Results

Phylogenetic analyses

BLAST searches based on ITS, BenA, CaM, and RPB2 sequences indicated that the eight isolates belonged to Penicillium section Lanata-Divaricata. Species-level sequence similarities based on the ex-type isolates of the three proposed species are summarized in Table 2.

Species | Ex-type isolate | Closest species | Reference /type strain | Series | ITS (%) | BenA (%) | CaM (%) | RPB2 (%)
P. longyangense | YMF 1.10573T | P. koreense | KACC 47721T | Janthinella | 99.01 | 98.98 | 98.94 | 98.52
P. longlingense | YMF 1.11137T | P. daleae | CBS 211.28T | Dalearum | 99.22 | 99.75 | 98.24 | 99.34
P. zhenyuanense | YMF 1.11149T | P. bissettii | CBS 140972T | Rolfsiorum | 97.55 | 97.45 | 97.49 | 96.65

To clarify their phylogenetic positions and species boundaries, a concatenated dataset of ITS, BenA, CaM, and RPB2 sequences was constructed, including 71 strains representing three series (Janthinella, Dalearum, and Rolfsiorum). The final alignment comprised 2,188 characters (ITS: 476 bp, BenA: 435 bp, CaM: 517 bp, and RPB2: 760 bp). Maximum likelihood and Bayesian inference produced congruent tree topologies with strong support values (Fig. 1). The phylogenetic tree assigned the eight isolates to three proposed species placed in three different series of section Lanata-Divaricata: (1) P. longyangense was placed in series Janthinella and formed a sister lineage to P. koreense S.B. Hong, D.H. Kim & Y.H. You (100% BS, 1.00 PP); (2) P. longlingense was assigned to series Dalearum and clustered with P. daleae K.W. Zaleski (100% BS, 1.00 PP); (3) P. zhenyuanense was recovered in series Rolfsiorum and grouped with P. bissettii Visagie & Seifert (100% BS, 1.00 PP).

Figure 1.: Phylogenetic tree of Penicillium species based on the combined ITS, BenA, CaM and RPB2 sequences, inferred using maximum likelihood. Bootstrap values (BS ≥ 75%) and Bayesian posterior probabilities (PP ≥ 0.90) are shown at the nodes. Branches with BS ≥ 75% and PP ≥ 0.90 are shown in bold. The branch marked “4 ×” was shortened to one-quarter of its original length for presentation. Penicillium boreae CBS 111717T was used as the outgroup. Newly described species are indicated in bold. An asterisk indicates BS = 100% or PP = 1.00; T indicates ex-type strain.

Taxonomy

Penicillium longyangense

Z. F. Yu & X. K. Zhang sp. nov.

C89D8F97-58B7-5DBC-B920-481B552DE125

863617

Fig. 2

Etymology.

The epithet refers to Longyang District, the type locality.

Figure 2.: Colonies and microscopic characteristics of Penicillium longyangenseYMF 1.10573. A–C. Colony obverse on MEA, YES and CYA after 7 d at 25 °C in darkness; D–H. Conidiophores and phialides; I. Conidia. Scale bars: 10 μm.

Type.

China • Yunnan Province, Baoshan City, Longyang District, Lujiangba. 24°56'34.8"N, 98°50'44.5"E, 916.7 m alt., isolated from agricultural soil in a kidney bean (Phaseolus vulgaris) field, Dec. 2024, Z.F. Yu & X.K. Zhang (holotype YMFD 1.10573, dried culture; ex-type culture YMF 1.10573 = GDMCC 3.1370).

Subgeneric classification.

Subgenus Aspergilloides, section Lanata-Divaricata, series Janthinella.

Description.

Conidiophores smooth-walled, thin-walled, 170–570 × 2–3 μm (mean = 288 × 2.5 μm, n = 30). Penicilli monoverticillate, compact, terminal, occasionally with a divaricate branch. Metulae absent. Phialides ampulliform, borne in terminal whorls of 8–12, 6.5–11.5 × 2–3.5 μm (mean = 8.7 × 2.8 μm, n = 30). Conidia globose to subglobose, smooth-walled, 2–3 μm diam. (mean = 2.8 μm, n = 30).

Colony diam.

(in mm) 7 days, 25 °C: MEA 19–21, YES 22–24, CYA 24–26.

Culture characteristics.

7 days, 25 °C: Colonies on MEA flat, white to pale yellowish, floccose at the margin, dominated by aerial hyphae, sporulation sparse, pale green at the centre, exudate and soluble pigments absent. Colonies on YES floccose, centrally raised, radially sulcate, white, margin distinct, regular, sporulation scarce, exudate and soluble pigments absent. Colonies on CYA dense, floccose to fluffy, wrinkled, radially sulcate, centrally depressed, white to pale buff; margin regular, slightly fimbriate, sporulation sparse, exudate and soluble pigments absent. Colour descriptions follow Rayner (1970).

