The genus Juglanconis (Diaporthales) on Pterocarya

Based on molecular phylogenetic analyses of a multigene matrix of partial nuSSU-ITS-LSU rDNA, cal, his, ms204, rpb1, rpb2, tef1 and tub2 sequences, recent European and Iranian collections of Melanconium pterocaryae from the type host, Pterocarya fraxinifolia, are shown to be distinct from the Japanese Melanconis pterocaryae from Pterocarya rhoifolia, and both are confirmed as closely related members of the recently described genus Juglanconis. Therefore, the new name Juglanconis japonica is proposed for Melanconis pterocaryae. As no type collection could be traced, Melanconium pterocaryae (syn. J. pterocaryae) is neotypified, described and illustrated, and it is recorded for Europe for the first time. During field surveys in natural stands of P. fraxinifolia in Guilan province (Iran), Juglanconis pterocaryae was consistently isolated from tissues affected by branch and trunk cankers, twig dieback and wood necrosis, indicating that it is the causal agent of these diseases. The external and internal symptoms associated with these trunk diseases are described and illustrated.


Introduction
The Diaporthales (Ascomycota, Sordariomycetes) comprise important plant pathogens, but the species diversity and host range of many phytopathologically important lineages are still imperfectly known. Recently, substantial progress was made to tackle the species diversity of several diaporthalean lineages involved in plant diseases by the application of multi-gene phylogenies in combination with morphological studies, e.g. in Coniella (Alvarez et al. 2016), Cytospora (Lawrence et al. 2018), Diaporthe (Guarnaccia et al. 2018) and Harknessia (Marin-Felix et al. 2019). These studies revealed a number of undescribed species on various plant hosts of economic importance in silvi-, agri-and horticulture, but also improved our knowledge on the circumscription and host range of already described species.
Based on morphology and molecular phylogenies, the genus Pterocarya is the closest relative of the genus Juglans in tribe Juglandinae, Juglandaceae (Manos et al. 2007;Xiang et al. 2016). The genus Pterocarya currently comprises about six accepted species, of which five occur in Eastern Asia (Vietnam, China, Korea and Japan), while one species, P. fraxinifolia, occurs widely disjunct in Western Asia from Anatolia via the southern Caucasus area to the Caspian forest of Iran (also known as Northern Iran) and Azerbaijan (Rix 2007). In Iran, P. fraxinifolia grows wildly in the three northern provinces Golestan, Guilan and Mazandaran, but in recent years, small populations have also been reported in two other western provinces, Lorestan (in the Zagros Mountains) and Ilam (bordering Iraq) (Nabavi et al. 2008). For a long time, native and local people have used young leaves of this tree as an anaesthetic agent for catching fish (Sadighara et al. 2009), for dyeing and as an antifungal agent (Hadjmohammadi and Kamyar 2006;Ebrahimzadeh et al. 2008Ebrahimzadeh et al. , 2009. Various parts of this plant are rich in phenolic and flavonoid compounds Nabavi et al. 2008) and may therefore provide interesting bioactive compounds. Although P. fraxinifolia is currently of little economic importance in forestry, it has been planted as an ornamental tree throughout Section Editor: Gerhard Rambold Europe mainly in large parks (Forrest 2006). So far, although Pterocarya species represent important components of Western and Eastern Asian forest ecosystems and are widely planted as ornamental trees, their mycobiota are poorly known and largely understudied. Voglmayr et al. (2017) recently described the new genus Juglanconis for four Melanconis species on hosts of tribe Juglandinae, viz. three species (Juglanconis appendiculata, J. juglandina, J. oblonga) on various Juglans species and one (J. pterocaryae) from Pterocarya spp. During these investigations, the taxonomy of J. pterocaryae proved to be a complex issue that could not be resolved with certainty, as it involved asexual and sexual morphs described from two different Pterocarya hosts, i.e. P. fraxinifolia and P. rhoifolia from Western Asia and Japan, respectively. As first species, the asexual Melanconium pterocaryae was described by Kuschke (1913) from P. fraxinifolia collected in the Georgian Republic (Abkhazia). The species apparently was not recollected again until Riedl and Ershad (1977) published a record from the same host from Iran. No sexual morph is known from this host, and no specimens or cultures were available for morphological investigations and sequencing. Based on a holomorphic collection from P. rhoifolia collected in Japan, Kobayashi (1970) described Melanconis pterocaryae, and he considered that his species represented the sexual morph of Melanconium pterocaryae, based on similar conidial sizes of the Japanese collection and the original description of M. pterocaryae by Kuschke (1913). This synonymy was also accepted by Voglmayr et al. (2017), who accordingly combined the older Melanconium pterocaryae into their new genus Juglanconis. However, at that time, this synonymy could only be based on morphological evidence, because DNA data were only available for the ex-type culture of the Japanese Melanconis pterocaryae, but not for isolates from P. fraxinifolia, the type host of the basionym.
Recently, fresh collections from the type host of Melanconium pterocaryae, P. fraxinifolia, were made in Austria, the Czech Republic and Iran. This enabled us to perform detailed morphological investigations as well as pure culture isolation for sequencing and molecular phylogenetic analyses to resolve the taxonomic status of Melanconium pterocaryae and Melanconis pterocaryae, the results of which are reported here.

