1Simultaneous effect of habitat remnancy, exotic species and anthropogenic disturbance on

2orchid diversity and abundance

3

4Martín-Forés, I.1,2, Bywaters, S. L. 1,2, Sparrow, B. 1,2 & Guerin, G. R. 1,2

5

61 School of Biological Sciences, The University of Adelaide, Adelaide, South 5005,

7Australia

82 Terrestrial Ecosystem Research Network (TERN), University of Adelaide, Adelaide, South

9Australia 5005, Australia

10*corresponding author: [email protected]

11

12

13

14

1 15ABSTRACT

16Orchids are potentially useful as ecological indicators because of their sensitivity to habitat

17fragmentation and anthropogenic disturbance. While many studies explore the effect of single

18factors on orchid diversity, few investigate how the extent, configuration and condition of

19surrounding habitat affect whole orchid communities. Here, we unravel the effect of biological

20invasions, anthropogenic disturbance (i.e. grazing pressure, ecological condition), habitat

21fragmentation and climate on an Australian orchid community. We sampled 39 plots across

22nine sites in the Mount Lofty Ranges, Australia. We recorded the number of orchid species and

23number of individuals per species in mid-winter, early-spring and late-spring to account for the

24effect of season on species visibility, with 115 surveys in total. We ranked grazing intensity and

25ecological condition, and estimated cover of exotic species. We analysed the response of

26richness and diversity through generalised linear mixed models, and differences in species

27composition through non-metric multidimensional scaling. We also explored fruiting success in

28two species associated with floral resources in the surrounding habitat. Habitat configuration

29in the surrounding landscape had different effects at increasing radii. Patch-level orchid

30diversity was positively correlated with habitat edges in the immediate area, and with habitat

31cohesion at medium scales, whereas diversity was negatively correlated with increasing

32habitat area across larger surrounding areas. Orchids co-existed with exotic species but were

33negatively affected once exotic cover exceeded 20%. Species composition was correlated with

34both exotic cover and level of disturbance. Fruiting success was unrelated to floral resources of

35the surrounding vegetation, although associated with certain bee-attracting species. Our

36findings reveal a complex relationship between orchid communities and their surrounding

37environments suggesting that while orchids benefit from a somewhat disturbed landscape,

38they fail to thrive once exotic cover exceeds 20%. These idiosyncratic responses suggest orchid

39diversity may be unreliable as early-warning indicators of habitat disturbance.

40

2 41INTRODUCTION

42Orchid species constitute the largest family of including over 700 genera and 27,000

43species (Chase et al. 2015; Govaerts 2016) occurring globally, except for the poles and

44extremely dry deserts (Jones 2006; Tsiftsis 2020). Due to the complexity of their symbiotic

45interactions with other species (e.g. dependence on specific associations with mycorrhizal

46fungi and pollinators; Hutchings 2010) and their narrow niche breadth and high levels of

47geographic endemism (Dixon et al. 2007; Orejuela-Gartner 2012), the consequences of global

48change appear to be particularly severe for orchid communities (Gale et al. 2018).

49 Indeed, orchid species are particularly sensitive to climate change (Seaton et al. 2010;

50Zhang et al. 2015), exotic species invasion (Chupp et al. 2015), anthropogenic disturbances

51(e.g. grazing pressure, fire regimes and illegal collection; Burgman et al. 2007; Roberts & Dixon,

522008, Newman et al. 2013), and land-use change (e.g. habitat fragmentation and land

53clearance; Kull & Hutchings 2006; Vogt-Schilb et al. 2016). Their sensitivity and reliance on

54specific interactions position orchids to be considered an early indicators of habitat

55disturbance and ecosystem degradation (Rose 1999).

56 The increase in mean annual temperature due to climate change can alter flowering

57times (Gallagher et al. 2009) and thereby decouple the phenological synchronicity of

58pollinator–orchid species and emergence times of their pollinators, leading to reduced

59reproductive success (Hutchings et al. 2018; Hoffmann et al. 2019). The effect of climate

60change on increasing the intensity of drought periods and wildfires may further restrict the

61suitable habitats available for orchids (Wraith & Pickering 2018). Most orchid species require

62specific temperature conditions, displaying narrow niche breadth; thus, extremes decrease

63orchid performance (Zhang et al. 2015; Djordjević & Tsiftsis 2020). Likewise, increased drought

64periods can produce variations in soil conditions including nutrient availability, moisture

65content and microorganism composition and as a result have an enormous impact on orchids

66in certain regions (Rasmussen et al. 2015; Djordjević & Tsiftsis 2020).

