Phylogenetic analysis of Xylaria based on nuclear ribosomal ITS1-5.8S-ITS2 sequences

Abstract

To elucidate phylogenetic relationships of Xylaria, nuclear ribosomal ITS1-5.8S-ITS2 regions from 22 strains of 18 species were sequenced. Members of Xylaria formed a monophyletic group and proved to be phylogenetically homogeneous except for Xylaria cubensis. Species of the section Xylorugosa were divided into three groups and X. cubensis belonging to the section Xyloglossa developed an independent lineage from the section Xylorugosa. Groupings were partially congruent with the morphological taxonomy. The stromal structure, ascal apex, ectostromal surface, perithecial structure, stipe differentiation and the germ slit of ascospores were phylogenetically significant characters in grouping Xylaria.

Ascomycete, Evolution, ITS, Phylogeny, Xylaria

1 Introduction

The genus Xylaria Hill: Schrank is classified in the family Xylariaceae of the class Pyrenomycetes. The Xylariaceae are a large and relatively well-known family which is representative of ascomycetes in most countries [1]. About 35 genera belong to the family Xylariaceae [2]. It is characterized by perithecial ascocarps bearing paraphyses and periphyses that are embedded in stroma. The asci of most species bear a ring at the apex that appears as a characteristic amyloid ascal plug when stained with iodine. Many species actively decay wood of living or dead angiosperms and are known to be saprobic in most cases [3].

Xylaria is a large and the first described genus of the Xylariaceae [4]. Xylaria species are saprobic or sometimes weakly to strongly parasitic on woody plants and usually have erect elongated stromata. Although they are found mostly on wood, certain stroma are found on sawdust, leaf, dung or soil. Species of Xylaria are difficult to identify and classify for a few reasons. First, stromata of a given species often vary greatly in color and size and sometimes in general shape [1]. They may be filiform, thick to sausage-shaped, simple or forked at the base or tip. These variations are associated with stages of development (immature, mature and senescent stages), locality and probably inherent variability. Thus, knowledge on the anamorph is required to separate closely related taxa in certain species complexes [1,5,6]. Second, many species of Xylaria are cosmopolitan and have been described more than once from different localities and often in different stages of maturity [7]. They are basically uniform and specialized in structure, but the extreme range in morphology has frequently led to considerable confusion in specific delimitation.

Martin [4] reported that the most important taxonomic character in Xylaria is probably the development of the ectostroma. He accepted a division of Xylaria into two sections: Xylorugosa and Xyloglossa, based on nature of the ectostroma. Species with smooth and continuous ectostroma are placed in Xyloglossa, and those with rough ectostroma in Xylorugosa. These two sections hardly overlap in characters and very few intermediate species exist.

The genus Xylaria shows great variation in morphology but, due to lack of appropriate applicable characters, few phylogenetic studies have been conducted to infer the relationships of taxa within the genus. Recently, molecular sequence data have been successfully applied for the study of evolutionary patterns and phylogenetic systematics in fungi. Nuclear small subunit ribosomal RNA gene regions are usually used as a molecular tool to analyze fungal taxa at a family or order level, and ITS regions are commonly used to examine phylogenetic positions or relationships at a species or intraspecies level. In this study, ITS1-5.8S-ITS2 region sequences were used to infer phylogenetic relationships among the species of Xylaria and the results were compared with the present taxonomy derived from morphological characters.

2 Materials and methods

2.1 Organisms studied

Three specimens (Daldinia sp. SFC 980601-10, Hypoxylon sp. SFC 971026-6, Xylaria longipes SFC 960725-6) and 19 strains of 16 species used in this study, and their sources and localities are listed in Table 1. Cultures were maintained on PDA (potato dextrose 2.4%, agar 1.5%) at 24 h under dark conditions. Mycelia grown on agar plates for 5–10 days or ascocarp pieces taken from dried specimens were used for DNA isolation.

