Joshee et al Podocarp endophytes

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Diversity and distribution of fungal foliar endophytesin New Zealand Podocarpaceae

Sucheta JOSHEEa,b, Barbara C. PAULUSa, Duckchul PARKa, Peter R. JOHNSTONa,*aLandcare Research, Private Bag 92170, Auckland 1142, New ZealandbBioDiscovery New Zealand, 24 Balfour Rd, Parnell, Auckland 1052, New Zealand

a r t i c l e i n f o

Article history:

Received 5 March 2008

Received in revised form

24 March 2009

Accepted 9 June 2009

Published online 17 June 2009

Corresponding Editor: Barbara Schulz

Keywords:

Dacrycarpus dacrydioides

Dacrydium cupressinum

Host specialisation

Kunzea ericoides

Multivariate analysis

Podocarpus totara

Prumnopitys ferruginea

a b s t r a c t

The diversity and distribution of fungal endophytes in the leaves of four podocarps (Dacry-

dium cupressinum, Prumnopitys ferruginea, Dacrycarpus dacrydioides, and Podocarpus totara, all

Podocarpaceae) and an angiosperm (Kunzea ericoides, Myrtaceae) occurring in close stands

were studied. The effects of host species, locality, and season on endophyte assemblages

were investigated. Host species was the major factor shaping endophyte assemblages.

The spatial separation of sites and seasonal differences played significant but lesser roles.

The mycobiota of each host species included both generalist and largely host-specialised

fungi. The host-specialists were often observed at low frequencies on some of the other

hosts. There was no clear evidence for family-level specialisation across the Podocarpaceae.

Of the 17 species found at similar frequencies on several of the podocarp species, 15 were

found also on Kunzea. Many of the endophytes isolated appear to represent species of fungi

not previously recognised from New Zealand.

ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction

Fungal endophytes live for all or a major part of their life cycle

within the healthy tissues of their host without causing any

symptoms of disease (Wilson 1995). Endophytes have been

isolated from almost all plants studied so far; bryophytes

(Davis et al. 2003), ferns (Petrini et al. 1992), monocotyledons

(Taylor et al. 1999), conifers (Carroll et al. 1977; Carroll & Carroll

1978; Petrini 1986; Petrini & Carroll 1981; Petrini & Muller 1979),

and various dicotyledons (Arnold et al. 2000; Johnston 1998),

from a wide range of habitats, such as coastal mangroves

(Kumaresan & Suryanarayanan 2001), temperate evergreen

forests (Espinosa-Garcia & Langenheim 1990), xeric regions

(Suryanarayanan et al. 2003), and tropical forests (Arnold

et al. 2000), and even from lichens (Petrini et al. 1990). Endo-

phytic fungi colonise all plant parts such as roots, stems,

leaves, bark and floral organs and in some cases can affect

both ecological and physiological processes of their host (Pet-

rini 1991; Schulz et al. 1999). However, biologically they are

diverse, and may include saprophytic and pathogenic fungi

with an ‘endophytic’ phase (Carroll 1988; Rodriguez & Redman

1997; Wilson 2000).

A highly diverse endophytic mycota has been demon-

strated in leaves of numerous hosts (e.g. Arnold et al. 2000;

Frohlich et al. 2000; Gamboa & Bayman 2001) although high en-

dophytic diversity may not be universal. For example, studies

in the dry forests of Tamil Nadu indicated a considerably

lower species richness (Suryanarayanan et al. 2003). The

* Corresponding author.E-mail address: [email protected]

journa l homepage : www.e l sev i er . com/ loca te /mycres

m y c o l o g i c a l r e s e a r c h 1 1 3 ( 2 0 0 9 ) 1 0 0 3 – 1 0 1 5

0953-7562/$ – see front matter ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.mycres.2009.06.004


distribution of endophytes seems to be governed by a variety

of host and environmental factors. A degree of host prefer-

ence has been observed for endophytic communities both in

temperate (e.g. Petrini 1996; Petrini & Fisher 1988, 1990) and

tropical plant species (e.g. Arnold et al. 2001). This host prefer-

ence may be expressed at various levels of the taxonomic hi-

erarchy, including at the level of plant family (Petrini & Carroll

1981), subgenus (Carroll & Carroll 1978), and species (Petrini &

Fisher 1990).

In addition to host-related factors, spatial heterogeneity has

been reported for fungal endophytic assemblages at different

scales. While endophyte assemblages in a particular host may

be similar throughout its continuous range (e.g. in Sequoia sem-

pervirens; Rollinger & Langenheim 1993) major differences ap-

pear when hosts are from geographically disjunct areas (Fisher

et al. 1994; Taylor et al. 1999). Other studies have also demon-

strated differences in species composition at smaller spatial

scales, for example at the level of sites of the same region

(Arnold et al. 2001; Vujanovic & Brisson 2002), between trees of

the same stand (Petrini et al. 1992; Johnston 1998), between

branches of the same tree (Espinosa-Garcia & Langenheim

1990), and even within areas of leaves (Johnston & Fletcher 1998).

The effect of seasonal changes on endophyte assemblages

remains unclear. Seasonal patterns have been detected

among taxa that were not host specific (Widler & Muller

1984), but other studies could not confirm any seasonality

among endophytes (e.g. Sieber & Hugentobler 1987).

The Podocarpaceae are a notable group of ancient conifers

with a largely Southern Hemisphere distribution (Hill & Bro-

dribb 1999). They are widespread in New Zealand where

they are represented by eight genera and at least 18 species,

all of which are endemic (Connnor & Edgar 1987; Molloy

1995, 1996). The species sampled in this study include several

of New Zealand’s most important canopy trees, Dacrycarpus

dacrydioides, Dacrydium cupressinum, Prumnopitys ferruginea,

and Podocarpus totara.

The aim of the present study is to explore the diversity and

distribution of fungal endophytes associated with members of

the Podocarpaceae with special reference to the effects of host

species, geographic range and season. In particular, we ask

whether fungal endophytes are more similar in comparisons

between members of the Podocarpaceae than in comparisons

between podocarps and an angiosperm, Kunzea ericoides (Myr-

taceae). Host-specific endophytes can occur on morphological

similar trees growing in a close stand with the host (Kowalski

& Kehr 1996). Hence, K. ericoides was selected as it has leaves of

similar size and grows in the vicinity of these podocarps. Spa-

tial relationships are also explored across sites within the

same region and at a more distant site. In addition, sampling

was undertaken in summer and winter to provide a prelimi-

nary indication of seasonality among endophytes.

