Four Tulasnella taxa associated with populations of the Australian evergreen terrestrial orchid Cryptostylis ovata

Journal Pre-proof Four Tulasnella species are associated with populations of the Australian evergreen terrestrial orchid Cryptostylis ovata D.Q. Nguyen, Hua Li, T.T. Tran, K. Sivasithamparam, M.G.K. Jones, S.J. Wylie PII:

S1878-6146(19)30148-5

DOI:

https://doi.org/10.1016/j.funbio.2019.10.006

Reference:

FUNBIO 1077

To appear in:

Fungal Biology

Received Date: 12 July 2019 Revised Date:

11 October 2019

Accepted Date: 15 October 2019

Please cite this article as: Nguyen, D.Q., Li, H., Tran, T.T., Sivasithamparam, K., Jones, M.G.K., Wylie, S.J., Four Tulasnella species are associated with populations of the Australian evergreen terrestrial orchid Cryptostylis ovata, Fungal Biology, https://doi.org/10.1016/j.funbio.2019.10.006. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

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Four Tulasnella species are associated with populations of the Australian evergreen

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terrestrial orchid Cryptostylis ovata

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D. Q. Nguyen, Hua Li, T. T. Tran, K. Sivasithamparam, M. G. K. Jones, S. J. Wylie*

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Plant Biotechnology Research Group - Virology, Western Australian State Agricultural

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Biotechnology Centre, School of Veterinary and Life Sciences, Murdoch University, 90

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South Street, Murdoch, WA6150, Australia

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Email: Duy Q. Nguyen, [email protected] Email: Hua Li, [email protected] Email: Thao T. Tran, [email protected] Email: Krishnapillai Sivasithamparam, [email protected] Email: Michael G. K. Jones, [email protected] *Corresponding author: Stephen J. Wylie. E-mail: [email protected] Tel: +61 89360 6600 Address: Plant Biotechnology Research Group - Virology, Western Australian State Agricultural Biotechnology Centre, School of Veterinary and Life Sciences, Murdoch University, 90 South Street, Murdoch, WA6150, Australia

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Abstract

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Of the more than 400 indigenous orchid species in Western Australia, Cryptostylis ovata is

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the only species that retains its leaves all year round. It exists as a terrestrial herb and

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occasionally as an epiphyte in forested areas. Like all terrestrial orchids, C. ovata plants

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associate with mycorrhizal fungi, but their identities have not previously been investigated.

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Fungi were isolated from pelotons in rhizomes collected from three southern and two

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northern populations of C. ovata on six occasions over two years. Phylogenetic analysis of

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ITS sequences temporally and spatially revealed that all the fungal isolates were of

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Tulasnella species of four distinct groups. One Tulasnella group was present only in the three

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southern orchid populations, and it closely resembled T. prima isolates previously described

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from Chiloglottis sp. orchids from eastern Australia. Isolates collected from plants in the two

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northern populations were of three undescribed Tulasnella groups. Analysis of intra-group

1

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diversity using inter-simple sequence repeat markers revealed that plants were usually

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colonised by a single genotype of Tulasnella at each sampling period, and this genotype

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usually, but not always, persisted with the host plant over both years tested.

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Keywords: Wild plants; symbiosis; orchid; mycorrhizal association; Tulasnella

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1. Introduction

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Most land plants maintain intimate symbiotic root associations with fungi, known as

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mycorrhizal associations. Orchids in particular are fully dependent on mycorrhizae at some or

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all stages of their life cycles. At the early protocorm development stage, all orchid species

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studied are parasites on fungi (mycoheterotrophs), on which they are completely dependent

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for carbon and nutrients. Even those that become photosynthetic later usually maintain fungal

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symbionts over the course of their lives, the exceptions being some epiphytic groups

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(Bayman et al. 2002). Some orchids are generalists, accepting mycorrhizae representing

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several different species or genera (Bonnardeaux et al. 2007), while others are specialists,

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undertaking relationships with only one or a few related species (Brundrett 2004). The

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generality or specificity of these symbiotic partnerships may or may not dictate distribution

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and abundance of orchids in the wild. Other factors, such as pollinator presence, are also

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important dictators of distribution and abundance (Davis et al. 2015). In some species of

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Drakaea (hammer) orchids in Australia, for example, mycorrhizal specificity is associated

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with rarity, but in other species it is not (Phillips et al. 2011). The indigenous Australian

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orchid Pheladenia deformis is a mycorrhizal specialist, associating with only one genotype of

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a Sebacina species, yet it occurs widely across the continent. In contrast, the highly

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endangered and localized North American orchid Piperia yadonii associates with a wide

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range of mycorrhizal species representing families Ceratobasidiaceae, Sebacinaceae and

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Tulasnellaceae (Pandey et al. 2013).

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In some orchid species, there is temporal variation in mycorrhizal species over the lifecycle

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(Oja et al. 2015), and this indicates ability by the plant to dictate acceptance or rejection of

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certain fungal partners. The mechanisms of this choice process is largely unknown

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(Dearnaley et al. 2013). Knowledge of temporal changes to mycorrhizal fungi associations

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may be crucial in understanding reproductive success or otherwise of orchid species (Swarts

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and Dixon, 2009; Barnes et al. 2016).