1

List of strains used in this study, their sources, localities and GenBank accession numbers

Species name	Sourcea	Locality	Accession number
C. militaris (L.: Fr.) Link	CBS 178.59 (KCTC 6862)	–	AF163020
D. concentrica (Bolt.: Fr.) Ces. et de Not.	CBS 139.73 (KCTC 6843)	Netherlands	AF163021
D. vernicosa (Schw.) Ces. et de Not.	CBS 157. 32 (KCTC 6998)	USA	AF163022
Daldinia sp.	SFC 980601-10	Korea	AF163023
Hypoxylon sp.	SFC 971026-6	Korea	AF163024
N. cinnabarina (Tode: Fr.) Fr.	CBS 279.48 (KCTC 6841)	–	AF163025
X. acuta Peck	ATCC 56487 (KCTC 6675)	MI, USA	AF163026
X. apiculata Cooke	CBS 365.81 (KCTC 6562)	Colombia	AF163027
X. arbuscula Saccardo	CBS 454.63 (KCTC 6563)	CA, USA	AF163028
	CBS 452.63 (KCTC 6564)	CA, USA	AF163029
X. castorea Berkeley	ATCC 76020 (KCTC 6679)	New Zealand	AF163030
X. cornu-damae (Schw.) Fr.	CBS 724.69 (KCTC 6565)	Ont., Canada	AF163031
X. cubensis (Montagne) Fr.	CBS 116.85 (KCTC 6566)	Japan	AF163032
X. enteroleuca (Spegazzini) Martin	CBS 651.89 (KCTC 6569)	HI, USA	AF163033
X. fioriana Saccardo	CBS 486.61 (KCTC 6570)	South Africa	AF163034
Xylaria hypoxylon (L.: Fr.) Greville	CBS 590.72 (KCTC 6574)	Netherlands	AF163036
	CBS 868.72 (KCTC 6840)	Netherlands	AF163037
X. longipes Nitschke	CBS 148.73 (KCTC 6575)	Netherlands	AF163038
	SFC 960725-6	Korea	AF163039
X. mali Fromme	CBS 385.35 (KCTC 6576)	–	AF163040
X. polymorpha (Pers.: Fr.) Greville	IFO 9780 (IMSNU 32067)	–	AF163041
	IFO 9786 (KCTC 6486)	–	AF163042

^aCBS, Centraalbureau voor Schimmelcultures, Baarn, The Netherlands; KCTC, Korean Collection for Type Cultures, Taejon, Korea; SFC, Seoul National University Fungus Collection, Seoul, Korea; ATCC, American Type Culture Collection, Rockville, MD; IFO, Institute for Fermentation, Osaka, Japan; IMSNU, Institute of Microbiology, Seoul National University, Seoul, Korea.

2.2 DNA extraction and PCR amplification

Total DNA was extracted by the rapid method for nucleic acid extraction [8] with some modifications [9]. Agar plate-grown mycelia or specimen pieces were recovered in an Eppendorf tube where 750 μl extraction buffer (100 mM Tris–HCl (pH 8.0), 1 mM EDTA (pH 8.0), 100 mM NaCl and 2% SDS) was added. The mixture was vortexed for 30 s, frozen in liquid nitrogen for 30 s and then warmed in a dry oven at 70°C for 30 s. This process was repeated until the complete breakdown of cells. Extracted DNA was purified through phenol, phenol-chloroform and chloroform extractions. Just before the chloroform extraction, RNA was incubated with and removed by RNase A at 37°C for 10 min. The purified DNA was precipitated with 1 volume of isopropanol and immediately centrifuged at 12 000 rpm at room temperature. The supernatant was removed and the pellet was washed twice in 70% ethanol, dried in air and then resuspended in 40 μl of sterile TE (pH 8.0).

ITS1-5.8S-ITS2 regions were amplified through PCR amplification from total DNA extracts using the forward primer NS7 and the reverse primer ITS4 [10]. Amplification was performed in a reaction mixture of 20 mM Tris–HCl (pH 8.8), 10 mM (NH₄)₂SO₄, 10 mM KCl, 0.1% Triton X-100, 2 mM MgSO₄, 0.2 mM of each dNTP, 0.5 μM of each primer with about 200 ng template DNA and 1 unit of the Taq DNA Polymerase (Poscochem). For relief of amplification inhibition in PCR, 0.3% bovine serum albumin (BSA) was added [11]. The total volume was adjusted to 50 μl and the cycling reactions were performed in a programmable thermal controller (Perkin Elmer) following the parameter of Ko et al. [12]. Amplified products were observed on 0.5% agarose gel containing ethidium bromide in Tris–acetate EDTA (TAE) buffer. The presence of a single bright band in each lane was a check for a successful amplification.