Materials and methods

Site and host characteristics

For our study, we selected four sites from the Waitakere

Ranges near Auckland and one close to the Urewera National

Park in the central North Island (Fig 1). The Waitakere Ranges

extend about 25 km north to south and 25 km west of central

Auckland in New Zealand. The topography is the result of

ancient volcanic eruptions and lava flows. The vegetation

typically comprises native forest in various stages of regener-

ation after extensive logging and farming in the last century.

The tea-tree species (Leptospermum scoparium and Kunzea eri-

coides) are the main early succession plants, being replaced

by kauri (Agathis australis), tanekaha (Phyllocladus trichoma-

noides), rimu (Dacrydium cupressinum), totara (Podocarpus

totara), miro (Prumnopitys ferruginea), kahikatea (Dacrycarpus

dacrydioides), hinau (Elaeocarpus dentatus), and rewarewa

(Knightia excelsa) after about 100 years (Esler & Astridge

1974). D. cupressinum is the most common emergent, while

shrubs like Myrsine australis, Geniostoma ligustrifolium, Cop-

rosma spp., Melicytus ramiflorus, and Pseudopanax arboreum,

along with tree ferns, form the understorey. At the fifth

site, further to the south, the podocarp–hardwood forest

has been highly modified by logging. The sampling site com-

prises a dense stand of regenerating D. cupressinum and

Beilschmiedia tawa, along with smaller shrubs and scattered

seedlings of the other podocarp species, with scattered

stands of K. ericoides around the margins. Seedlings and sap-

lings of the four podocarp species we sampled co-occurred in

close stands.

The four podocarps species we sampled demonstrate vari-

able leaf morphology. D. cupressinum has imbricate leaves

(4–7� 0.5–1 mm), D. dacrydioides has bilaterally flattened

leaves in juvenile stages (3–7� 0.5–1 mm) that become imbri-

cate with age, P. totara has larger bifacially flattened leaves

(15–30� 3–4 mm) that are helically arranged, and P. ferruginea

has bifacially flattened leaves (15–30� 2–3 mm) with petioles

twisted to arrange them in a two dimensional plane along

the shoot axis. The leaves of K. ericoides are also flattened,

about 4–12� 1–2 mm in size. Most of the leaves of K. ericoides

remain attached for two years, although a few are lost within

that time. All of the podocarp species sampled retain all of

their leaves for more than two years.

Sampling and isolation strategy

Sampling was carried out in the winter months (July–August

2004) and the following summer (January 2005). Two trees

per site were sampled for each of the five host species, and

the same trees were sampled in each season. For each sea-

son, a total of 2500 leaves (10 000 leaf segments) were sam-

pled; this represents 500 leaves (or 2000 leaf segments) for

each tree species. Five branches were arbitrarily selected

and cut from each tree, kept cool in polythene bags until

they were brought to the laboratory. Isolations were mostly

undertaken within 4–6 h of collection, always within 12 h.

From each branch, ten healthy leaves from the previous sea-

son’s growth were carefully excised and surface sterilised

(1 min in 95 % ethanol, 3 min in bleach solution (approxi-

mately 14 % free chlorine), 30 s in 95 % ethanol, then rinsed

in sterile distilled water). Leaves of Dacrydium cupressinum,

Dacrycarpus dacrydioides, and Kunzea ericoides were cut into

four pieces. For the larger leaves of Podocarpus totara and

Prumnopitys ferruginea, four separate slices approximately 1–

2 mm� 3–4 mm were taken from along the length of the

leaf. The leaf pieces were plated on Petri dishes containing

1004 S. Joshee et al.


1.25 % malt extract (Difco) and 2 % agar. The inoculated

plates were incubated in daylight at room temperature. Petri

dishes were checked regularly for the growth of fungi for one

month. Fungi growing from the leaf pieces were subcultured

onto potato dextrose agar, oatmeal agar (Difco) and/or water

agar, sometimes amended with small pieces of sterile host

tissue in an attempt to stimulate sporulation. The endo-

phytes were grouped into morphospecies, based on cultural

appearance together with conidial and ascospore morphol-

ogy. Sequences of the internally transcribed spacer regions

of nuclear rDNA (ITS) were generated for representatives of

the ten most frequently isolated groups for each host species

that could not be identified to genus by morphological char-

acters. The methods used were the same as those of John-

ston & Park (2005). Sequences were compared with those

deposited in Genbank using a BLAST search, and directly

with sets of authentic sequences from published studies of

taxa such as Xylariaceae (Guo et al. 2003), Pezicula (Verkley

et al. 2003), and Mycosphaerella (Crous et al. 2001). Representa-

tive isolates of each group have been deposited in the Inter-

national Collection of Micro-organisms from Plants (ICMP,

maintained by Landcare Research, Auckland).

Fig 1 – Five collection sites at two geographical regions in the North Island, New Zealand. Adjacent Waitakere Ranges sites

are approximately 3–4 km apart; the Waitakere and Urewera sites are approximately 300 km apart. Site 1, Upper Nihotupu

Dam Walk, 36 56.221� S, 174 33.523� E, 300 m asl; Site 2, Home Track, 36 57.183� S, 174 30.924� E, 312 m asl; Site 3, Puke-

matekeo Summit Track, 36 53.008� S, 174 32.090� E, 336 m asl; Site 4, Fairy Falls Track, 36 54.940� S, 174 32.746� E, 300 m asl;

Site 5, Urewera, Mangapae Stream, 38 38.285� S, 176 52.048� E, 600 m asl. Mean average annual rainfall 1971–2000, Waitakere

sites approximately 1600–1800 mm, Urewera site approximately 1800–2000 mm (Anonymous 2004a). Mean average tem-

perature Waitakere sites approximately 13–14 �C, Urewera site approximately 10–11 �C (Anonymous 2004b).

