Seven New Species of Eurotiales (Ascomycota) Isolated from Tidal Flat Sediments in China

Tidal flats have been reported to contain many microorganisms and play a critical role in maintaining biodiversity. In surveys of filamentous fungi from tidal flat sediments in China, seven new species of Eurotiales were discovered and described. Morphological characteristics and DNA sequence analyses of combined datasets of the BenA, CaM, and RPB2 regions support their placements and recognition as new species. Aspergillus liaoningensis sp. nov. and A. plumeriae sp. nov. belong to sections Candidi and Flavipedes of subgenus Circumdati, and A. subinflatus sp. nov. is a member of section Cremei of subgenus Cremei. Penicillium danzhouense sp. nov., P. tenue sp. nov., and P. zhanjiangense sp. nov. are attributed to sections Exilicaulis and Lanata-Divaricata of subgenus Aspergilloides. Talaromyces virens sp. nov. is in section Talaromyces. Detailed descriptions and illustrations of these novel taxa are provided. Their differences from close relatives were compared and discussed.

During the examinations of filamentous fungi isolated from tidal flat sediments in different provinces in China, seven undescribed taxa were encountered.Judging by the cultural and microscopic characteristics, they belong to Aspergillus, Penicillium, and Talaromyces.Their taxonomic placements were further confirmed by carrying out multilocus phylogenetic analyses of β-tubulin (BenA), calmodulin (CaM), and the second-largest subunit of RNA polymerase II (RPB2).The distinctions between the novel species and their close relatives were compared.

Sampling and Fungal Isolation
Tidal flat sediment samples were collected from Guangdong, Hainan, and Liaoning provinces from August to October 2020.Sediment samples were kept at 4 • C until used.Strains were isolated using a 3% sea salt medium with the dilution plate method and were preserved in the China General Microbiological Culture Collection Center (CGMCC).Dried cultures were deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS).

Morphological Observations
Colony characteristics were observed and described following the method of Visagie et al. [14].Four standard growth media were used: Czapek yeast autolysate agar (CYA, yeast extract Oxoid), malt extract agar (MEA, Amresco), yeast extract agar (YES), and potato dextrose agar (PDA) [48][49][50].A twenty-five percent lactic acid (LA) solution was used as the mounting medium for the microscopic examinations of structures and measurements of the conidial head, stipe, phialide, vesicle, and conidia.The methods for inoculation, morphological observation, and digital recordings were performed following previous studies [51,52].

Phylogenetic Analyses
The newly generated sequences and those retrieved from GenBank are listed in Table 1.They were assembled and aligned with BioEdit 7.0.5 [58] and manually edited.To evaluate statistical congruence amongst the loci BenA, CaM, and RPB2, the partition homogeneity test (PHT) was performed in PAUP*4.0b10[59] with 1000 replicates.To determine the positions of these strains, the datasets of these regions belonging to Aspergillus sect.Candidi, Cremei and Flavipedes, Penicillium sect.Exilicaulis and Lanata-Divaricata, and Talaromyces sect.Talaromyces were compiled and analyzed by the maximum likelihood (ML) and Bayesian inference (BI) methods.ML analysis was performed with the default GTRCAT model using RAxML [60].The BI analysis was conducted by MrBayes 3.2.5 [61].Nucleotide substitution models were determined by MrModeltest 2.3 [62].Dendrogram trees were visualized and edited using TreeView v. 1.6.6 [63] and FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/ figtree/ (accessed on 25 November 2018)).A Bayesian inference posterior probability (BIPP) greater than 90% and a maximum likelihood bootstrap support (MLBS) greater than 70% were shown at the nodes.

Phylogenetic Analyses
To determine the positions of the Aspergillus strains, Hamigera avellanea Stolk & Samson and Penicillium expansum Link were used as outgroup taxa.The partition homogeneity tests (p = 0.01 and 0.25, respectively) indicated that the individual partitions were not highly incongruent [64]; thus, these three loci were combined for the phylogenetic analyses.The phylogenetic trees showed that strains CGMCC 3.25201 and 3.25202 were located in sect.Candidi and Flavipedes, respectively (Figure 1).

