Mycol Progress DOI 10.1007/s11557-016-1218-4 ORIGINAL ARTICLE Reassessment of Allantonectria, phylogenetic position of Thyronectroidea,andThyronectria caraganae sp. nov. Hermann Voglmayr1 & Olexander Yu. Akulov2 & Walter M. Jaklitsch1,3 Received: 6 May 2016 /Revised: 10 July 2016 /Accepted: 14 July 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract The genus Allantonectria is synonymised with Thyronectria caraganae is described as a new species from Thyronectria, based on morphological and molecular phylo- herbarium specimens of Caragana arborescens collected in genetic considerations. Investigations of types and fresh col- the Ukraine. It is characterised by morphology of the sexual lections revealed that Tubercularia concentrica is an earlier morph and by DNA sequence data, which place it within the name for Sphaeria (syn. Allantonectria) miltina and is thus T. austroamericana - T. rhodochlora clade with high support. combined in Thyronectria. Allantonectria yuccae is This is also supported by its morphology, specifically recognised as a distinct species and transferred to ascomata partly embedded within a stroma, muriform asco- Thyronectria, as well as the recently described A. zangii. spores not budding within the ascus, becoming yellowish to Descriptions and illustrations are provided for T. concentrica rosy at maturity. and T. yuccae. A recent collection of the North American Thyronectroidea chrysogramma, the generic type of Keywords Ascomycota . Hypocreales . Nectriaceae . Thyronectroidea, was studied with respect to morphology of Phylogenetic analysis . Sordariomycetes . Taxonomy its sexual morph in fresh condition and of its asexual morph produced in pure culture. Molecular phylogenies based on six loci (ITS and LSU regions of nuc rDNA, act1, rpb1, Introduction rpb2, tef1 and tub2 genes) place T. chrysogramma within Thyronectria,confirmingsynonymyofThyronectroidea with The genus Thyronectria was recently re-instated by Jaklitsch Thyronectria, but remarkably a relationship to European spe- and Voglmayr (2014), as it takes precedence over the younger cies with green to brown spores (former genus Mattirolia) genus Pleonectria. Based on detailed morphological as well receives no support, and its closest relatives remain unclear. as molecular phylogenetic analyses, they synonymised Mattirolia, Pleonectria and Thyronectroidea with Thyronectria and combined all respective epithets in Section Editor: Gerhard Rambold Thyronectria. This was also implemented by Lombard et al. (2015) in their overview of genera of the Nectriaceae. * Hermann Voglmayr [email protected] Morphologically, the genus Thyronectria is mainly characterised by nectriaceous ascomata with a yellow scurf at the outer surface in combination with long, more or less 1 Division of Systematic and Evolutionary Botany, Department of persistent, apical paraphyses (Jaklitsch and Voglmayr 2014). Botany and Biodiversity Research, University of Vienna, Rennweg 14, 1030 Wien, Austria Ascospores within the genus are highly diverse in size, shape, colour and septation; they can be ellipsoid, oblong, fusiform, 2 V. N. Karazin Kharkiv National University, Maidan Svobody 4, 61022 Kharkiv, Ukraine globose, clavate or vermiform, with eusepta and/or distosepta, one- to several-septate or muriform, smooth or striate, and 3 Institute of Forest Entomology, Forest Pathology and Forest Protection, Department of Forest and Soil Sciences, their colour can be hyaline, yellowish, rosy, green or brown. BOKU-University of Natural Resources and Life Sciences, In several species, ascospores are budding in the ascus to Hasenauerstraße 38, 1190 Wien, Austria produce oblong to allantoid, 1-celled, hyaline ascoconidia. Mycol Progress Ecologically, several species have commonly been found in lack of significant support for a sister group relationship of association with effete pyrenomycetes, indicating that they are Thyronectria to Allantonectria in recent molecular phyloge- fungicolous. nies (Jaklitsch and Voglmayr 2014; Checa et al. 2015)called Subsequently, Checa et al. (2015) described two new for a re-evaluation of the generic status of Allantonectria.We Thyronectria species with olivaceous to green-brown performed detailed morphological studies of Allantonectria muriform ascospores from Spain. In the phylogenetic analy- specimens from Agave and Yucca spp. for comparison with ses, the four known European Thyronectria species with green Thyronectria and to evaluate the species status of A. miltina to brown muriform ascospores (T. asturiensis, T. giennensis, and A. yuccae. In addition, we examined the types and fresh T. pistaciae and T. roseovirens) formed a highly supported collections of Tubercularia concentrica and A. miltina to in- subclade, together