2.3 DNA sequencing and phylogenetic analyses

Amplified DNAs were purified using Wizard PCR preps (Promega), and then direct sequencing was applied by the thermal cyclic termination method with ³⁵S-labeled dATP [13]. Sequencing reactions were executed for both strands using primers ITS2, ITS3, ITS4 and ITS5 [10]. Sequenced ITS1-5.8S-ITS2 regions were aligned using an alignment algorithm CLUSTAL W [14] with gap open penalty of 7.0 and gap extension penalty of 4.0. Due to some variation in areas of ITS1 and ITS2 regions, alignment was manually verified and 48 ambiguous sites out of 648 aligned nucleotides were removed from the following process. Phylogenetic analyses of data sets were done using both distance and parsimony methods. In all analyses, Cordyceps militaris and Nectria cinnabarina were used as an outgroup. For parsimony and distance analyses, the heuristic search option and the Neighbor-joining method [15] of PAUP* 4.0 [16] were used respectively. All characters were weighted equally and gaps were treated as missing data. Strengths of internal branches of resulting trees were statistically tested by the bootstrap analysis of 500 replications [17].

3 Results and discussion

Phylogenetic relationships inferred from ITS1-5.8S-ITS2 region sequences of species of Xylaria and related genera are shown in Fig. 1, which is one of six equally parsimonious trees (tree length=673 steps, CI=0.6642, and RI=0.6878) inferred by heuristic search. Six equally parsimonious trees showed some differences in the branching pattern for positions of Xylaria cornu-damae and Xylaria polymorpha (asterisked branches in Fig. 1) which were supported by low bootstrap values. The tree constructed by the Neighbor-joining method [15] was topologically identical to the parsimony tree except for branch-supporting values. Both trees indicated that species of Xylaria merged into a single clade except for Xylaria cubensis, and the clade was supported by bootstrap values of 97 and 100% in parsimony and Neighbor-joining trees, respectively. Species of Xylaria were divided into three groups and phylogenetic separation of these groups was also supported by a set of signature nucleotides of ITS1-5.8S-ITS2 sequences (Table 2).

1

Phylogenetic tree inferred from the analysis of nuclear ribosomal ITS1-5.8S-ITS2 sequences of 22 strains. This tree is one of six equally parsimonious trees (tree length=673 steps, CI=0.6642 and RI=0.6878) inferred by the heuristic search option and is topologically identical to the tree constructed by the Neighbor-joining method of PAUP* 4.0 except for branch-supporting values. C. militaris and N. cinnabarina were used as an outgroup. Bootstrap values by the heuristic search option are indicated on corresponding branches supported by more than 50%, bold lines were used where branches are significantly supported by more than 90% and the values by the Neighbor-joining method are entered to the right with parentheses. The numbers following ‘d’ indicate decay indices calculated up to three additional steps and branches with asterisks collapse in the strict consensus tree.

2

Signature nucleotide positions of the ITS1-5.8S rDNA-ITS2 region that separate three groups of Xylaria

Positionsa

ITS1

5.8S rDNA ITS2

86

87

95

124

129

131

135

174

204

217

365

461

466

597

Group A

T

C

A

C

C

G

G

T

A

T

T

C

G

G

Group B

T

C

A

C

C

G

G

C

–/Cb

C/Ab

T

A/Tc

–/Cb

A

Group C

C

T

–

G

G

T

A

C

T

G

C

C

T

A

^aPositions for aligned sequences before adjustment.

^bExceptional nucleotides for the sequence of X. fioriana.

^cAn exceptional nucleotide for the sequence of X. cornu-damae.

Group A consisted of Xylaria arbuscula, Xylaria mali and Xylaria apiculata. The group was supported by a somewhat strong bootstrap value of 84% in the parsimony tree but was weakly supported in the Neighbor-joining tree (Fig. 1). All members of Group A have more or less gregarious stroma, sterile stromal apex and smooth ectostromata. And their stromal surfaces are yellow, becoming black at maturity [18]. Spores with a straight germ slit are also characteristic of Group A. Members except for X. mali are characterized by conidiophores of a palisade form and stipitate fruitbodies without producing a hypoxyloid form.

Presence or morphology of a subiculum showed an established evolutionary pattern in Group A. The subiculum is absent only in X. arbuscula[4] and, from the viewpoint of evolutionary pathway, it is inferable that absence of subiculum in X. arbuscula could be a derived character. As for X. mali, its hyphae of subiculum is reticulate, while those of the others are ropy [4], which fact possibly suggests that its reticulate hyphae came from ropy hyphae.