Phylogenetic Analyses
To determine the positions of the Aspergillus strains, Hamigera avellanea Stolk & Samson and Penicillium expansum Link were used as outgroup taxa.The partition homogeneity tests (p = 0.01 and 0.25, respectively) indicated that the individual partitions were not highly incongruent [64]; thus, these three loci were combined for the phylogenetic analyses.The phylogenetic trees showed that strains CGMCC 3.25201 and 3.25202 were located in sect.Candidi and Flavipedes, respectively (Figure 1).In the phylogenetic analyses of Penicillium sect.Exilicaulis, H. avellanea and A. glaucus (L.) Link served as outgroup taxa.The partition homogeneity test (p = 0.01) indicated that the individual partitions were not highly incongruent [64]; thus, these three loci were combined for the phylogenetic analyses.The phylogenetic trees showed that strains CGMCC 3.25204 and 3.25205 were located in ser.Erubescentia (BIPP/MLBS = 100%/93%) (Figure 3).The strain CGMCC 3.25204 was clustered with P. canis S.W. Peterson, while strain CGMCC 3.25205 was grouped with P. striatisporum Stolk, both receiving full support.the individual partitions were not highly incongruent [64]; thus, these three loci were combined for the phylogenetic analyses.The phylogenetic trees showed that strains CGMCC 3.25204 and 3.25205 were located in ser.Erubescentia (BIPP/MLBS = 100%/93%) (Figure 3).The strain CGMCC 3.25204 was clustered with P. canis S.W. Peterson, while strain CGMCC 3.25205 was grouped with P. striatisporum Stolk, both receiving full support.In the phylogenetic analyses of Penicillium sect.Lanata-Divaricata, H. avellanea and P. glabrum (Wehmer) Westling were used as outgroup taxa.The partition homogeneity test (p = 0.01) indicated that the individual partitions were not highly incongruent [64]; thus, these three loci were combined for the phylogenetic analyses.The strain CGMCC 3.25206 was placed in ser.Janthinella and clustered with P. janthinellum Biourge with high supporting values (BIPP/MLBS = 100%/100%) (Figure 4).In the phylogenetic analyses of Penicillium sect.Lanata-Divaricata, H. avellanea and P. glabrum (Wehmer) Westling were used as outgroup taxa.The partition homogeneity test (p = 0.01) indicated that the individual partitions were not highly incongruent [64]; thus, these three loci were combined for the phylogenetic analyses.The strain CGMCC 3.25206 was placed in ser.Janthinella and clustered with P. janthinellum Biourge with high supporting values (BIPP/MLBS = 100%/100%) (Figure 4).[64]; thus, these three loci were combined for the phylogenetic analyses.The phylogenetic tree showed that strain CGMCC 3.25207 was grouped with the     Colony characteristics: On CYA 25 °C, 7 days: Colonies irregular, plain, cracked; margins narrow, nearly entire; mycelia white to cream; texture velutinous to floccose; sporulation dense; conidia en masse cream; no exudate, no soluble pigments; reverse cream to light yellow, white at periphery.On CYA 37 °C, 7 days: Colonies irregular, slightly protuberant in centers; margins narrow to moderately wide; mycelia cream; texture velutinous to floccose; sporulation dense; conidia en masse white to cream; no exudate, no soluble pigments; reverse dark cream, brown at centers.On MEA 25 °C, 7 days: Colonies nearly circular, slightly protuberant in centers; margins narrow to moderately wide, nearly entire; mycelia white to light cream; texture velutinous to floccose; sporulation dense in center, light cream; no exudate, no soluble pigments; reverse cream, brown at centers, white at periphery.On PDA 25 °C, 7 days: Colonies irregular, plain, cracked; Colony characteristics: On CYA 25 • C, 7 days: Colonies irregular, plain, cracked; margins narrow, nearly entire; mycelia white to cream; texture velutinous to floccose; sporulation dense; conidia en masse cream; no exudate, no soluble pigments; reverse cream to light yellow, white at periphery.On CYA 37 • C, 7 days: Colonies irregular, slightly protuberant in centers; margins narrow to moderately wide; mycelia cream; texture velutinous to floccose; sporulation dense; conidia en masse white to cream; no exudate, no soluble pigments; reverse dark cream, brown at centers.On MEA 25 • C, 7 days: Colonies nearly circular, slightly protuberant in centers; margins narrow to moderately wide, nearly entire; mycelia white to light cream; texture velutinous to floccose; sporulation dense in center, light cream; no exudate, no soluble pigments; reverse cream, brown at centers, white at periphery.On PDA 25 • C, 7 days: Colonies irregular, plain, cracked; margins narrow, nearly entire; mycelia white to grey; texture velutinous to floccose; sporulation dense; conidia en masse grey; no exudate, no soluble pigments; reverse cream, brown at centers, white at periphery.On YES 25 • C, 7 days: Colonies nearly circular, protuberant in centers, edges irregular; margins narrow, nearly entire; mycelium light grey at center, white at margin; texture velutinous to floccose; sporulation moderately dense; no exudate, no soluble pigments; reverse white, light yellow at centers.

