, a new species of basidiomycetous aquatic hyphomycetes from southwest China


, based on multiple gene sequence analyses. Morphologically, it is characterised by the apical, hyaline, obclavate or navicular conidia with several hair-like lateral appendages and by its holoblastic and monoblastic conidiogenesis, with a flat un-thickened conidiogenous locus. Clamp connections and haustorial branches were often observed in culture.

Keywords: Research Article
Keywords: Basidiomycota, Classiculomycetes
Keywords: Taxonomy
Keywords: Asia
Keywords: fresh water fungi, mycoparasites, , taxonomy
Introduction

Aquatic hyphomycetes constitute a dominant mycoflora on submerged decaying plant debris, both in lotic and lentic systems (Khan 1987 (Shearer 2007). Most known basidiomycetous aquatic hyphomycetes have been reported from North America (Marvanová 1977, Marvanová and Barlocher 1988, 1998, 2000, Marvanová and Suberkropp 1990, Raj 1981), Australia (Shaw 1972), Asia (Hudson and Ingold 1960, Nawawi 1985, Marvanová and Bandoni 1987, Kirschner 2013) and Europe (Scheuer 2008).

) and these comprise about one-third of all described basidiomycetes (Aime et al. 2006 has been reviewed and revised multiple times. Based on sequences at large and small subunits of the nuclear rDNA, Aime et al. (2006) (Schell et al. 2011 (Wang et al. 2015, with only two monotypic fungi,  H. J. Huds. & Ingold and  R. Bauer, Begerow, Oberw. & Marvanová. As early as 1960,  was erected to accommodate a single anamorphic fungal species,  H. J. Huds. & Ingold. The genus is characterised by having narrow and delicate conidiophores and obclavate conidia with 0–3 hair-like lateral appendages (Hudson and Ingold 1960 Marvanova & Bandoni was established with  Marvanova & Bandoni (synomy of ) as the type species (Marvanová and Bandoni 1987). Bauer et al. (2003) observed the basidial stage of  and connected it to the teleomorphic state .  was recommended over  because  is the base of the higher level taxonomy (Aime et al. 2018). The conidia of  are similar to those of  in shape.  is characterised by the production of clamped hyphae with tremelloid haustorial cells and binucleate fusoid conidia with 3–4 bristle-like lateral branches (Marvanová and Bandoni 1987). Bauer et al. (2003) defined the phylogenetic positions of genera  and  (Schell et al. 2011).

During a study of aquatic hyphomycetes on submerged decaying leaves collected from a stream in south-western China, we encountered two fungi which resembled species in the genus . Combining the morphological and phylogenetic analyses, we identified that the fungi belonged to ).


Materials and methods

Collection of samples, isolation and characterisation

).


DNA extraction, PCR and sequencing

) and incubated at 27 °C for about 10 days. Fungal mycelia were harvested and transferred to a 2.0 ml Eppendorf tube. Total DNA was extracted using a CTAB method as described by Pratibha et al. (2014).

) were amplified: Primer pairs ITS4 and ITS5 (White et al. 1990 (Vilgalys and Hester 1990 gene (Rehner and Buckley 2005). PCR amplifications were performed using the methods described previously (Wang et al. 2014). The PCR products were then sent to the Beijing Tsingke Biotechnology Co. of China, Ltd and sequenced on both strands with the same primers that were used for amplification.


Sequence alignment and phylogenetic analysis

 gene sequences of the new isolates indicated that they had a close phylogenetic relationship with sequences from the genera  only includes two known species,  and , another 8 classes were chosen to carry out phylogenetic analysis. Four sequences of each representative strain of 8 classes were combined with those from our own cultures. (see Table 1 for all GenBank accession numbers).