Group B included X. cornu-damae, X. longipes, Xylaria acuta, Xylaria castorea, Xylaria enteroleuca and Xylaria fioriana, forming a conflicted branch, which collapsed in the strict consensus tree. Slightly moderate to rather strong bootstrap values were replicated for branches within Group B and indicated that the group is made of relatively robust branches. Verrucose and scabrous ectostromal surface and brown color of stroma are typically believed to characterize Group B. In addition, all species belonging to Group B usually have unbranched stromata. However, X. cornu-damae and X. fioriana sometimes have stromata which are dichotomous or with up to four branches. Perithecia of X. cornu-damae and X. fioriana are completely to vaguely evident, while those of the others belonging to Group B are immersed. However, the ectostroma of X. fioriana is different from those of others in Group B, which forms polygonal crusts [4]. Fertility of the stromal apex, however, may not be a character supporting Group B, although sterile apices are a shared character in Group A. In Group B, stromal apices of species like X. acuta and X. castorea are sterile, while those of the others are fertile, and this inconsistency is also the case with Group C. Thus, the fertility of the stromal apex seems to be a flexible character in groups of Xylaria.

In Group B, both X. acuta and X. longipes have ascospores of ellipsoid-inequilateral shape [18,19], while all of X. castorea, X. enteroleuca and X. fioriana have oval and more equilateral ones [4]. The shape of germ slits, along with fertility of stromal apices, is also different between those two subgroups. Phylogenetic trees inferred from molecular data supported their relationships moderately. These facts can be indirect evidence that some morphological characters occurred many times in the evolution of Xylaria and certain characters thus have only limited taxonomic significance for given taxa of Xylaria. X. cornu-damae has ectostromata composed of broadly polygonal or linear crusts, which is characteristic of Group B, but its conidium is uniquely oblong fusoid, while conidia of most xylariaceous fungi are always unicellular with a flattened secession scar at one end. The position of this species is poorly supported by bootstrap analysis and is loosely bound to Group B as a basal taxon to the other species of Group B, suggesting that its phylogenetic position is rather uncertain. In addition, Group B itself appears statistically fragile due to easily collapsing branches and thus phylogenetic relationships of above-mentioned taxa within Group B are still poorly defined.

X. polymorpha and X. hypoxylon constituted Group C which was completely supported by the bootstrap analysis (100% in both trees). X. polymorpha is an extremely variable and complicated species showing interfaces and intergradations with numerous other taxa from which it is very difficult to separate X. polymorpha alone [20]. It has been most frequently confused with X. longipes[19]. It can be distinguished from other Xylaria species in having rugulose to rugose stromata with relatively large ascospores featured by short, straight to somewhat oblique germ slits. Xylaria hypoxylon has ascospores without germ slits but sometimes with visible gelatinous sheaths [4], which is different from those of X. polymorpha. However, these two species constitute a same group and share unbranched stromata, perithecial characters and poorly developed stipes.

X. cubensis is included in section Xyloglossa, while the other Xylaria species used in this study are classified into section Xylorugosa[4]. X. cubensis is separated from other Xylaria species by the small, predominantly brown or brownish-black, clavate stromata with rounded apices and unwrinkled to somewhat wrinkled surfaces. And it has small, dark ascospores usually without an apparent germ slit [21]. In the phylogenetic trees deduced from ITS1-5.8S-ITS2 sequences (Fig. 1), X. cubensis appeared to be separated from other Xylaria species. It is thus possible to infer that sections Xyloglossa and Xylorugosa are distinct taxa. In this study, however, only one species in section Xyloglossa was included. Xylaria myosurus was described as a member of section Xyloglossa by Martin [4], and Rogers [21] reported that X. allantoidea and X. poitei are closely related to X. cubensis. Phylogenetic relationships between Xyloglossa and Xylorugosa will become more obvious when above-mentioned species of section Xyloglossa are included in the future study.

Daldinia vernicosa and Daldinia concentrica formed a monophyletic group and then made another monophyletic group with Daldinia sp., which was somewhat weakly to strongly supported in turn with Hypoxylon sp. Daldinia along with Hypoxylon was grouped as a sister taxon of Xylaria, which was congruent with the result of Spatafora and Blackwell [22].

A few characters of ascospores, perithecia and stromata support the grouping of Xylaria inferred from molecular data, but there seems to be no character of universal significance that can justify the present phylogenetic results. It may indicate that convergent evolution of characters occurred many times within Xylaria. Such possible changes in convergent evolution, along with variations associated with developmental stages of fruitbodies might have caused confusions in identifying and classifying Xylaria species. Phylogenetic analyses based on molecular data such as ITS sequences of the present study proved to be very practical for taxonomic investigations at specific or generic levels in identification or classification of fungi of highly variable morphology like Xylaria.

Acknowledgements

The authors are grateful to Dr. K.S. Bae of KCTC (Korean Collection for Type Cultures), KRIBB (Korea Research Institute for Bioscience and Biotechnology), who kindly provided fungal strains for research collaboration. This work was supported by the Brain Korea 21 Project.