Colony characteristics:
On CYA 25 • C, 7 days: Colonies nearly circular, plain, slightly protuberant in centers; margins narrow to moderately wide, nearly entire; mycelia white to cream; texture velutinous; sporulation dense, conidia en masse white; no exudate, no soluble pigments; reverse light yellow.On MEA 25 • C, 7 days: Colonies nearly circular, wrinkled, slightly protuberant in centers, radially sulcate; margins moderately wide, nearly entire; mycelia white to cream; texture velutinous; sporulation dense in center, cream to light yellow; no exudate, no soluble pigments; reverse yellowish-brown.On PDA 25 • C, 7 days: Colonies nearly circular, wrinkled, slightly concave at centers, radially sulcate; margins moderately wide, nearly entire; mycelia white, bright yellow at center; texture velutinous to floccose; sporulation dense in center, bright yellow; no exudate, no soluble pigments; reverse light-brown at centers, white at periphery.On YES 25 • C, 7 days: Colonies nearly circular, wrinkled, slightly concave at centers, radially sulcate; margins moderately wide, nearly entire; mycelium white to cream at center, white at margin; texture velutinous; sporulation moderately dense, white to cream; no exudate, no soluble pigments; reverse white to light yellow.
Note: Among the known species of Aspergillus, A. plumeriae is distinct because of its yellowish-white colony on PDA.It is phylogenetically related to A. movilensis (Figure 1), but the latter differs in its ability to grow at 37 • C and production of pyriform vesicles with smaller sizes (5.0-16 µm in diam.)[65].Colony characteristics: On CYA 25 • C, 7 days: Colonies nearly circular, wrinkled, protuberant in centers, radially sulcate; margins narrow to moderately wide, nearly entire; mycelia white to grey; texture velutinous; sporulation dense; conidia en masse grey; no exudate, no soluble pigments; reverse light yellow.On CYA 37 • C, 7 days: Colonies nearly circular, lain; mycelia light pink to white; texture velutinous; no sporulation; no exudate, no soluble pigments; reverse light pink to white.On MEA 25 • C, 7 days: Colonies nearly circular, slightly wrinkled, slightly protuberant in centers; margins wide to moderately wide, nearly entire; mycelia white to grey; texture velutinous to floccose; sporulation dense in center, grey; no exudate, no soluble pigments; reverse yellowish-brown, yellow to white at centers.On PDA 25 • C, 7 days: Colonies nearly circular, slightly wrinkled, slightly protuberant in centers; margins moderately wide, nearly entire; mycelia white to light grey; texture velutinous to floccose; sporulation dense in center, light grey; no exudate, no soluble pigments; reverse pale yellow.On YES 25 • C, 7 days: Colonies nearly circular, wrinkled, protuberant in centers; margins narrow, nearly entire; mycelium white, light grey at center; texture velutinous to floccose; sporulation moderately dense, white; no exudate, no soluble pigments; reverse yellow, white at periphery.
Note: This species is phylogenetically related to P. striatisporum (Figure 3), but the latter differs in its ellipsoidal to ovoid and striate conidia [68].Sequence comparisons between the ex-type cultures of the two species revealed that 8 bp, 12 bp, and 3 bp divergences were detected for the BenA, CaM, and RPB2 regions.
Fungal Names: FN 571618.Etymology: The specific epithet refers to the type locality "Zhanjiang City" of the fungus.
Colony characteristics: On CYA 25 °C, 7 days: Colonies nearly circular, concave at centers, protuberant at margins, wrinkled, radially sulcate; margins narrow to moderately wide, nearly entire; mycelia white, light grey at center; texture velutinous to floccose; sporulation sparse; conidia en masse cream to light grey; no exudate, no soluble pigments; reverse yellowish-brown, light green to blackish-green at centers, white at periphery.On CYA 37 °C, 7 days: Colonies nearly circular, concave at centers, protuberant at margins, Colony characteristics: On CYA 25 • C, 7 days: Colonies nearly circular, deep, protuberant at centers, radially sulcate; margins narrow, entire; mycelia white, light yellow at center; texture velutinous to floccose; no