Foliar endophytes in Podocarpaceae 1005


Statistical analysis of data

The percentage of leaf pieceswith single and multiple infections

was computed in Microsoft Excel, and Shannon’s Diversity In-

dex (H0) in PRIMER v.5 (Clarke & Warwick 2001). In addition to

the total dataset a second datasetwas created that excludedsin-

gleton species. Expected species accumulation curves based on

the Mao Tau estimator were computed for both datasets in Esti-

mateS v.8.0 (Colwell 2006) and plotted for individual tree species

in each season separately using Microsoft Excel.

We used non-parametric approaches of data analysis be-

cause the endophyte assemblage data were skewed and con-

tained many zero counts, making parametric analysis

unsuitable (Anderson 2001). Data were square-root trans-

formed to reduce the influence of the most abundant species

(Clarke & Warwick 2001). Bray–Curtis similarity indices were

calculated (Bray & Curtis 1957), and patterns from the resulting

similarity matrix were examined using Nonmetric Multidi-

mensional Scaling (NMDS) ordination in PRIMER v.5. An anal-

ysis of similarities (ANOSIM) was carried out using PRIMER

v.5 to assess the statistical significance of differences between

and within hosts and sites, using the same similarity matrix

calculated for NMDS. Data were square-root transformed to re-

duce the influence of the most abundant species.

The R value in ANOSIM gives an absolute measure of how

separated the groups are, on a scale of 0 (indistinguishable)

to 1 (all similarities within groups are less than any

similarities between groups) (Clarke & Gorley 2001). Permuta-

tion tests established the statistical significance (P) of the R

values.

Variability in the endophyte assemblages at different spatial

scales, i.e. among sites, trees within sites, and branches within

trees, was measured explicitly using a semi-parametric permu-

tational multivariate analysis of variance (PERMANOVA;

Anderson 2001; McArdle & Anderson 2001). This approach al-

lows a direct additive partitioning of variation among individual

terms using the multivariate analogue of the ANOVA compo-

nent estimators (e.g. Searle et al. 1992). For each season a three

factor nesteddesign wasapplied: sites (five levelscorresponding

to each site), trees (two trees per genus nested within sites), and

branches (five branches per tree nested within trees). Data were

square-root transformed, and Bray–Curtis dissimilarities were

used in all analyses. The mean squares derived from PERMA-

NOVA were used to calculate components of multivariate varia-

tion following the methods in Anderson et al. (2005).

Results

Diversity of endophytes

In winter, a total of 4922 endophyte strains representing 479

morphotypes and in summer, 4045 endophyte strains repre-

senting 495 morphotypes were isolated. Table 1 provides the

Table 1 – Number of isolates from each host in each season, and their relative diversity

Host D. cupressinum P. ferruginea P. totara D. dacrydioides K. ericoides

Season (w¼winter; s¼ summer) w s w s w s w s w s

% leaf pieces infected (n¼ 2000) 42.9 45.8 74.2 67.6 49.8 44.3 48.3 61.4 84.3 81.6

% leaf pieces with multiple infections 4.5 4.3 3.2 3.0 1.9 1.8 2 1.7 7.7 11.0

Total number of isolates 872 806 1197 1117 557 702 838 1140 1458 1280

Total species of endophytes 137 162 75 155 88 53 141 131 100 88

Shannon’s diversity index H0 3.96 3.54 2.66 3.09 2.74 2.97 3.18 3.39 2.40 2.57

Fig 2 – Expected endophyte species accumulation curves for each host species based on the Mao Tau estimator calculated

using EstimateS v.8.0 (Colwell 2006).(A) Winter sample. (B) Summer sample.



number of leaf pieces with single and multiple infections, the

number of species, and Shannon’s diversity indices for each

host from each season. All hosts contained a high number of

taxa that were observed only once (singletons); 360 (75.2 %)

in winter, 423 (85.5 %) in summer. Shannon’s diversity indices

reveal that diversity was highest in the endophyte assemblage

of Dacrydium cupressinum (3.52 and 3.96 in summer and winter

respectively) and low in Kunzea ericoides (2.57 and 2.40 in

Table 2 – Number of isolates of the ten most frequently isolated endophytes per host species. Fungi showing an apparenthost preference (more than 75 % of the isolates are from one host) are indicated in bold