Field survey and sample collection
During 2013-2017, natural forests in Guilan province (Northern Iran) were surveyed for endophytic fungal pathogens associated with trunk diseases of Pterocarya fraxinifolia. Symptomatic branches (1-4 samples from each tree) from trees showing canker and dieback were collected randomly from Asalem (Talesh), Chobar (Shaft), Jirdeh (Shaft), Masal, Rezvanshar (Talesh), Rudbar, Shaft and Talesh. Cross sections of symptomatic branches were examined in order to investigate development of wood necrosis in the wood and the type of necrosis was recorded. For fungal isolations, small wood fragments (5-8 mm) were cut from the margin between healthy and affected wood tissues. Wood discs were surface disinfected by immersion in 2% sodium hypochlorite (NaOCl) for 2 min and rinsed twice in sterile distilled water (SDW). Then they were dried under sterile airflow in the laminar hood and were placed on Petri dishes containing malt extract agar (MEA: 2% malt extract, Merck, Darmstadt, Germany) supplemented with 100 mg/l streptomycin sulphate (MEAS). Petri dishes were incubated at 25°C for 5-15 days. Growth of endophytic fungi from the tissue segments were subcultured onto fresh MEA plates and incubated at 25°C. In most cases, cankers and twigs with dieback symptoms were covered with black conidiomata (acervuli). Fungal isolations were made also from conidiomata formed on cankers and twigs. During 2017-2018, cankered branches of P. fraxinifolia bearing black conidiomata were also collected in landscape parks in Austria and the Czech Republic and pure cultures isolated from conidia.

Sample sources
Of the 12 isolates of Juglanconis pterocaryae from P. fraxinifolia included in the morphological and molecular phylogenetic analyses, 10 originated from conidia of fresh specimens and 2 were isolated from diseased host tissues (IRNHM-K116 = IRNHM-JP116 and IRNHM-K151 = IRNHM-JP151). Details of the strains including NCBI GenBank accession numbers of gene sequences used to compute the phylogenetic trees are listed in Table 1. Strain acronyms other than those of official culture collections are used here primarily as strain identifiers throughout the work. Representative isolates have been deposited at the Westerdijk Fungal Biodiversity Centre, Utrecht, The Netherlands (CBS culture collection). Details of the specimens used for morphological investigations are listed in the Taxonomy section under the respective descriptions. Herbarium acronyms are according to Thiers (2018). Specimens have been deposited in the Fungarium of the Department of Botany and Biodiversity Research, University of Vienna (WU).

