Phylogenetic analyses
The strains of Moelleriella (18 strains for LSU, 17 strains for TEF and 15 strains from RPB1) were included in the phylogenetic analyses. A phylogenetic tree using a combined dataset of seventy specimens including allied genera, such as Aschersonia, Conoideocrella, Helicocollum, Orbiocrella, Regiocrella and Samuelsia with Purpureocillium lilacinum (CBS 431.87), P. lilacinum (CBS 284.36) as outgroup is shown in Fig. 1.
The numbers on the branches represent bootstrap values from the RAxML analysis and posterior probability from the Bayesian inference. The results revealed that seven major clades representing various genera found in Clavicipitaceae, such as Aschersonia, Conoideocrella, Helicocollum, Moelleriella, Orbiocrella, Regiocrella and Samuelsia are independently formed. The Moelleriella clade segregated into two distinct clades, Clade A and Clade B. Clade A segregated in two sister clades, subclade I and subclade II. Clade A subclade I includes five new species, namely M. chiangmaiensis, M. flava, M.Kanchanaburiensis, M. nanensis and M.nivea, and seven known species, viz. M. gracilispora, M. mollii, M. ochracea, M. phukhiaoensis, M. pongdueatensis, M. sinensis and M. zhongdongii. Most of the species in subclade I were from the Old World (OW), except M. ochracea and M. zhongdongii that were found from the New World (NW). Clade A subclade I species have mostly flattened stromata, except for M. pongdueatensis and M. zhongdongii with stud-shaped and thick-pulvinate stromata, respectively. Most species in subclade I have whitish stromata, except M.flava, M. phukhiaoensis and M.pongdueatensis which have pale yellow to yellow stromata. Subclade II includes ten known species, seven of which were found in the NW, namely M. basicystis, M. disjuncta, M. evansii, M. libera, M. phyllogena, M. rhombispora and M. umbospora. Only three were found from the OW, such as M. alba, M. chumphonensis and M. raciborskii. Five species in subclade II produce perithecia in tubercles, viz. M. alba, M. chumphonensis, M. evansii, M. libera and M. raciborskii, while other species have semi-immersed to immersed perithecia in stromata, such as M.basicystis, M. disjuncta, M.phyllogena, M. rhombispora and M.umbospora. Furthermore, most of the species in this subclade II have pale yellow to yellow stromata, except for M.alba with whitish stromata. Clade B includes nine known species with fully embedded perithecia, the species in this clade can be found both in the OW (M. africana, M. insperata, M. reineckiana, M. schizostachyi) and the NW (M. boliviensis, M. epiphylla, M. macrostroma, M. sloaneae, M. turbinata). Additionally, the phylogenetic analyses of our study revealed that the species in each clade show no correlation to the different geographic areas.
Taxonomy
Moelleriella chiangmaiensis Khonsanit, Noisripoom, Phosrithong & Luangsa-ard, sp. nov. Fig. 2
MycoBank. MB835903
Anamorph. Aschersonia-like.
Etymology. Refers to the locality where the type specimen was found, Chiang Mai Province.
Typification. THAILAND. Chiang Mai Province, Doi Inthanon National Park, KM. 25 Nature Trail; 18°35'15.66"N, 98°29'11.69"E; on scale insect (Coccidae) found on the underside of dicotyledonous leaves; 30 Jun. 2005; B. Thongnuch, K. Tasanathai, N. Boonyuen, P. Srikitikulchai, R. Ridkaew, S. Mongkolsamrit, W. Chaygate (holotype BBH13024, ex-type living culture BCC18029)
Description.Anamorphic stromata convex, flattened, globose to subglobose, hat-shaped, raised, 1-7 mm in diam, white, pale greenish yellow (160C) to moderate yellow (160A) conidial masses. Conidiomata scattered, irregularly in shaped, widely open orifice, rim around the orifice thick and raised, 400-500 × 150-270 µm. Phialides hyaline, cylindrical, (8-)9.5-15(-20) × 1-2 µm. Conidia hyaline, (7-)8-10(-11) × 1.5-2 µm. Paraphyses present, hyaline, filiform, up to 130 µm long, 1-1.5 µm wide. Teleomorphic stromata thin-umbonate, raised, with globose to subglobose base, 4-7 mm diam, white to moderate yellow (162A). Perithecia semi-embedded in stromata, most perithecia fused together, occasionally developing singly in stromata, numerous perithecia per stroma (≤10), obpyriform, 450-500 × 260-360 µm. Ostioles deep orange yellow (163A). Asci hyaline, cylindrical, (102-)120-167(-220) × (4-)4.5-6.5(-7) µm. Asci caps convex, 2 × 4 µm. Ascospores hyaline, disarticulating into part-spores, cylindrical with rounded ends, (7-)9-11(-12) × 1-2 µm. The teleomorph and anamorph could be present in the same stroma.