Host D. cupressinum P. ferruginea P. totara D. dacryioides K. ericoides

Season (w¼winter; s¼ summer) w s w s w s w s w s

Agaricomycotina

Agaricomycetes, Agaricomycetidae, Agaricales

Cylindrobasidium sp.a 25 0 0 0 0 0 0 0 0 0

Pezizomycotina

Dothideomycetes, Dothideomycetidae, Mycosphaerellales

Mycosphaerella sp. 1a 0 0 0 0 0 2 1 2 678 249



Phyllosticta sp. 1 3 30 38 125 9 3 0 27 8 5

Phyllosticta sp. 2 3 2 0 17 0 12 111 209 0 6

Phyllosticta sp. 3 0 0 0 0 0 0 143 0 0 0

Leotiomycetes, Helotiales

Helotiales sp. 1a 25 46 0 0 0 0 0 2 0 0

Helotiales sp. 2a 46 28 0 1 0 1 0 0 0 0

Pezicula sp. 1a 53 193 0 6 0 1 0 12 0 1

Pezicula sp. 2a 0 0 458 195 0 0 0 10 0 0

Cryptosporiopsis actinidiaea 4 5 0 27 6 27 0 19 6 13

Neofabraea sp. 1a 0 0 0 0 119 50 0 0 0 0

Torrendiella sp. 0 0 0 0 0 0 0 0 192 209

Sordariomycetes

Sordariomycetes sp. 1a 5 1 0 12 3 0 2 0 0

Sordariomycetes sp. 2a 36 3 0 0 0 0 0 0 0 0

Sordariomycetes, Hypocreomycetidae, Glomerellaceae

Colletotrichum sp. 1 (C. gloeosporioides group) 0 0 123 0 0 0 0 0 0 0



Colletotrichum sp. 4 (C. boninense group) 22 0 0 138 0 0 0 62 0 0






Sordariomycetes, Sordariomycetidae, Diaporthales

Ophiognomonia sp. 15 11 62 48 34 48 22 13 33 33

Phomopsis sp. 2 2 8 19 0 21 15 32 15 28

Sordariomycetes, Sordariomycetidae, Sordariales

Lasiosphaeria sp.a 24 63 60 68 19 88 0 28 6 9

Sordariomycetes, Xylariomycetidae, Xylariales

Amphisphaeriaceae sp. 1a 0 0 0 0 0 0 0 48 0 0

Amphisphaeriaceae sp. 2a 130 48 15 16 0 1 7 7 1 4

Rosellinia sp. 1a 0 0 0 0 0 29 0 28 0 19

Xylaria sp. 1a 30 35 10 178 4 45 51 139 0 35

Xylaria sp. 2a 1 23 0 9 0 16 0 12 0 9

Xylaria sp. 3a 0 0 0 5 4 3 54 16 0 1

Xylariaceae sp. 1a 14 25 81 56 10 78 0 56 12 0

Xylariaceae sp. 2a 4 39 0 0 0 0 0 0 0 0

Xylariaceae sp. 3a 2 0 8 0 4 7 79 33 0 3

Xylariaceae sp. 4 2 0 28 0 0 0 0 0 2 0

Xylariaceae sp. 5a 24 2 13 1 0 25 0 31 0 11

a Identification based on comparison with ITS sequences in Genbank (vouchers listed in Table 3). This is a cumulative list, many species rep-

resented in the top ten of more than one host. Names for higher taxa follow Hibbett et al. (2007).



summer and winter) as compared with other hosts. Species

accumulation curves did not reach an asymptote when single-

ton species were included in the analysis (not shown). When

singletons were removed, all curves approximated an asymp-

tote (Fig 2).

The ten most frequently isolated species from each host

are listed in Table 2. Genbank accession numbers for ITS se-

quences generated for the species in this list which could

not be identified morphologically are given in Table 3.

Differences between host species

The endophyte assemblages of the podocarps and Kunzea eri-

coides are strongly shaped by the host species, shown by en-

dophyte assemblages of the same host species clustering

together in the NMDS graph (Fig 3). Stress levels of 0.18 and

0.19 indicate that the NMDS plots provide a satisfactory rep-

resentation of the data (Podani 1994). The statistical signifi-

cance of assemblage differences in host species was

confirmed by ANOSIM (Global R¼ 0.825, P¼ 0.001) (Table 4).

Endophyte assemblages in each host species also differed

significantly in all pair-wise comparisons. The average R

value of the podocarp–podocarp comparisons was slightly

lower than the podocarp–K. ericoides comparisons, 0.795 ver-

sus 0.862 respectively.

Several of the most frequently isolated fungal taxa were

common to all plant species, including Cryptosporiopsis actini-

diae, Ophiognomonia sp., Lasiosphaeria sp., Phomopsis sp., some

species of Colletotrichum, and some xylariaceous species (Table

2). Among these taxa there was limited evidence of endophyte

specificity at the family level; only two of the frequently

isolated species shared by different hosts (Sordariomycetes sp.

1 and Colletotrichum sp. 4) were restricted to members of the

Podocarpaceae.

Differences between sites

Endophyte assemblages from sites in the Waitakere Ranges

clustered together in NMDS plots and were often separated

from those of the Urewera site (Fig 3). Significant differences

in endophyte assemblages were detected in a global compari-

son of sites using ANOSIM (Global R¼ 0.523, P¼ 0.001) (Table 5).

Most sites also differed significantly in pair-wise comparisons,

except for comparisons between the Waitakere sites involving

site 3 (Table 5). In addition, pair-wise comparisons suggest

a greater similarity for sites within the Waitakere Ranges

(R¼ 0.23–0.75) than for comparisons between the Waitakere

sites and the Urewera site (R¼ 0.70–0.90).

Differences between seasons

The differences in endophyte assemblages between summer

and winter were less pronounced than those observed for

both host species and sites, indicated by a lower Global R

value, but these differences were still statistically significant

(Global R¼ 0.367, P¼ 0.001). Some of the endophytes were

dominant in summer (e.g. Amphisphaeriaceae sp. 1, Xylaria

sp. 2) and others in winter (e.g. Cylindrobasidium sp., several

of the Colletotrichum spp., Phyllosticta sp. 3 and Xylariaceae sp.

4). Most of the taxa observed at higher frequencies were found

at similar levels in both seasons (Table 2).

Table 3 – Representative isolates of taxa listed in Table 2 with ITS sequences generated as part of this study. Identificationsbased on comparison with sequences deposited in GenBank

Endophyte species ICMP voucher number(s) Genbank accession number(s)

Amphisphaeriaceae sp. 1 16120, 16023 EU482201, EU482202, EU482203

Amphisphaeriaceae sp. 2 16130 EU482204, EU482205

Sordariomycete sp. 1 16009, 16011 EU482206, EU482207

Sordariomycete sp. 2 15976, 16021 EU482208, EU482209

Colletotrichum boninense group-species 17319, 17320, 17321, 17322 EU482210, EU482211, EU482212, EU482213

Colletotrichum gloeosporioides group-species 17323, 17324, 17325, 17326 EU482214, EU482215, EU482216, EU482217

Colletotrichum sp. 17327, 17328 EU482218, EU482219

Cryptosporiopsis actinidiae 15963, 15964, 15978, 15970 EU482220, EU482221, EU482222, EU482223

Cylindrobasidium sp. 16027 EU482224

Helotiales sp. 1 16015, 16121, 16016, 16013, 16014 EU482225, EU482226, EU482227, EU482228, EU482229

Helotiales sp. 2 16134, 16012 EU482230, EU482231, EU482232, EU482233

Lasiosphaeria sp. 16019 EU482234

Mycosphaerella sp. 1 16478 EU482235, EU482236, EU482237

Mycosphaerella sp. 2 16122, 16123 EU482238, EU482239, EU482240

Mycosphaerella sp. 3 16124, 16025 EU482241, EU482242

Neofabraea sp. 1 16020, 16127 EU482243, EU482244

Pezicula sp. 1 16024, 15983 EU482245, EU482246, EU482247, EU482248

Pezicula sp. 2 15980, 15981, 15971, 15982 EU482249, EU482250, EU482251, EU482252

Rosellinia sp. 1 15984, 16033, 16032 EU482253, EU482254, EU482255

Xylaria sp. 1 16132, 15985, 15990, 16129, 15972 EU482256, EU482257, EU482258, EU482259, EU482260