3 67 Biological invasions can heavily alter ecological interactions (Pyšek et al. 2012).

68Invasive species can trigger competition for light and space and act as pollination

69generalists (Bartomeus et al. 2008), potentially dominating the diet of pollinators (Morales &

70Traveset 2009; Pyšek et al. 2011) and modifying dynamics in plant-pollinator networks (Vila et

71al. 2009; Vanbergen et al. 2017).

72 Despite these threats, land-use change, including land clearing and habitat

73fragmentation are recognised as the main threats to orchids diversity, having contributed to

74significant population declines in several parts of the world (Wraith & Pickering, 2019).

75Configuration of the surrounding landscape pattern (e.g. vegetation heterogeneity, area of

76suitable habitat, edge length, and degree of cohesion) is likely to influence species

77assemblages (Fischer & Lindenmayer 2007). The species-area relationship, hypothesizing that

78the greater the size of the habitat, the greater the biodiversity, has proved true in several

79orchid studies (e.g. Schödelbauerová et al. 2009; Traxmandlová et al. 2018). As such, many

80orchid species are at a higher risk because they are confined to habitats that have been

81fragmented or undergone loss of area (Brummitt et al. 2015). Moreover, fragmented patches

82are more likely to have poor ecological condition and increased levels of disturbance (McIntyre

83& Hobbs, 1999). Habitat fragmentation, together with increasing ecological disturbance can

84lead to the interruption of interspecific interactions such as the ones established by orchids

85(Faast 2010; Parra-Tabla et al. 2011), pollination failure and decreased reproductive success

86(Aguilar et al. 2006; Newman et al. 2013). Habitat fragmentation indirectly enhances grazing

87intensity as it affects the proportion of edges around patches and accessibility for herbivores

88(Faast & Facelli 2009). Additionally, proximity to roads and urban nuclei enhances the risk of

89orchids being collected and harvested illegally further threatening the conservation of orchid

90diversity in remnant habitats.

91 Orchid reproduction mainly depends on pollination success, normally by insects,

92although there are some orchid species that can undergo auto-pollination or asexual

4 93propagation by forming clonal populations (Roberts & Dixon 2008). Orchid pollination is often

94one-sided and specialized, with the orchid relying more on its pollinator than the pollinator on

95the orchid (Roberts & Dixon 2008). Orchid pollination can be based on nectar rewards or

96partial rewards (Shrestha et al. 2020a), although one-third of orchid species have developed

97deceit strategies, including food and sexual deception, in which orchids mimic a reward

98(mimicry of floral features and production of a copy of the insect pheromone or even

99resembling insect forms, respectively; Jersáková et al. 2006; Roberts & Dixon 2008). is an

100example of orchid species pollinated by a presumed deceptive mechanism, whereas Glossodia

101has a more generalist pollination strategy (Beardsell et al. 1986; Bates & Weber 1990; Faast

1022010). Both are reported to produce nectar (Shrestha et al. 2020b). Pollination success may be

103impacted by habitat disturbance, with pollinator activity typically enhanced by greater density

104and diversity of floral resources in the surrounding vegetation (Johnson et al. 2003; Faast

1052010).

106 The rapid decline of orchid communities suggests urgent conservation measures are

107required. From the orchid species that have already been assessed for the IUCN Global Red List

108to date, more than half have been included under some form of threatened or near-

109threatened category (IUCN, 2020; Wraith & Pickering 2018). Thus, from a conservation

110perspective, orchids constitute a key but complicated group (Reiter et al. 2016; Swarts & Dixon

1112017), for which process-based conservation actions or those preserving the habitat might be

112more appropriate than species-focused ones (Fay 2018). Therefore, to design successful

113conservation strategies for this plant family, we need to fully understand orchid dynamics by

114studying simultaneously multiple variables affecting their survival (Fay 2018).

115 Australian orchids provide a sound model for understanding the simultaneous effect of

116different threats on orchids diversity. Australia is a large and megadiverse continent that

117contains a high diversity of endemic orchid species (approximately 1800 species, 95% endemic;

118Backhouse 2007; Wraith & Pickering 2019). Orchids are over-represented among Australia's

5 119threatened species, making up 17% of nationally threatened plants (Faast & Facelli 2007). The

120susceptibility of orchids to environmental change is believed to be due to a complex set of

121threatening processes, including small population sizes, habitat modification and

122fragmentation, invasive species, and grazing and disruption of pollination interactions

123(Quarmby 2010; Wraith & Pickering 2019).