Additional materials examined.

China • Yunnan Province, Baoshan City, Longyang District, Lujiangba; isolated from agricultural soil in a kidney bean (Phaseolus vulgaris) field; Dec. 2024; Z.F. Yu; living culture YMF 1.11657.

Notes.

Phylogenetically, P. longyangense is closely related to P. koreense within the series Janthinella (Fig. 1). It differs from P. koreense by its slower growth on CYA and MEA after 7 d at 25 °C (24–26 mm and 19–21 mm, respectively, vs. 32–34 mm and 42–48 mm); paler colonies with sparse to scarce sporulation and absence of exudates and soluble pigments, whereas P. koreense produces grey-green conidia en masse, shows moderate to strong sporulation, and produces clear exudate droplets on CYA; shorter conidiophores (170–570 × 2–3 μm vs. 200–800 × 2–3 μm), generally narrower phialides (6.5–11.5 × 2–3.5 μm vs. 8–11.5 × 3–4 μm), and globose to subglobose conidia (2–3 μm diam. vs. 2.5–3.5 × 2–3 μm, globose to broadly ellipsoidal) (You et al. 2014). These phylogenetic and morphological differences support the recognition of P. longyangense as a distinct species.

Penicillium longlingense

Z. F. Yu & X. K. Zhang sp. nov.

95C6DE3C-6FEF-5ADF-B7B8-F2E96A6D79D1

863618

Fig. 3

Etymology.

The epithet refers to Longling County, the type locality.

Figure 3.: Colonies and microscopic characteristics of Penicillium longlingenseYMF 1.11137. A–C. Colony obverse on MEA, YES and CYA after 7 d at 25 °C in darkness; D–H. Conidiophores and phialides; I. Conidia. Scale bars: 10 μm.

Type.

China • Yunnan Province, Baoshan City, Longling County, Longjiang. 24°49'12"N, 98°44'34"E, 1,919.47 m alt., isolated from soil in a sweet potato (Ipomoea batatas) field, Sep. 2025, Z.F. Yu & X.K. Zhang (holotype YMFD 1.11137, dried culture; ex-type culture YMF 1.11137 = GDMCC 3.1371).

Subgeneric classification.

Subgenus Aspergilloides, section Lanata-Divaricata, series Dalearum.

Description.

Conidiophores smooth-walled, thin-walled, hyaline, mostly unbranched, occasionally furcate or branched near the apex, 80–190 × 2.5–4 μm (mean = 127 × 3 μm, n = 30). Penicilli biverticillate, compact to irregularly branched. Metulae divergent to appressed, 10–20 × 2–3.5 μm (mean = 15 × 2.9 μm, n = 30). Phialides ampulliform, borne in whorls of 4–6, 5–9 × 2–3.5 μm, with inconspicuous collarettes (mean = 6.9 × 2.6 μm, n = 30). Conidia globose to subglobose, finely roughened, 2–4 μm diam. (mean = 2.8 μm, n = 30).

Colony diam.

(in mm) 7 days, 25 °C: MEA 16–18, YES 15–17, CYA 14–16.

Culture characteristics.

7 days, 25 °C: Colonies on MEA are floccose to lanose, sporulation is prolific in the centre, coloured leek green, sporulation density decreases progressively from the centre to the periphery, the colour becoming paler, and a well-defined white margin develops at the extreme edge, exudate and soluble pigments absent. Colonies on YES low to slightly raised, white, weakly radially sulcate to wrinkled; margin well defined and regular, sporulation sparse to nearly absent, exudate and soluble pigments absent. Colonies on CYA white, dense, floccose, low to slightly raised, margin regular, sporulation sparse, exudate and soluble pigments absent. Colour descriptions follow Rayner (1970).

Additional materials examined.

China • Yunnan Province, Baoshan City, Longling County, Longjiang; isolated from soil in a sweet potato (Ipomoea batatas) field; Sep. 2025; Z.F. Yu; living cultures YMF 1.11153, YMF 1.11156, YMF 1.11658.

Notes.

Phylogenetically, P. longlingense is closely related to P. daleae (Fig. 1), but differs in several diagnostic cultural and morphological characters. In culture, P. longlingense grows more slowly on CYA and MEA after 7 d at 25 °C (14–16 mm and 16–18 mm, respectively) than P. daleae (30–34 mm and 25–35 mm, respectively) and lacks exudates and soluble pigments on both media; by contrast, P. daleae may produce clear brown exudates on CYA and typically produces yellow to reddish brown soluble pigments on MEA. Micromorphologically, P. longlingense has generally wider and slightly longer conidiophores (80–190 × 2.5–4 μm vs. 35–170 × 2.3–2.7 μm). Phialide dimensions partially overlap, although P. longlingense generally produces shorter and less variable phialides (5–9 × 2–3.5 μm vs. 6–7(–12) × 2.4–3.0 μm in P. daleae). In addition, P. longlingense produces globose to subglobose, finely roughened conidia (2–4 μm diam., mean = 2.8 μm), whereas P. daleae has spheroidal, conspicuously spinose conidia (2.6–3.2 × (2–)2.5–3 μm) (Cho et al. 2005). These differences distinguish P. longlingense from P. daleae and support its recognition as a distinct species in series Dalearum.