Morphology
Microscopic observations were made in tap water except w h e r e n o t e d . M e t h o d s o f m i c r o s c o p y i n c l u d e d stereomicroscopy using a Nikon SMZ 1500 equipped with a Nikon DS-U2 digital camera, and Nomarski differential interference contrast (DIC) using a Zeiss Axio Imager.A1 compound microscope equipped with a Zeiss Axiocam 506 colour digital camera. Images and data were gathered using the NIS- Table 1 Strains and NCBI GenBank accessions used in the phylogenetic analyses of the combined multigene matrix of Juglanconis; accessions of J. pterocaryae for which only the ITS-LSU was sequenced were not included in the phylogenetic analyses. Sequences formatted in bold were generated during the present study   Elements D v. 3.22.15 or Zeiss ZEN Blue Edition software packages. Measurements are reported as maxima and minima in parentheses, and the range representing the mean plus and minus the standard deviation of a number of measurements given in parentheses. Due to poor or untypical sporulation in pure culture, conidial and conidiophore morphology was only studied in detail from natural substrates.
Culture preparation, DNA extraction, PCR and sequencing Single conidium isolates were prepared and grown on MEA or on 2% corn meal agar plus 2% w/v dextrose (CMD). Growth of liquid culture and extraction of genomic DNA was performed as reported previously (Voglmayr and Jaklitsch 2011;Jaklitsch et al. 2012) using the DNeasy Plant Mini Kit (QIAgen GmbH, Hilden, Germany).
The following eight loci were amplified and used for phylogenetic analyses: partial nuSSU-ITS-LSU rDNA, cal, his, ms204, rpb1, rpb2, tef1 and tub2; for details on loci and primers see Table 2. PCR products were purified using an enzymatic PCR cleanup (Werle et al. 1994) as described in Voglmayr and Jaklitsch (2008). DNA was cycle-sequenced using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit v. 3.1 (Applied Biosystems, Warrington, UK) and the PCR primers; in addition, primers ITS4, LR2R-A and LR3 were used as internal sequencing primers for the ITS-LSU rDNA region and TEF1_INTF and TEFD_iR for tef1 (Table 2). Sequencing was performed on an automated DNA sequencer (ABI 3730xl Genetic Analyser, Applied Biosystems).

Data analysis
The newly generated sequences were aligned to the sequence alignments of Voglmayr et al. (2017), and a combined matrix of the eight loci (partial SSU-ITS-LSU rDNA, cal, his, ms204, rpb1, rpb2, tef1 and tub2) was produced for phylogenetic analyses, with two accessions of Melanconis stilbostoma added as the outgroup. The GenBank accession numbers of sequences used in these analyses are given in Table 1.
Maximum parsimony (MP) analyses were performed with PAUP v. 4.0a163 (Swofford 2002). All molecular characters were unordered and given equal weight; analyses were performed with gaps treated as missing data; the COLLAPSE command was set to MINBRLEN. MP analysis of the combined multilocus matrix was done using 1000 replicates of heuristic search with random addition of sequences and subsequent TBR branch swapping (MULTREES option in effect, steepest descent option not in effect). Bootstrap analyses with 1000 replicates were performed in the same way, but using 10 rounds of random sequence addition and subsequent branch swapping during each bootstrap replicate.
Maximum likelihood (ML) analyses were performed with RAxML (Stamatakis 2006) as implemented in raxmlGUI 1.5 (Silvestro and Michalak 2012), using the ML + rapid bootstrap setting and the GTRGAMMA substitution model with 1000 bootstrap replicates. The matrix was partitioned for the different gene regions.

