Phyllosticta hubeiensis a . Spots on living leaves of Viburnum odoratissimum . b – c . Pycnidia on living leaves. d . Colony on PDA (front). e . Colony on PDA (reverse). f – g . Pycnidia on colonies. h – i . Conidia on living leaves. j . Conidiogenous cells. k . Spermatiophore. l . Spermatia on colonies. m – n . Conidia on Colonies. o . Conidium with a falling appendage. p . Conidia without appendage. q – r . Shedding appendages. Bars: c, f, g 0 100 μ m, h – r 0 10 μ m 

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... styracicola K. Zhang & L. Cai, sp.nov. ( Fig. 3) Mycobank: MB 801465 Distribution: Yunnan Province, China. Host: Styrax grandiflorus (Styracaceae). Etymology: styracicola , derived from the genus name of the host plant. Description: Leaf lesions spread radially from the apex, dark brick colour, surrounded by a darker brown margin. Pycndia on leaves iron gray to black, subglobose to globose, 60 – 130 μ m in diameter. Conidiophores cylindrical to ampulli- form, reduced to 2 conidiogenous cells. Conidiogenous cells 2 – 3.5×8 – 12.5 μ m ( x 1⁄4 2 : 7 Æ 0 : 4 Â 10 : 1 Æ 1 : 39 μ m , n 0 20), holoblastic, hyaline, cylindrical. Conidia 9.5 – 13×6.5 – 9 μ m ( x 1⁄4 11 : 5 Æ 0 : 9 Â 7 : 9 Æ 0 : 4 μ m , n 0 30), 1-celled, ellipsoidal to subglobose, surrounded by a thick mucilaginous layer, with a long and slender apical appendage measuring 12 – 24(36) μ m ( x 1⁄4 17 : 9 Æ 5 : 6 μ m , n 0 30). Culture characters: Colonies reach 3 cm diam. in 10 days on PDA, first olive green, becoming greenish black at maturity, olive green in reverse. Stromata forming olive green to greenish black masses. Pycndia visible after 20 days, with milky white exuded tendrils. Specimen examined . CHINA , Yunnan, Xishuangbanna Tropical Botanical Garden of Chinese Academy of Sciences. Living leaves of Styrax grandiflorus , 01 Sep. 2011, F. Liu, HMAS 243474 (holotype); ex-type culture CGMCC3.14985; ibid LC1655, living culture CGMCC3.14989. Note: Styrax grandiflorus is found in eastern and south- eastern Asia, where it is often used as incense, ornamental plant and for medicinal purposes. Phyllosticta styracina Miura (1957) has been reported from Styrax japonicas , which is the only record of Phyllosticta from Styraceae. As Miura (1957) did not provide a Latin diagnosis, the name P. styracina was regarded invalid (van der Aa 1973). The description provided for this taxon made its determination uncertain as it could represent a species in the genus Asteromella , Phoma or Phomopsis , but not Phyllosticta (van der Aa 1973). Phyllosticta styracicola is phylogenetically most closely related to P. ampelicida (teleomorph Guignardia bidwellii ) with 96 % max identity in ITS sequence, and P. styracicola clustered in the same clade in multi-locus tree with P. ampelicida and P. schimae . In morphology, P. ampelicida produces shorter conidiogenous cells (3×6 μ m vs. 2 – 3.5×8 – 12.5 μ m), and apical appendages (ca. 5 – 12 μ m vs. 12 – 36 μ m) than P. styracina (van der Aa 1973). Phyllosticta schimae produces longer conidiogenous cells (8 – 30×2 – 4 μ m vs. 2 – 3.5×8 – 12.5 μ m) and shorter apical appendages (4 – 10 μ m vs. 12 – 36 μ m) than P. styracina (Su and Cai 2012). Phyllosticta hubeiensis K. Zhang & L. Cai, sp.nov. (Fig. 4) Mycobank: MB 801466 Distribution: Hubei province, China. Host: Viburnum odoratissimum (Adoxaceae) Etymology: hubeiensis , derived from the distribution of this species. Specimen examined. CHINA , Hubei, Shennongjia forest region. Living leaves of Viburnum odoratissimum , 01 Aug. 2011, K. Zhang, HMAS 24395 (holotype); ex-type culture CGMCC3.14986; ibid LC1653, living culture CGMCC3. 14987; ibid LC1654, culture CGMCC3.14988. Description: Leaf spot irregularly circular, surrounded by dark brick margin, with sky grey center sometimes. Pycndia black, single, subglobose to globose, 100 – 170 μ m diam. on leaves; 140 – 220 μ m on colonies. Conidiogenous cells 6 – 12.5×3 – 5 μ m ( x 1⁄4 9 : 6 Æ 1 : 8 Â 4 : 3 Æ 0 : 6 μ m , n 0 20) holoblastic, hyaline, cylindrical or conical. Conidia 10 – 14.5×6 – 9 μ m ( x 1⁄4 1 2 7 0 9 7 1 0 5 m , n 30), 1-celled, ellipsoid to obovoid, truncate at the base sometimes, surrounded by a thin mucilaginous layer, containing numerous minute guttules, with long and slender apical appendage measuring 12 – 24 μ m ( x 1⁄4 17 : 5 Æ 2 : 6 μ m , n 0 30), usually fall off from conidia at maturity. Spermatia 5.5 – 10×1.2 – 2.3 μ m ( x 1⁄4 8 : 3 Æ 1 : 1 Â 1 : 7 Æ 0 : 2 μ m , n 0 30) cylindrical to dumbbell- shaped, with one guttule at each end. Culture characters: Colonies reach 3.5 cm diam. in 20 days on PDA, first white, becoming brown to black at maturity, greenish black in reverse, forming a dense crust, white, margin regular. Pycndia visible after 20 days, with milky white to sulphur yellow exuded tendrils. Note: The host of Phyllosticta hubeiensis, Viburnum, belongs to the Adoxaceae, one of two major lineages in the Dipsacales with Caprifoliaceae (Donoghue et al. 2001) . Only one species, Phyllosticta adoxae have been associated with plants