Raw sequences were aligned using CLUSTAL W 1.6 (Thompson et al. 1994); then manually adjusted to minimise the number of uninformative gaps and to improve alignments using MEGA 6.06 (Kumar et al. 2012, 18S and 28S matrices were concatenated with BIOEDIT 7.5.0.3. The tree construction procedure was performed in MrBAYES 3.2 (Ronquist et al. 2012). Maximum likelihood was performed with MEGA 6.06.  sp. and  were used as outgroups. Phylogenetic trees were imported into FIGURETREE 1.4.2 and exported as SVG vector graphics for Figure assembly in ADOBE ILLUSTRATOR CS6. The phylogenetic analyses of different datasets were performed using Bayesian and maximum likelihood algorithms.


Table: Table 1.


Table: The species used in the phylogenetic analyses. Also included in the Table are the representative isolate name of each species and the GenBank accession numbers for each of the four analysed gene fragments of each isolate.

	Class	Species	Isolate No.	GenBank accession No.	Reference
	 	28S	18S	TEF
	  	 	AS 2.2310 AY233339	  AY233339	  AY233339	  KJ707751	 Wang et al. 2003; 2015	
 	 	CBS 7811 AF444553	  AF177406	  AY665775	  KJ707749	 Scorzetti et al. 2002; Wang et al. 2015	
 	 	CBS 7430 AF444599	  AF177407	  U77662	  KJ707883	 Scorzetti et al. 2002; Wang et al. 2015	
 	 	AS 2.1945 AF444563	  AF189887	  D38234	  KF706486	 Scorzetti et al. 2002; Wang et al. 2015	
 	 	JCM 10409 AF444594	  AF177408	  KJ708424	  KJ707893	 Scorzetti et al. 2002; Wang et al. 2015	
 	 	AS 2.1933 AF444548	  AF177412	  AB021688	  KJ707900	 Scorzetti et al. 2002; Wang et al. 2015	
 	 	CBS 11308 KJ778629	  KJ708456	  KJ708370	  KJ707882	  Wang et al. 2015	 
 	 	JCM 1602 AB038053	  KP216512	  KP216516	  KJ707852	  Wang et al. 2015	 
 	 	CBS 8352 AF444562	  AF189901	  KJ708417	  KJ707905	 	Scorzetti et al. 2002
	 	 sp.	MP 3490 DQ631900	  DQ631898	  DQ631899	  DQ631902	 	Matheny et al. 2006
 	 	CBS 8477 DQ911616	  DQ831020	  KP216515	  KF706487	  Wang et al. 2015	 
 	 	JCM 8772 AF444578	  AF189980	  D66880	  KJ707798	 	Scorzetti et al. 2002
 	 	JCM 3795 AF444597	  AF189972	  D13459	  KP216514	 	Scorzetti et al. 2002
 	 	JCM 10338 AB035721	  AB082572	  AB035721	  KJ707777	 	Hamamoto et al. 2002
 	 	JCM 8977 AF444523	  AF189920	  AB126644	  KJ707762	 Scorzetti et al. 2002; Wang et al. 2015	
 	 	JCM 10725 AF444567	  AF189910	  AB055193	  KJ707829	 Scorzetti et al. 2002; Wang et al. 2015	
 	 	CBS 274.82 KJ778626	  KJ708454	  KJ708360	  KJ707830	  Wang et al. 2015	 
 	 	AS 2.1923 AF444522	  AF189899	  D12803	  KJ707776	 Scorzetti et al. 2002; Wang et al. 2015	
	  	  CFB22246	  DQ200930	  AY700192	  AY657009	  DQ435789	 	Matheny et al. 2007
 	  CFB22250	  DQ206982	  AY700193	  AY665785	  DQ234567	 	Matthias et al. 2004
 	 	CBS 278.51 AY292429	  AY254179	  U77064	  EF100614	 	Matthias et al. 2004
 	  IFO32431	  DQ234556	  AY629314	  DQ234563	  DQ056288	 	Matheny et al. 2007
 	 	CRL75-36-700-3/ECS AF468044	  AF522177	  AY125409	 XM_003333	024	Weber et al. 2003
 	 	TDB183-1	– AF522169	  AY123292	 	– Wang et al. 2015	 
 	 	DUKE:DAH(323) DQ241446	  DQ241479	  DQ241410	 	–	Henket al. 2007
	  	 	CBS 164.67 GQ329853	  KF258732	  JF779647	  JF779645	  Schell et al. 2011	 
	 sp.	CBS 473.93 JF779664	  JF779649	  JF779650	  JF779651	  Schell et al. 2011	 
	 sp.	CBS 265.96 JF779668	  JF779652	  JF779653	 	- Schell et al. 2011	 
	  	 	CBS 9802 DQ831010	  DQ831009	  D14163	  KJ707837	 	Matheny et al. 2006
	  	 	JCM 9052 AF444495	  AF070419	  X53499	  KJ707788	 Scorzetti et al. 2002; Wang et al. 2015	
 	 	CBS 179.24 KJ708463	  KJ708463	  KJ708394	  KJ707807	  Wang et al. 2015	 
 	 	CBS 143.21 KJ708462	  KJ708462	  KJ708388	  KJ707811	  Wang et al. 2015	 
 	 	AS 2.2285 AY274824	  AY274823	  KJ708405	  KJ707934	 	Zhao et al. 2003
 	 	JCM 9279 AF444542	  AF070420	  AB073270	  KJ707874	 Scorzetti et al. 2002; Wang et al. 2015	
 	 	CBS 10977 EU872492	  EU872490	  KJ708384	  KJ707854	  Wang et al. 2015	 
	  	 	FO 36341	– AY512849	  AY124476	 	– Bauer et al. 2003	 
 	 	KW 1540	– L20282	  U78043	 	–	Berres et al. 1995
 	 	DB 1280	– AY512872	  AY124480	 	– Bauer et al. 2003	 
	  	 	ATCC 64713	– AY512838	  AY124478	 	– Schell et al. 2011	 
 	   1.04613	  KY548838	  KY548836	  KY468515	  MG787169	 	This study
 	   1.04389	  KY548837	  KY548835	  KY468514	  MG787170	 	This study
 	 	CCM 8127	– AY512853	  AY124477	 	–	Schell et al. 2014
	 	 sp.	AFTOL-ID 676 DQ200918	  AY634277	  DQ234542	  DQ408144	 	Schell et al. 2014
 	 	AFTOL-ID 626 AY854066	  AY635772	  AY665772	  AY881026	 	Schell et al. 2014