71 2

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Cryptostylis R. Br. are commonly known as slipper orchids or tongue orchids. Cryptostylis,

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along with the monotypic genus Coilochilus, create the Cryptostylidinae subtribe of

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Diuridaea within family Orchidaceae. Cryptostylis contains about 25 described species

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distributed in Australia, New Zealand, several islands in Melanesia and the Pacific, Papua

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New Guinea, Philippines, Vietnam, Malaysia, Thailand, Taiwan, Sri Lanka, India, and

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Indonesia (Pridgeon et al. 2001). The lifestyles of Cryptostylis species vary greatly. For

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example, C. hunteriana Nicholls is a leafless saprophytic herb that lacks chlorophyll and

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relies completely on its association with its (undescribed) mycorrhizal partner (Bell 2001). It

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produces flowers in winter and is listed as vulnerable across its range from Victoria to

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Queensland (Wyong Shire Council, 2000). In contrast, C. subulata (Labill.) Rchb.f. is

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photosynthetic and usually produces flowers from summer to autumn (November to April),

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but some individuals flower over the whole year. It is widely distributed in South Australia,

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Queensland, New South Wales, Victoria, Tasmania, and New Zealand (Biodiversity

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Conservation Unit 2008), and is not considered vulnerable.

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Cryptostylis ovata R.Br. is an evergreen orchid endemic to the south-west of Western

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Australia (Brown et al. 2008), a region of high indigenous plant biodiversity (Hopper and

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Gioia, 2004). Its habitat is mainly terrestrial, occurring on rock outcrops, forest floors, dead

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tree stumps and trunks, and infrequently it is epiphytic on live trees (Brundrett, 2014).

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Besides its evergreen habit, it is unusual among most other orchids of the region in that it

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produces flowers in mid-summer when temperatures can reach over 40oC and it rarely rains.

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C. ovata is not listed as threatened. Pollination of C. ovata, and the other four Australian

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species of Cryptostylis, is achieved by the sexual deception of males of one widespread

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species of ichneumon wasp (Lissopimpla excelsa), that are attracted by volatile pheromones

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emitted by the orchid flower that mimic those of female wasps (Brundrett 2014). This orchid

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usually grows in clonal groups linked by rhizomatous roots (Brundrett 2014). Thus,

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reproduction of C. ovata is often by vegetative means (eMonocot 2014). Germination from

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seed, either in artificial or natural conditions, has not been reported for any Cryptostylis

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species, although copious numbers of seed are produced when pollination is successful. The

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existence of widely-separated plants in the field suggests natural distribution by viable seed.

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The lifespan of individual plants is unknown, but the authors have observed individual plants

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for over three decades, and they may persist for much longer periods. Individual leaves on

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plants persist for several years (Brundrett 2014).

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Identification of mycorrhizal fungi associated with Cryptostylis species is largely unknown.

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Previous work by Warcup (1981) in Australia revealed that plants of an unidentified

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Cryptostylis species were associated with Tulasnella asymmetrica and an unidentified

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Tulasnella sp. based on characteristics of sporophore morphology. In Australia, orchids

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generally maintain mycorrhizal associations with Rhizoctonia-like fungi such as Tulasnella,

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Sebacina and Ceratobasidium (Linde et al. 2014).

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Tulasnella is a genus in the fungal family Tulasnellaceae, order Cantharellales. Several

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Tulasnella species are recorded as forming symbiotic associations with orchids outside

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Australia, including Serapias vomeracea in Europe (Fochi et al. 2017), Dendrobium

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officinale in China (Tan et al. 2014), and Goodyera pubescens, Liparis lilifolia, and Tipularia

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discolor in the USA (McCormick et al. 2004). This genus contains approximately 50

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described species (Kirk et al. 2008), and more have been described from barcode sequences

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but not formally named (Cruz et al. 2014; Jacquemyn et al. 2012; Jacquemyn et al. 2011; Oja

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et al. 2015; Pandey et al. 2013; Smith et al. 2010). A recent study found that ITS regions of

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Tulasnella isolates collected from several genera of Australian terrestrial orchids, but not

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including Cryptostylis, were discriminatory between species (Linde et al. 2014).

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Orchids from other parts of the world associate with a broad range of other fungi, including

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Psathyrella, Coprinus (Yamato et al. 2005), Leptodontidium orchidicola, Epulorhiza repens,

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E. anaticula, Ceratorhiza goodyerae-repentis. Sebacina, Sistotrema and Moniliopsis

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anomala (Currah et al. 1990). Identification of mycorrhizal fungi associated with orchids

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generally involves identification from fungal pelotons, coils of fungi within specialist

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root/rhizome cells. High throughput sequencing approaches have identified large numbers of

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fungi associated with orchids, for example sequences corresponding to 205 genera of fungi

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were associated with healthy Phalaenopsis roots (Huang et al. 2014), although whether these

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were genuine symbionts with ecological roles in orchid biology was uncertain.

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The aim of this study was to identify fungi associated with root pelotons in C. ovata, and to

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compare genetic diversity of the fungi with respect to host plant, population location, and

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collection time over a period of two years.

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2. Materials and Methods

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2.1 Cryptostylis ovata

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Five collection sites were chosen to represent some of the different climatic and ecological

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conditions under which C. ovata exists (Table 1). The Bertram site is a remnant Banksia and

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Eucalyptus forest system surrounded by urban development. Of the sites sampled, it received

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the highest mean maximum temperatures in 2016 and 2017. The Jarrahdale site is a

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previously-milled Eucalyptus marginata (Jarrah) forest. Although it is located only 19 km to

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the east of Bertram, its mean temperatures are lower, its rainfall is higher, and the soil type

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differs. Stratham and Ludlow are located 20 km apart, and have quite similar climatic

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conditions. The two sites differ in the vegetation cover. The Ludlow site is an exotic Pinus

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radiata plantation. Stratham is a remnant fragment of forest dominated by E. gomphocephala

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(Tuart) trees. Pemberton is distinct from the other sites in that it is dominated by E.