Field survey and isolation
In the field surveys in the natural forests in Guilan province (Iran), declining trees of P. fraxinifolia showed branch and trunk canker, extensive dieback of terminal and lateral branches and death (Fig. 1b, c). Examination of branches from symptomatic trees revealed seven types of wood discolouration in cross sections: brown to black wood streaking, black spots, arch-shaped necrosis, central necrosis, irregular wood necrosis, water necrosis and wedge-shaped necrosis ( Fig. 1g-k). Some collected samples showed multiple lesion types on the same sample in cross sections (Fig. 1g, i, j). A fungus morphologically resembling the genus Juglanconis (Voglmayr et al. 2017) was consistently isolated from wood lesions of affected trees (eight isolates). Among those isolates, seven (i.e. one from each different wood lesion type) were selected as representative isolates for further detailed studies. All of these isolates showed the same pure culture, conidioma and conidial characters. Two of these isolates, IRNHM-JP116 and IRNHM-JP151, were also selected for molecular studies. IRNHM-JP116 was isolated from infected tissue of a tree from Masal showing dieback and irregular wood necrosis in cross section, while IRNHM-JP151 was isolated from a tree from Asalem (Talesh) showing branch canker and irregular wood necrosis in cross section. During this work, 24 Iranian and three Austrian isolates were also recovered from conidiomata produced on twigs showing dieback (Fig. 1d-f). All these isolates had the same pure culture, conidioma and conidial characters like the isolates from lesions. In addition to Juglanconis, two isolates of Phaeoacremonium alvesii

Molecular phylogeny
The combined multilocus matrix used for phylogenetic analyses comprised 8441 characters, of which 748 were parsimony informative (112 from SSU-ITS-LSU, 41 from cal, 34 from his, 64 from ms204, 35 from rpb1, 178 from rpb2, 173 from tef1 and 111 from tub2). The MP analysis revealed 30 MP trees 1090 steps long, one of which is shown in Fig. 2. Tree topologies of all MP trees were identical except for minor differences within Juglanconis appendiculata and J. pterocaryae. The ML tree revealed by RAxML was identical to the MP tree shown. M e l a n c o n i s p t e ro c a r y a e f r o m P. rh o i f o l i a a n d J. pterocaryae from P. fraxinifolia were revealed as distinct species; the two species were not closest relatives, but the latter was placed basal to the clade containing M. pterocaryae, J. juglandina and J. oblonga with maximum support. Due to the same species epithet, a new name needs to be proposed for Melanconis pterocaryae. All five species of Juglanconis received maximum support in both analyses, as well as the relationships between the species. Etymology: referring to its occurrence in Japan. 1 SSU-ITS-LSU, partial nuclear 18S rDNA, internal transcribed spacers and intervening 5.8S rDNA and 28S rDNA amplified and sequenced as a single fragment; cal, calmodulin; his, histone H3; ms204, guanine nucleotide-binding protein subunit beta; rpb1, DNA-directed RNA polymerase II largest subunit; rpb2, DNA-directed RNA polymerase II second largest subunit; tef1, translation elongation factor 1-alpha; tub2, β-tubulin 2 Internal sequencing primers Holotype: Japan, Shizuoka, Fuji, on corticated twigs of Pterocarya rhoifolia, 5 Aug. 1968, T. Kobayashi (TFM FPH2623!); ex-type culture MAFF 410079.

Taxonomy
Notes: When describing Melanconis pterocaryae from P. rhoifolia collected in Japan, Kobayashi (1970) considered his species to represent the sexual morph of Melanconium pterocaryae from P. fraxinifolia, based on similar conidial sizes. This synonymy was also accepted by Voglmayr et al. (2017), who combined the older M e l a n c o n i u m p t e ro c a r y a e i n t o t h e n e w g e n u s Juglanconis. However, the current molecular phylogenies reveal Melanconis pterocaryae to represent a clearly distinct species, which therefore needs a new name. Morphologically, the conidial size of J. japonica is similar to that of J. pterocaryae, with slightly narrower conidia (11-20 × 5-9 μm vs. 11-22 × 6-11 μm in J. pterocaryae); however, the conidia of J. japonica usually have in average a distinctly higher length/width ratio, (1. Sexual morph unknown. Conidiomata on natural substrate acervular, 0.8-2.2 mm diam, embedded in bark tissues, blackish, inconspicuous, scattered, with central or eccentric conical olivaceous grey stromatic column 300-850 μm wide at the base; at maturity covered by blackish discharged conidial masses forming black spots 0.2-2.5 mm diam or sometimes long cirrhi on the cortex. Conidiophores (11-)17-30(−48) × (3.0-)3.5-4.7(−5.5) μm (n = 74), narrowly cylindrical, simple or branched at the base, smooth, subhyaline to pale brown. Conidiogenous cells annellidic with distinct a n n e l l a t i o n s , i n t e g r a t e d . C o n i d i a ( 11 . 2 -) 1 3 . 3 -16.8(−22.3) × (6.0-)7.5-9.3(−11.0) μm, l/w = (1.3-)1.5-2.1(−3.0) (n = 980), unicellular, hyaline when immature, medium to dark brown when mature, variable in shape, ellipsoid to elongate, sometimes pip-shaped, often truncate with an abscission scar at the base, densely multiguttulate, thick-walled; wall ca. 0.5-0.8 μm, with distinct ornamentation on the inside of the wall consisting of small irregular confluent verrucae 0.3-0.7 μm diam, with ca. 0.5-1 μm wide gelatinous sheath.
Despite extensive enquiries, no type collection of Melanconium pterocaryae could be traced in Russian or Georgian herbaria. In the apparent lack of an extant type, we here propose a well-developed Austrian collection, for which a culture and sequences are available, as neotype. Although the neotype collection does not originate from the area from where the species was described, we consider this justified, as the P. fraxinifolia accessions (and therefore also its associated Juglanconis) grown in Central Europe likely originate from the Caucasus area, the conidial sizes of the neotype collection and the protologue agree well, and the conspecific Austrian and Iranian Juglanconis accessions confirm a wide distribution of the species that likely corresponds with the distribution of its host.