124 The aim of this study was to unravel the effect of the above factors on orchid

125community diversity in . The factors included: i) climatic and edaphic gradients,

126ii) habitat configuration in the surrounding landscape (including vegetation remnancy at

127increasing radii; adapting the method suggested by Parkes et al. 2003), and iii) local

128disturbance, including grazing intensity, ecological condition, and exotic species cover. We also

129explored how iv) species composition of surrounding floral resources influenced reproductive

130success (i.e. fruit set). Regarding habitat configuration in the surrounding landscape, we

131hypothesized that greater habitat remnancy and connectivity of patches would correlate with

132higher orchid richness, abundance and diversity. Regarding local patch condition, we

133hypothesized that orchid diversity would be higher in plots with lower levels of disturbance,

134i.e. where ecological condition is good, and there is low intensity of grazing and low cover of

135exotic species. Finally, regarding fruiting success rate, we expected floral resources (abundance

136and diversity) to be positively related to the number of bees’ visits and therefore with orchid

137fruit set (Beardsell et al. 1986). We further hypothesized that fruiting in the species Diuris

138pardina (Lindl.) would be enhanced by the presence of the legume Pultenaea largiflorens

139(F.Muell.) due to putative mechanisms of floral mimicry to resemble the orange native pea

140flowers (Beardsell et al. 1986).

141

142METHODS

143Study area

6 144The study was conducted in the Mount Lofty Ranges, South Australia, which are a biodiversity

145hotspot and climatic refugium at the continental scale due to their heterogeneity of climatic

146and topographic conditions (Crisp et al. 2001; Byrne 2008; Guerin & Lowe 2012). The area has

147a Mediterranean-climate. Mean annual precipitation ranges from 460 to 990 mm, whereas

148minimum and maximum annual mean temperatures range from 7.8 to 10.3 °C and from 18.1

149to 21.7 °C, respectively (Harwood et al. 2016). The Mount Lofty Ranges, like other

150Mediterranean-climate ecosystems around the world, have been subjected to intensive

151disturbance, including land clearing for agrarian purposes, habitat fragmentation, species

152introductions, and regular wildfire regimes (Westphal et al. 2007; Martín-Forés 2017; Martín-

153Forés et al. 2017; Casado et al. 2018).

154

155Data collection

156We selected nine sites from existing monitoring networks (Guerin et al. 2014; Bond et al. 2019)

157corresponding to remnant vegetation patches. Sampling took place between winter 2019 and

158spring 2020. Depending on the size of the patch, between three and five 30x30 m plots were

159established at each site, with a total of 39 plots collected. Plots comprised a gradient of land-

160use from heavily disturbed to relatively undisturbed areas. We surveyed each plot three times,

161in mid-winter, early-spring and late-spring, to ensure the most complete composition sampling

162possible across the main orchid flowering season, during which many species flower for short,

163specific periods. Due to logistic constraints, six plots were only visited twice and three were

164only visited once; thus, a total of 115 surveys comprised the final dataset.

165 In each plot, pairs of observers searched for orchids along 5m-wide panels. We

166recorded and photo-vouchered each orchid species and counted the number of individuals per

167species per plot. Records were uploaded to the WOW iNaturalist project for confirmation of

168species identifications. We differentiated clonal and non-clonal species to analyse potential

169differences in their abundance and response. For each plot, we ranked grazing intensity from 0

7 170to 3, we scored ecological condition based on Keighery Condition Scale (Keighery, 1994;

171Supplemental table S1) from 0 (completely degraded) to 1 (very good), and we estimated the

172percentage of cover occupied by exotic species.

173 To explore the effect of floral resources on fruit set, we first determined the fruiting

174success of two common orchid species and related the proportion of recorded flowers that

175developed into fruits, to both the density of flowers and the abundance and diversity of floral

176resources in the plot. For this, we systematically surveyed 18 30x30 m plots at Spring Gully

177Conservation Park. In early-mid September 2020, during the peak flowering period, where we

178counted the number of open flowers of the orchid species Diuris pardina and Glossodia major

179(R.Br.) (counting ~3,000 individuals; Fig. 4). To test the idea that fruiting success relates to the

180floral resources provided to pollinators in the surrounding vegetation at the time of flowering,

181we set a floral transect up in each plot in order to quantify the diversity and species

182composition of other floral resources available to insect pollinators. The transect consisted of a

1831.8 m wide strip along the western edge of the plot (54m2); we recorded number of individuals

184and the number of open flowers was scored for all surrounding vegetation except for non-

185insect pollinated species such as grasses and sedges. Flowers were either counted or, where

186necessary, estimated by counting the flowers on branchlets and multiplying by number of

187branchlets. We returned to the same plots in late October to count the proportion of orchid

188flowers that had set fruit.