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diversicolor (Karri) trees, it has the lowest mean maximum temperature and the highest

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annual rainfall (Table 1), and is the least disturbed site.

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2.2 Fungal collections

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Rhizome samples from C. ovata were collected at approximately three-monthly intervals

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from August 2016 to November 2017. Non-lethal sampling was done by carefully exposing

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the end of a rhizome and removing a 2-4 cm length. C. ovata plants often exist as groups of

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clonally propagated shoots covering several square metres. Thus, it was often difficult to

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decide whether shoots located within short distances of one another were from distinct plants

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or if they were clones connected by underground rhizomes. Five plants we believe were

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distinct from one another were sampled at each of the five populations. Usually, the same

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five plants were sampled at each sampling occasion. Exceptions occurred at the two northern

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populations, one located at Jarrahdale, where a forest fire destroyed the plant labels during

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the November 2016 to February 2017 summer period, and the other at Bertram, where a

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firebreak was constructed by a bulldozer through the small C. ovata population we were

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sampling, destroying our labeled plants in February 2017. In both cases, new plants were

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labelled and subsequently sampled. The sampled populations were broadly divided into the

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two northern populations of Bertram, Jarradale, and the three southern populations of

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Ludlow, Stratham and Pemberton (Table 1, 2, Fig 1). Samples were stored in polythene zip-

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lock bags and kept cool until analysis.

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2.3 Isolation and culture of mycorrhizal fungi

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Cryptostylis ovata rhizome sections were washed and surface-sterilized by dipping in 2%

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(w/v) sodium hypochlorite solution for 3 min, then in 70% ethanol for 10 sec, and then rinsed

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twice in sterile water. The pelotons (fungal coils inside rhizome cells) (Fig 2) were released

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by crushing sections of rhizome in a 1.5 mL centrifuge tube containing 300 µL sterile water

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with a sterile pestle. A compound microscope was used to identify individual pelotons, which

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were placed onto soil solution equivalent (SSE) agar medium (0.40 g L-1 NH4NO3, 0.0136 g

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L-1 KH2PO4, 0.61 g L-1 MgCl.6H2O, 0.058 g L-1 NaCl, 0.861 g L-1 CaSO4.2H2O, 0.073 g L-1

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FeNaEDTA, 0.2 g L-1 MES buffer, 2.5 g L-1 sucrose, 8.0 g L-1 agar, and 0.1 g L-1

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streptomycin sulfate) in petri dishes (Hollick 2004). Pelotons were incubated on SSE agar

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medium in the dark for 10-14 days at 24oC. Sub-culturing to fresh SSE agar or liquid SSE

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medium (as above but lacking agar) was done by cutting a 5 mm x 5 mm block of agar taken

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from the edge of the fungal colony. SSE liquid medium cultures were incubated as above, but

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on a shaker.

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2.4 DNA analysis

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DNA extraction

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DNA was extracted from approximately 100 mg of macerated fungal tissue. Mycelium of

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each sample was harvested, washed with distilled water and then dried by pressing between

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absorbent water towels. Mycelium was transferred to a mortar and ground thoroughly in

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liquid nitrogen to a fine powder. The powder was transferred to 1.5 mL centrifuge tubes to

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which was added 450 µL extraction buffer (0.1 M NaCl, 50 mM Tris pH 8.0, 0.5 mM EDTA

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pH 8.0, 1% SDS, 1% PVPP, 0.1% β-mercaptoethanol) and 450 µL phenol:chloroform pH 8.0

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(1:1) saturated with TE. The mixture was vortexed for 5 min and centrifuged for 2 min (24oC,

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10,000 xG). The aqueous phase mixed with an equal volume of phenol:chloroform before

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mixing for 5 min and centrifugation for 2 min. The aqueous phase (300 µL) was removed to a

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new tube containing 58 µL of absolute ethanol and approximately 200 mg cellulose powder

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(Whatman CF11), was added to the tube before vortexing 5 min. The tube was centrifuged 2

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min before 250 mL of the supernatant was removed to a new tube that was then added 20 µL

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3M NaOAC pH 5.2 and 625 µL cold absolute ethanol. The mixture was chilled 30 min at -

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20oC before centrifugation 20 min at 10,000 xG, and the supernatant was poured off. The

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pellet was washed two times with 200 mL of 70% ethanol and dried. The pellet containing

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DNA was suspended 100 µl of buffer EB (Qiagen) then stored at -20oC.

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PCR amplification and DNA sequencing

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Universal primer pairs ITS1 and ITS4 and/or ITS5 and ITS4 (Table S2) were used to amplify

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the internal transcribed spacer (ITS) region and 5.8s rDNA genes (White et al. 1990) of each

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fungal isolate. PCRs were done in 20 µL reaction volumes that included 10 µL of GoTaq®

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Green 2X PCR Master Mix (Promega Corp), 8 µL of H2O, 1 µL of fungal DNA, 0.5 µL of

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ITS4, and 0.5 µL of either ITS1 or ITS5. Primers ITS1 and ITS4 were used in PCRs under

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the following conditions: denaturation at 95oC for 3 min followed by 35 cycles of 94oC for 50

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sec, 56oC for 50 sec, and 72oC for 1 min, and a final extension step at 72oC for 10 min. When

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amplification using primers ITS1 and ITS4 was unsuccessful, primers ITS5 and ITS4 were

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used with the above settings except that annealing was at 48oC for 30 sec (Raja et al. 2017).