Discussion
Previous molecular phylogenetic analyses had shown that Melanconis species on Juglans and Pterocarya form a highly supported lineage that is distinct from Melanconis sensu stricto, and the new genus Juglanconis was established for them (Voglmayr et al. 2017), which was classified in the new family Juglanconidaceae. However, in this previous study, only a single Eastern Asian isolate from Pterocarya rhoifolia could be included, but none from the Western Asian P. fraxinifolia. The current molecular phylogenetic analyses (Fig. 2) clearly show that Juglanconis accessions from P. fraxinifolia and P. rhoifolia represent two distinct species, J. pterocaryae and J. japonica, respectively. This is not surprising, as high host specificity in combination with vicariant speciation has been commonly reported in Diaporthales on woody hosts, e.g. in Coryneum (Jiang et al. 2018), Cryptosporella (Mejía et al. 2008(Mejía et al. , 2011a, Melanconiella pterocaryae. e, f Dead branches with J. pterocaryae acervuli, some with conidial cirrhi (spore tendrils). g Co-occurrence of arch-shaped necrosis (a) and young wedge-shaped necrosis (b). h Extensive central necrosis. i Co-occurrence of watery necrosis (a), irregular necrosis (b) and black wood streaking (c). j Co-occurrence of wedgeshaped necrosis (a), black spots (b) and arch-shaped necrosis (c). k Irregular wood necrosis MP and ML bootstrap support above 50% are given at the first and second position, respectively, above or below the branches. Strain numbers are given following the taxon names; strains formatted in bold were isolated and sequenced in the present study , Melanconis (Fan et al. 2016), Plagiostoma (Mejía et al. 2011b;Walker et al. 2014), Stegonsporium and Stilbospora Jaklitsch 2008, 2014). In many of these lineages, morphological species identification can be difficult due to lack of a clear morphological distinction, while molecular data but also host ranges are highly diagnostic on the species level. However, in the Juglanconis species on Juglans, host specificity was shown to be rather on the genus than on the species level, as both European species, J. appendiculata and J. juglandina, were reported from various hosts (the indigenous Juglans regia as well as the naturalised North American J. nigra), and the North American and Eastern  (CMD, 25 d, 16°C). e-h Conidiophores (annellides; in e, g with young conidia). i-e1 Vital conidia with gelatinous sheath. f1 Squashed conidium showing the densely verruculose inner conidial wall. All in water (a-c, i-m, f1 WU 39981, neotype; d WU 39983; e, f, n-x WU 39982; g, h WU 39985b; y-d1 WU 39986b; e1 WU 39987a). Scale bars a 500 μm; b, c 200 μm; e-e1 10 μm; f1 5 μm