bootstrap values in the nLSU tree, the phylogenetic relationships between subclades of U. ramanniana and closely related species were undetermined. As mentioned by Ogawa et al. [27,28], it is more reasonable to make the taxonomic treatment when species relationships are clarified.
In the present study, the six-loci phylogeny of Umbelopsis was reconstructed and obtained strong support values. The U. ramanniana complex is polyphyletic and can be divided into six clades. The first two clades U. curvata (C1) and U. ramanniana (C2) were lo-cated in a well-supported lineage that included U. angularis, U. gibberispora, U. heteropsproa and U. wiegerinckiae. The last two species in this lineage are recently reported, of which, U. wiegerinckiae is characterized by forming umbellate sporangiophores, and U. heterosporus produces varied sporangiophores and columellae [48,49]. The other four species can be distinguished from each other by their unique sporangiospores. In detail, they are the biggest and unilaterally thickened oblong-ellipsoidal sporangiospores in U. gibberispora, bigger and oblong-ellipsoidal with slight curved to one side in U. curvata, smaller and ellipsoidal without any appendage in U. ramanniana and smallest and polygonal in U. angularis. Variations of sporangiospores seem to confirm the hypothesis that tight sporangial walls physically limit the free expansion of sporangiospores and consequently several kinds of shape of sporangiospores could evolve [15]. Moreover, it appears that the tighter sporangial walls seem to result in the deeper sporangial and colonial color. The color of colonies is pale vinaceous in U. gibberispora, brownish vinaceous to light russet-vinaceous in U. curvata and U. ramanniana, and etruscan red to prussian red in U. angularis. The acquisition of a tight sporangial wall should be a main evolution strategy for taxa in this lineage, which supports the hypothesis that relatively few mutations are required to determine a tight sporangial wall [15]. Hence, the differentiation of the lineage is probably a recent event in the evolution of the Umbelopsis. The other four clades U. dura (C4), U. macrospora (C6), U. microsporangia (C3) and U. oblongiellptica (C5) were clustered together with U. swartii and U. westeae. These species formed another well-supported lineage. In this lineage, the sporangiospore shape of the above mentioned six species also varies from appendage to smooth and subglobose to oblong-ellipsoidal. In detail, the sporangiospores are subglobose to ovoid in U. microsporangia, ovoid in U. dura, ellipsoidal to oblong-ellipsoidal with irregularly slight curved to one side in U. macrospora and U. oblongielliptica, and appendaged in U. swartii and U. westeae. It is suggested that variations in sporangiospores shape and size are acquired independently in those two lineages. Moreover, U. dura and U. microsporangia possess a similar sporangiospore shape and size but differ in their maximum growth temperature. Additionally, the maximum growth temperature increased gradually from 31-32 • C to 38 • C in the clades U. microsporangia (C3) to U. macrospora (C6) ( Table S2). It is indicated that at least two strategies occurred in the evolution of the lineage of U. swartii through to U. microsporangia.
As shown in previous studies, the ITS barcode was not sufficient in achieving species level identification in Umbelopsis [27,32]. Secondary barcodes, usually protein-coding genes, have been introduced for species discrimination [50,51]. The extensively used loci, such as beta-tubulin (ßtub), translation elongation factor 1-alpha (TEF1), the largest subunit of RNA polymerase II (RPB1) and the second largest subunit of RNA polymerase II (RPB2), were tested, but resulted in multiple copies [32]. The present study preliminarily suggests that the MCM7 should be a suitable secondary barcode, but this needs to be further studied.

Conclusions
The multi-gene (nSSU, ITS, nLSU, act1, MCM7 and cox1) phylogeny were proved to be reliable indications of taxon differentiation for the Umbelopsis ramanniana complex. Five species are newly described from the species complex: U. curvata, U. dura, U. macrospora, U. microsporangia and U. oblongielliptica. Those species can be distinguished by morphological traits in combination with the speed of growth and their maximum growth temperature.