Molecular Phylogenetics and Evolution 54 (2010) 957–969 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Phylogenetic status of Xylaria subgenus Pseudoxylaria among taxa of the subfamily Xylarioideae (Xylariaceae) and phylogeny of the taxa involved in the subfamily Huei-Mei Hsieh a, Chun-Ru Lin a, Mei-Jane Fang a, Jack D. Rogers b, Jacques Fournier c, Christian Lechat d, Yu-Ming Ju a,* a Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei 115, Taiwan Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430, USA Las Muros, 09420 Rimont, France d AscoFrance, 64 route de Chizé, 79360 Villiers-en-Bois, France b c a r t i c l e i n f o Article history: Received 3 June 2009 Revised 15 December 2009 Accepted 18 December 2009 Available online 24 December 2009 Keywords: Ascomycota Macrotermitine termites Molecular phylogeny Systematics Xylariaceae Xylarioideae a b s t r a c t To infer the phylogenetic relationships of Xylaria species associated with termite nests within the genus Xylaria and among genera of the subfamily Xylarioideae, b-tubulin, RPB2, and a-actin sequences of 131 cultures of 114 species from Xylaria and 11 other genera of the subfamily were analyzed. These 11 genera included Astrocystis, Amphirosellinia, Discoxylaria, Entoleuca, Euepixylon, Kretzschmaria, Nemania, Podosordaria, Poronia, Rosellinia, and Stilbohypoxylon. We showed that Xylaria species were distributed among three major clades, TE, HY, and PO, with clade TE—an equivalent of the subgenus Pseudoxylaria—encompassing exclusively those species associated with termite nests and the other two clades containing those associated with substrates other than termite nests. Xylaria appears to be a paraphyletic genus, with most of the 11 genera submerged within it. Podosordaria and Poronia, which formed a distinct clade, apparently diverged from Xylaria and the other genera early. Species of Entoleuca, Euepixylon, Nemania, and Rosellinia constituted clade NR, a major clade sister to clade PO, while those of Kretzschmaria were inserted within clade HY and those of Astrocystis, Amphirosellinia, Discoxylaria, and Stilbohypoxylon were within clade PO. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Xylaria, under which more than 700 epithets were listed in Index Fungorum (CABI Bioscience, 2009), is likely the largest genus in the family Xylariaceae. Nonetheless, due to lack of a world monographic treatment for the genus, the actual species number of Xylaria remains unclear. Xylaria possesses cardinal features of the Xylariaceae, including massive stromatal tissue, cylindrical asci with an apical apparatus bluing in an iodine solution, and dark, unicellular ascospores with a slit-like germination site. The majority of Xylaria species are characterized by producing upright, cylindrical to clavate, multiperitheciate stromata and Geniculosporium-like conidiophores that are arranged into dense palisades. However, some Xylaria species, e.g., X. cranioides and X. berteri, deviate in featuring stunted, sessile stromata and are often referred to as penzigioid species because Petch (1924) and Miller (1961) considered these as belonging to genus Penzigia, which is considered untenable among modern mycologists (Ju and Rogers, 2001). Kretzschmarioid Xylaria species, such as X. heliscus, were previously associated with the genus Kretzschmaria due to their * Corresponding author. E-mail address: yumingju@gate.sinica.edu.tw (Y.-M. Ju). 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.12.015 crowded aggregated stromata with the fertile parts tending to be united into a crust. Most Xylaria species produce stromata on decayed dicot wood. Many fewer fruit on dead woody monocots, fallen fruits and seeds, fallen leaves and petioles, and termite nests mainly of macrotermitine termites. These substrate types are of plant origin, including the last substrate type, where plant debris is ingested and deposited into termite nests by macrotermitine termite workers after quickly passing through their digestive system. There are approximately 2600 species of termites in the world, 330 of which belong to the subfamily Macrotermitinae (Kambhampati and Eggleton, 2000). Macrotermitine termites build fecal-based fungus gardens in their nests where they cultivate mycelia of the mushroom genus Termitomyces, with which the termites form an obligate ectosymbiotic mutualism (e.g., Aanen and Boomsma, 2006). Stromata of Xylaria are frequently reported to emerge from those fungus gardens that are no longer attended by termites (e.g., Petch, 1906; Batra and Batra, 1979). Xylaria species have also been found from nests of non-macrotermitine termites but are much less in number. There are 20 described Xylaria species undoubtedly or likely associated with termite nests (Rogers et al., 2005; Ju and Hsieh, 2007). These species share a suite of common teleomorphic traits, 958 H.-M. Hsieh et al. / Molecular Phylogenetics and Evolution 54 (2010) 957–969 including stromatal crust entirely lacking or highly reduced, ostioles coarsely conic-papillate, ascospores tiny and lacking an enclosing sheath, ascospore germ slits spore length or nearly so in most species, and stromatal stipes with a tortuose rooting base (Ju and Hsieh, 2007). In fact, Xylaria species associated with termite nests have been considered to belong to a separate genus or a distinct subgeneric taxon of Xylaria, as respectively done by Boedijn (1959), who erected the monotypic genus Pseudoxylaria Boedijn for one of these fungi, X. nigripes, and Dennis (1961), who categorized Xylaria species inhabiting termite nests as a distinct subgenus of Xylaria (as Xylosphaera Dumortier). On the other hand, the variability of stromata and anamorphs among Xylaria species associated with termite nests is surprisingly high and quite comparable to that of the vast majority of Xylaria species associated with other substrate types (Ju and Hsieh, 2007). Stromata of Xylaria species associated with termite nests are remarkably diversified, ranging from unbranched to branched once to several times, light-colored to dark-colored on the surface, acuminate to blunt at apices, and darkening or remaining pale at center. Their perithecial contours are inconspicuous or so prominent as to appear that the perithecia are naked. Cultures are highly variable in colony growth rates and appearances. Conidiophores are arranged in dense palisades, in loose palisades, or solitary on the stromatal surface, and conidiogenous cells are strongly geniculate or straight. Therefore, it is also tantalizing to speculate that these Xylaria species have established themselves in termite nests more than once during evolutionary time and represent convergence of several phylogenetic lines on termite nests. Xylaria species have been included in a number of molecular phylogenetic studies concerning the Xylariaceae. Most of these studies were exclusively or mainly ITS-based (e.g., Lee et al., 2000; Okane and Nakagiri, 2007; Peláez et al., 2008; Peršoh et al., 2009) or combined ITS with nuclear ribosomal and/or protein-coding loci (e.g., Tang et al., 2007; Okane et al., 2008; Visser et al., 2009). However, Xylaria species associated with termite nests have only been included in Okane and Nakagiri (2007) and Visser et al. (2009). Okane and Nakagiri (2007) included X. angulosa J. D. Rogers et al. and Geniculisynnema termiticola Okane & Nakagiri, perhaps an anamorphic Xylaria species, in their ITS-based analysis. These two fungi were somehow nested within the cluster composed of Nemania species, which formed a branch within a clade comprising Xylaria species associated with substrates other than termite nests. Visser et al. (2009) obtained 16 anamorphic Xylaria taxa from fungus combs collected in South Africa. In their analysis based on the large subunit of ribosomal DNA sequences, these Xylaria taxa were mixed with three species of three different genera of the subfamily Xylarioideae, namely Astrocystis cocoes (Henn.) Læssøe & Spooner, N. maritima Y.