Culture characteristics. Colonies on PDA forming a compact mycelium, convex to pulvinate, attaining a diam of 8-10 mm in 2 wk, white, pale yellow green (155A) conidial masses covering stromatic colonies. Colony reverse brownish orange (164A), pale yellow (164D) at the margins.
Habitat. On scale insects (Coccidae) found on the underside of dicotyledonous leaves.
Additional specimens examined. THAILAND, Chiang Mai Province, Doi Inthanon National Park, KM. 25 Nature Trail; 18°35'15.66"N, 98°29'11.69"E; on scale insects (Coccidae) found on the underside of dicotyledonous leaves; 30 Jun. 2005; B. Thongnuch, K. Tasanathai, N. Boonyuen, P. Srikitikulchai, R. Ridkaew, S. Mongkolsamrit, W. Chaygate (BBH13024, BCC18030, BCC18031, BCC18064); Krabi Province, Jiranan Techaprasan’s house (Thap Prik); 8°10'53.31"N, 98°52'38.66"E; on scale insects (Coccidae), found on the underside of plant leaves (Poaceae); 12 Dec. 2012; J. Techaprasan (BBH33051); on scale insect (Coccidae) found on the underside of dicotyledonous leaves; 1 Jan. 2013; J. Techaprasan (BBH33697); Nakhon Ratchasima Province, Khao Yai National Park, Pha Kluaimai Waterfall Nature Trail; 14°26'21.46"N, 101°22'20.20"E; on scale insects (Coccidae) found on the underside of dicotyledonous leaves; 10 Jan. 2013; D. Thanakitpipattana, K. Tasanathai, P. Srikitikulchai (BBH33709, BCC60941)
Notes. Phylogenetically, M. chiangmaiensis is closely related to M. nanensis, M. nivea, M. mollii, M. ochracea, and M. sinensis (Fig. 1). Morphologically, M. chiangmaiensis is similar to M. ochracea in having flattened, globose to subglobose base, white stromata, widely open orifices. However, some specimens containing the anamorphic stromata of M. chiangmaiensis are hat-shaped, producing pale yellow conidial masses (Fig. 2 a), while M. ochracea produces yellowish orange to orange conidial masses (Chaverri et al. 2008). Additionally, the perithecia in M. chiangmaiensis are slightly wider than those reported for other species (Table 3) and have various shapes and position of the conidiomata in the stromata (Fig. 2 a-d). It distinctly produces cream masses of conidia on the colonies on PDA (Fig. 2 n).
Moelleriellaflava Khonsanit, Noisripoom, Phosrithong & Luangsa-ard, sp. nov. Fig. 3
MycoBank. MB835904
Anamorph. Aschersonia-like.
Etymology. From the Latin ‘flava’ meaning yellow. Refers to the yellow stromata of the specimens.
Typification. THAILAND. Nakhon Ratchasima Province, Khao Yai National Park, Mo Sing To Nature Trail; 14°26'21.46"N, 101°22'20.20"E; on scale insects (Coccidae), found on the underside of Ardisia sanguinolenta leaves; 9 Jan. 2013; A. Khonsanit, S. Mongkolsamrit, W. Noisripoom (holotype BBH33722, ex-type living culture BCC60924).