189 We combined observations of orchid community composition, including richness,

190abundance and diversity metrics with spatial data and climatic and edaphic variables. We

191calculated ‘fragstats’ from spatial data with the function ClassStat from the SDMTools R

192package (VanDerWal et al. 2015) and the package raster (Hijmans 2020). We calculated habitat

193configuration (Jaeger 2020), and assessed the effect of the area of the remnant native

194vegetation, total edge length, perimeter/area ratio and habitat cohesion on orchids at

195increasing spatial scales (i.e. increasing radii around the plots; Hirao et al. 2008). We extracted

8 196data for 25 climatic variables from Harwood et al. (2016) and for twelve edaphic variables from

197the CSIRO Data Access Portal (https://data.csiro.au/, accessed in May 2020; Viscarra Rossel et

198al. 2014) (Supplementary material S2 and S3).

199

200Data analyses

201We conducted Principal Component Analyses for both climatic and edaphic variables and

202extracted the first axes (hereafter PC1_climate and PC1_edaphic), which explained 71.1% and

20350% of the variance, respectively. We used this approach because we were interested in

204overall environmental effects rather than focusing on the effect of individual bioclimatic

205predictors. The first climatic axis, PC1_climate, was positively correlated with precipitation,

206and negatively correlated with temperature.

207 We tested for the effect of season, and climatic and edaphic conditions in orchid

208richness, abundance and Shannon diversity index. We included season (i.e. the time during the

209main orchid flowering season in which the survey took place: winter, early spring and late

210spring), PC1_climate and PC1_edaphic as fixed factors. We also included the interaction

211between PC1_climate and season, as it is known that different climatic conditions affect orchid

212performance more when they occur in late spring.

213 We tested for differences in orchid species richness, abundance and Shannon diversity

214index associated with the effect of habitat configuration in the surrounding landscape. For

215orchid species richness and abundance, we conducted a suite of Generalized Linear Mixed

216Models (GLMM) fitted to a Poisson error distribution whereas for Shannon diversity index we

217used Linear Mixed Effects Models (LMM) fitted to a gaussian error distribution. To check for

218those effects at patch scale, we included season, patch area and patch length as fixed effects.

219To check for those effects at landscape scale, we conducted comparisons of sets of mixed-

220effects models at four different landscape scales, including circular areas around the plot of

221one, two, five, and ten kilometres radii. Fixed effects included season, area of the remnant

9 222vegetation, edge length, perimeter/area (ratio) and habitat cohesion (VanDerWal et al. 2015).

223We included plot nested within site as a random effect to account for potential spatial

224autocorrelation. To test for the effect of disturbance in species richness, abundance and

225Shannon diversity index, we conducted the same suite of models but including season, grazing,

226ecological condition and cover of exotic species as fixed effects. We included plot nested

227within site as random effects.

228 Kruskal-Wallis tests were performed to look for differences among the three survey

229times. In every case we extracted the best subset of models based on ΔAICc ≤ 2. Afterwards,

230we calculated marginal (i.e. the proportion of variance explained by fixed effects) and

231conditional (i.e. the proportion of variance explained by fixed and random effects) R2 for the

232LMMs (i.e. Shannon diversity models) with the ‘MuMIn’ R package (Barton 2018). We

233estimated R2 for the species richness and abundace GLMMs with the delta method from a

234model with a Poisson distribution also with the ‘MuMIn’ R package. For the best subsets of

235models, we computed ANOVA-like tables testing the inclusion of random-effect terms in the

236models; we used the function ranova from the ‘lmerTest’ R package for the LMEMs. For the

237GLMEMs, we compared the models with and without the inclusion on the random effect, with

238and without nesting the effect of plot within site, and with only one (either site or plot) as

239random effect with an anova function.