Penicillium zhenyuanense

Z. F. Yu & X. K. Zhang sp. nov.

32018D5F-E0FF-577D-802D-21B87E6347EF

863619

Fig. 4

Etymology.

The specific epithet refers to Zhenyuan County, the type locality of this species.

Figure 4.: Colonies and microscopic characteristics of Penicillium zhenyuanenseYMF 1.11149. A–C. Colony obverse on MEA, YES and CYA after 7 d at 25 °C in darkness; D–H. Conidiophores and phialides; I. Conidia. Scale bars: 10 μm.

Type.

China • Yunnan Province, Pu’er City, Zhenyuan Yi, Hani and Lahu Autonomous County, isolated from soil in a tea (Camellia sinensis) plantation, 23°56'29.8"N, 101°29'54.4"E, 2363.7 m alt, Sep. 2025, Z.F. Yu & X.K. Zhang (holotype YMFD 1.11149, dried culture; ex-type culture YMF 1.11149 = GDMCC 3.1372).

Subgeneric classification.

Subgenus Aspergilloides, section Lanata-Divaricata, series Rolfsiorum.

Description.

Conidiophores smooth-walled, thin-walled, 80–300 × 2–4 μm (mean = 147 × 3 μm, n = 30), simple or occasionally branched near the apex. Penicilli mostly monoverticillate, occasionally with short apical or subapical branches, appearing reduced biverticillate, compact to irregularly branched. Metulae, when present, short, poorly differentiated. Phialides ampulliform to slightly lageniform, borne in terminal whorls or on short apical and subapical branches, usually 4–9 per whorl, 7–12 × 1.5–3.5 μm (mean = 9.1 × 2.6 μm, n = 30). Conidia globose to subglobose, smooth to finely roughened, 2.5–3.5 μm diam. (mean = 2.9 μm, n = 30).

Colony diam.

(in mm) 7 days, 25 °C: MEA 20–22, YES 13–15, CYA 15–17.

Culture characteristics.

7 days, 25 °C: Colonies on MEA floccose to lanose, with dense sporulation at the centre, grey-green, becoming paler towards the margin, margin well defined, white, exudates and soluble pigments absent. Colonies on YES low to slightly raised, white, weakly radially sulcate to wrinkled, margin well defined, regular, sporulation sparse to nearly absent, exudate and soluble pigments absent. Colonies on CYA dense, floccose, white to pale buff, low to slightly raised, weakly radially sulcate, margin regular, sporulation sparse, exudate and soluble pigments absent. Colour descriptions follow Rayner (1970).

Additional materials examined.

China • Yunnan Province, Pu’er City, Zhenyuan Yi, Hani and Lahu Autonomous County; isolated from soil in a tea (Camellia sinensis) field; Sep. 2025; Z.F. Yu; living culture YMF 1.11656.

Notes.

Phylogenetically, P. zhenyuanense is closely related to P. bissettii within series Rolfsiorum (Fig. 1), but differs from P. bissettii by its markedly slower growth after 7 d at 25 °C on CYA, MEA and YES (15–17, 20–22 and 13–15 mm diam., respectively, vs. 42–43 mm on CYA, 35–43 mm on MEA and 47–52 mm on YES). In addition, P. zhenyuanense forms paler colonies, with grey-green sporulation mainly restricted to the colony centre on MEA and sparse to nearly absent sporulation on CYA and YES, whereas P. bissettii produces more rapidly spreading colonies with moderately dense sporulation on CYA and YES, pinkish red submerged mycelium on MEA, and brown to dark brown or brownish orange reverses on CYA and YES. Micromorphologically, P. zhenyuanense differs from P. bissettii in having smooth-walled, shorter conidiophores (80–300 × 2–4 μm vs. rough-walled stipes, 190–670 × 2.5–3.5 μm), mostly monoverticillate to occasionally reduced biverticillate penicilli, and short, poorly differentiated metulae when present, rather than more complex biverticillate to terverticillate penicilli with well-developed metulae. Conidia of P. zhenyuanense are globose to subglobose, smooth to finely roughened and slightly larger on average (2.5–3.5 μm diam.) than those of P. bissettii (2–3 μm diam.) (Visagie et al. 2016b). Together, these phylogenetic, cultural and morphological differences support the recognition of P. zhenyuanense as a distinct species in series Rolfsiorum.