Description.Anamorphic stromata flat to umbonate, globose to subglobose, 1-4.5 mm in diam, pale greenish yellow (2D) to brilliant greenish yellow (1A), strong orange yellow conidial masses (163B). Only one conidioma per stroma, narrowly U-shaped, widely open orifice, 370-600 × 100-300 µm. Phialides hyaline, cylindrical, (10-)13-20(-22) × 1.5 µm. Conidia hyaline, fusiform, (9-)10.5-12.5(-14) × (1-)1.5-2 µm. Paraphyses present, hyaline, filiform, up to 173 µm long, 1-1.5 µm wide. Teleomorphic stromata flat to umbonate, globose to subglobose, 2.5-4.5 mm in diam, pale greenish yellow (1D) to brilliant greenish yellow (1A). Perithecia scattered or fused together, semi-embedded, numerous perithecia per stroma (³10), occasionally developing singly in stroma, obpyriform, 260-430 × 90-170 µm. Ostioles strong orange (N136B) to dark orange (N163A). Asci hyaline, cylindrical, (138-)158-206(-250) × 5-7 µm. Ascicaps convex, 3-5 × 4-7 µm. Ascospores hyaline, disarticulating into part-spores, cylindrical with truncated ends (6-)7-10.5(-11) × 1-1.5 µm. The teleomorph and anamorph are rarely found in the same stroma.
Culture characteristics. Colonies on PDA forming a compact mycelium, pulvinate, wrinkled surface, attaining a diam of 8.5-11 mm in 2 wk, white, pale yellow green (4D) conidial masses. Colony reverse moderate yellow (160A), pale yellow in the margins (160D).
Habitat. On scale insects (Coccidae), found only on underside of Ardisia sanguinolenta leaves.
Additional specimens examined. THAILAND, Nakhon Ratchasima Province, Khao Yai National Park, Mo Sing To Nature Trail; 14°26'21.46"N, 101°22'20.20"E; on scale insects (Coccidae), found on the underside of Ardisia sanguinolenta leaves; 9 Jan. 2013; A. Khonsanit, S. Mongkolsamrit, W. Noisripoom (BBH33722, BCC60925; BBH33723, BCC60926; BBH33724, BCC60927, BCC60928; BBH38474, BCC60929, BCC60930).
Notes.Moelleriella flava differs from other related species in producing yellow stromata (Fig. 3 a-d) and found only one side of conidioma on the stromata (Fig. 3 a-b, e-f). Moelleriella flava produces narrow obpyriform perithecia (Fig. 3 j-k) and longer asci than other species (Table 3). It is found on scale insects feeding only on Ardisia sanguinolenta leaves.
Moelleriella kanchanaburiensis Khonsanit, Mongkolsamrit, Noisripoom, Phosrithong Luangsa-ard sp. nov. Fig. 4
MycoBank. MB835906
Anamorph. Aschersonia-like.
Etymology. Refers to the place where the type specimen was found, Kanchanaburi Province.
Typification. THAILAND .Kanchanaburi Province, Takhian Thong Waterfall Nature Trail; 15.314651"N, 98.442993"E; on scale insects (Coccidae), found on the underside of bamboo leaves; 20 Nov. 2014; A .Khonsanit, D. Thanakitpipattana, K. Tasanathai, P. Srikitikulchai, .R Promharn, S. Mongkolsamrit, S. Wongkanoun, W. Noisripoom (holotype BBH40814, ex-type living culture BCC75981)
Description.Anamorphic stromata flat to umbonate, globose to subglobose, 1–7 in diam, 1–1.5 mm in thick, white, light yellow (17D) to strong orange yellow (17A) conidial masses. Conidiomata scattered around the center of stromata, widely open orifice, irregular or U-shaped, 200-350 × 150-250 µm. Phialides hyaline, cylindrical, (10-)11.5-36.5(-50) × 1-1.5 µm. Conidia hyaline, narrowly fusiform with thickened wall at ends, 10-12.5(-14) × 1.5-2 µm. Paraphyses present, hyaline, filiform, up to 110 µm long, 1-1.5 µm wide. Teleomorphic stromata raised, 2–3 mm in diam, white. Perithecia scattered, semi-embedded, numerous perithecia per stroma (³5). Ostioles pale green (151D). The teleomorph of M. kanchanaburiensis is immature, we could not observe the size and shape of perithecia, asci, asci caps and ascospores. The teleomorph and anamorph are occasionally found in the same stroma.