-M. Ju & J. D. Rogers, and Rosellinia corticium (Schwein.) Sacc., to form a paraphyletic clade. The phylogenetic relationships between Xylaria species associated with termite nests and other Xylaria species as well as other genera in the Xylariaceae remain unsolved. In this study, we used sequences of three nuclear protein-coding loci, namely b-tubulin, RPB2, and a-actin genes, to test whether the Xylaria species associated with termite nests have a single or multiple origins as well as their phylogenetic relationships with other Xylaria species. Despite the use of the ITS region in several phylogenetic studies involving Xylaria species and the fact that ITS is useful in suggesting the identity of a given Xylaria species, it does not appear suitable for addressing the phylogenetic questions that we intended to ask. The highly variable nature of the ITS region presented great difficulties in aligning the ITS sequences of the species that we studied. This is one of the major problems about ITS-based phylogenetic studies discussed by Bruns (2001). Herein we include in our phylogenetic analyses nine of the 20 described species and five undescribed species from termite nests, as well as representatives from most of the major species aggregates of Xylaria. The substrate types with which Xylaria species are associated were also taken into account. We were also interested in knowing if Xylaria stands as a cohesive genus in relation to other genera of the subfamily Xylarioideae. Therefore, we added to the analyses representatives from Astrocystis Berk. & Broome, Amphirosellinia Y.-M. Ju et al., Discoxylaria J. C. Lindq. & J. E. Wright, Entoleuca Syd., Euepixylon Füisting, Kretzschmaria Fr., Nemania S. F. Gray, Podosordaria Ellis & Holw., Poronia Willd., Rosellinia De Not., and Stilbohypoxylon Henn. 2. Materials and methods 2.1. Taxon sampling Included in the phylogenetic analyses were 131 isolates of 114 taxa of the family Xylariaceae (Table 1), which comprised 12 genera of the subfamily Xylarioideae, including three taxa of Astrocystis, two of Amphirosellinia, one of Discoxylaria, one of Entoleuca, one of Euepixylon, seven of Kretzschmaria, nine of Nemania, two of Podosordaria, one of Poronia, five of Rosellinia, two of Stilbohypoxylon, 77 of Xylaria, and three outgroup taxa of the subfamily Hypoxyloideae—Annulohypoxylon cohaerens, Biscogniauxia arima, and B. mediterranea. We selected many of the Xylaria taxa from commonly recognized species aggregates of the genus and other taxa that were not obvious members of a species aggregate. These species aggregates included the X. arbuscula aggregate, the X. coccophora aggregate, the X. corniformis aggregate, the X. cubensis aggregate, the X. heliscus aggregate, the X. hypoxylon aggregate, the X. ianthinovelutina aggregate, and the X. polymorpha aggregate (Table 2). Taxon sampling from various substrate types were also considered (Table 3). The majority of the included Xylaria species have typical upright stromata (Fig. 1A), except for six penzigioid species, ‘‘Penzigia” cantareirensis, X. areolata, X. berteri, X. cranioides (Fig. 1B), X. crozonensis, and X. frustulosa, which are characterized by sessile stromata. Taxa from the 11 genera that are closely related to Xylaria were included to test the cohesiveness of Xylaria. These genera are considered to have close affinities to Xylaria mainly because of their Geniculosporium-like anamorphs (Ju and Rogers, 1996; Ju et al., 2007), except for Discoxylaria and Poronia, which have Hypocreodendron Henn. and Lindquistia Subram. & Chandrashekara anamorphs, respectively. These two genera were included in this study because of their Xylaria-like teleomorphic features, notably the erect, stipitate stromata with a white to light-colored, soft interior. Most of these generic allies of Xylaria have kretzschmarioid (1C), ustulinoid (1D), hypoxyloid (1E), or rosellinioid (1F) stromata, except for Discoxylaria, Podosordaria, and Poronia, which have Xylaria-like stromata. The stromata of Discoxylaria are also characterized by having a unique anamorphic apical disc. Kretzschmarioid, ustulinoid, and hypoxyloid stromata contain numerous perithecia, whereas rosellinioid stromata are highly reduced to contain only one perithecium in most cases. Rosellinioid stromata are found in Amphirosellinia, Astrocystis, Rosellinia, and Stilbohypoxylon. Hypoxyloid stromata, which are found in Entoleuca, Euepixylon, and Nemania, differ from kretzschmarioid and ustulinoid stromata in being attached to substrates with the entire base. Kretzschmarioid and ustulinoid stromata are mainly found in Kretzschmaria. Although both types of stromata are densely aggregated, stromata of kretzschmarioid type still have discernable fertile parts, whereas those of ustulinoid type are seamlessly fused into a crust and attached to substrates with multiple narrow connectives. Morphology-based identification keys including the genera discussed in the present study are available in Ju and Rogers (1996) and the online source http://mycology.sinica.edu.tw/ Xylariaceae. Table 1 Taxa included in the present study. Note that sequences of those taxa in boldface were generated in this study. Origin Collecting data b-Tubulin gene a-Actin gene RPB2 gene Amphirosellinia fushanensis Y.-M. Ju et al. Amphirosellinia nigrospora Y.-M. Ju et al. Annulohypoxylon cohaerens (Pers.: Fr.) Y.-M. Ju et al. Astrocystis bambusae (Henn.) Læssøe & Spooner Astrocystis mirabilis Berk. & Broome Taiwan Taiwan France HOLOTYPE (Ju et al., 2004) HOLOTYPE (Ju et al., 2004) Fournier 03041 (Hsieh et al., 2005) GQ495950 GQ495951 AY951655 GQ452360 GQ452361 AY951766 GQ848339 GQ848340 GQ844766 Taiwan GQ495942 GQ449239 GQ844836 GQ495941 GQ449238 GQ844835 Astrocystis sublimbata (Durieu & Mont.) G. C. Hughes Biscogniauxia arima San Martín et al. Biscogniauxia mediterranea (De Not.) Kuntze Discoxylaria myrmecophila J. C. Lindq. & J. E. Wright Entoleuca mammata (Wahlenb.) J. D. Rogers & Y.-M. Ju Euepixylon sphaeriostomum (Schwein.) Y.M. Ju & J. D. Rogers Kretzschmaria clavus (Fr.: Fr.) Sacc. Kretzschmaria guyanensis J. D. Rogers & Y.M. Ju Kretzschmaria lucidula (Mont.) Dennis Kretzschmaria megalospora J. D. Rogers & Y.M. Ju Kretzschmaria neocaledonica (Har. & Pat.) J. D. Rogers & Y.-M. Ju Kretzschmaria pavimentosa (Ces.) P. Martin Kretzschmaria sandvicensis (Reichardt) J. D. Rogers & Y.-M. Ju Nemania abortiva J. D. Rogers et al. Taiwan GQ495940 GQ449236 GQ844834 Mexico France Mexico Tainan Co., Hsin-hua, on bamboo culms, 19 February 2000, Ju, Y.-M. & Hsieh, H.-M. 89021904 (HAST) Miao-li Co., Tai-an, Jin-shui, on bamboo culm, 8 July 2005, Ju, Y.-M. & Hsieh, H.-M. 94070803 (HAST) Taipei City, Nankang Dist., on bamboo culm, 22 March 2000, Ju, Y.-M. & Hsieh, H.-M. 89032207 (HAST) ISOTYPE (Ju et al., 1998; Hsieh et al., 2005) Candoussau 366 (Ju et al., 1998; Hsieh et al., 2005) Moreno 713 (Rogers et al., 1995) AY951672 AY951684 GQ487710 AY951784 AY951796 GQ438747 GQ304736 GQ844765 GQ844819 France Pyrenees Atlantiques, Oloron, Bois de Larbaig, 1997, on Fagus, Candoussau, F. 5254 (JDR) GQ470230 GQ398230 GQ844782 USA Wisconsin, Dane Co., Madison, on Fraxinus wood, 24 May 1995, Huhndorf, S. M. 1447 (JDR) GQ470224 GQ389696 GQ844774 French Guiana Taiwan EF025611 GQ478214 EF025596 GQ408901 GQ844789 GQ844792 French Guiana Malaysia Huhndorf 803 (Rogers and Ju, 1998; Ju et al., 2007) Taipei Co., Wu-lai, on bark of Machilus thunbergii, 29 June 2000, Ju, Y.-M. & Hsieh, H.-M. 89062903 (HAST) Huhndorf 677 (Rogers and Ju, 1998; Ju et al., 2007) Whalley, M. FH 64-97 (Ju et al., 2007) EF025610 EF025609 EF025595 EF025594 GQ844790 GQ844791 Taiwan Kao-hsiung Co., Liu-kui, Shan-ping, on bark, 10 March 2005, Guu, J.-R. 94031003 (HAST) GQ478213 GQ398236 GQ844788 Taiwan USA, Hawaiian Islands USA, Hawaiian Islands French West Indies Taiwan Wang 511 (Rogers and Ju, 1998) Rogers/4 January 1996 (Rogers and Ju, 1998) GQ478212 GQ478211 GQ398235 GQ398234 GQ844787 GQ844786 HOLOTYPE (Rogers et al., 2006) GQ470219 GQ374123 GQ844768 Martinique, St. Esprit, Morne David, on bark, 23 August 2004, Lechat, C. CLL2039 (HAST, JF) GQ470222 GQ389694 GQ844772 Tai-tung Co., Lan-yuh, on bark, 6 August 2001, Ju, Y.-M. & Hsieh, H.-M. 90080610 (HAST) GQ470221 GQ389693 GQ844771 I-lan Co., Yuan-shan, Fu-shan, on bark of Castanopsis carlesii var. sessilis, 4 February 2002, Ju, Y.-M. & Hsieh, H.-M. 91020401 (HAST) Tsai, S.-J. (Ju et al., 2007) HOLOTYPE (Ju and Rogers, 2002) GQ470220 GQ389692 GQ844769 EF025608 GQ470226 EF025593 GQ389698 GQ844770 GQ844776 HOLOTYPE (Ju and Rogers, 2002) HOLOTYPE (Ju et al., 2005, 2007) Barron, G. (Petrini and Rogers, 1986, as Hypoxylon) GQ470225 EF025607 GQ470223 GQ389697 EF025592 GQ389695 GQ844775 GQ844767 GQ844773 Guadeloupe, on bark, November 2005, Lechat, C. CLL5437 (HAST, JF) GQ478220 GQ408907 GQ844798 San Martín 6013T (Rogers et al., 1998) HOLOTYPE (Rogers et al., 1998) EPITYPE (Ju and Rogers, 2001) Ariege, Coume de Roux, on Buxus sempervivens, 12 April 1992, Candoussau, F. (JDR) Ju & Hsieh 89112602 (Ju et al., 2007) Ping-tung Co., Heng-chun, Ken-ting, on bark, 26 November 2000, Ju, Y.