in this family. While in the description, the characters showed that it is actually an Asteromella species (van der Aa and Vanev 2002). Several species of Phyllosticta have been reported on Caprifoliaceae, including P. akaisiana , P. alpi- gena , P. caprifolii , P. diervillae , P. ebuli , P. lantanae , P. lantanoidis , P. lonicerae , P. nitidula , P. opuli , P. punctata , P. roumegueri , P. roumeguerii , P. sambuci , P. sambucicola , P. sydowii , P. symphoricarpi , P. tinea , P. tineola , P. viburni , P. viburni-opuliae and P. vulgaris according to USDA fungal database (Farr et al. 2012). All of them have been reclassified as Mycosphaerella , Phoma or Phomopsis by van der Aa and Vanev (2002). Currently, there are no accepted Phyllosticta species reported on Adoxaceae or Caprifoliaceae. A Blast search in GenBank using ITS sequences showed that the most similar species to P. hubeiensis is P. concentrica (teleomorph Guignardia philoprina , strain number CBS 447.68), with 94 % max identity. In morphology, P. hubeiensis produces larger conidiogenous cells (6 – 12.5×3 – 5 μ m vs. 4 – 10×1.5 – 4 μ m), and smaller spermatia (5.5 – 10×1.2 – 2.3 μ m vs. 6 – 15× 1..5 – 3 μ m) than P. concentrica (van der Aa 1973). There are 14 facultative synonyms and three variants listed under P.con- centrica Sacc. (www.mycobank.org), but none of them para- sitize on Adoxaceae plants. In the phylogenetic tree based on the ITS sequence, P. hubeiensis showed close relationship with P. gaultheriae (teleomorph G. gaultheriae ) and P. concentrica (teleomorph Guignardia philoprina ) (Fig. 1). In multi-locus phylogenetic tree, P. hubeiensis clustered in a same clade with P. brazilianine and P. ilicis - aquifolii . Morphologically, P. gaultheriae produces smaller conidia than P. hubeiensis (4 – 9×4 – 7 μ m vs. 10 – 14.5×6 – 9 μ m), while P. brazilianine produces smaller conidia (8 – 11×6 μ m vs. 10 – 14.5×6 – 9 μ m) and shorter appendages (4 – 9×4 – 7 μ m vs. 10 – 14.5×6 – 9 μ m) than P. hubeiensis (Glienke et al. 2011). P. ilicis-aquifolii produces longer conidiogenous cells (12 – 17× 3 – 4 μ m vs. 6 – 12.5×3 – 5 μ m) than this new species. It is noteworthy that in P. hubeiensis , the appendages tend to detach from the conidia, which indicates that appendages may be caducous in fresh specimens and pure cultures, as reported by van der Aa (van der Aa 1973). In this study, two new species, Phyllosticta styracicola from Styrax grandiflorus and P. hubeiensis from Viburnum odoratissimum , are reported and characterised. Both species are typical of Phyllosticta as they produce conidia bearing mucilaginous layers and apical appendages (van der Aa 1973; van der Aa and Vanev 2002). Van der Aa and Vanev (2002) accepted 190 species among more than 2000 records in this genus. Since then 13 new species have been described (Motohashi et al. 2008; Wulandari et al. 2009; Wulandari et al. 2010; Glienke et al. 2011; Wang et al. 2011; Su and Cai 2012). Currently only a few species of Phyllosticta have ex-type living cultures and derived DNA sequences, thus the phylogenetic relationships within the genus is mostly unknown. Although van der Aa ’ s generic concept is widely accepted (van der Aa 1973; van der Aa and Vanev 2002; Wang et al. 2011; Wikee et al. 2011), species delimitation in the genus is still prob- lematic due to the limited morphological characters and the wide host range of some endophytic species, e.g. P. capitalensis (Okane et al. 2001; Wikee et al. 2011). Pathogenic species, on the other hand, are often specific to host genus or family (Hudson 1987; Wang et al. 2011; Wulandari et al. 2011). Recently, the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) has been widely applied in species delimitation in Phyllosticta . Morphologically less dis- tinguishable species are increasingly recognized as indepen- dent species. For example, P. citricarpa , is the pathogen of citrus black spot, and is subjected to phytosanitary legislation in the EU and US ( Recently P. citriasiana and P. citrichinaensis from Citrus maxima are revealed phylogenetically well separated from P. citricarpa and thus have been described as new species (Wulandari et al. 2009; Wang et al. 2011). Further research is needed to determine their distribution, host range, pathogenecity and quarantine significance of these morphologically similar cryptic species (Shivas and Cai 2012). In this study, plant pathogenic species of Phyllosticta were morphologically compared with phylogenetically related species. The conidial appendages have been regarded as a deci- sive character in distinguishing Phyllosticta and other related genera such as Asteromella , Phoma and Phompsis (van der Aa and Vanev 2002). The appendages are often elongated or detached from conidia, and are an unreliable taxonomic character (Jin 2011; Su and Cai 2012). In this study, caducous appendages were often observed in pure cultures. This indicates the necessity of checking fresh cultures periodically when studying Phyllosticta to reduce the risk of false ...