Culture characteristics. Colonies on PDA forming a compact mycelium, convex to pulvinate, flat in the margins, attaining a diam of 9.5-11 mm in 2 wk, white with brilliant greenish yellow (1B) conidial masses. Colony reverse brilliant greenish yellow (1B), white in the margins.
Habitat. On scale insects (Coccidae), found on the underside of bamboo and dicotyledonous leaves.
Additional specimens examined. THAILAND. Kanchanaburi Province, Takhian Thong Waterfall Nature Trail; 15°18'1.21"N, 98°26'54.90"E; on scale insect (Coccidae), found on dicotyledonous leaves; 20 Nov. 2014; A. Khonsanit, D. Thanakitpipattana, K. Tasanathai, P. Srikitikulchai, R. Promharn, S. Mongkolsamrit, S. Wongkanoun, W. Noisripoom (BBH40813, BCC75979, BCC75980).
Notes. Moelleriella kanchanaburiensis is found occurring on scale insects on the underside of bamboo and dicotyledonous leaves. The anamorph produces narrowly fusiform conidia with thickened ends appearing as yellow conidial masses in culture. This species can be easily distinguished from other Moelleriella species in producing multiple conidiomatal orifices on around the center of stromata surrounding the scale insects. Additionally, the phialides in M. kanchanaburiensis are longer than other Moelleriella species (Table 2).
Moelleriella nanensis Khonsanit, Noisripoom, Phosrithong & Luangsa-ard, sp. nov. Fig. 5
MycoBank. MB835907
Anamorph. Aschersonia-like.
Etymology. Refers to the locality where the type specimen was found, Nan Province.
Typification. THAILAND. Nan Province, Doi Mongkhon Nature Trail; 18°46'32.27"N, 100°46'22.95"E; on scale insects (Coccidae), found on the upper side of dicotyledonous leaves; 25 Jul. 2013; A. Khonsanit (holotype BBH36571, ex-type living culture BCC66303).
Description.Anamorphic stromata flattened to raised, globose to subglobose, 2-6 mm in diam, white, pale greenish yellow conidial masses (160C). Conidiomata scattered around the margins of stromata, irregular in shape, widely open orifices, 350-600 × 160-500 µm. Phialides hyaline, cylindrical, (9-)10-13.5(-19) × 1-1.5 µm. Conidia hyaline, fusiform, (8-)8.5-10.5(-11) × (1-)1.5-2 µm. Paraphyses present, hyaline, filiform, up to 110 µm long. 1-1.5 µm wide. Teleomorphic stromata raised to convex, globose to subglobose, 3-6 mm in diam, white. Perithecia scattered, semi-embedded, perithecia fused together, numerous perithecia per stroma (³20), obpyriform, 310-550 × 110-180 µm. Ostioles moderate yellow (160A) to strong greenish yellow (151A). Asci hyaline, cylindrical, (105-)161.5-224.5(-238) × (5-)5.5-9.5(-12) µm. Asci caps umbonate, 2-4 × 3.5-6 µm. Ascospores hyaline, disarticulating into part-spores, cylindrical with rounded ends, slightly indented in the middle, (7-)8-13(-18) × (1-)1.5-2 µm. The teleomorph and anamorph are present in the same stroma.
Culture characteristics. Colonies on PDA forming a compact mycelium, convex, surface wrinkled and flat in the margins, attaining a diam of 8-9 mm in 3 wk, white, pale yellow (160D) conidial masses. Colony reverse light greyish olive (197C), greyish yellow green (197D) at the margins.
Habitat. On scale insects (Coccidae), found on the upper side of dicotyledonous leaves.
Additional specimens examined. THAILAND. Nan Province, Doi Mongkhon Nature Trail; 18°46'32.27"N, 100°46'22.95"E; on scale insects (Coccidae), found on the upper side of dicotyledonous leaves; 25 July 2013; A. Khonsanit (BBH36571, BCC66304, BCC66305).