-M. & Hsieh, H.-M. 89112601 (HAST) GQ844840 GQ844839 GQ502720 GQ470228 EF025604 GQ470229 GQ455451 GQ455450 GQ455449 GQ398228 EF025589 GQ398229 GQ853039 GQ853038 GQ853037 GQ844780 GQ844778 GQ844781 Nemania beaumontii (Berk. & M. A. Curtis) Y.-M. Ju & J. D. Rogers Nemania bipapillata (Berk. & M. A. Curtis) Pouzar Nemania diffusa (Sowerby) S. F. Gray Nemania illita (Schwein.) Pouzar Nemania macrocarpa Y.-M. Ju & J. D. Rogers Nemania maritima Y.-M. Ju & J. D. Rogers Nemania primolutea Y.-M. Ju et al. Nemania serpens (Pers.: Fr.) S. F. Gray ‘‘Barron isolate” ‘‘Penzigia” cantareirensis (Henn.) J. H. Miller Podosordaria mexicana Ellis & Holw. Podosordaria muli J. D. Rogers et al. Poronia pileiformis (Berk.) Fr. Rosellinia buxi Fabre Rosellinia lamprostoma Syd. & P. Syd. Rosellinia merrillii Syd. & P. Syd. Taiwan Taiwan USA USA, Hawaiian Islands Taiwan Taiwan Canada French West Indies Mexico Mexico Taiwan France Taiwan Taiwan GenBank accession number H.-M. Hsieh et al. / Molecular Phylogenetics and Evolution 54 (2010) 957–969 Taxon (continued on next page) 959 960 Table 1 (continued) Origin Collecting data GenBank accession number b-Tubulin gene a-Actin gene RPB2 gene Rosellinia necatrix (R. Hartig) Berl. Rosellinia sanctaecruciana Ferd. & Winge Taiwan Taiwan EF025603 GQ470227 EF025588 GQ389699 GQ844779 GQ844777 Stilbohypoxylon elaeicola (Henn.) L. E. Petrini Stilbohypoxylon elaeicola (Henn.) L. E. Petrini Stilbohypoxylon quisquiliarum (Mont.) J. D. Rogers & Y.-M. Ju Stilbohypoxylon quisquiliarum (Mont.) J. D. Rogers & Y.-M. Ju Xylaria acuminatilongissima Y.-M. Ju & H.M. Hsieh Xylaria adscendens (Fr.: Fr.) Fr. French Guiana Taiwan EF025616 GQ495933 EF025601 GQ438754 GQ844826 GQ844827 French Guiana Ju & Hsieh 89062904 (Ju et al., 2007) Ping-tung Co., Heng-chun, Ken-ting, on fronds of Arenga engleri, 29 July 2001, Ju, Y.-M. & Hsieh, H.M. 90072903 (HAST) Huhndorf 928 (Rogers and Ju, 1997, as S. moelleri; Ju et al., 2007) Tainan Co., Nan-hsi, Mei-lin, on fronds of Areca catechu, 26 August 2005, Ju, Y.-M. & Hsieh, H.-M. 94082615 (HAST) Huhndorf 940 (Rogers and Ju, 1997; Ju et al., 2007) EF025605 EF025590 GQ853020 Taiwan Ju & Hsieh 89091608 (Ju et al., 2007) EF025606 EF025591 GQ853021 Taiwan HOLOTYPE (Ju and Hsieh, 2007) GQ502711 GQ853046 GQ853028 French West Indies Thailand Guadeloupe, ravine Blondeau, on wood, 4 September 2005, Lechat, C. CLL5347 (HAST, JF) GQ487708 GQ438745 GQ844817 Nakorn Nayok, Wangtakrai, on wood, 7 July 1996, Bandoni, R. J., Bandoni, A. A. & Flegel, T. W. 12017 (JDR) I-lan Co., Yuan-shan, Fu-shan, on trunk, 29 April 2005, Ju, Y.-M. & Hsieh, H.-M. 94042903 (HAST) Guadeloupe, ravine Blondeau, on dead leaves, 4 September 2005, Lechat, C. CLL5352 (HAST, JF) GQ487709 GQ438746 GQ844818 GQ502692 GQ478218 GQ452377 GQ408905 GQ848356 GQ844796 Tai-tung Co., Lan-yuh, on bark, 8 August 2001, Ju, Y.-M. & Hsieh, H.-M. 90080804 (HAST) GQ495930 GQ438751 GQ844823 Ping-tung Co., Heng-chun, Ken-ting, on bark, 12 April 2000, Ju, Y.-M. & Hsieh, H.-M. 89041211 (HAST) Ping-tung Co., Heng-chun, Ken-ting, on wood, 28 August 2004, Ju, Y.-M. & Hsieh, H.-M. 93082814 (HAST) Guadeloupe, Grand Etang, 6 September 2005, on bark, Chabrol, J. CLL5372 (HAST, JF) GQ478226 GQ421286 GQ844805 GQ478225 GQ421285 GQ844804 GQ478215 GQ408902 GQ844793 HOLOTYPE (Ju and Hsieh, 2007) GQ502713 GQ853048 GQ853030 I-lan Co., Yuan-shan, Fu-shan, on bark of Machilus thunbergii, 12 November 2002, Ju, Y.-M. & Hsieh, H.-M. 91111214 (HAST) I-lan Co., Yuan-shan, on bamboo culm, 1 July 2006, Ju, Y.-M. & Hsieh, H.-M. 95070101 (HAST) HOLOTYPE (Ju and Rogers, 1999; Hsieh et al., 2005) Surat Thani, Khao Sok, Tree Tops River Huts, trail to falls, on bamboo culm, 6 October 1995, Bandoni, R. J. & A. A. et al. (JDR) Kauai, Kokee Park, on bark, 9 January 1996, Rogers, J. D. K-1 (JDR) GQ495953 GQ452363 GQ848342 GQ495939 AY951762 GQ478223 GQ449235 AY951873 GQ408910 GQ844833 GQ844802 GQ844801 GQ502698 GQ455442 GQ848363 Ju & Hsieh 90112623 (Hsieh et al., 2005) HOLOTYPE (Ju and Hsieh, 2007) AY951763 GQ502706 AY951874 GQ853041 GQ848362 GQ853023 Samuels 85-75 (PDD 47417) Nan-tou Co., Tsui-fong, on bark, 23 September 2002, Ju, Y.-M. & Hsieh, H.-M. 91092303 (HAST) EPITYPE (Ju and Hsieh, 2007) Sinnamary, Exploitation forestière du CIRAD, Paracou, on wood, 26 February 2007, Lechat, C. CLL7056 (HAST, JF) Wen 712 (Ju and Rogers, 2001) Finistère, Lanvéoc, Kerguéréon, on bark of Quercus, 18 July 2004, Mornand, F. JF04151 (HAST, JF) North Carolina, Great Smoky Mountains National Park, Big Creek, on wood, 9 September 2005, Rogers, J. D. (JDR) Van der Gucht & De Meester 92-521 (Van der Gucht, 1995). GQ502703 GQ502704 GQ502707 GQ487701 GQ455447 GQ455448 GQ853042 GQ421289 GQ853018 GQ853019 GQ853024 GQ844809 GQ478210 GQ502697 GQ502700 GQ398233 GQ455441 GQ455444 GQ844785 GQ848361 GQ848365 GQ502702 GQ455446 GQ853017 Primorsky Territory, reserve Ussurisky, on log, 17 August 2005, Vasilyeva, L. N. (HAST, VLA) GQ502699 GQ455443 GQ848364 Martinique, Prise d’eau, Fond Bourlet, on bark, 21 August 2005, Lechat, C. CLL5121 (HAST, JF) GQ502701 GQ455445 GQ848366 On pod, Whalley, M. F.NH9 (JDR) GQ495935 GQ438756 GQ844829 Xylaria adscendens (Fr.: Fr.) Fr. Xylaria allantoidea (Berk.) Fr. Xylaria amphithele San Martín & J. D. Rogers Xylaria apoda (Berk. & Broome) J. D. Rogers & Y.-M. Ju Xylaria arbuscula Sacc. Taiwan French West Indies Taiwan Xylaria arbuscula var. plenofissura Y.-M. Ju & S.-S. Tzean Xylaria areolata (Berk. & M. A. Curtis) Y.-M. Ju & J. D. Rogers Xylaria atrodivaricata Y.-M. Ju & H.-M. Hsieh Xylaria atrosphaerica (Cooke & Massee) Callan & J. D. Rogers Xylaria badia Pat. Xylaria bambusicola Y.-M. Ju & J. D. Rogers Xylaria bambusicola Y.-M. Ju & J. D. Rogers Taiwan Xylaria berteri (Mont.) Cooke Taiwan French West Indies Taiwan Taiwan Taiwan Taiwan Thailand USA, Hawaiian Islands Taiwan Taiwan Xylaria berteri (Mont.) Cooke Xylaria brunneovinosa Y.-M. Ju & H.-M. Hsieh Xylaria castorea Berk. Xylaria cf. castorea Berk. Xylaria cirrata Pat. Xylaria coccophora Mont. New Zealand Taiwan Taiwan French Guiana Xylaria cranioides (Sacc. & Paol.) Dennis Xylaria crozonensis P. Leroy & Mornand Xylaria cubensis (Mont.) Fr. Taiwan France USA Xylaria cubensis (Mont.) Fr. Papua New Guinea Russian Far East French West Indies Thailand Xylaria cubensis (Mont.) Fr. Xylaria cubensis (Mont.) Fr. Xylaria culleniae Berk. & Broome H.-M. Hsieh et al. / Molecular Phylogenetics and Evolution 54 (2010) 957–969 Taxon Xylaria curta Fr. Xylaria curta Fr. French West Indies Taiwan Xylaria digitata (L.: Fr.) Grev. Ukraine Xylaria enterogena (Mont.) Fr. French Guiana Xylaria escharoidea (Berk.) Fr. Xylaria feejeensis (Berk.) Fr. Taiwan French West Indies Taiwan Xylaria feejeensis (Berk.) Fr. Xylaria feejeensis (Berk.) Fr. Xylaria fimbriata C. G. Lloyd Xylaria fissilis Ces. Xylaria grammica (Mont.) Fr. Xylaria griseosepiacea Y.-M. Ju & H.-M. Hsieh Xylaria haemorrhoidalis Berk. & Broome French West Indies French West Indies Taiwan Taiwan Taiwan Xylaria cf. heliscus (Mont.) J. D. Rogers & Y.M. Ju Xylaria hypoxylon (L.: Fr.) Grev. Taiwan Xylaria hypoxylon (L.: Fr.) Grev. Xylaria ianthinovelutina (Mont.) Fr. Taiwan French West Indies Taiwan Xylaria intracolorata (J. D. Rogers et al.) J. D. Rogers & Y.-M. Ju Xylaria intraflava Y.-M. Ju & H.-M. Hsieh Xylaria juruensis Henn. Xylaria laevis C. G. Lloyd Belgium Xylaria laevis C. G. Lloyd Taiwan Taiwan French West Indies Taiwan Xylaria liquidambar J. D. Rogers et al. Taiwan Xylaria luteostromata C. G. Lloyd var. macrospora J. D. Rogers & Samuels Xylaria meliacearum Læssøe Xylaria microceras (Mont.) Fr. French West Indies Puerto Rico French West Indies French West Indies French West Indies USA, Hawaiian Islands Xylaria montagnei Hamme & Guerrero Xylaria multiplex (Kunze: Fr.) Fr. Xylaria multiplex (Kunze: Fr.) Fr. GQ495937 GQ449233 GQ844831 GQ495936 GQ438757 GQ844830 GQ495949 GQ449245 GQ848338 GQ502685 GQ452370 GQ848349 GQ502709 GQ495945 GQ853044 GQ449241 GQ853026 GQ848334 GQ495947 GQ449243 GQ848336 GQ495946 GQ502705 GQ449242 GQ853040 GQ848335 GQ853022 GQ470231 GQ398231 GQ844783 GQ495943 GQ449237 GQ844837 GQ495944 GQ449240 GQ844838 GQ495956 GQ452366 GQ848345 GQ502684 GQ452369 GQ848348 GQ487704 GQ502714 GQ427197 GQ853049 GQ844813 GQ853031 GQ502683 GQ452368 GQ848347 GQ502691 GQ452376 GQ848355 GQ260187 GQ427196 GQ844812 GQ487703 GQ495934 GQ427195 GQ438755 GQ844811 GQ844828 GQ502690 GQ452375 GQ848354 HOLOTYPE (Ju and Hsieh, 2007) Taipei Co., Wu-lai, on Arenga engleri, 25 April 2003, Ju, Y.-M. & Hsieh, H.