sporulation; exudate yellow, no soluble pigments; reverse yellow, deepens in the center.On CYA 37 • C, 7 days: Colonies nearly circular, concave at centers, protuberant at margins, slightly wrinkled, radially sulcate; margins narrow; mycelia white, light yellow at centers; texture velutinous to floccose; sporulation sparse; exudate yellow to yellowish-brown, no soluble pigments; reverse yellow, yellowishbrown in centers.On MEA 25 • C, 7 days: Colonies nearly circular, protuberant at centers; margins narrow to moderately wide, nearly entire; mycelia white, light yellow at center; texture velutinous to floccose; sporulation sparse; no exudate, no soluble pigments; reverse yellowish-brown, white at periphery.On PDA 25 • C, 7 days: Colonies nearly circular, protuberant at centers; margins narrow, nearly entire; mycelia white to yellow; texture velutinous to floccose; sporulation sparse; exudate yellow to yellowish-brown, no soluble pigments; reverse light yellow.On YES 25 • C, 7 days: Colonies nearly circular, deep, concave at centers, protuberant at margins; margins narrow, entire; mycelium white; texture velutinous to floccose; no sporulation; no exudate, no soluble pigments; reverse yellowish-brown, white at center and periphery.
Note: This species is phylogenetically related to P. striatisporum (Figure 3), but the latter differs in its ellipsoidal to ovoid and striate conidia [68].Sequence comparisons between the ex-type cultures of the two species revealed that 8 bp, 12 bp, and 3 bp divergences were detected for the BenA, CaM, and RPB2 regions.nearly circular, deep, concave at centers, protuberant at margins, slightly wrinkled, radially sulcate; margins narrow, nearly entire; mycelium white to cream; texture velutinous to floccose; sporulation sparse; no exudate, no soluble pigments; reverse yellow, white at periphery.
Note: This species is phylogenetically related to P. janthinellum (Figure 4), but the latter differs in its faster growth rate on YES (44-46 mm) at 25 °C, while a slower growth rate was observed on CYA at 37 °C (20-30 mm) [69].Colony characteristics: On CYA 25 • C, 7 days: Colonies nearly circular, concave at centers, protuberant at margins, wrinkled, radially sulcate; margins narrow to moderately wide, nearly entire; mycelia white, light grey at center; texture velutinous to floccose; sporulation sparse; conidia en masse cream to light grey; no exudate, no soluble pigments; reverse yellowish-brown, light green to blackish-green at centers, white at periphery.On CYA 37 • C, 7 days: Colonies nearly circular, concave at centers, protuberant at margins, wrinkled, radially sulcate; margins narrow; mycelia pink, white to yellowish-grey at centers; texture velutinous; texture velutinous to floccose; sporulation sparse; exudate pink to pinkish-brown, no soluble pigments; reverse pink to dark pink, yellow at periphery.On MEA 25 • C, 7 days: Colonies nearly circular, concave at centers, protuberant at margins, wrinkled, radially sulcate; margins narrow to moderately wide, nearly entire; mycelia cream, brown at center; texture velutinous to floccose; sporulation dense; conidia en masse brownish-yellow; no exudate, no soluble pigments; reverse yellowish-brown, deepens in the center.On PDA 25 • C, 7 days: Colonies nearly circular, concave at centers, protuberant at margins, slightly wrinkled, radially sulcate; margins narrow to moderately wide, nearly entire; mycelia light yellow, brown at center; texture velutinous to floccose; sporulation dense; conidia en masse brownish-yellow; no exudate, no soluble pigments; reverse light green to dark green, deepens in the center.On YES 25 • C, 7 days: Colonies nearly circular, deep, concave at centers, protuberant at margins, slightly wrinkled, radially sulcate; margins narrow, nearly entire; mycelium white to cream; texture velutinous to floccose; sporulation sparse; no exudate, no soluble pigments; reverse yellow, white at periphery.
Note: This species is phylogenetically related to P. janthinellum (Figure 4), but the latter differs in its faster growth rate on YES (44-46 mm) at 25 • C, while a slower growth rate was observed on CYA at 37 • C (20-30 mm) [69].