Notes. Phylogenetically, M. nanensis is closely related to M. nivea (Fig. 1) and is found on the upper side of dicotyledonous leaves. It differs from M. nivea and other species in producing longer obpyriform perithecia (Fig. 5 j), umbonate asci caps (Fig. 5 l) and ascospores that are slightly indented in the center (Fig. 5 m). The conidiomatal orifice in M. nanensis could be found surrounding the outer parts of the stroma (Fig. 5 b-c) while in most species the orifices are situated in the center of stromata.
Moelleriella nivea Khonsanit, Mongkolsamrit, Noisripoom, Phosrithong & Luangsa-ard, sp. nov. Fig. 6
MycoBank. MB835908
Anamorph. Aschersonia-like.
Etymology. From the Latin ‘nivea’ meaning white. Refers to the white stromata of the specimens.
Typification.THAILAND. Surat Thani Province, Khao Sok National Park, Wing Hin Waterfall Nature Trail; 8°54'55.64"N, 98°31'42.25"E, on scale insects (Coccidae), found on dicotyledonous leaves; 28 Nov. 2012; A. Khonsanit, D. Thanakitpipattana, K. Tasanathai, P. Srikitikulchai, W. Noisripoom (holotype BBH33037, ex-type living culture, BCC60891).
Description.Anamorphic stromata flat to umbonate, subglobose, 1-5 mm in diam, 1–1.5 mm in thick, white, brilliant yellow (14C) conidial masses. Conidiomata aggregated on the center of stromata, few conidiomata per stromata (2–5), U-shaped, rim around the orifice thick, narrow widely open orifices, 280–420 × 120–300 μm. Phialides hyaline, cylindrical, 11–15 × 1 μm. Conidia hyaline, fusiform, (7–)8–9.5(–10) × (1–)1.5–2 μm. Paraphyses present, hyaline, filiform, up to 57 µm long, 1–1.5 µm wide. Teleomorphic stromata flat to umbonate, subglobose, 3-3.5 mm in diam, white. Perithecia scattered, semi-embedded, occasionally developing singly in stromata, numerous perithecia per stroma (³5), obpyriform, 260–420 × 110–230 μm. Ostioles vivid yellow (13A). Asci hyaline, cylindrical, 130–185 × 5–7 μm. Asci caps hemispherical, 4–5 × 5–7 µm. Ascospores hyaline, disarticulating into part-spores, cylindrical with rounded ends, (5–)7–10(–10.5) × 1–1.5(–2) µm. The teleomorph and anamorph are present in the same stroma.
Culture characteristics. Colonies on PDA forming a compact mycelium, convex to pulvinate, flat in the margins, attaining a diam of 8-9 mm in 2 wk, white, yellowish white (N155A) conidial masses. Colony reverse pale yellow (162D) with white at the margins.
Habitat. On scale insects (Coccidae), found on dicotyledonous leaves.
Additional specimens examined. THAILAND. Surat Thani Province, Khao Sok National Park, Wing Hin Waterfall Nature Trail; 8°54'55.64"N, 98°31'42.25"E, on scale insect (Coccidae), found on dicotyledonous leaves; 28 Nov. 2012; A. Khonsanit, D. Thanakitpipattana, K. Tasanathai, P. Srikitikulchai, W. Noisripoom (BBH33037, BCC58525; BBH33046, BCC58543, BCC58544)
Notes. Moelleriella nivea and M. nanensis are phylogenetically close but M. nivea differs from M. nanensis in having smaller stromata. The perithecia, asci and part-spores of M. nivea are also shorter than reported in M. nanensis (Table 3). In M. nanensis, the perithecia are fused together in the stromata (Fig. 5 d) while in M. nivea the perithecium develops singly in stromata (Fig. 6 c-d). M. nivea produces hemispherical asci caps (Fig. 6 k), while in M. nanensis it is umbonate (Fig. 5 k-l). The conidiomata in M. nivea are aggregated in the center of stromata (Fig. 6 a-b), almost like a bended tubercle with a cylindrical cavity and a narrow orifice while in M. nanensis the conidiomata are scattered around the margins with irregular in shape (Fig. 5 b-c).