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PCR products were purified using AxyPrep Mag PCR Clean-up kit (Axygen). Sequencing

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reactions were done in both directions using the primers that amplified them. The products

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were sequenced by Sanger sequencing using BigDye® version 3.1 terminator mix (Applied

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Biosystems). Sequences were edited using Geneious 10.2.6 (Biomatters). Sequences were

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then submitted to BlastN 2.8.1 (Altschul et al. 1997) searches of GenBank to identify

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matches.

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Phylogenetic analysis

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Geneious 10.2.6 (Biomatters) was used to manually trim low-quality data at the start and

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end of sequences. Edited sequences were aligned with similar sequences available at

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GenBank. Phylogenetic reconstruction of ITS sequences was done using the Maximum

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Likelihood method based on the Kimura 2-parameter model within Mega7 (Kumar et al.

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2016).

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2.5 ISSR markers

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University of British Columbia (UBC) primer set 9, consisting of 100 primers, was tested on

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fungal isolates to identify primers that gave polymorphic banding patterns. Five primers

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generated reproducible polymorphic banding patterns: UBC 808, 842, 861, 862 and 880

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(Table S3), and these were used in subsequent experiments. Amplification of genomic DNA

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was done in a 20 µl volume composing 10 µL GoTaq® Green 2X PCR Master Mix (Promega

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Corp), 1 µL of purified fungal DNA (approx. 300 ng), 8 µL H2O, and 1 µL UBC primer.

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PCR amplification was done after an initial denaturation cycle of 2 min at 95oC. Annealing

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temperatures differed depending on the primer used. Amplification occurred over 35 cycles

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of 30 seconds at 94oC in all cases; then 30 seconds at 46oC (for UBC 808), at 47oC (for UBC

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842), at 54oC (for UBC 861 and UBC 862), and at 42oC (for UBC 880); followed in all cases

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by 1 min at 72oC. There was a final incubation at 72oC for 8 min. PCR products were

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separated on 2% agarose gels stained with SYBR®safe. All ISSR reactions were repeated

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using the original DNA sample, and amplicons were separated on another 2% agarose gel.

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ISSR amplification products were recorded as being either present (1) or absent (0) for each

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isolate. Where bands were very faint and/or were not reproducible between the two

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amplification reactions, they were ignored. ISSR data was analysed using GenAlEx v6.5

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software (Peakall and Smouse, 2012). Nei’s genetic distance (Nei 1978) was calculated to

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establish degrees of distance between isolates and populations. Genetic diversity of each

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population was determined by Shannon’s diversity index (Shannon and Weaver, 1949).

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GenAIEx v6.5 was used for analysis of molecular variance (AMOVA) to examine the source

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of observed genetic variation. Dendrograms were generated using the Maximum Composite

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Likelihood model with Unweighted Pair Group Method with Arithmetic Mean (Sneath and

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Sokal, 1973) in Mega7 (Kumar et al. 2016).

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3. Results

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3.1 Identification of fungi

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Ninety fungal isolates (Table 2) were identified from sequences of PCR amplicons generated

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from ITS1 and ITS4 or ITS5 and ITS4 primer pairs from five plants at five populations.

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Analysis of pairwise alignments of ITS sequences revealed that they clustered into four

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distinct groups. Some Tulasnella ITS sequences in this study shared high sequence identity to

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sequences of T. prima and T. sphagneti isolates collected from other species of indigenous

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orchids in Australia (Linde et al. 2017; Roche et al. 2010). Twenty-two ITS sequences from

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Tulasnella species collected in Australia and the USA (Table S1) were compared to the C.

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ovata fungal sequences.

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Fungal isolates were named as follows: C = Cryptostylis ovata. The second letter corresponds

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to the collection site: B = Bertram, J = Jarrahdale, L = Ludlow, P = Pemberton, S = Stratham.

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The first number is the sample collection time: 1 = first sampling time (8/2016), 2 = second

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sampling time (11/2016), 3 = third sampling time (2/2017), 4 = fourth sampling time

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(5/2017), 5 = fifth sampling time (8/2017), 6 = sixth sampling time (11/2017). The second 8

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number is the individual plant number. The number in brackets is the peloton (culture)

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number (Table 2).

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Analysis of ITS sequences of isolates from the three southern populations at Ludlow,

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Stratham and Pemberton revealed that these isolates were highly similar, clustering together

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in one group with isolates of T. prima isolated from Chiloglottis species from the Australian

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Capital Territory and New South Wales. Together, all members of this group shared 98-100%

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pairwise sequence identity (Table 3, Fig 3). Sequences of isolates from the two northern

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populations of Bertram and Jarrahdale differed between populations. All isolates from

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Bertram fell into one clade with 97% bootstrap support (Fig 3), which was not associated

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with previously-described Tulasnella species. ITS sequences of fungal isolates from Bertram

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at all collection times that fell into one clade and shared 93-95% pairwise identity with other

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populations, representing a distinct group. Isolates from Jarrahdale fell into two groups

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(Jarradale 1 and 2) with 99% bootstrap support (Fig 3). Neither group is associated with

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previously-described Tulasnella species. All isolates within the Jarrahdale 1 group were

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collected from two C. ovata plants (CJ11 and CJ13) in August 2016. Isolates from Jarrahdale

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2 were collected at three different times: one isolate, CJ35(15), was sampled in February

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2017, two isolates, CJ45(12) and CJ45(16), were collected from one plant in May 2017, and

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13 isolates, CJ51(3, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 17, 19), were isolated from one plant in

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August 2017. Isolates from Jarrahdale 1 and Jarrahdale 2 groups shared 93-94% pairwise

292

identity. Within each phylogenetic group, pairwise identities were within the 98-100% range,

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whereas between groups, identities were 95% or less. The fungal isolates from northern

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clades Jarrahdale 1, Jarrahdale 2 and Bertram shared 93% to 95% pairwise identity. Isolates

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from clades Jarrahdale 1, Jarrahdale 2, and Bertram shared 89-94% identity with isolates in

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the southern group and T. prima clade (Table 3). All C. ovata isolates shared 78-92% identity

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with ITS sequences of T. sphagneti, T. secunda, T. tomaculum, and T. warcupii (Table 3).