Results

Phylogenetic analysis

In our Bayesian and maximum likelihood analyses (Figure 1), our isolates representing the new species named  was a sister group to  and consistently had  as the next closest relative with a strong statistical support. The close relationship between  and  was supported with a posterior probability of 1.00 in the Bayesian analysis and with a bootstrap value of 0.93 in the maximum likelihood analysis. Phylogenetic relationships amongst the taxa inferred from the combined four gene sequences are in general agreement with those based on SSU rDNA and LSU rDNA D1/D2 domains by Wang et al. (2015). Although there are some minor variations in the relationships amongst the classes between the two studies, taxa within each class still formed a single clade.

 regions of our  by Bauer et al. (2003) sequences for species confirmation with those in . Sequences of accession numbers AY512838 and AY512853 of  R. Bauer, Begerow, Oberw. & Marvanová and  by Wang et al. (2015).

Figure 1.

 sequences.  sp. and  are used as outgroups. Clades and taxa are labelled according to Schell et al. (2011). Bayesian posterior probabilities, greater than 0.95, are given above the nodes (out of 100). Maximum likelihood bootstrap values, greater than 75%, are given below the nodes (out of 100). The scale bar shows the expected changes per site.


Taxonomy





819813

Figure 2 

Etymology.

Sinensis refers to the country in which this species was found.


Diagnosis.

 differs from  by having fusiform conidiogenous cells growing from the hyphae directly.


Type.

CHINA, ca. 750 m elev.


Description.