Xylaria sp. 2 15989, 16128 EU482261, EU482262

Xylaria sp. 3 15991 EU482263

Xylariaceae sp. 1 15987, 16131, 16030, 15988, 15986 EU482264, EU482265, EU482266, EU482267, EU482268

Xylariaceae sp. 2 16034 EU482269

Xylariaceae sp. 3 16031 EU482270

Xylariaceae sp. 5 16029 EU482271, EU482272



Differences within a host species

The results of a PERMANOVA analysis based on a Bray–Curtis

dissimilarity measuring differences in endophyte assem-

blages at the scale of site, tree, branch, and leaf (residual) for

each host/season combination are tabulated in Table 6. Endo-

phyte assemblages differed significantly at most spatial levels

for all hosts. The size of the component of multivariate varia-

tion between endophyte assemblages was only slightly lower

for branches within the same tree (12.8–22.6 %) compared with

conspecific trees at the same site (variation 14.6–27.4 %) (Fig 4).

Discussion

Diversity of endophytes

Members of the New Zealand Podocarpaceae and Kunzea

ericoides host a rich diversity of foliar fungal endophytes

(Table 1). Between 53 and 162 morphotypes were recovered

for individual tree species during the summer (a total of 479

morphotypes) and between 75 and 141 morphotypes during

winter (a total of 495 morphotypes). General species richness

patterns were similar to those of extensive studies of foliar

fungal endophytes in conifers from the Northern Hemisphere.

For example, 100 endophytic species were isolated from Nor-

way Spruce needles (Sieber 1988), 48 taxa from Pinus abies nee-

dles (Lorenzi et al. 2004), and up to 110 species (mean value 60)

for individual hosts among a number of conifers studied (Pet-

rini 1986). Fungal endophytes in tropical dicotyledonous trees

appear even more diverse. For example, Arnold et al. (2000)

detected 242 morphotypes in Heisteria concinna and 259 in Our-

atea lucens in a study that utilised approximately half the num-

ber of leaf segments per tree species compared to the present

study. A considerably lower diversity was reported from dry

tropical forests of Tamil Nadu, India (Suryanarayanan et al.

2003), which may suggest that endophyte diversity is influ-

enced to some extent by climatic factors.

The total diversity of endophytes in the tree species stud-

ied was not captured in the present study, as morphotypes

continued to accumulate with each additional sampling

unit. However, accumulation curves levelled off when single-

ton species were removed (Fig 3). This pattern agrees with that

observed for fungal endophytes in tropical trees (Arnold et al.

2001) and suggests that although additional sampling would

have yielded more morphotypes, these would have primarily

comprised ‘rarer’ taxa. In the present study, a high percentage

of morphotypes were observed as singletons (75.2 % in winter

and 85.5 % in summer). The reasons for the high number of

singleton species detected remain unclear, but may primarily

be the result of chance events rather than ecological con-

straints (Magurran & Henderson 2003). Just as many of the

characteristic, dominant, host species-specialised podocarp

endophytes were occasionally isolated from the other podo-

carps sampled, it is likely that some of the rarely isolated spe-

cies could have been dominant on other unsampled plant

species from the same forests.

Distribution of endophytes – host-related factors

Endophyte assemblages in the four New Zealand members of

the Podocarpaceae studied and in Kunzea ericoides are strongly

shaped by host species. This is indicated by the clear separa-

tion of groups corresponding to host taxa in NMDS plots (Fig

3), and by significant differences in global (Global R¼ 0.825,

P¼ 0.001) and pair-wise comparisons of endophyte assem-

blages in different hosts (Table 4). Our results agree with those

of other studies, which also indicated a degree of host prefer-

ence among endophyte assemblages (e.g. Petrini 1991).

We observed between one and six endophyte taxa strongly

dominant in each host species. Many of these taxa were also

isolated in lower frequencies from other hosts, where they

may represent chance infections (Table 2). Some of these ap-

parently host-restricted taxa belong to genera such as Colleto-

trichum, Mycosphaerella, and Phyllosticta, which include known

host-specific plant pathogens as well as endophytes. Helotia-

ceous fungi were also strongly represented among the appar-

ently host-restricted taxa, including two species of Pezicula,

Neofabraea sp., Torrendiella sp., and two unidentified

Fig 3 – Nonmetric Multidimensional Scaling plot based on

Bray–Curtis similarities, comparing endophyte assem-

blages across host species and site. Each symbol represents

a single tree, 2 trees from each site, with site number indi-

cated on each data point. Sites 1–4 in the Waitakere Ranges

(each site approximately 3–4 km apart), Site 5 at Urewera

(about 300 km from the Waitakere sites). (A) Winter sample.

(B) Summer sample.



helotialean genera (Table 2). Within this group, at least two

genera (Pezicula and Neofabraea) include known pathogens,

and Pezicula has also been reported as a widespread endo-

phyte of shrubs and trees from the northern temperate zone

(Abeln et al. 2000). Species of Torrendiella are strongly host-spe-

cialised, and some have been reported as having an endo-

phytic stage to their life cycle (Cabral 1985; Johnston 1998).

Among the most frequently isolated xylariaceous taxa,

four appeared host-restricted while five infected three or

more hosts (Table 2). Xylariaceae are well-known endophytes

of a wide range of plants, from liverworts to angiosperms

(Davis et al. 2003). Data from Anonymous (2001–2007) shows

most of New Zealand’s xylariaceous species exhibit little

host preference, although there are exceptions. Examples of

putatively host-restricted species include several species of

Hypoxylon on Nothofagus wood, Rosellinia rhopalostilicola on

fronds of the palm Rhopalostylis, and Hypoxylon torrendii on

leaves of the epiphytic lily Astelia. The fruiting bodies of Xylar-

iaceae have never been found on leaves of Podocarpaceae or

K. ericoides in New Zealand (data from Anonymous 2001–

2007). Whether or not the species we isolated as endophytes

are the same that develop fruiting bodies on fallen wood

and dead leaves in the same forests is unknown (see Discus-

sion below).