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3.2 Intra-group diversity

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ISSR markers were used to assess genetic diversity of fungal isolates collected from the same

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plants over time, and between plants of the same population. Forty-nine fungal isolates

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representing the five orchid populations sampled were assessed, of which 13 fungal isolates

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were from the Pemberton population, 10 from Ludlow, 10 from Stratham, seven from

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Bertram, and nine from Jarrahdale (Table 5). Fifty-four fragments were generated by five

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ISSR markers and fragments ranged from approximately 320 - 1,800 bp in length. The 9

306

number of fragments amplified per primer was nine (UBC 880) to twelve (UBC 861 & 862)

307

(Table S3). The percentage of polymorphic loci (% P) were from 31.5% in Bertram to 53.7%

308

in Ludlow (Table 5). Shannon’s information indices (I) of Bertram (I = 0.18) and Jarrahdale

309

(I = 0.23) were significantly lower than those of southern populations (from 0.24 to 0.30)

310

(Table 5). This measure confirmed that the genetic diversity of fungal isolates from Stratham,

311

Ludlow and Pemberton was more uniform than isolates at the two northern populations.

312 313

Like the ITS-based analysis, ISSR analysis placed the two Jarrahdale groups close together,

314

yet distinct. Unlike the ITS-based analysis, ISSR markers placed the Jarrahdale groups within

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the southern group that resembled T. prima. The Bertram population was clearly distinct

316

under both analyses (Fig 4, Table 4).

317 318

The banding patterns of ISSR markers showed that fungal isolates collected from the same

319

plant at the same time were often of the same genotype (Fig 4). This indicated that individual

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plants were usually associated with only one fungal genotype over the study period. For

321

example, genetically similar isolates CJ51(3), CJ51(9) and CJ51(19) were collected from the

322

same Jarrahdale plant at the same time in August 2017. Similarly, genetically similar isolates

323

CJ35(15) and CJ45(12) and CJ45(16) were collected from the same Jarrahdale plant in

324

February and May 2017, evidence of a stable association over at least three months. At the

325

Ludlow population, genetically similar isolates CL14(9), CL24(1), CL44(17) and CL54(10)

326

were collected from one plant over one year, again indicative of an association between the

327

plant and fungus being stable over at least one year. However, this was not always the case.

328

In the Pemberton population, the fungi associated with plant 1 showed greater diversity.

329

There were six isolates collected from plant 1 in both 2016 and 2017, which were of two

330

genotypes. One group included isolates CP11(3) collected in August 2016 and CP21(1)

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collected in November 2016, and the other group consisted of isolates CP51(4), CP51(5),

332

CP51(12) collected in August 2017, and CP61(11) collected in November 2017. The group

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collected in 2016 had higher identities than the group collected in 2017, possibly indicative of

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two related genotypes of the same Tulasnella species colonizing the same plant. Although

335

care was taken to collect rhizome tissue from the same plant each time, plants occurred in

336

clumps and so it is possible roots from another plant were inadvertently collected.

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Bertram was the most unusual group. Bertram isolates exhibited more diversity under ISSR

339

analysis than they did under ITS analysis (Fig 3, 4). However, under ISSR analysis, the

340

isolates from Jarrahdale more closely resembled those from the southern group (Table 4).

341 342

The genetic diversity within the southern population was less than that of the northern group,

343

where three potential species occurred. Nei’s genetic distances of isolates from Bertram and

344

Jarrahdale, from 0.250 - 0.311 and 0.234 - 0.295, respectively, were higher than other

345

populations (Table 4). This supports their classification as distinct taxa.

346 347

4. DISCUSSION

348

Attempts to confirm that the fungal isolates were mycorrhizal by germinating C. ovata seed

349

in their presence were unsuccessful (unpublished). Therefore, we refer to these isolates from

350

rhizome pelotons as being ‘associated’ with C. ovata plants. The fact that all the fungal

351

isolates were of genus Tulasnella, and that others have also found members of this group

352

associated with orchids in Australia and the USA is further evidence that they are, in fact,

353

mycorrhizal symbionts of C. ovata. Tulasnella fungi have not previously been identified as

354

pathogens in any study, so their association with orchids is not likely to be of a pathogenic

355

nature. Most known orchid mycorrhizae belong to a taxonomic group within the Rhizoctonia

356

alliance, a large group of fungi with multiple ecological roles. Orchid-associated members

357

include species from clades Ceratobasidium, Sebacina, Epulorhiza, and Tulasnella (Stalpers

358

and Andersen, 1996).

359 360

Tulasnella species have been identified from both terrestrial and epiphytic Dendrobium

361

orchids. Tulasnella species identified in Australia include T. allantospora, T. asymmetrica, T.

362

calospora, T. cruciata, T. deliquescens, T. irregularis and T. violea (Perkins et al. 1995;

363

Warcup 1971, 1973, 1981, 1990; Warcup and Talbot, 1967, 1971, 1980). Furthermore, Linde

364

and colleagues (2017) recently described four new species of Tulasnella from Australian

365

terrestrial orchids: T. prima and T. sphagneti were isolated from Chiloglottis species, T.