with T. obscura, which has hyaline vestigate whether they are conspecific. Based on these inves- muriform, budding ascospores. tigations, we conclude that T. concentrica and A. miltina are The lack of fresh collections and cultures precluded the synonymous, A. yuccae represents a distinct species and inclusion of the North American Thyronectroidea Allantonectria should be merged with Thyronectria. chrysogramma, which also has greenish to brown muriform ascospores, in molecular phylogenetic analyses. Based on morphological investigations, it was included in Mattirolia Materials and methods by Checa et al. (2013), while Jaklitsch and Voglmayr (2014) provided convincing arguments for a placement within Morphological observations Thyronectria. A recent Canadian collection from the type host, Ulmus americana, enabled us to document its sexual Microscopic preparations were mounted in water, 3 % potas- morph in fresh condition, to study its asexual morph produced sium hydroxide (KOH) or lactic acid (LA). Methods of mi- in pure culture and to assess its phylogenetic position by DNA croscopy included stereomicroscopy using a Nikon SMZ sequence data. 1500 and Nomarski differential interference contrast (DIC) In the course of a revision of herbarium specimens at the V. using the Zeiss Axio Imager.A1 compound microscope. N. Karazin Kharkiv National University, Ukraine (CWU), Images and data were gathered using the Nikon DS-U2 or several specimens of an unidentified nectriaceous fungus were Zeiss Axiocam 506 color digital cameras and measured by revealed on Caragana arborescens. All specimens were orig- using the NIS-Elements D v.3.0 or Zeiss ZEN Blue Edition inally deposited at the M.G. Kholodny Institute of Botany, softwares. For certain images of ascomata the stacking soft- NAS of Ukraine, Kyiv (KW). Although their characters ware Zerene Stacker v.1.04 (Zerene Systems, Richland, WA, matched the genus Thyronectria, they could not be identified USA) was used. Measurements are reported as maxima and with the keys of Jaklitsch and Voglmayr (2014) and Checa minima in parentheses and the range representing the mean et al. (2015). DNA sequence data subsequently obtained from plus and minus the standard deviation of a number of mea- ascomata confirmed an affiliation with Thyronectria, and fol- surements given in parentheses. lowing molecular phylogenetic and morphological analyses, it is here described as a new species. Culture observations The genus Allantonectria (Earle 1901) has been revealed as closely related to Thyronectria in molecular phylogenies with Due to lack of fresh material of Thyronectria caraganae,no high support (Hirooka et al. 2012; Jaklitsch and Voglmayr cultures could be obtained. Cultures of T. chrysogramma and 2014; Checa et al. 2015; Lombard et al. 2015). Hirooka T. concentrica were prepared and maintained as described et al. (2012)acceptedAllantonectria as a separate monotypic previously (Jaklitsch 2009) except that 2 % malt extract agar genus, characterized by minute, one-celled allantoid to rod- (MEA) was used for isolation. Germinating ascospores/ shaped ascospores and growth on monocotyledonous hosts as conidia were placed on MEA and CMD (CMA: Sigma, St diagnostic characters. In previous works (Höhnel and Weese Louis, MI, USA; supplemented with 2 % (w/v) D(+)-glu- 1910;Lowen1991; Hirooka et al. 2012), the generic type of cose-monohydrate) or 2 % malt extract agar (MEA; 2 % w/v Allantonectria, A. yuccae from Yucca spp., was regarded as a malt extract, 2 % w/v agar-agar; Merck, Darmstadt, synonym of A. miltina, which was described from Agave Germany). Cultures used for the study of asexual morph americana. However, none of these authors mentioned micro-morphology were grown on 2 % MEA or CMD at room Tubercularia concentrica, although this species was consid- temperature (RT; 22 ± 3 °C) under alternating 12 h daylight ered to be the asexual morph of A. miltina in the older litera- and 12 h darkness. Microscopic observations were made in ture (e.g., Saccardo 1878, 1883) and which, in case of tap water except where noted. The plates were sealed with conspecificity, would take precedence due to priority. laboratory film and incubated at room temperature. A culture The description of A. zangii from Populus sp. (Zeng and of T. chrysogramma was deposited at CBS-KNAW Fungal Zhuang 2012, 2013), a non-monocotyledonous host, and the Biodiversity Centre, Utrecht, The Netherlands (CBS). Mycol Progress DNA extraction, PCR and sequencing analyses were performed with gaps treated as missing data; the COLLAPSE command was set to MAXBRLEN. The extraction of genomic DNA, PCR and sequencing of Bootstrap analysis with 1000 replicates was performed in segments of six loci, i.e., the nuc rDNA