Moelleriellasinensis Qiu & Chen, Phytotaxa 429(4): 289-296 (2020). Fig. 7
The description below is based on Thai materials.
Description.Anamorphic stromata flat to umbonate, cottony, globose to subglobose, slightly convex with wide base, 2-6 mm in diam, white with vivid yellow (17C) conidial masses. Conidiomata scattered in the middle of stromata, elongate flask shaped, narrow orifices, 180-300 × 100-160 µm. Phialides hyaline, cylindrical, 11-21 × 1-1.5 µm. Conidia hyaline, fusiform, (6.5-)7.5-10(-12) × (1-)1.5-2 µm. Paraphyses present, hyaline, filiform, up to 100 µm long. 1-1.5 µm wide. Teleomorphic stromata flat to umbonate, globose to subglobose, slightly convex with wide base, 3-6 mm in diam, white to pale yellow (8D). Perithecia scattered, semi-embedded, most perithecia fused together, occasionally developing singly in stromata, numerous perithecia per stroma (³15), obpyriform, 270-480 × 130-260 µm. Ostioles white greenish yellow (7D) to brilliant yellow (20A). Asci hyaline, cylindrical, (161-)178.5-211.5(-238) × (6-)6.5-9(-11) µm. Asci caps hemispherical, 3-5 × 4-6.5 µm. Ascospores hyaline, disarticulating into part-spores, cylindrical with rounded ends, (7-)8-14.5(-26) × 1-2 µm. The teleomorph and anamorph are present in the same stroma.
Culture characteristics. Colonies on PDA forming a compact mycelium, convex to pulvinate, flat in the margins, wrinkled surface, attaining a diam of 9-10 mm in 2 wk, white, light greenish yellow (5D) conidial masses. Colony reverse pale yellow (160A), pale greenish yellow (160C) at the margins.
Habitat. On scale insects (Coccidae), found on the upper side of dicotyledonous leaves.
Specimens examined. THAILAND, Nakhon Ratchasima Province, Khao Yai National Park, Mo Sing To Nature Trail, 14°26'21.46"N, 101°22'20.20"E; on scale insects (Coccidae), found on the underside of dicotyledonous leaves; 9 Jan. 2013; D. Thanakitpipattana, K. Tasanathai, P. Srikitikulchai, R. Promharn (BBH33725, BCC 60931, BCC 60932); Chiang Mai Province, Doi Inthanon National Park, Mae Chaem Junction (KM.38) Nature Trail; 18°35'15.66"N, 98°29'11.69"E; on scale insects (Coccidae), found in the underside of dicotyledonous leaves; 28 Oct. 2013; A. Khonsanit, D. Thanakitpipattana, K. Tasanathai, P. Srikitikulchai, W. Noisripoom (BBH38467, BCC69128; BBH38468, BCC69129); Kanchanaburi Province, Thung Yai Naresuan Wildlife Sanctuary (West), Krathon Ruesi Nature Trail, 15°20'0.11"N, 98°54'59.34"E; on scale insects (Coccidae), found on the underside of dicotyledonous leaves; 18 Nov. 2014; A. Khonsanit, D. Thanakitpipattana, K. Tasanathai, P. Srikitikulchai, R. Promharn, S. Mongkolsamrit, S. Wongkanoun, W. Noisripoom (BBH40812, BCC75977)
Notes. Moelleriellasinensis was first proposed by Chen et al. (2020) on whitefly nymphs (Hemiptera) found from Wu Yi Mountain, Fujian province, southeastern China. Only the anamorphic state was described and illustrated therein. This species was nested in a group in Moelleriella clade A subclade I (Fig. 1). In our study, we describe and illustrate the teleomorphic state of M. sinensis found on scale insects (Coccidae). Phylogenetically, M.sinensis is closely related to M. nivea, M. nanensis, M. ochracea and M. mollii. Moelleriellasinensis differs from other closely related species in producing longer part spores (see Table 3) and elongate flask-shaped conidiomata with narrow orifices in the center of stromata, while in the other species the orifices are widely open.