366

secunda was isolated from Drakaea and Caleana species, and T. warcupii was isolated from

367

Arthrochilus oreophilus.

368 369

ITS sequences placed the fungi into sequence-associated groups that corresponded to

370

collection location. ITS sequence divergence thresholds between taxa were within the range

371

of 3-5% accepted for delimiting Tulasnella species (Girlanda et al. 2011; Jacquemyn et al. 11

372

2011). A recent examination of Tulasnella from Australian orchids demarcated 4 species

373

based on 6.3% difference in ITS sequences (Linde et al. 2017). Here, ITS sequences of

374

Tulasnella isolates from two northern C. ovata populations diverged into three groups,

375

designated Jarrahdale 1, Jarrahdale 2 and Bertram, based on 5-7% ITS sequence divergence.

376

They diverged by 6-11% from T. prima, T. sphagneti, and isolates from the southern sites

377

(Table 3). Divergence of >6% in ITS regions suggest that members of clades Jarrahdale 1,

378

Jarrahdale 2 and Bertram represent three novel taxa within Tulasnella. Tulasnella from plants

379

collected at the three southern sites all fell into the same ITS-based taxon, and sequence

380

identity placed them within the same group as T. prima isolates described from Chiloglottis

381

species collected from New South Wales and the Australian Capital Territory sites located at

382

least 3000 km away. This study suggests that C. ovata associates primarily, perhaps

383

exclusively, with Tulasnella species, but it is not restricted to a single Tulasnella species. It

384

also indicates the geographical range of Tulasnella species associating with C. ovata between

385

northern and southern populations.

386 387

Of the around 400 described species of terrestrial orchids from Western Australia, only C.

388

ovata remains photosynthetic all year round (Brundrett 2014). Other terrestrial orchids live

389

much of their lives as primordia and storage organs below ground, emerging when soil

390

moisture and other conditions are suitable. In such cases, mycorrhizal associations are

391

probably re-established at emergence every year (Bonnardeaux et al. 2007), so the potential

392

exists for different genotypes to establish in the plant from year to year. In contrast, a plant of

393

C. ovata potentially associates with only one Tulasnella genotype during the course of its

394

lifespan.

395 396

The isolated nature of the Bertram and Jarrahdale populations and their distinct fungal

397

associates should be considered when preserving them. The Bertram site in particular is the

398

most northern population of C. ovata known, and it is threatened by encroaching

399

urbanisation, weeds and feral animals.

400 401

Fungi associated with C. ovata plants have not previously been identified. In this study, only

402

Tulasnella was found associated with this orchid species, suggesting that it specialises in

403

associations with Tulasnella, but whether it associates with other fungal genera is unknown.

404

Further collections of C. ovata rhizomes from other populations are needed to confirm this.

405

We acknowledge that only fungi from pelotons that grew on nutrient media were identified. It 12

406

is possible that other species, non-culturable on nutrient media, exist in C. ovata pelotons.

407

Direct amplification and sequencing of DNA from individual pelotons should clarify whether

408

such species are present. Warcup (1981) described two Tulasnella species from an unnamed

409

Cryptostylis species, but no work on eastern Australian species or those from other countries

410

has been published subsequently. Of special interest are the associations with the

411

achlorophilic C. hunteriana, which relies entirely on fungal sources of energy.

412 413

Acknowledgements

414

Nguyen DQ and Tran TT each received a scholarship provided jointly by Vietnam

415

International Education Development (VIED) and Murdoch University. The authors thank

416

Prof. David Blair and Prof. Li Ju for access to their property near Pemberton where orchid

417

samples were collected.

418 419

Figure legends

420 421

Fig 1. Map of south-western Australia showing approximate locations of the five populations

422

of Cryptostylis ovata sampled (red numbers). 1. Bertram; 2. Jarrahdale; 3. Stratham; 4.

423

Ludlow; 5. Pemberton.

424 425

Fig 2. Pelotons inside rhizome cortex of a Cryptostylis ovata.

426

A. Pelotons of C. ovata

427

B. Single peloton of C. ovata

428 429

Fig 3. Maximum likelihood tree of ITS sequences from Tulasnella isolates associated with

430

Cryptostylis ovata and other orchids. Species names or isolate codes are shown with

431

GenBank accessions. The ITS sequences of Tulasnella isolates from C. ovata are shown in

432

four named groups. The southern isolates contained sequences that resembled those of

433

isolates of T. prima and undescribed isolates. Bootstrap values >60 generated after 1000

434

replications are shown.

435 436

Fig 4. Dendrogram of ISSR gel patterns of 49 Tulasnella isolates isolated from five

437

populations of Cryptostylis ovata. Analysis was based on Unweighted Pair Group Method

438

with Arithmetic Mean (UPGMA) analysis of ISSR polymorphisms. The isolate names in the

439

same colour were from the same plant. 13

440 441

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16

Table 1. Climatic and ecological conditions at collection sites Location

GPS coordinates

Mean maximum temperature 2016a

Mean maximum temperature 2017a

Annual rainfall 2016 (mm)a

Annual rainfall 2017 (mm)a

Soil texture

Bertram

32o14’27.6”S 115o50’53.2”E

861.6 Anketell (5.1km away)

Loam

32o19’23.9”S 116o04’13.1E

1091.2 Jarradale station

1197.4 Jarradale station

Clay loam

57 km south

Stratham

33o26’23.1”S 115o36’29.9E

679.2 Bunbury station (12.5 km away) 639.0 Ludlow station

191 km south

33o36’32.0”S 115o29’56.6E

801.8 Bunbury station (12.5 km away) 874.4 Ludlow station

Silt loam

Ludlow

Sandy loam

210 km south

Pemberton

34o29’40.3”S 115o55’42.1E

24.8 Jandakot Aero station (16.9 km away) 22.5 Karnet (11.1 km away) 23.4 Bunbury station (12.5 km away) 23.1 Busselton Aero station (11.3 km away) 21.1 Pemberton station