240 We explored orchid species composition as a response to disturbance (including

241ecological condition, grazing intensity and percentage of cover of exotic species) by conducting

242non-metric multidimensional scaling. We used as input a matrix of species abundances per

243survey. We calculated Bray-Curtis dissimilarities among each sampling event (i.e. plot and

244survey) with the vegdist function of the vegan package (Oksanen et al. 2019). We explored the

245ideal number of dimensions to consider with the NMDS.scree function from vegan and

246afterwards set the number of dimensions to three (displaying stress <0.1).

10 247 We conducted NMDS with the metaMDS function setting the maximum random starts

248to 100. We fitted environmental factors (i.e. ecological condition, grazing intensity, cover of

249exotic species) as correlation vectors onto the obtained ordination with 999 permutations to

250test significance of associations. We obtained the projections of points onto vectors that had

251maximum correlation with corresponding environmental variables, the squared correlation

252coefficient of these vectors with the obtained ordination and the associated p-values.

253 To determine the fruiting success, we calculated the proportion of recorded flowers

254that develop into fruit. We related the fruiting success of D. pardina and G. major to the

255species richness and Shannon diversity of floral resources and the abundance of flowers in the

256plot through Pearson correlations. We explored surrounding floral resources affecting fruiting

257success by conducting pairwise correlations for D. pardina and G. major with the abundances

258of each of the recorded species. For those species that we observed greater values of

259correlation coefficients (i.e. r > 0.35), we conducted linear models between D. pardina and G.

260major fruiting success rates and the species abundance to check for the significance of the

261effect of that particular flowering species on enhanced pollination.

262 All statistical analysis and calculations were performed using R (R Core Team 2020)

263employing the packages vegan (Oksanen et al. 2019), SDMTools (VanDerWal et al. 2015),

264corrplot (Wei & Simko, 2017), Hmisc (Harrel Jr 2020), xtable (Dhal et al. 2019), ggplot2

265(Wickham 2016), lme4 (Bates et al. 2015), AICcmodavg (Mazerolle 2019), lmerTest (Kuznetsova

2662017) and MuMIn (Barton 2019).

267

268RESULTS

269Effect of environmental conditions

270We recorded a total of 68 orchid species. The best models for both orchid richness and

271abundance included season, PC1_climate and the interaction between them as fixed effects

272(i.e. Richness ~ Season*PC1_climate + (1|Site/Plot) and Abundance ~ Season*PC1_climate +

11 273(1|Site/Plot)). Regarding orchid Shannon diversity index, among the best subset of models, the

274most parsimonious one only included the effect of season (See supplementary materials S4 &

275S5 for details of the best subsets of models, and tables with the estimates, statistics and

276significance for the fixed effects of the best models, respectively). The effect of season is

277discussed in Supplementary material S6. The inclusion of the random effects significantly

278improved the model (see details in Supplementary material S7).

279

280Effect of spatial configuration

281The effect of habitat configuration in the surrounding landscape differed among spatial scales.

282The best models of the effect of habitat configuration in an area of 1 km radius richness

283included season and edge, with edge having a significant effect on species richness and

284abundance, whereas for orchid Shannon diversity, the best model only included season. The

285best models for habitat configuration in an area of 2 km radius always included season and a

286positive and significant effect of habitat cohesion. At 5 km, we obtained a similar result for

287Shannon diversity, whereas the best models for richness and abundance included also a

288negative effect of the total area of the habitat. However, at greater spatial scale (i.e. 10 km

289radius) the best models for the diversity metrics included season and total habitat area, with

290the last one displaying a significantly negative effect on species richness and Shannon diversity

291index. Only season was included in the best model. (Fig 1; Supplementary materials S4, S5 &

292S7).

293

294Effect of ecological disturbance

295The best model for orchid species richness and Shannon index with regards to the effect of

296ecological disturbance included season and exotic species; with the effect of exotic species in

297both cases being significantly negative (Supplementary materials S4 & S5). The best and most

298parsimonious models for orchid abundance included either the effect of season and exotic

12 299species, or the effect of season and ecological condition. Exotic species had a negative impact

300on orchid diversity in any case, whereas ecological condition had a positive one

301(Supplementary materials S4, S5 & S7). However, although the effect of exotic species cover

302was negative overall, the most diverse orchid communities coexisted with intermediate levels

303of exotic cover (i.e. 15-20%; Fig. 2); when the cover of exotic species surpassed 25%, the

304impact on orchids became stronger with highly invaded sites displaying few or no orchids (Fig.