In contrast to host-restricted species, twelve of the fre-

quently isolated fungal taxa were detected not only in all

four podocarp species, but also from K. ericoides (Table 2).

These included species of Xylariaceae, Colletotrichum, Phyllos-

ticta, and Pezicula (as the anamorphic state Cryptosporiopsis

actinidiae), all families or genera which included some host-re-

stricted species in our study. A small number belonged to

other ascomycetous genera, such as Lasiosphaeria and Ophiog-

nomonia. Some studies have shown endophyte assemblages to

reflect host relationships above the level of species. For exam-

ple, Carroll & Carroll (1978) showed that taxonomic affinities

within subgenera of Abies were mirrored closely by the degree

of similarity among their endophytic assemblages. Petrini &

Carroll (1981) suggested there may be host preference of foliar

endophytes at the level of host family in Cupressaceae, as sev-

eral of the observed fungal taxa had been previously reported

from other hosts within the family (Petrini & Carroll 1981). In

contrast, the phylogenetic relationship among Podocarpaceae

was barely reflected in the degree of similarity among their

endophyte assemblages when compared to K. ericoides (Fig 2,

Table 4). Only two of the common fungal endophytes occur-

ring in three or more podocarp species were absent from K. eri-

coides, Colletotrichum sp. 4 and Sordariomycete sp. 1 (Table 2),

and the ANOSIM R values differed only slightly between podo-

carp–podocarp comparisons compared with podocarp–K. eri-

coides comparisons (Table 4).

Although our study has answered the question of host-

preference of endophytes at the level of host species and fam-

ily, more work is required to elucidate fungal-plant affiliations

at the generic level of the host plant. The inclusion of a second

species of Prumnopitys in our study, Prumnopitys taxifolia, was

hampered by the absence or the rare occurrence of this conge-

neric tree species at the study sites.

Distribution of endophytes – other factors

While host-related factors were the strongest determinant,

geographic separation and seasonal effects also modulated

fungal endophytic assemblages, albeit to a lesser extent. Over-

all, species assemblages differed significantly between the

sites studied (Table 5) and greater geographic distances, i.e.

between the Waitakere and Urewera sites separated by ap-

proximately 300 km, were reflected in lower similarities be-

tween endophyte assemblages (Fig 3, Table 5). These results

contrast with a study of endophytic assemblages in Sequoia

sempervirens, which were similar across the natural range of

the tree species (Rollinger & Langenheim 1993). Although situ-

ated on the same island, the sites in the Waitakere Ranges and

Urewera are in disjunct stretches of native forest, separated

not only by distance but also by farmland. The observed differ-

ences might indicate either barriers to dispersal, or other fac-

tors, such as climate or site history.

Table 4 – Analysis of similarity (ANOSIM) pair-wisecomparisons of endophyte assemblages between eachhost. Global R value [ 0.825 (P [ 0.001). The R value givesan absolute measure of how separated the groups are, ona scale of 0 (indistinguishable) to 1 (all similarities withingroups are less than any similarities between groups)(Clarke & Gorley 2001)

Pair-wise comparisonsbetween hosts

R value P

D. cupressinum, P. ferruginea 0.8 0.008

D. cupressinum, P. totara 0.8 0.004

D. cupressinum, D. dacrydioides 1 0.004

D. cupressinum, K. ericoides 1 0.004

P. ferruginea, P. totara 0.65 0.012

P. ferruginea, D. dacrydioides 0.9 0.004

P. ferruginea, K. ericoides 0.9 0.004

P. totara, D. dacrydioides 0.625 0.021

P. totara, K. ericoides 0.55 0.021

D. dacrydioides, K. ericoides 1 0.004

Table 5 – Analysis of similarity of (ANOSIM) pair-wisecomparisons of endophyte assemblages between eachsite. Sites 1–4 in the Waitakere Ranges with adjacent sitesapproximately 3–4 km apart, and site 5 in near theUrewera National Park, about 300 km distant fromWaitakere. Global R value [ 0.523 (P [ 0.001). The R valuegives an absolute measure of how separated the groupsare, on a scale of 0 (indistinguishable) to 1 (all similaritieswithin groups are less than any similarities betweengroups) (Clarke & Gorley 2001)

Pair-wise comparisonsbetween sites

R value P

1, 2 0.5 0.037

1, 3 0.225 0.148

1, 4 0.5 0.012

1, 5 0.7 0.008

2, 3 0.2 0.255

2, 4 0.75 0.004

2, 5 0.9 0.004

3, 4 0.4 0.074

3, 5 0.65 0.008

4, 5 0.95 0.004



Endophyte assemblages were also influenced by season.

Seasonal differences, although statistically significant, were

less pronounced than those observed for host species and sites

across the total endophyte assemblage (R¼ 0.367, P¼ 0.001).

Seasonal patterns differed for individual taxa with a few spe-

cies being present only either in summer or winter, such as

Cylindrobasidium sp. More commonly, there were differences

in isolation frequency across seasons (Table 2). Because the

winter and summer samples were selected from the same co-

hort of leaves, the variation observed suggests that for most en-

dophytes, individual infections are not persistent. Rather, over

time there may be a high and ongoing turnover of endophyte

populations within a single leaf. A preferential loss of infected

leaves may also explain this result for Kunzea ericoides, but there

was no leaf loss from the one season old twigs of the podocarps.

Phylogenetic diversity and biology of the endophytes isolated

Stone et al. (2004) listed taxa commonly isolated as endophytes

from woody plants, and most of the broad groups in their list

are represented amongst the fungi we found frequently (Table

2). Several of the ascomycetes we isolated were not represented

in Genbank with ITS sequences, and their identity remains un-

known. Basidiomycetes are generally recorded only rarely as

foliar endophytes, although Crozier et al. (2006) reported large

numbers from trunks of mature trees. The single basidiomy-

cete species in our survey was found only on Dacrydium cupres-

sinum in winter, but at that time the fungus was isolated from

all eight trees sampled from the Waitakere Ranges. The ITS se-

quence of this basidiomycete is only 4 bp different from a fun-

gus isolated from Podocarpus falcatus seeds in Ethiopia (Gure

et al. 2005). Although the Ethiopian fungus was identified as Pol-

yporus gayanus on the basis of cultural characters (Gure et al.