841.6 Anketell station (5.1km away)

Jarradale

24.0 Jandakot Aero station (16.9 km away) 21.9 Karnet (11.1 km away) 22.4 Bunbury station (12.5 km away) 22.3 Busselton Aero station (11.3 km away) 20.8 Pemberton station

1444.4 Walters Farm station

1122.4 Pemberton station

Sandy loam

326 km south

a

Weather station from which data was collected

Distance by road from Perth CBD 39 km south

Table 2. Tulasnella isolates from plants of five populations of Cryptostylis ovata identified in Western Australia. No.

Isolates

Time of collection

Population

Genbank accession

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70

CB13(1) CB14(1) CB14(3) CB15(5) CB15(6) CB15(10) CB43(6) CB43(9) CB55(16) CJ11(1) CJ11(3) CJ11(4) CJ11(8) CJ13(3) CJ13(6) CJ35(15) CJ45(12) CJ45(16) CJ51(3) CJ51(4) CJ51(5) CJ51(6) CJ51(7) CJ51(8) CJ51(9) CJ51(12) CJ51(13) CJ51(14) CJ51(15) CJ51(17) CJ51(19) CL12(5) CL13(3) CL13(4) CL13(6) CL14(9) CL24(1) CL35(19) CL44(17) CL51(16) CL51(17) CL51(19) CL53(1) CL53(2) CL53(6) CL54(5) CL54(10) CL54(13) CP11(3) CP12(4) CP12(5) CP13(2) CP14(2) CP14(3) CP14(6) CP21(1) CP32(4) CP32(5) CP33(1) CP33(8) CP51(4) CP51(5) CP51(12) CP61(11) CP63(13) CS11(3) CS11(4) CS11(6) CS11(8) CS12(3)

8/2016 8/2016 8/2016 8/2016 8/2016 8/2016 5/2017 5/2017 8/2017 8/2016 8/2016 8/2016 8/2016 8/2016 8/2016 2/2017 5/2017 5/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2016 8/2016 8/2016 8/2016 8/2016 11/2016 2/2017 5/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2016 8/2016 8/2016 8/2016 8/2016 8/2016 8/2016 11/2016 2/2017 2/2017 2/2017 2/2017 8/2017 8/2017 8/2017 11/2017 11/2017 8/2016 8/2016 8/2016 8/2016 8/2016

Bertram Bertram Bertram Bertram Bertram Bertram Bertram Bertram Bertram Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Jarrahdale Ludlow Ludlow Ludlow Ludlow Ludlow Ludlow Ludlow Ludlow Ludlow Ludlow Ludlow Ludlow Ludlow Ludlow Ludlow Ludlow Ludlow Pemberton Pemberton Pemberton Pemberton Pemberton Pemberton Pemberton Pemberton Pemberton Pemberton Pemberton Pemberton Pemberton Pemberton Pemberton Pemberton Pemberton Stratham Stratham Stratham Stratham Stratham

MK430438 MK430439 MK430440 MK430441 MK430442 MK430443 MK430444 MK430445 MK430447 MK430448 MK430449 MK430450 MK430451 MK430452 MK430453 MK430454 MK430455 MK430456 MK430457 MK430458 MK430459 MK430460 MK430461 MK430462 MK430463 MK430464 MK430465 MK430466 MK430467 MK430468 MK430469 MK430470 MK430471 MK430472 MK430473 MK430474 MK430475 MK430476 MK430477 MK430478 MK430479 MK430480 MK430481 MK430482 MK430483 MK430484 MK430485 MK430486 MK430487 MK430488 MK430489 MK430490 MK430491 MK430492 MK430493 MK430494 MK430495 MK430496 MK430497 MK430498 MK430499 MK430500 MK430501 MK430502 MK430503 MK430504 MK430505 MK430506 MK430507 MK430508

71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

CS32(1) CS32(3) CS32(4) CS32(5) CS34(4) CS34(5) CS34(6) CS34(17) CS34(19) CS35(1) CS35(2) CS35(4) CS43(17) CS51(1) CS51(2) CS51(4) CS51(5) CS51(6) CS51(8) CS51(9)

2/2017 2/2017 2/2017 2/2017 2/2017 2/2017 2/2017 2/2017 2/2017 2/2017 2/2017 2/2017 5/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2017 8/2017

Stratham Stratham Stratham Stratham Stratham Stratham Stratham Stratham Stratham Stratham Stratham Stratham Stratham Stratham Stratham Stratham Stratham Stratham Stratham Stratham

MK430509 MK430510 MK430511 MK430512 MK430513 MK430514 MK430515 MK430516 MK430517 MK430518 MK430519 MK430520 MK430521 MK430522 MK430523 MK430524 MK430525 MK430526 MK430527 MK430528

Table 3. Pairwise sequence identities of ITS regions of Tulasnella sequences from four phylogenetic groups isolated from Cryptostylis ovata, compared to described species of Tulasnella isolated from other Australian orchids.