-M. 92042501 (HAST) Martinique, Prêcheur, Anse Couleuvre on dead wood, 28 August 2004, Lechat, C. CLL2179 (HAST, JF) Ping-tung Co., Heng-chun, Ken-ting, on bark, 29 July 2006, Ju, Y.-M. & Hsieh, H.-M. 95072910 (HAST) I-lan Co., Da-tung, Chi-lan-shan, on fallen fruits of Liquidambar formosana, 7 September 2004, Ju, Y.-M. & Hsieh, H.-M. 93090701 (HAST) Martinique, Prêcheur, Anse Couleuvre, on dead wood, 18 August 2005, Lechat, C. CLL5020 (HAST, JF) Lodge, D. J. PR-894 (JDR) (Læssøe and Lodge, 1994) Guadeloupe, Route forestière de Jules, 01 September 2004, Lechat, C. CLL2265 (HAST, JF) GQ502718 GQ495932 GQ502695 GQ853053 GQ438753 GQ455439 GQ853035 GQ844825 GQ848359 GQ502696 GQ455440 GQ848360 GQ487702 GQ421290 GQ844810 GQ502688 GQ452373 GQ848352 GQ478219 GQ478221 GQ408906 GQ408908 GQ844797 GQ844799 Martinique, Case Pilote, Bois Laroche, on dead wood, 22 August 2005, Lechat, C. CLL5131 (HAST, JF) GQ495948 GQ449244 GQ848337 Martinique, Anse Noire, on dead wood, 30 August 2005, Lechat, C. CLL5287 (HAST, JF) GQ487705 GQ427198 GQ844814 Hawaii, Kole Kole Beach Park, on wood of Hibiscus tiliaceus, 8 September 1989, Hemmes, D. E. Xy-7 (JDR) GQ487706 GQ438743 GQ844815 Ping-tung Co., Heng-chun, Ken-ting, on bark, 20 September 2003, Ju, Y.-M. & Hsieh, H.-M. 92092013 (HAST) An endophyte isolated from teak leaves, Whalley, M. UN515 (JDR) Martinique, Prêcheur, Anse Couleuvre, on a termite nest, 18 August 2005, Lechat, C. CLL5010 (HAST, JF) Martinique, Forêt de Colson, on bark, 6 September 2003, Lechat, C. CLL0928 (HAST, JF) Guadeloupe, Petit Bourg, Bois Sergent, on bark, 21 November 2006, Lechat, C. CLL6002 bis (HAST, JF) Ping-tung Co., Heng-chun, Ken-ting, on bark, 20 September 2003, Ju, Y.-M. & Hsieh, H.-M. 92092010 (HAST) Martinique, La Pirogue, on fruit of Swietenia macrophylla, 26 August 2004, Lechat, C. CLL2128 (HAST, JF) Guadeloupe, Capesterre Belle Eau, 3ème Chute du Carbet, on bark, 23 November 2006, Lechat, C. CLL6033 (HAST, JF) Nan-tou Co., Lu-ku, Shi-tou, on wood, September 2005, Chen, G.-T. (HAST) HOLOTYPE (Ju and Hsieh, 2007) Ping-tung Co., Heng-chun, Ken-ting, on bark, 12 April 2000, Ju, Y.-M. & Hsieh, H.-M. 89041207 (HAST) Ping-tung Co., Heng-chun, Ken-ting, on bark, 30 November 1999, Ju, Y.-M. & Hsieh, H.-M. 88113010 (HAST) Ottignies-Louvain-la-Neuve, campus of Catholic Univ. of Leuven, on wood, October 1995, Ju, Y.-M. (HAST) Taichung Co., Ho-ping, Bi-lu-hsi, on wood, 20 August 2006, Guu, J.-R. 95082001 (HAST) Martinique, Forêt de Montravail, on fallen fruit of Swietenia macrophylla, 07 December 2005, Lechat, C. CLL5599 (HAST, JF) Taipei Co., Sun-shei, on bark, 4 August 2001, Ju, Y.-M. & Hsieh, H.-M. 90080402 (HAST) 961 (continued on next page) H.-M. Hsieh et al. / Molecular Phylogenetics and Evolution 54 (2010) 957–969 Xylaria frustulosa (Berk. & M. A. Curtis) Cooke Xylaria frustulosa (Berk. & M. A. Curtis) Cooke Xylaria cf. glebulosa (Ces.) Y.-M. Ju & J. D. Rogers Xylaria globosa (Spreng. ex Fr.: Fr.) Mont. Thailand French West Indies French West Indies French West Indies Taiwan Martinique, Prêcheur, Anse Couleuvre, on dead wood, 19 August 2005, Lechat, C. CLL5044 (HAST, JF) Ping-tung Co., Heng-chun, Ken-ting, on bark, 20 September 2003, Ju, Y.-M. & Hsieh, H.-M. 92092022 (HAST) Kharkov District, Zmiev Area, National Nature Park ‘‘Gomolshanskie Lesa”, vicinity of Velicaya Gomolsha Village, 13 July 2007, Prilutsky, O. (CWU [Myc] AS2438, HAST) Sinnamary, Exploitation forestière du CIRAD, Paracou, on wood, 26 February 2007, Lechat, C. CLL7043 (HAST, JF) EPITYPE (Ju and Hsieh, 2007) Martinique, Anse Noire, on dead wood, 10 December 2005, Lechat, C. CLL5653 (HAST, JF) 962 Table 1 (continued) Origin Collecting data GenBank accession number b-Tubulin gene a-Actin gene RPB2 gene Xylaria muscula C. G. Lloyd French West Indies Taiwan Taiwan Guadeloupe, Petit Bourg, Vallon de la Rivière Tambour, on dead branch, 3 September 2005, Lurel, D. CLL5323 (HAST, JF) Chou, K.-H. 94053001 (Ju and Hsieh, 2007) HOLOTYPE (Ju and Hsieh, 2007) GQ478222 GQ408909 GQ844800 GQ502710 GQ502717 GQ853045 GQ853052 GQ853027 GQ853034 French Guiana Taiwan Sinnamary, Piste St. Elie, on dead wood, 25 February 2007, Lechat, C. CLL7031 (HAST, JF) Ping-tung Co., Heng-chun, Ken-ting, on bark, 28 August 2004, Ju, Y.-M. & Hsieh, H.-M. 93082805 (HAST) Washington State, Seattle, on fallen seeds of Crataegus monogyna, September 2007, Yeomans, R. (HAST, JDR) On palm fruits, Samuels, G. J. 85-83 (PDD) Tainan Co., Hsin-hua, on wood, 19 February 2000, Ju, Y.-M. & Hsieh, H.-M. 89021903 (HAST) Guadeloupe, Trace de Sofa, on dead leaves, 1 September 2005, Lechat, C. CLL5302 (HAST, JF) GQ487700 GQ495955 GQ421288 GQ452365 GQ844808 GQ848344 GQ495927 GQ438748 GQ844820 GQ495929 GQ487707 GQ495938 GQ438750 GQ438744 GQ449234 GQ844822 GQ844816 GQ844832 Nan-tou Co., Yu-chee, Lien-hwa-chee, on trunk of Machilus zuihoensis, 24 December 2002, Ju, Y.M. & Hsieh, H.-M. 91122401 (HAST) West Virginia, Tucker Co., Blackwater Falls State Park, on wood, 26 August 2008, Rogers, J. D. (HAST, JDR) Maharashtra, Western Ghats, Pune, on log of Ficus racemosa, 22 October 2007, Gailawad, S. AMH 9204 (HAST) Ping-tung Co., Heng-chun, Ken-ting, on bark, 20 July 2003, Ju, Y.-M. & Hsieh, H.-M. 92072001 (HAST) Ping-tung Co., Heng-chun, Ken-ting, on bark, 20 September 2003, Ju, Y.-M. & Hsieh, H.-M. 92092023 (HAST) Martinique, Prêcheur, Anse Couleuvre, on dead wood, 19 August 2005, Lechat, C. CLL5025 (HAST, JF) Ping-tung Co., Heng-chun, Ken-ting, on fallen leaves, 16 July 2001, Ju, Y.-M. & Hsieh, H.-M. 90071613 (HAST) Tainan Co., Hsin-shih, Tan-ting, on ground of mango orchard, 20 May 2006, Ju, Y.-M. & Hsieh, H.-M. 95052006 (HAST) Tainan Co., Nan-hsi, on ground of mango orchard, 23 May2006, Chou, K.-H. 95052301 (HAST); immature Tainan Co., Hsin-shih, Tan-ting, on ground of bamboo plantation, 5 June–5 October 2006, Chou, K.H. 95060503 (HAST) Tainan Co., Shen-hua, I-min-liao, fungus comb, 20 July 2006, Chou, K.-H. 95072001 (HAST); stroma emerging directly from fungus comb in laboratory condition, bearing anamorph only Tainan Co., Hsin-shih, Tan-ting, on ground of bamboo plantation, 12 July 2006, Chou, K.-H. 95071201 (HAST); anamorph only Hawaii, Leilaris Estates, on fallen leaves of Tibouchina semidecandra, 2 January 1996, Hemmes, D. E. DEH-1052 (JDR) Ping-tung Co., Heng-chun, Ken-ting, on fallen fruits of Reevesia formosana, 16 July 2001, Ju, Y.-M. & Hsieh, H.-M. 90071609 (HAST) Martinique, Prêcheur, Anse Couleuvre, on Magnolia fruit, 5 December 2005, Lechat, C. CLL5539 (HAST, JF) Yunnan Prov., on branch of Punica granatum, 2006, Leu, L.-S. (HAST) Martinique, Rivière Rouge, on dead wood, 29 August 2004, Lechat, C. CLL2224 (HAST, JF) GQ502689 GQ452374 GQ848353 GQ495954 GQ452364 GQ848343 GQ502694 GQ452379 GQ848358 GQ502693 GQ452378 GQ848357 GQ495957 GQ452367 GQ848346 GQ495952 GQ452362 GQ848341 GQ478216 GQ408903 GQ844794 GQ502719 GQ853054 GQ853036 GQ502708 GQ853043 GQ853025 GQ502712 GQ853047 GQ853029 GQ502715 GQ853050 GQ853032 GQ502716 GQ853051 GQ853033 GQ478217 GQ408904 GQ844795 GQ495928 GQ438749 GQ844821 GQ495931 GQ438752 GQ844824 GQ478224 GQ502686 GQ421284 GQ452371 GQ844803 GQ848350 I-lan Co., Yuan-shan, Fu-shan, on bark, 19 August 2001, Ju, Y.-M. & Hsieh, H.-M. 90081901 (HAST) Martinique, La Pirogue, on dead wood, 29 August 2004, Lechat, C. CLL2146 (HAST, JF) GQ502687 GQ478209 GQ452372 GQ398232 GQ848351 GQ844784 Ju & Hsieh 94080508 (Ju et al., 2007) EF025617 EF025602 GQ844806 Ping-tung Co., Heng-chun, Ken-ting, on bark, 30 November 1999, Ju, Y.-M. & Hsieh, H.-M. 88113002 (HAST) GQ487699 GQ421287 GQ844807 Xylaria nigripes (Klotzsch: Fr.) Fr. Xylaria ochraceostroma Y.-M. Ju & H.-M. Hsieh Xylaria oligotoma Sacc. & Paol. Xylaria ophiopoda Sacc. Xylaria oxyacanthae Tul. & C. Tul. USA Xylaria palmicola G. Winter Xylaria papulis C. G. Lloyd Xylaria phyllocharis Mont. Xylaria plebeja Ces. New Zealand Taiwan French West Indies Taiwan Xylaria polymorpha (Pers.: Fr.) Grev. USA Xylaria regalis Cooke India Xylaria regalis Cooke Taiwan Xylaria schweinitzii Berk. & M. A. Curtis Taiwan Xylaria scruposa (Fr.: Fr.) Fr. French West Indies Taiwan Xylaria sicula Pass. & Beltr. f. major Ciccarone Xylaria sp. 1 (from termite nests) Taiwan Xylaria sp. 2 (from termite nests) Taiwan Xylaria sp. 3 (from termite nests) Taiwan Xylaria sp. 4 (from termite nests) Taiwan Xylaria sp. 5 (from termite nests) Taiwan Xylaria sp. 6 (from leaves) USA, Hawaiian Islands Taiwan Xylaria sp. 7 (from fruits) Xylaria sp. 8 (from fruits) Xylaria striata Pat. Xylaria telfairii (Berk.) Fr. Xylaria telfairii (Berk.) Fr. Xylaria tuberoides Rehm Xylaria venosula Speg. Xylaria venustula Sacc. French West Indies China French West Indies Taiwan French West Indies USA, Hawaiian Islands Taiwan H.