Discussion
The genus Aspergillus is divided into six subgenera with 27 sections [13,22].Our new species A. liaoningensis was well-located in the ser.Candidi of sect.Candidi (BIPP/MLBS = 100%/100%) (Figure 1).Many species within this section have been reported as producers of secondary metabolites, such as taichunamides, shikimic acid derivatives, and terpenederived taichunins [71][72][73].Studies on the metabolic application of A. liaoningensis are surely our future goal.Aspergillus plumeriae belongs to ser.Spelaei of sect.Flavipedes, which is in accordance with the growth rate at 37 °C [65].Aspergillus subinflatus was classified as a member of ser.Inflati, sect.Cremei of subgen.Cremei, and is most related to A. inflatus in both phylogeny and morphology.However, sequence comparisons revealed that there were 69 bp, 74 bp, and 88 bp unmatched loci detected in the BenA, CaM, and RPB2 regions between them.
Both P. danzhouense and P. tenue form monoverticillate conidiophores, consistent with

Colony characteristics:
On CYA 25 • C, 7 days: Colonies nearly circular, protuberant in centers; margins narrow to moderately wide, nearly entire; mycelia white; texture velutinous; sporulation dense; conidia en masse dark olive green; no exudate, no soluble pigments; reverse light khaki, light brown at centers, light yellow and white at periphery.On CYA 37 • C, 7 days: Colonies nearly circular, deep, wrinkled, deeply concave in centers; margins narrow; mycelia white; texture velutinous; sporulation dense; conidia en masse grey; no exudate, no soluble pigments; reverse grey or white.On MEA 25 • C, 7 days: Colonies nearly circular, slightly protuberant in centers; margins wide, nearly entire; mycelia white; texture velutinous; sporulation dense in center, olive-drab; no exudate, no soluble pigments; reverse light yellow, light orange at centers.On PDA 25 Note: This species is morphologically and phylogenetically related to T. xishaensis (Figure 5).However, the latter has greyish-green colonies on CYA, yellowish-green colonies on MEA, and grey to bluish-green colonies on YES [70].