Southern group and isolates of T. prima Bertram Jarrahdale 1 Jarrahdale 2 T. sphagneti T. secunda T. tomaculum T. warcupii

Southern group and isolates of T. prima (n = 8) 98-100

Bertram

Jarradale 1

Jarradale 2

93-94 91-92 89-91 90-92 79-81 80-82 77-81

100 94-95 93 91-92 81-82 83 79-80

100 93-94 90-91 79-80 81-82 79-80

99-100 90-91 79-80 81-83 78-80

n = number of isolates

T. sphagneti (n = 6)

T. secunda (n = 3)

T. tomaculum (n = 2)

T. warcupii (n = 3)

99-100 80 81-82 79-80

98-100 85 82-83

100 84-85

99

Table 4. Pairwise population matrix of Nei’s genetic distance. Population

Pemberton

Ludlow

Stratham

Bertram

Jarrahdale

Pemberton Ludlow Stratham Bertram Jarrahdale

0.000 0.055 0.061 0.311 0.295

0.000 0.072 0.282 0.263

0.000 0.292 0.234

0.000 0.250

0.000

Table 5. Analysis of genetic variation as measured by ISSR primers in five Cryptostylis ovata populations Population Pemberton Ludlow Stratham Bertram Jarrahdale

Percentage polymorphic loci (%P) 51.85% 53.70% 51.85% 31.48% 37.04%

Sample size (N) 13 10 10 7 9

Shannon's Information Index (I) 0.24 0.28 0.30 0.18 0.23

CS43(17) MK430521 CS51(9) MK430528 CS51(8) MK430527 CS51(6) MK430526 CS51(5) MK430525 CS51(4) MK430524 CS51(2) MK430523 CS51(1) MK430522 CS35(4) MK430520 CS35(2) MK430519 CS35(1) MK430518 CS34(19) MK430517 CS34(17) MK430516 CS34(5) MK430514 CS34(4) MK430513 CS32(5) MK430512 CS32(4) MK430511 CS32(3) MK430510 CS32(1) MK430509 CS12(3) MK430508 CS11(8) MK430507 CS11(6) MK430506 CS11(4) MK430505 CS11(3) MK430504 CP61(11) MK430502 CP51(12) MK430501 CP51(5) MK430500 CP51(4) MK430499 CP21(1) MK430494 CP11(3) MK430487 CL54(13) MK430486 CL54(10) MK430485

Southern group

CL54(5) MK430484 CL51(19) MK430480 CL51(17) MK430479 CL51(16) MK430478 CL44(17) MK430477 CL24(1) MK430475 CL14(9) MK430474 CS34(6) MK430515 CL12(5) MK430470 CL13(3) MK430471 CL35(19) MK430476 CL53(1) MK430481 CL53(2) MK430482 CL53(6) MK430483 CP14(2) MK430491 CP14(3) MK430492 CP14(6) MK430493 CL13(4) MK430472 CL13(6) MK430473 KF476556 T. prima HM196807 T. prima KF476565 Tulasnella sp. 99

KF476550 T. prima KF476559 Tulasnella sp. CP12(4) MK430488 CP12(5) MK430489 CP13(2) MK430490 CP32(4) MK430495 87 CP32(5) MK430496

CP33(1) MK430497 CP33(8) MK430498 CP63(13) MK430503 HM196790 T. prima HM196789 T. prima 73

91 KF476553 Tulasnella sp.

CB14(1) MK430439 61

CB14(3) MK430440 CB13(1) MK430438

CB43(9) MK430445

Bertram

97 CB55(16) MK430447

CB43(6) MK430444 CB15(10) MK430443 CB15(6) MK430442 CB15(5) MK430441 CJ13(3) MK430452 CJ13(6) MK430453

Jarrahdale 1

99 CJ11(8) MK430451

69

CJ11(4) MK430450 CJ11(3) MK430449 CJ11(1) MK430448

CJ51(17) MK430468

99

CJ45(12) MK430455 CJ45(16) MK430456

93

CJ35(15) MK430454 CJ51(4) MK430458 CJ51(5) MK430459 CJ51(6) MK430460 CJ51(8) MK430462 99 CJ51(9) MK430463

CJ51(12) MK430464 CJ51(13) MK430465 CJ51(14) MK430466 CJ51(15) MK430467 CJ51(19) MK430469 CJ51(3) MK430457 CJ51(7) MK430461 KY445928 T. sphagneti KY445929 T. sphagneti 97

KY445924 T. sphagneti KY445926 T. sphagneti 95 KY445923 T. sphagneti

KY445927 T. sphagneti 95 99

KF476586 T. secunda KF476593 T. secunda KF476568 T. secunda

99 AY373296 T. tomaculum

KC152380 T. tomaculum KF476598 T. warcupii 99

KF476596 T. warcupii KF476600 T. warcupii

0.05

Jarrahdale 2

CL44(17) CL54(10) CL14(9) CL24(1) CP61(11) CP51(12) CP51(4) CP51(5) CP63(13) CL13(4) CP13(2) CP12(5) CP33(1)

Southern group 1

CP32(4) CP32(5) CS12(3) CP11(3) CP21(1) CS11(3) CS11(8) CS32(3) CS51(8) CS51(1) CS51(5) CJ11(3) CJ13(3)

Jarrahdale 1

CJ11(1) CJ51(9) CJ51(3) CJ51(19) CJ45(16)

Jarrahdale 2

CJ45(12) CJ35(15) CL35(19) CL12(5) CS43(17) CS35(2) CS34(5) CP14(3) CL51(17) CL53(1) CL53(6) CB43(6) CB43(9) CB35(1) CB55(16)

Bertram

CB14(3) CB13(1) CB15(6) 1.2

1.0

0.8

0.6

0.4

0.2

0.0

Southern group 2