2005), comparison with other ITS sequences in Genbank sug-

gest it is more likely to be a Cylindrobasidium sp. The Ethiopian

fungus is pathogenic to germinating seeds of P. falcatus (Gure

et al. 2005). There is no information on podocarp seed patho-

gens in New Zealand, and the biology of our Dacrydium endo-

phyte remains unknown, although it is intriguing that these

two closely related fungi share a host in the Podocarpaceae.

Table 6 – Permutational multivariate analyses of variance (PERMANOVA) based on Bray–Curtis dissimilarity measure forsquare-root transformed abundance data of all endophyte species for each host/season combination. MS values were usedto calculate the proportion of variance for each component (see Fig 4)

Source df D. dacrydioides summer D. dacrydioides winter

MS F P MS F P

Site 4 78 322.8 3.5523 0.0120 112 439.7 3.5530 0.0030

Tree (Si) 5 22 048.5 3.3291 0.0010 31 646.6 5.5436 0.0010

Branch (Tr(Si)) 40 6622.9 1.9502 0.0010 5708.7 2.0607 0.0010

Residual 450 3396.4 2770.2

Total 499

P. ferruginea summer P. ferruginea winter

MS F P MS F P

Site 4 102 596.2 3.1938 0.0070 120 983.4 4.4956 0.0060

Tree (Si) 5 32 123.1 5.8673 0.0010 26911.5 3.5095 0.0010

Branch (Tr(Si)) 40 5474.9 1.8310 0.0010 7668.2 3.0061 0.0010

Residual 450 2990.1 2550.9

Total 499

P. totara summer P. totara winter

MS F P MS F P

Site 4 40 989.7 2.3501 0.0330 70 390.2 1.6424 0.1130

Tree (Si) 5 17 441.4 2.5889 0.0010 42 858.3 8.1070 0.0010

Branch (Tr(Si)) 40 6736.9 1.8179 0.0010 5286.6 1.9011 0.0010

Residual 450 3705.9 2780.9

Total 499

D. cupressinum summer D. cupressinum winter

MS F P MS F P

Site 4 42 636.8 2.0145 0.0440 60 665.8 2.9169 0.0010

Tree (Si) 5 21 165.4 2.8616 0.0010 20 797.9 4.0431 0.0010

Branch (Tr(Si)) 40 7396.5 2.0603 0.0010 5144.1 1.4686 0.0010

Residual 450 3589.9 3502.8

Total 499

K. ericoides summer K. ericoides winter

MS F P MS F P

Site 4 60 805.5 2.3857 0.0260 53 919.0 1.5072 0.1160

Tree (Si) 5 25 487.4 5.2612 0.0010 35 774.9 6.5125 0.0010

Branch (Tr(Si)) 40 4844.4 1.8119 0.0010 5493.3 2.4209 0.0010

Residual 450 2673.7 2269.1

Total 499

F¼ pseudo F statistic (McArdle & Anderson 2001).



Xylariaceae were frequently isolated in our study, and these

fungi are also common on fallen wood in the forests we sam-

pled. However, the fruiting bodies of Xylariaceae are found

rarely on leaves, raising a question about the biological signif-

icance of the leaf endophyte infections. Despite their strong

lignolytic enzymatic capability (Pointing et al. 2003) and ability

to cause various types of disease primarily through extensive

tissue degradation, several studies have indicated that some

xylariaceous species isolated as endophytes may not be latent

saprophytes (Bayman et al. 1998; Griffith & Boddy 1990; Laessøe

& Lodge 1994), but rather exist solely as mutualistic endo-

phytes (Davis et al. 2003; Rogers 2000). There is insufficient mo-

lecular data for New Zealand Xylariaceae to confirm whether

the species we isolated as leaf endophytes are the same as

those fruiting on fallen wood in the same forests. However,

a comparison with taxa in the Xylariaceae tree published by

Guo et al. (2003) suggests that some at least some may not be.

Four of our Xylariaceae groups belong in the WMS15 clade of

Guo et al. (2003). All isolates in this clade are known only as

leaf endophytes and it could perhaps represent an ecologically

specialised group of Xylariaceae, biologically and genetically

distinct from the species which form ascomata on wood.

Pezicula and Neofabraea are two closely related genera of in-

operculate discomycetes that have been reported rarely from

New Zealand native forests (Anonymous 2001–2007), but were

surprisingly diverse as podocarp endophytes. Host-special-

ised species were isolated frequently from D. cupressinum

(two species), Prumnopitys ferruginea, and Podocarpus totara. As-

suming the leaf endophyte infections were initiated from as-

cospores, New Zealand has at least four species of Pezicula

awaiting discovery. In addition, the Pezicula anamorph Crypto-

sporiopsis actinidiae, first described as a pathogen of Actinidia in

orchards (Johnston et al. 2004), was commonly isolated in our

study, but it was not host specialised.

We used ITS sequences to compare several of the endo-

phytic Colletotrichum species with isolates from fruits of horti-

cultural crops, previously intensively studied in New Zealand

(e.g. Johnston & Jones 1997; Johnston et al. 2005; Lardner et al.

1999). Most of the endophyte isolates were members of the

Colletotrichum gloeosporioides (e.g. Genbank deposit numbers

EU482214 – EU482217) and Colletotrichum boninense (e.g. Gen-

bank deposit numbers EU482210 – EU482213) clades (sensu

Johnston et al. 2005), and Colletotrichum acutatum was also iso-

lated. Colletotrichum isolates from fruits also fall commonly

into these three clades. However, one of the less frequently

isolated Colletotrichum species (e.g. Genbank deposit numbers

EU482218 and EU482219) was genetically distinct from all the

fruit-inhabiting Colletotrichum species known from New Zea-

land, was also distinct from all species included in the analy-

sis of Farr et al. (2006), and may represent an indigenous,

forest-inhabiting species. This species was isolated from sev-

eral different podocarps and was morphologically distinctive

in having colonies with restricted growth, the surface of the

colonies densely covered with orange conidial ooze and al-

most no aerial mycelium, setae lacking, conidia about 20–

25� 4–4.5 mm, gently curved, tapering toward each end.