-M. Hsieh et al. / Molecular Phylogenetics and Evolution 54 (2010) 957–969 Taxon H.-M. Hsieh et al. / Molecular Phylogenetics and Evolution 54 (2010) 957–969 963 Table 2 Commonly recognized species aggregates of Xylaria and their representative taxa included in this study. Species aggregate Included taxa Uniting characters X. arbuscula aggregate X. coccophora aggregate X. corniformis aggregate X. cubensis aggregate X. arbuscula, X. arbuscula var. plenofissura, X. bambusicola, X. striata, X. venosula Pointed apex on cylindrical stromata; striped, persisting peeling layer on stromatal surface Pointed apex; ochraceous peeling layer that gradually falls off while maturing Finely cracked outer layer of stromata; wrinkled surface; ascospores mostly 8–16 lm long Thick carbonaceous layer immediately beneath stromatal surface, which is not wrinkled Turbinate to peltate stromata highly aggregated into crust Pointed apex; longitudinal, long striped peeling layer X. heliscus aggregate X. hypoxylon aggregate X. ianthinovelutina aggregate X. polymorpha aggregate X. coccophora, X. oligotoma, X. venustula X. curta, X. feejeensis, X. montagnei, X. plebeja X. allantoidea, X. berteri, X. castorea, X. crozonensis, X. cubensis, X. laevis, X. regalis X. apoda, X. cf. heliscus, X. intracolorata, X. luteostromata var. macrospora X. adscendens, X. hypoxylon, X. liquidambar, X. multiplex X. culleniae, X. ianthinovelutina, X. juruensis, X. sp. 8 X. atrosphaerica, X. cf. glebulosa, X. globosa, X. haemorrhoidalis, X. ophiopoda, X. polymorpha, X. schweinitzii, X. scruposa Pubescent on entire stromatal surface; entirely exposed perithecial mounds Finely cracked outer layer of stromata; wrinkled surface; ascospores mostly 17–35 lm long Table 3 Substrate types and their inhabiting taxa. Substrate typesa Inhabiting taxa Termite nests X. acuminatilongissima, X. atrodivaricata, X. brunneovinosa, X. cirrata, X. escharoidea, X. fimbriata, X. griseosepiacea, X. intraflava, X. nigripes, X. sp. 1, X. sp. 2, X. sp. 3, X. sp. 4, X. sp. 5 Discoxylaria myrmecophila Podosordaria mexicana, P. muli, Poronia pileiformis X. amphithele, X. meliacearum, X. phyllocharis, X. sicula f. major, X. sp. 6 Ant nests Dung Fallen leaves/ petioles Fallen fruits/seeds Monocots a b X. culleniae, Xylaria cf. glebulosa, X. ianthinovelutina, X. liquidambar, X. oxyacanthae, X. palmicolab, X. sp. 7, X. sp. 8 Astrocystis bambusae, A. mirabilis, A. sublimbata, Stilbohypoxylon elaeicola, X. badia, X. bambusicola, X. juruensis, X. palmicolab The other species included in this study but not listed here are associated with dicot wood. Growing on fallen seeds of a monocot. Fig. 1. Major stromatal types found in the Xylarioideae. (A) Xylarioid stromata of X. schweinitzii. The stroma on the right is sectioned vertically. (B) Penzigioid stromata of X. cranioides. The lower stroma is sectioned vertically. (C) Kretzschmarioid stromata of Kretzschmaria cetrarioides. (D) Ustulinoid stromata of K. sandvicensis. (E) Hypoxyloid stroma of Nemania primolutea. (F) Rosellinioid stromata of Rosellinia necatrix. Scale bars represent 5 mm. 964 H.-M. Hsieh et al. / Molecular Phylogenetics and Evolution 54 (2010) 957–969 2.2. Fungal culturing and DNA extraction Cultures were grown in 100 mL of malt extract broth (20 g Difco malt extract per liter of water) on a rotary shaker at 120 rpm for 7– 14 d. Mycelia were harvested by filtration through Whatman No. 1 filter paper, freeze-dried, and stored at 20 °C. Total DNA from a dried mycelium was extracted by using: (i) a modified cetyltrimethylammonium bromide (CTAB) method (Hsieh et al., 2005) or (ii) a MX-16 automatic nucleic acid extractor (Compacbio Sciences, Burlingame, CA) with MAXWELL 16 Tissue DNA purification kit (PROMEGA Corp., Madison, WI). 2.3. Amplifying, cloning, and sequencing b-tubulin, a-actin, and RPB2 genes Sequences of b-tubulin, a-actin and RPB2 genes were obtained with either direct sequencing or cloning. PCR amplifications of btubulin and a-actin genes were described in detail in Hsieh et al. (2005). Part of the gene encoding RNA polymerase II second largest subunit (RPB2) was amplified with the primer pair fRPB2-5F/ fRPB2-7cR (Liu et al., 1999). PCR condition for RPB2 was according to Liu et al. (1999) with an annealing temperature of 44–52 °C. Reaction components for PCR were 0.1–0.2 ng lL 1 of total DNA, 0.2 lM of each primer, 200 lM dNTP, 0.025 U lL 1 of Taq polymerase with the addition of 1.5 mM MgCL2 (Invitrogen Inc., Carlsbad, CA) or 0.05 U lL 1 of Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA), and 1 standard PCR buffer supplied with the Taq or Pfu Turbo DNA polymerase. PCR products were cleaned with PCR-Advanced™ clean-up system (Viogene-Biotek Corp., Shijr, Taipei County, Taiwan) following the manufacturer’s protocol. DNA cloning was carried out according to the description in Hsieh et al. (2005), or using the pCRII plasmid vector or Zero Blunt TOP10 plasmid vector (Invitrogen Inc., Carlsbad, CA). The plasmids were extracted with a plasmid DNA extraction kit Mini Plus™ (Viogene-Biotek Corp.). Sequencing of b-tubulin and a-actin genes was as described in Hsieh et al. (2005), and this sequencing method was also used for RPB2 genes. The internal sequencing primer pair RPB2-6F/RPB2-6R (Liu et al., 1999) was used for RPB2. 2.4. Phylogenetic analyses b-Tubulin, a-actin and RPB2 gene sequences were aligned initially using the alignment program ClustalX version 1.81 (IntelliGenetics, Inc., Mountain View, CA; Thompson et al., 1997) with the ‘‘gap penalty” and ‘‘gap extension penalty” set to 10 and 0.1, respectively, for pairwise alignment and 10 and 0.2, respectively, for multiple alignment. The alignments were then improved visually. To determine whether analyses of combined sequences can be conducted, statistical congruence was tested using a partition homogeneity test (PHT) (Farris et al., 1995; Huelsenbeck et al., 1996). The PHT was performed in PAUP (Swofford, 2003) using 100 replicates and the heuristic standard search options, and the Goloboff fit criterion (K = 2) was employed to reduce the effect of homoplasy on the tree (Goloboff, 1993). The partition homogeneity test was conducted as a simultaneous three-way test and as each possible pairwise comparison. Four datasets, including aligned sequences of RPB2 gene (RPB2), b-tubulin gene (TUB), a-actin gene (ACT), and their combined sequences (RPB2–TUB–ACT), were generated for subsequent phylogenetic analyses, which were performed by using MrBayes 3.0b4 (Huelsenbeck and Ronquist, 2003) for Bayesian (BA) analyses and using PAUP* version 4.0b10 (Swofford, 2003) for maximum parsimony (MP) analyses. For BA analyses, the data were first analyzed with MrModeltest v2.1 software (Nylander, 2004) to find the most appropriate model of DNA substitution using akaike information criterion (AIC). The number of generations was set to one million, and one tree was saved per 100 generations. The first 20% of the trees were excluded from construction of the consensus tree. The cladogram and posterior credibility values for the clades found were based on the outcome of the last 0.8 million generations. In the MP analyses, the heuristic searches were performed with ADDSEQ set to ‘‘RANDOM”, MAXTREES to ‘‘AUTO”, STEEPEST to ‘‘YES”, and SWAP to ‘‘TBR.” Bootstrapping for each MP analysis was performed using 1000 replicates, each with one heuristic search with the same parameter settings. All characters were assessed as independent, unordered, and equally weighted. Gaps were treated as missing characters. Annulohypoxylon cohaerens, Biscogniauxia arima, and B. mediterranea were used as the outgroup taxa for both BA and MP analyses of the RPB2, TUB, ACT, and RPB2–TUB–ACT datasets. 