Colonies on CMA reach about 10 mm diameter after incubating for 7 days at 27 °C. Colony effuse, mycelium partly superficial, partly immersed in substratum, composed of hyaline, branched, thin-walled, septate, smooth, binucleated hyphae, 1.5–4.8 µm wide, often 1.8–2.7 µm wide. Clamp connection and haustorial branches on hyphae present. Haustorial branches with basal clamps, tapering distally or obclavate, 9–14.2 (–16.5) µm long, 1.2–2.6 µm wide, one or two terminal filaments of 3–8.5 × 1.3 µm located on the top of it. Conidiophores absent. Conidiogenous cells fusiform, monoblastic, 7.5–11×2–2.8 µm, attaching directly on the hyphae, solitary or in aggregates of two. Conidia solitary, acrogenous, navicular or obclavate, attenuating upwards, 25–38 (–42) µm long, 3.8–6.2 µm wide, 1.3–3.4 µm wide at the truncate base, (0–) 2–5 (–7) septa appear in those conidia without cytoplasm, with 1–5 (mainly 3–4) lateral appendages, attaching to the upper part of conidia, opposite or verticillate, filiform, smooth, divergent, pendulous or straight (–7) 13–21 (–25) µm long, 0.8–1.2 µm wide, 0–2(–3) septate. Occasionally, 1(–2) appendages also arise from apex of the main axis. Sometimes clamp connections appear at the top of conidia.

Figure 2.

Microscopic features of  1.04613). a, b Conidia c empty conidia d clamp connection on conidia e Haustorial branches with basal clamps on hyphae f Conidiogenous cells (black arrow) and clamp connection on hyphae (white arrow). Scale bar: 10 µm (a–f).





Discussion

 is phylogenetically related to  and morphologically similar to the latter. When  was established, Hudson and Ingold (1960) did not mention clamp connections or haustorial cells. Later, Matsushima (1987) observed clamps on hyphae from  (isolate MFC 12864), but did not see haustorial cells. Bauer et al. (2003) reported that  also presented tremelloid haustorial cells similar to . In fact, both  and  are very similar in having navicular conidia with 3–4 distal setose branches. However, conidiogenous cells of  are discrete fusiform, differentiated obviously and those of  are integrated. Conidiogenous cells of  are also integrated, but in the analysis of concatenated dataset of four sequences,  and  formed a well-supported clade separated from , so we treated our strains as a member of the genus .

 is similar to  in having haustorial branches and obclavate or navicular conidia with hair-like lateral and apex appendages. However, their conidiophores and conidiogenous cells were totally different. First,  has no conidiophore and its conidiogenous cells grow from hyphae directly, while conidiophores of  are determinate, micronematous to semi-macronematous. Second, typical conidiogenous cells of  are discrete fusiform formed successively, clamped basally, but  has no clamps at the base of conidiogenous cells and conidiogenous cells of  are integrated, which resemble that of . Besides the main differences described above, conidia of  are shorter and wider [(18–)25– 32(–45) × (4–)5–6.5(–9)] than those of , lateral branches of  are 2–3, while 4 lateral branches often appear in . Furthermore, coralloid structures were interpreted as appressoria in  but were not observed in  (Bauer et al. 2003).

 is similar to  in conidia form, but conidia of the latter grow on the tip of long micronematous conidiophores, while that of  grow from conidiogenous cells directly produced on hyphae. Besides, conidia of  are longer than those of  (type strain: 35–55 × 5–7, MFC-12864:35–56 × 4–6 µm). Septa of conidia without cytoplasm were not mentioned in the type strain of . In strain MFC-12864, there is a septum obscurely presented at the attenuated part, while conidia of  have 3–4 septa after cytoplasm drained out of the conidia.

A combination of morphological and molecular characters was used to establish . Conidiogenous cells of  and  are needed to circumscribe genus characteristics of  better and in more detail.


Supplementary Material

 


Acknowledgements

This work was financed by the National Natural Science Foundation Program of PR China (31770026, 31570023). We are grateful to two reviewers for critically reviewing the manuscript and providing helpful suggestions to improve this paper.


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, a new species of basidiomycetous aquatic hyphomycetes from southwest China