Several Phyllosticta and Mycosphaerella species were fre-

quently isolated. Both genera are known to have many unde-

scribed species associated with plants in New Zealand’s

native forests (PRJ, unpubl. data).

It remains unknown whether the podocarp and Kunzea en-

dophytes form a true mutualistic association with their hosts

or whether they are latent pathogens, commensalistic or

have a putatively neutral relationship (Cabral et al. 1993; Rodri-

guez & Redman 1997). In a concurrent study, fungal hyphae in

leaves of Kunzea ericoides were visualised using a fluorescent la-

belling method (Johnston et al. 2006), and three different host

reactions to fungal hyphae were observed: (1) no reaction

with fungal hyphae extending deep into the leaf within inter-

cellular spaces; (2) callose formation and restriction of fungus

to stomatal cavity and intercellular spaces, indicating a plant

defence reaction; and (3) a hypersensitive plant defence reac-

tion with intracellular penetration and death of a single plant

cell. These data suggest different fungal life styles for the fungi

present in K. ericoides, and a similar diversity is likely amongst

the podocarp-associated fungi. The hypersensitive plant

Fig 4 – Sizes of components of multivariate variation in endophyte assemblages between sites, trees, branches, and leaves

(residual), as multivariate analogues to the univariate variance components, obtained using mean squares from the PER-

MANOVA results in Table 6. The values plotted are the square root of the sizes of the components of variation, the values

thus matching the scale of the original Bray–Curtis dissimilarities (expressed as a percentage difference between assem-

blages). a–e are the values for hosts Dacrycarpus dacrydioides, Prumnopitys ferruginea, Podocarpus totara, Dacrydium cupressi-

num, and Kunzea ericoides respectively. (A) Winter sample. (B) Summer sample.



defence reaction is a common expression of disease resistance

in plants, controlled by interactions between pathogen aviru-

lence genes and plant resistance genes (Heath 2000). The other

reactions are difficult to interpret as responses to either path-

ogenic or endophytic invasions of the leaf, with Schulz et al.

(1999) describing a range of putatively defensive plant reac-

tions to hyphae of both pathogenic and endophytic fungi.

Practical sampling considerations

Observed fungal species composition in studies using cultur-

ing methods to assess endophyte diversity can be affected

by the size of the leaf segments sampled, as multiple infec-

tions per leaf segment may bias species composition in favour

of more competitive or fast growing strains (Carroll 1995;

Gamboa et al. 2002). Only 1.7–4.5 % of leaf segments from

podocarp leaves and 7.7–11.0 % of leaf segments from Kunzea

ericoides leaves had multiple infections, and on average about

45 % of leaf pieces from the podocarps and 17 % of leaf pieces

from K. ericoides remained uninfected (Table 1). Furthermore,

slow growing morphotypes were commonly isolated among

both the abundant and rarer taxa. Together, this suggests

the degree of bias introduced by species competition is likely

to have been low in the present study.

Sampling strategies for studies on endophyte diversity

must take into account natural patchiness in the distribution

of fungal species across the landscape. For example, Johnston

(1998) illustrated between-tree variation in the distribution of

some endophytes in even-aged stands of Leptospermum scopa-

rium. The hierarchical sampling structure used in this study

allows the proportion of the variances between branches,

trees, and sites to be estimated. We applied a PERMANOVA

(Anderson 2005; Anderson et al. 2005) and partitioned the var-

iation among three nested components: individual sites, trees,

and branches. For each host species/season combination the

variability in overall patterns of diversity between branches

was similar to, or less than, that between trees (Fig 4). Thus,

our within-tree sampling strategy of selecting ten leaves

from each of five branches, rather than the less practical se-

lection of 50 leaves across the whole tree, was unlikely to

have biased the results to a major extent.

All isolations were carried out within a few hours of the

branches being picked, minimising the impact that leaf senes-

cence following picking may have had on the diversity ob-

served (Johnston 1998; Millar & Richards 1974).

An attempt was made to define taxa at about the level of

species. For those fungi that did not sporulate in culture, this

involved initial morpho-taxa groupings that were subse-

quently tested using ITS sequences. In most cases, the origi-

nal morpho-taxa were well supported, but initial groupings

were sometimes modified on the basis of the sequencing re-

sults. A few of the original morpho-taxa were combined, and

one xylariaceous taxon was removed from the analysis after

it was shown to be polyphyletic. When initially defining the

morpho-taxa, allowance was made for expected natural var-

iation in cultural appearance. This is a particular issue in

some taxa such as members of the Helotiales and Colletotri-

chum, and previous experience with these groups (e.g. John-

ston & Gamundı 2000; Johnston & Park 2005; Lardner et al.

1999; PRJ, unpubl.) proved valuable. Despite this, it is probable

that if all the singleton isolates had been sequenced, some

would have been found to match some of the morpho-taxa.

Naming of sterile morpho-taxa, and decisions on species

limits, were based on an initial BLAST search followed by in-

corporation of the sequence of the unknown endophyte into

the alignment of an appropriate published phylogeny. In only

one instance did the endophyte sequence match exactly an

existing sequence deposited in Genbank, this for Cryptospor-

iopsis actinidiae, a species originally described from New Zea-

land. For those that could be identified to the level of genus

and could be compared to taxa in a published phylogeny,

species-level variation was defined on the basis of the level

of between-species genetic variation generally accepted for

that taxon. Mechanical decisions on taxon limits based on

pre-determined levels of sequence similarity were avoided.

Several taxa could be identified only to the level family, order,

or class.

Acknowledgements

We wish to thank Drs Marti Anderson (Institute of Informa-

tion and Mathematical Sciences, Massey University), Margaret

Stanley, and Greg Arnold (Landcare Research) for helping with

statistical analyses. P.W. Wilkie, M. Fletcher, K. McDermott

and M. Sue are thanked for technical assistance, and R.E.

Beever for helpful discussions. Auckland Regional Council

are thanked for permission to collect the Waitakere samples

and the T�uhoe Tuawhenua Trust for allowing collections in

their native forests. Funds for this research were provided

by Landcare Research through its Investment Fund, and the

New Zealand Foundation for Research, Science and Technol-

ogy through the Defining New Zealand’s Land Biota OBI and

Agrochemicals From Microbes (FRST Contract number

BIDX0201).

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