Discussion
The genus Aspergillus is divided into six subgenera with 27 sections [13,22].Our new species A. liaoningensis was well-located in the ser.Candidi of sect.Candidi (BIPP/MLBS = 100%/100%) (Figure 1).Many species within this section have been reported as producers of secondary metabolites, such as taichunamides, shikimic acid derivatives, and terpene-derived taichunins [71][72][73].Studies on the metabolic application of A. liaoningensis are surely our future goal.Aspergillus plumeriae belongs to ser.Spelaei of sect.Flavipedes, which is in accordance with the growth rate at 37 • C [65].Aspergillus subinflatus was classified as a member of ser.Inflati, sect.Cremei of subgen.Cremei, and is most related to A. inflatus in both phylogeny and morphology.However, sequence comparisons revealed that there were 69 bp, 74 bp, and 88 bp unmatched loci detected in the BenA, CaM, and RPB2 regions between them.
Both P. danzhouense and P. tenue form monoverticillate conidiophores, consistent with the other members of ser.Erubescentia in sect.Exilicaulis [7, 48,74,75].The phylogenetic results indicate that P. danzhouense is closely related to P. catenatum with high statistical support values (Figure 3); however, the latter differs in having larger phialides (8.0-12 × 2.5-3.0 µm vs. 4.6-8.7 × 1.7-2.2µm) [67].Moreover, there were 20 bp and 56 bp divergences in the BenA and CaM regions between them.Our results also showed that P. tenue was grouped with P. striatisporum, receiving full support (Figure 3), but the latter possesses ellipsoidal to ovoid and striate conidia [68].Penicillium dravuni, a marine-derived species belonging to this section, was not included in our phylogenetic analysis because no sequence data are available at present.However, it can be easily distinguished from P. danzhouense (white to light pink colonies) and P. tenue (white to light yellow colonies) because it forms yellow-gray colonies and has faster growth rates (25-35 mm vs. 21-24 mm, and 11-12 mm in CYA at 25 • C) [76].
Species of the genera Penicillium, Aspergillus, and Talaromyces have been isolated from various substrates, including dust, soil, dung, cloth, human tissue, plants, and insects [4,11].Due to the special ecological habitat, the fungi of these groups within tidal flats have high biodiversity and are an important source of active natural products [77][78][79][80].Recently, three novel taxa of Penicillium were reported in tidal flats [28].Similarly, the present study introduces three species of Aspergillus as well as three taxa of Penicillium and one of Talaromyces derived from tidal flat sediments.With the extensive use of molecular approaches, large-scale surveys in these unexplored tidal flats regions will significantly improve our knowledge of fungal species diversity in special ecological environments.

Conclusions
The filamentous fungi from tidal flat sediments in China were surveyed, and seven novel taxa of the genera Aspergillus, Penicillium, and Talaromyces were discovered.With the joining of the new species, the phylogenetic relationships among species of these three genera were updated.

Figure 2 .
Figure 2. BI tree generated from analyses of combined BenA, CaM, and RPB2 sequences of Aspergil-

Figure 4 .
Figure 4. BI tree generated from analyses of combined BenA, CaM, and RPB2 sequences of Penicillium sect.Lanata-Divaricata species.BIPP ≥ 90% (left) and MLBS ≥ 70% (right) are indicated at nodes.To determine the position of the Talaromyces strain, T. trachyspermus (Shear) Stolk & Samson and T. chongqingensis X.C. Wang & W.Y. Zhuang were used as outgroup taxa.The partition homogeneity test (p = 0.01) indicated that the individual partitions were not highly incongruent[64]; thus, these three loci were combined for the phylogenetic analyses.The phylogenetic tree showed that strain CGMCC 3.25207 was grouped with the

Figure 4 .
Figure 4. BI tree generated from analyses of combined BenA, CaM, and RPB2 sequences of Penicillium sect.Lanata-Divaricata species.BIPP ≥ 90% (left) and MLBS ≥ 70% (right) are indicated at nodes.To determine the position of the Talaromyces strain, T. trachyspermus (Shear) Stolk & Samson and T. chongqingensis X.C. Wang & W.Y. Zhuang were used as outgroup taxa.The partition homogeneity test (p = 0.01) indicated that the individual partitions were not highly incongruent[64]; thus, these three loci were combined for the phylogenetic analyses.The phylogenetic tree showed that strain CGMCC 3.25207 was grouped with the others of sect.