DOI: 10.5061/dryad.vhhmgqnsv
title: Seven new Serendipita species associated with Australian terrestrial orchids

description: Serendipita is one of the main fungal genera that form mutualistic
 associations with species of orchids (Orchidaceae). Seven new Serendipita
 species associated with various Australian orchid genera are described.
 These Serendipita species were originally characterized by multilocus DNA
 sequence species delimitation analyses (three mtDNA and four nuclear
 genes) and confirmed as distinct with addition of further isolates and
 re-analysis of the nuc rDNA ITS1-5.8S-ITS2 (ITS) and nuc 28s rDNA (28S).
 Morphology and micro-features of cultures of each of the species are
 described. Three of the new species are binucleate, whereas the other four
 are multinucleate. For the ITS region, the seven species have within
 species sequence divergence between 1.07 and 4.31 % and all but one of the
 species pairs are separated by interspecific divergence of at least 4.35
 %. The newly described Serendipita species, S. australiana, S. communis,
 S. occidentalis, S. rarihospitum, S. secunda, S. talbotii, and S. warcupii
 are shown to be separate species to S. vermifera on the basis of
 comparison against a sequence from the type. Isolates originally
 identified by Warcup as Sebacina “vermifera” from Caladenia orchids are
 reidentified as belonging to three of the species newly described here.
 Some non-Caladenia isolates identified by Warcup as S. “vermifera” are
 also shown to be non-conspecific with the type of S. vermifera. On the
 basis of ITS sequences, 346 isolates from 26 other studies, previously
 identified under provisional designations, are accomodated under the novel
 species. The species of Serendipta described here associate with the
 Australian orchid genera Caladenia, Cyanicula, Elythranthera,
 Ericksonella, Eriochilus, Glossodia, and Pheladenia. Most of the novel
 Serendipita species occur widely across Australia, often with widely
 distributed hosts, but one species, Serendipita rarihospitum, associates
 with narrowly distributed orchid species.
description: <b><i>Fungal
 isolation.</i></b><b><i>- </i></b>To
 broaden the breadth of sampling for some <i>Serendipita</i>
 OTUs (B, D, E, F, H) identified in Whitehead et al. (2017), additional
 fungal isolates were obtained from several orchid species including
 <i>Glossodia major </i>(Australian Capital Territory, New
 South Wales, Queensland)<i>, Elythranthera brunonis
 </i>(Western Australia)<i>, Caladenia attingens</i>
 (Western Australia), and <i>C. cairnsiana </i>(Western
 Australia)<i>. </i>Fungal pelotons were isolated from the
 collar regions of orchid roots by cutting this region into sections in
 sterile double-distilled
 water (ddH<sub>2</sub>O) using a scalpel blade.
 Sections containing pelotons were serially rinsed in four changes of
 sterile dd.H<sub>2</sub>O and torn apart in the last rinse
 with sterile forceps. The pelotons were then transferred to half-strength
 Fungal Isolation Medium agar (½ FIM) (Clements and Ellyard 1979) using a
 pipette, after which plates were sealed and incubated at 23 C for 2–4 d.
 The growing mycelium from the pelotons were then subcultured onto
 quarter-strength Potato Dextrose Agar (¼ PDA) or ½ FIM for approximately
 two wks. All cultures were stored and maintained in Mili-Q
 H<sub>2</sub>O at 7 C. Voucher specimens of the fungi, as
 living cultures stored in a metabolically inactive state, were lodged at
 the Royal Botanic Gardens Victoria Fungal Culture Collection (RBGV-FCC)
 and the culture collection of the Department of Agriculture, Victoria
 (VPRI). In addition, specimens prepared by freeze-drying cultures grown in
 ½ FIM broth were lodged at the National Herbarium of Victoria
 (MEL). <em><strong>DNA
 extraction, PCR, Sequencing, and phylogenetic
 analysis.-</strong></em> Protocols for DNA extraction,
 polymerase chain reaction (PCR), and sequencing followed Oktalira et al.