3. Results 3.1. DNA sequences and phylogenetic analyses GenBank accession numbers of RPB2, b-tubulin, and a-actin gene sequences are listed in Table 1. No significant incongruence was found among RPB2, TUB, and ACT datasets in three-way test and each pairwise comparison (p > 0.05 in all comparisons, Table 4), and we thus combined them into the dataset RPB2–TUB–ACT. RPB2, TUB, ACT, and RPB2–TUB–ACT datasets each consisted of 131 aligned sequences of 114 taxa, including the three sequences of the outgroup taxa, and contained 1268, 2430, 391, and 4089 alignable characters, 548, 1049, 126, and 1723 constant characters, and 638, 1135, 229, and 2003 phylogenetically informative characters, respectively. Trees generated from BA and MP analyses of the four datasets were highly similar in topology except for those of ACT dataset, which had lower resolutions primarily because the characters contained in ACT dataset were much fewer than those in the other datasets. Since these trees are congruent, unless otherwise noted, only the tree generated from BA analysis of the RPB2–TUB–ACT dataset (Fig. 2) is presented and further described herein. The tree generated from MP analysis of RPB2–TUB–ACT dataset is provided in the online supplementary material. Poronia and Podosordaria formed a branch separated early from the branch comprising Xylaria and other genera, where four major clades, TE, HY, NR, and PO, were well identified and all of them received 100% posterior probability values and respective bootstrap values 82%, 96%, 92%, and 80%. TE denotes the clade containing termite Xylaria species; HY the clade represented by X. hypoxylon; NR the clade composed primarily of species of Nemania and Rosellinia; and PO the clade represented by X. polymorpha. All of the Xylaria species associated with termite nests formed a monophyletic clade (TE), which diverged away from the rest of Xylaria species. Xylaria fimbriata, the only non-Asian species included in the study, branched off the first internode within clade TE and was sister to the Asian species. Xylaria species associated with other substrate types were distributed within either clade HY or clade PO. The Xylaria taxa grouped in clade HY mainly belonged to the X. arbuscula aggregate, the X. coccophora aggregate, and the X. hypoxylon aggregate, with their stromata featuring a sterile, pointed apex. Each of these species aggregates formed a monophyletic clade except for the X. hyp- Table 4 Significance values from pairwise partition homogeneity tests. RPB2 ACT TUB RPB2 ACT — p = 0.54 p = 0.22 All three together: p = 0.44 — — p = 0.88 — TUB H.-M. Hsieh et al. / Molecular Phylogenetics and Evolution 54 (2010) 957–969 965 Fig. 2. A phylogenetic tree generated by using BA analysis from the RPB2–TUB–ACT dataset. Numbers at internodes represent posterior probability values of a 50% majorityrule consensus tree from a one-million generation Markov Chain Monte Carlo analysis. These are immediately followed by the bootstrap values that are above 50%. Annulohypoxylon cohaerens, Biscogniauxia arima, and B. mediterranea were used as the outgroup taxa. Taxa associated with dicot wood are in black; those with termite nests are in brown; those with fallen leaves/petioles are in green; those with fallen fruits/seeds are in purple; those with monocots are in blue; those with dung are in orange; and the one with ant nests is in red. Genera and species aggregates listed in Table 2 are indicated on the right. When taxa of a particular genus or species aggregate did not form a coherent group, each of its different segregates is followed by a number within parentheses. Asterisk denotes X. palmicola growing on seeds of a monocot. 966 H.-M. Hsieh et al. / Molecular Phylogenetics and Evolution 54 (2010) 957–969 oxylon aggregate, with which X. grammica and X. papulis were clustered despite their more robust stromata frequently with blunt apices. Three penzigioid Xylaria taxa, including ‘‘Penzigia” cantareirensis, X. areolata, and X. cranioides, were immersed in clade HY, but they were not near one another. In addition to the woodinhabiting species, four foliicolous/caulicolous taxa, X. amphithele, X. meliacearum, X. sicula f. major, X. sp. 6, one fructicolous/seminicolous species, X. liquidambar, and one bambusicolous species, X. bambusicola, were also included in clade HY. Although the branch containing the four foliicolous/caulicolous taxa and three woodinhabiting species, ‘‘Penzigia” cantareirensis, X. microceras, and X. muscula was supported by BA analysis with a 93% posterior probability, it was not resolved by MP analysis. Clade PO contained Xylaria species mostly from the X. corniformis aggregate, the X. cubensis aggregate, the X. heliscus aggregate, the X. ianthinovelutina aggregate, and the X. polymorpha aggregate. Most species have stromata with a fertile, blunt apex except for the fructicolous/seminicolous, foliicolous/caulicolous, kretzschmarioid, and penzigioid species. Taxa within the X. polymorpha aggregate formed a monophyletic group, whereas those within the X. ianthinovelutina aggregate formed another with Stilbohypoxylon elaeicola. Taxa within each of the X. corniformis aggregate, the X. cubensis aggregate, and the X. heliscus aggregate did not form a coherent group but were segregated into four, two, and two branches, respectively. The seven fructicolous/seminicolous species were segregated into three discrete groups: with one group comprising X. oxyacanthae, X. palmicola, and X. sp. 7, one comprising X. culleniae, X. ianthinovelutina, and X. sp. 8, and one comprising X. cf. glebulosa only. Xylaria phyllocharis was the only foliicolous/ caulicolous species in clade PO. Xylaria badia and X. juruensis were the species inhabiting monocots but did not belong to the same branch. Like their penzigioid counterparts in clade HY, X. berteri and X. frustulosa were not positioned next to each other. Kretzschmarioid species, i.e., species of the X. heliscus aggregate, formed a group together with X. plebeja, with X. apoda as the only exception. Except for Podosordaria and Poronia, other genera apparently closely related to Xylaria were immersed within Xylaria, which thus appears to be paraphyletic. Species of Kretzschmaria formed a subclade next to X. cranioides within clade HY. Species of Astrocystis, Amphirosellinia, and Discoxylaria formed three discrete subclades within clade PO, where the two species of Stilbohypoxylon were also inserted but they were widely separated from each other. Amphirosellinia was grouped together with X. montagnei and X. digitata in the BA tree but not resolved in the MP tree, whereas the relationships between Astrocystis and other studied taxa in clade PO remains unresolved. The monotypic genus Discoxylaria was sister to X. oxyacanthae, X. palmicola, and X. sp. 7, three fructicolous Xylaria species. Species of Entoleuca, Euepixylon, Nemania, and Rosellinia constituted clade NR, which formed a sister group to clade PO. Species of Nemania and Rosellinia constituted the two major subclades within clade NR. The presence of Entoleuca and Euepixylon within Rosellinia and Nemania, respectively, made the latter two genera paraphyletic. 4. Discussion 4.1. Xylaria species associated with termite nests Xylaria species associated with termite nests are substrate-specific, having not been found on substrates other than termite nests (Rogers et al., 2005; Ju and Hsieh, 2007; Visser et al., 2009). There are 20 described species, which are greatly variable in their macroscopic features but quite uniform microscopically. Seventeen out of the 20 species are known from the distribution range of macrotermitine termites in tropical and subtropical Africa and Asia. Xylaria brasiliensis, X. fimbriata, and X. rhizomorpha are distributed outside this range and thus likely associated with nests of non-macrotermitine termites. We included in the study 10 of the described species and five undescribed species, three of which lack teleomorphic elements. All of these species were from Asia, except for X. fimbriata, which is known only from West Indies thus far. The studied species formed a monophyletic clade (TE), which was segregated early from the Xylaria species associated with other substrate types. Within clade TE, X. fimbriata formed a separate branch from the Asian species, implying perhaps that the event where Xylaria came to be associated with termite nests might have occurred before the separation of Macrotermitinae from other members of the termite family Termidae. Our study clearly suggests that Xylaria species have successfully established themselves within termite nests only once. This is consistent with the tree based on the large subunit rDNA sequences in Visser et al. (2009), where 16 South African anamorphic Xylaria taxa isolated from fungus combs clustered within a clade. Their termite-associated clade, however, was paraphyletic with Astrocystis cocoes, Nemania maritima, and Rosellinia corticium distributed among its consisting Xylaria taxa. In the present study, species of Astrocystis were distributed within clade PO, whereas those of Nemania and Rosellinia were within clade NR. Xylaria escharoidea, unlike the other species where the ascospores have a slit-like germination site, is the only known species from termite nests characterized by having a pore-like germination site on the ascospores. Pore-like germination site appeared to be a derived character because X. escharoidea did not form a separate branch but were nested within those species with a slit-like germination site in clade TE. It is interesting to note that Xylaria species with penzigioid stromata (Fig. 1B) were not found in clade TE but in clades HY and PO. It would be a disadvantage in spore dispersal for Xylaria species emerging from subterrestrial termite nests to have penzigioid stromata. Boedijn (1959) erected a new genus Pseudoxylaria based on a single species X. nigripes. Curiously, one of the main characteristics used by him for recognizing Pseudoxylaria was the putative lack of an ascospore germination site, which, in fact, does exist in X. nigripes (Rogers et al., 2005). Dennis (1961) accepted Pseudoxylaria but treated it as a subgenus of Xylaria (as Xylosphaera Dumortier). Our phylogenetic analyses conflicted neither with the Boedijn hypothesis, because clade TE formed a lineage distinct from that comprising other Xylaria species, nor with the Dennis hypothesis, because clade TE shared a common root with the lineage containing other Xylaria species. In taking account of the fact that Xylaria species from termite nests still share major teleomorphic, anamorphic, and colony traits with those species from other substrate types and the fact that all of the Xylaria species shared common ancestry, we agree with Dennis in accepting Pseudoxylaria at subgeneric level for Xylaria species associated with termite nests. Xylaria species associated with termite nests seem to have occupied a unique niche where they may have been successful in avoiding competition with other Xylaria species. Their teleomorphic, anamorphic, and colony morphologies are apparently highly diversified to such a degree that is nearly comparable with the morphological diversifications found among Xylaria species associated with substrates other than termite nests. In this aspect, Xylaria species associated with termite nests are analogous to the marsupials in Australasia where, without the company of placentals, these pouched mammals have evolved independently into various species that are greatly variable in morphology. 4.2. Xylaria species associated with substrates other than termite nests Xylaria species distributed in clades HY and PO were rather ambiguous concerning their substrates. Dicot wood is the substrate H.-M. Hsieh et al. / Molecular Phylogenetics and Evolution 54 (2010) 957–969 type with which most Xylaria species are associated. The species growing on dicot wood were distributed in clades HY and PO, as were those growing on fallen leaves/petioles, fallen fruits/seeds, and monocots. Except for X. phyllocharis which was located in clade PO, the species associated with fallen leaves/petioles were found mostly in clade HY, sharing a branch with ‘‘Penzigia” cantareirensis, X. microceras, and X. muscula, three wood-inhabiting species, on the BA tree; this grouping, however, was not supported by MP analysis. The species associated with fallen fruits/seeds were mostly found in clade PO with X. liquidambar as the only exception. Nonetheless, those species in clade PO were segregated into three disjunct groups: X. culleniae, X. ianthinovelutina, and X. sp. 8 formed a branch with two monocot-inhabiting species, X. juruensis and Stilbohypoxylon elaeicola; X. oxyacanthae, X. palmicola, and X. sp. 7 formed a branch next to Discoxylaria myrmecophila; and X. cf. glebulosa was nested within the X. polymorpha aggregate. The four species from monocots were not grouped together, with X. bambusicola in clade HY and X. badia, X. juruensis, and X. palmicola in three different lineages of clade PO. We included eight commonly recognized Xylaria species aggregates (Table 2) in this study. The X. arbuscula aggregate, the X. coccophora aggregate, and the X. hypoxylon aggregate belong to clade HY, whereas the X. corniformis aggregate, the X. cubensis aggregate, the X. heliscus aggregate, the X. ianthinovelutina aggregate, and the X. polymorpha aggregate belong to clade PO. In general, species of a species aggregate clustered together. However, species of the X. heliscus aggregate did not form a coherent group, neither did those of the X. corniformis aggregate and the X. cubensis aggregate. This is indeed a surprise since members of each of the three species aggregates share a suite of common characteristics. Species of the X. heliscus aggregate have nail-shaped, kretzschmarioid stromata; those of the X. corniformis aggregate are characterized by a wrinkled stromatal surface that is overlain with a finely cracked outer layer; and those of the X. cubensis aggregate feature a more or less smooth stromatal surface, immediately beneath which a thick carbonaceous layer is formed. Although X. castorea has been considered allied to or frequently confused with X. corniformis and its like (Miller, 1942; Rogers and Samuels, 1986), it grouped within the X. cubensis aggregate. Unlike the species of the X. corniformis aggregate, it has a thick crust and a slightly wrinkled stromatal surface. Crust thickness seems a differentiating character between the X. corniformis aggregate and the X. cubensis aggregate. Delimitation between clade HY and clade PO is rather vague. In general, it is more frequent to encounter Xylaria species with a pointed or sterile stromatal apex in clade HY and those with a blunt or fertile stromatal apex in clade PO. However, exceptions to this generalization are not uncommon. For example, ‘‘Penzigia” cantareirensis, Xylaria areolata, X. cranioides, X. grammica, and X. tuberoides were distributed within clade HY but lack a pointed or sterile apex. Xylaria juruensis and all of the fructicolous/seminicolous species within clade PO, namely X. culleniae, X. ianthinovelutina, X. oxyacanthae, X. palmicola, X. sp. 7, and X. sp. 8, have a pointed or sterile apex despite the fact that most of the Xylaria species distributed within clade PO have a blunt or fertile apex. Most of the six penzigioid species included in this study were not grouped together, with ‘‘Penzigia” cantareirensis, X. areolata, and X. cranioides distributed in clade HY but X. berteri, X. crozonensis, and X. frustulosa in clade PO. 4.3. The genera Podosordaria and Poronia Podosordaria and Poronia formed a branch sister to the other genera of the Xylarioideae included in the present study. Many species of these two genera are coprophilous, while some are plant debris-inhabiting. It is difficult to separate these two genera from Xylaria by morphological criteria (Dennis, 1957; Rogers et al., 967 1998). Currently, we accept in Poronia only those species producing a Lindquistia anamorph. Podosordaria is like Xylaria in having a Geniculosporium-like anamorph but is generally differentiated from the latter by its capitate stromata and coprophilous nature. The two Podosordaria species and one Poronia species that we included in the study were collected from dung. Our analyses based on limited sampling of these two genera agree with the current concept in recognizing Podosordaria, Poronia, and Xylaria as distinct genera (Rogers et al., 1998). The results also suggest that the Lindquistia anamorph of Poronia was derived from the Geniculosporiumlike anamorph after the common ancestors of Podosordaria and Poronia had diverged from the ancestors of Xylaria. 4.4. The genus Discoxylaria Stromata of this monotypic genus are in general Xylaria-like, but they are unique in terminating at the top with a discoid or subcupulate receptacle, where the Hypocreodendron anamorph is born (Rogers et al., 1995). The percurrently proliferating conidiogenous cells also set Discoxylaria apart from Xylaria species, where the conidia are produced sympodially. Discoxylaria myrmecophila is known from ant nests, but it does not appear closely related to those species associated with termite nests in our trees, being immersed among Xylaria species in clade PO. Xylaria micrura Speg. was reported to be associated with cast-out nest substrate of an attine ant in Argentina (Weber, 1938), sharing with D. myrmecophila an ascospore germ slit conspicuously shorter than the spore length as well as a hyaline sheath enclosing the ascospore proper. These microscopic features are also shared by three termite nest-inhabiting Xylaria species, X. brasiliensis, X. readeri, and X. tolosa, which, interestingly, were collected outside the distribution range of macrotermitine termites, with X. brasiliensis from South America and the other two species from Australia. It remains to be tested whether these Xylaria species and D. myrmecophila share close affinities. 