 (2019). The 11 additional <em>Serendipita </em>samples
 isolated in this study (SUPPLEMENTARY TABLE 1) were sequenced for the nuc
 rDNA ITS1-5.8S-ITS2 (ITS) using universal primers ITS1 and ITS4 (White et
 al. 1990). Sequences obtained in the present study were checked and edited
 using Sequencher 5.4.1 (Gene Codes Corp., Ann Arbor, Michigan). These new
 sequences were deposited in GenBank –(SUPPLEMENTARY TABLE 1).
 Three analyes based on different sets of taxa were carried out to
 investigate the phylogenetic
 position and species delimitation of isolates of
 <i>Serendipita</i> from Australian orchids. First,
 we analysed the position of Australian
 orchid isolates in relation to the diversity in the family Serendipitaceae
 by constructing an alignment of the ITS-28S region including
 (1) up to three sequences representative of each of the eight OTUs defined
 by Whitehead et al. (2017), (2) sequences from described
 <i>Serendipita</i> species where available
 (S<i>. herbamans, S. indica</i>, <i>S.
 restingae</i>,<i> S. vermifera, S.
 whamiae</i>, and <i>S. williamsii</i>)<i>,
 </i>(3) sequences from isolates originally identified as <i>S.
 vermifera</i> by Warcup, (4) sequences representing the major
 subclades of the genus <i>Serendipita</i> from previous
 studies (Weiss et al. 2011; Weiss et al. 2016), and (5) sequences of
 several <i>Helvellosebacina</i> and <i>Sebacina
 </i>representating Sebacinaceae. <i>Auricularia
 auricula-judae</i> (Bull.) Quél was used as the outgroup
 following Weiss et al. (2011). While we attempted to only
 include taxa for which both ITS and 28S sequences were available, both
 sequences were not always available for key reference taxa (19/79 taxa
 lacked an ITS sequence, while the 28S was not available for six taxa)
 (FIG. 1, SUPPLEMENTARY TABLE 2).  For the second and
 third analyses, we placed additional isolates into the species framework
 generated by the first analysis, by analysing ITS (SUPPLEMENTARY FIG. 1)
 and 28S (SUPPLEMENTARY FIG. 2) alignments separately because
 <i>Serendipita</i> sequences from Australian orchids were
 mostly available for ITS only. The separate ITS and 28S analyses
 incorporated sequences from the study of Whitehead et al. (2017) and
 <i>Serendipita</i> sequences isolated from Australian orchids
 from GenBank (SUPPLEMENTARY TABLE 3), selected from BLAST searches against
 each OTU as identified in Whitehead et al. (2017) matching at least 92 %
 sequence similarity for ITS or 90 % for 28S. For ITS, we additionally
 incorporated 11 sequences newly obtained in this study (SUPPLEMENTARY
 TABLE 1). For 28S, the 123 sequences from
 Whitehead et al. (2017) are identified by numbers preceded by “CLM”
 (prefix for orchid fungal isolates in the culture collection of Celeste
 Linde Mycorrhizal collection – Australian National University) and the
 sequences were retrieved from Dryad (https://doi.org/10.5061/dryad.2hf40). In total there were 510 ITS sequences and 195 28S sequences. Alignments were constructed in ClustalW (Thompson et al. 1994) and manually checked. Phylogenetic trees were estimated using both maximum likelihood (ML) and Bayesian inference (BI). A ML phylogenetic analysis with 1000 replicate bootstraps was performed using RAxML (Stamatakis et al. 2008). The BI analysis was performed with MrBayes 3.2.6 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003) with node support assessed by Bayesian posterior probability (BPP) and calculated after running two parallel runs of four heated chains. Convergence of runs was confirmed when the average standard deviation of the split frequencies reached &lt; 0.01 and estimated sampling sizes were (ESS) &gt; 200. For both ML and BI analyses, the best-fit substitution model was selected by ModelFinder <b>(</b><strong>Kalyaanamoorthy et al. </strong>2017<b>)</b> in IQ-TREE 2.0 according to the Bayesian Information Criterion (Nguyen et al. 2014) for each locus and partition.  The concatenated ITS-28S alignment was partitioned by each locus.