4.5. The genus Kretzschmaria and kretzschmarioid Xylaria species Kretzschmaria species feature either kretzschmarioid (Fig. 1C) or ustulinoid (Fig. 1D) stromata (Rogers and Ju, 1998). The hard stromatal texture is primarily due to the thick, carbonaceous subsurface layer. The stromatal interior usually disintegrates when overmature. The seven studied species of Kretzschmaria formed a coherent group next to X. cranioides within clade HY. Among these species, K. pavimentosa and K. sandvicensis have ustulinoid stromata, while the others have kretzschmarioid stromata. The Kretzschmaria species with ustulinoid stromata were not grouped together, neither were the species with kretzschmarioid stromata. Thus, it seems problematic to divide Kretzschmaria into subgroups based on stromatal types only. The close relationship between Kretzschmaria and Xylaria have long been recognized by various mycologists (e.g., Dennis, 1961; Martin, 1970; Læssøe, 1994; Rogers et al., 1998). In fact, certain Xylaria species were originally or would have been described in Kretzschmaria because their kretzschmarioid stromata. Three of these Xylaria species, i.e., X. apoda, X. cf. heliscus, and X. intracolorata, were included in this study, but they are only remotely related to Kretzschmaria, being positioned in clade PO rather than HY. These three kretzschmarioid Xylaria did not form a cluster, with X. cf. heliscus and X. intracolorata grouped together and with X. apoda placed separately. 4.6. The genera Entoleuca, Euepixylon, Nemania, and Rosellinia Species in these four genera constituted clade NR, which formed a sister group to clade PO. Entoleuca, Euepixylon, and Nemania have hypoxyloid stromata (Fig. 1E), whereas Rosellinia has rosellinioid 968 H.-M. Hsieh et al. / Molecular Phylogenetics and Evolution 54 (2010) 957–969 stromata (Fig. 1F). Stromata of stunted Xylaria species and ustulinoid Kretzschmaria species may be confused with those of Nemania and Euepixylon but differ in being attached to the substrates via narrow connectives. Most species of these two genera were or would have been treated as Hypoxylon species in the subsection Primocinerea by Miller (1961). Euepixylon sphaeriostomum nests within Nemania and renders it a paraphyletic genus. Euepixylon has a poroid ascospore germination site and perithecia buried directly in the substrate beneath a clypeate, carbonaceous stromatal layer; it is mainly by these two characteristics that Euepixylon is separated from Nemania (Ju and Rogers, 2002). Nemania maritima deviates from other species of the genus in enclosing one to several perithecia within a stroma, inhabiting rotten wood in mangrove forests, and lacking an anamorph. Nonetheless, it was well placed among typical Nemania species. This agrees with the ITS-RPB2 tree in Tang et al. (2007), where the fungus also clustered with typical Nemania species. However, their ITS tree showed that N. maritima took an isolated position from other Nemania species. Typical Rosellinia species, in addition to having uniperitheciate stromata, commonly have subicular hyphae, within which the stromata are partially buried. The limited number of Rosellinia species that we included in this study formed a coherent group next to the branch containing Nemania species. Rosellinia appeared to be a paraphyletic genus with Entoleuca mammata nested in the cluster comprising Rosellinia species. The close relatedness between Entoleuca and Rosellinia has been noted by Læssøe and Spooner (1994) and Læssøe (1994) who even suggested the synonymy of Entoleuca and Rosellinia. However, Rosellinia and Entoleuca may be kept as distinct genera, with the former being considered paraphyletic, because Entoleuca differs from Rosellinia in having hypoxyloid stromata, a carbonaceous stroma overlying and partially encasing mature perithecia, and an anamorph in nature usually borne upon bark-rupturing pillars or specialized synnemata (Rogers and Berbee, 1964; Rogers and Ju, 1996). Also, unlike most Rosellinia species which have the teleomorph developed within subicular hyphae, the teleomorph of E. mammata is initiated in the base of the bark-rupturing pillars (Rogers and Berbee, 1964). 4.7. The genera Astrocystis, Amphirosellinia, and Stilbohypoxylon Although Astrocystis, Amphirosellinia, and Stilbohypoxylon share rosellinioid stromata (Fig. 1F) and synnematous anamorphs, they do not appear closely related. In the past, certain species within each of these genera were entangled with Rosellinia due to their uniperitheciate nature. For instance, Astrocystis bambusae, Amphirosellinia evansii (Læssøe & Spooner) Y.-M. Ju et al., and Stilbohypoxylon elaeicola were first described as Rosellinia species. Ju and Rogers (1990) even reduced Astrocystis to be a subgeneric taxon of Rosellinia. Our analyses revealed that the three genera are distributed in clade PO, not sharing close affinities with Rosellinia, which belongs to clade NR. Species of Astrocystis and Amphirosellinia formed two discrete monophyletic groups, but the two Stilbohypoxylon species were not resolved as a monophyletic group, with S. elaeicola, the type species of the genus, forming a branch next to X. juruensis and S. quisquiliarum next to the X. cubensis aggregate. The resemblance between X. juruensis andS. elaeicola is noteworthy: both inhabit monocots and have the ascospore enclosed within a hyaline sheath. In addition, X. juruensis frequently has the stroma highly reduced to contain only one perithecium. Species of Stilbohypoxylon may eventually be accepted as uniperitheciate Xylaria species. 5. Conclusion Our phylogenetic analyses were based on sequences of three nuclear protein-coding loci obtained from a large number of repre- sentatives in the genus Xylaria and its allied genera in the subfamily Xylarioideae. Our study suggests that Pseudoxylaria is a monophyletic taxon, being recognized as a subgenus of Xylaria to comprise species associated with termite nests. Results from previous studies based on nuclear ribosomal loci failed to show the monophyletic nature of Pseudoxylaria among various Xylaria species. To further test the monophyly of Pseudoxylaria, it will be of great significance to add sequences of protein-coding loci to the analyses from more Xylaria species associated with termite nests, especially those from African macrotermitine termites and nonmacrotermitine termites, where their associated Xylaria species are severely underrepresented. Collecting activities on Xylaria species associated with termite nests have been mostly carried out in Asia. African species and records of those species collected from non-macrotermitine termites are extremely meager. Pseudoxylaria, Podosordaria, and Poronia are taxa that diverged from the others of the Xylarioideae early in the evolution of the subfamily. These three taxa are largely associated with fecal substrates and signify a possible origin in animal associations for the Xylarioideae. Our sampling from Podosordaria and Poronia was far from being representative. Further analyses to test this hypothesis should include more species of these two genera and species from other coprophilous genera of the Xylariaceae, including Areolospora S. C. Jong & E. E. Davis, Hypocopra (Fr.) J. Kickx fil., and Wawelia Namyslowski. It is indeed a surprise to see those plant-associated genera of the Xylarioideae and the ant nest-associated Discoxylaria have their ancestry in Xylaria, which is therefore a paraphyletic genus. Our analyses also revealed the paraphyly of Nemania and Rosellinia. Stilbohypoxylon is likely a polyphyletic assemblage, and its harboring species may eventually be assigned to more than one genus or accepted as rosellinioid Xylaria species. Acknowledgments This study was supported by National Science Council of ROC Grant NSC 95-2311-B-001-048-MY3 to Y.-M.J. We greatly appreciate Alex Akulov, Françoise Candoussau, Don Hemmes, Sabine Huhndorf, Felipe San Martín, S.-J. Tsai, Larissa Vasilyeva, and Margaret Whalley for contributing culturable specimens. Régis Courtecuisse is thanked for having organized the collecting activities in West Indies and French Guiana. Our gratitude is also extended to two anonymous reviewers for improving our manuscript substantially. Appendix A. 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