3052). Similarly, sites with very low values of ecological condition (i.e. ranked condition < 0.4) did

306not harbour any orchids (Fig. 2).

307

308Floristic differences among sites and among surveys

309NMDS showed that orchid species composition differed among sites but also within them.

310Within some sites, such as LIT and to a lesser extent HOR, there was a lot of variability in

311orchid species composition among plots, whereas plots within some other sites such as BLA or

312MAC, were quite similar (Fig. 3). Additional overlap in species composition was associated with

313season (Supplementary material S6).

314 Both condition and exotic species were significantly correlated to the NMDS ordination

315configuration (Condition: NMDS1 = -0.97, NMDS2 = -0.25, R2 = 0.10, p-value < 0.01; Exotic

316species: NMDS1 = 0.43, NMDS2 = 0.90, R2 = 0.08, p-value < 0.05), whereas grazing intensity

317was not (Condition: NMDS1 = 0.34, NMDS2 = 0.94, R2 = 0.02, p-value > 0.1).

318

319Fruiting success

320Fruit set in Diuris pardina and Glossodia major was low (0–18%) and not obviously related to

321either the density of flowering orchids, nor the amount or composition of floral resources in

322the surrounding habitat. Glossodia major and D. pardina exceeded 5% of fruit success only in 5

323and 2 plots, respectively (Fig. 4). Fruiting success for D. pardina was especially low, with eleven

324plots showing flowers but not fruits (Fig. 4; Supplementary material S9). The success rates of

13 3250–18% appeared random with respect to surrounding floral resources at time of flowering.

326Diuris pardina fruiting success was correlated with its flower density (adjusted r-square = 0.39,

327p-value < 0.01), but there was no apparent trend between flowering and fruiting G. major

328individuals (adjusted r-square = -0.05, p-value = 0.71).

329 Contrarily to our predictions, fruiting success for D. pardina was not significantly

330correlated with the abundance of Pultenaea largiflorens (adjusted r-square = 0.02, p-value =

3310.28), although it appeared positively correlated with the abundance of Lavandula stoechas

332(adjusted r-square = 0.23, p-value < 0.05). Fruiting success for G. major was significantly

333correlated to Wurmbea dioica flowers (adjusted r-square = 0.24, p-value < 0.05), and

334marginally to Drosera auriculata (adjusted r-square = 0.14, p-value < 0.1).

335 In addition, fruiting success for both species was neither significantly correlated to

336overall floral resources (i.e. number of flowers from all the species present in the plot), nor to

337overall floral diversity in terms of both, species richness and Shannon diversity.

338

339DISCUSSION

340Contrary to our expectations, the area of remnant vegetation in the surrounding landscape did

341not have a positive effect on orchid diversity. The effect of habitat configuration in the

342surrounding landscape on orchid diversity varied with spatial scale. Orchid diversity was

343positively correlated with the total edge of the habitat in surrounding areas of 1 km radius,

344while when considering medium-sized surrounding areas (2-5 km radii) orchids were favoured

345by habitat cohesion. At larger surrounding areas (5-10 km radii), we observed a negative effect

346of the area of the remnant vegetation on the patch-level diversity. As expected, high

347disturbance negatively affected orchid diversity, with exotic weed cover > 20% being the main

348factor impacting orchid diversity. However, orchid communities appeared to be most diverse

349in patches that had some level of disturbance. Finally, fruiting success appeared to be low and

350unrelated to surrounding floral resources for the orchid species studied, although it was

14 351associated with the presence of certain species in the surrounding vegetation that are known

352to attract bees, and for D. pardina was positively correlated with number (density) of flowering

353plants.

354

355Effect of environmental conditions

356The effect of climate varied among the three biodiversity metrics. Environmental conditions

357differentially affected orchids richness and abundance among seasons. For example, it is

358known that low temperatures typical from winter in Mediterranean-type ecosystems can

359inhibit orchid seeds germination and flowering (Acharya et al. 2011; Djordjevic & Tsiftsis 2020).

360Likewise, pollinators associated with orchid reproduction may be inactive during periods of

361cold winter temperatures (Zhang et al. 2015). Moreover, increased precipitation in winter and

362higher temperatures in late-spring can be associated with the ability of seeds to germinate and

363to increased photosynthetic efficiency and flower production, respectively (Djordjević &

364Tsiftsis, 2020). Regarding our results for orchid abundance, it is necessary to keep in mind that

365clonal orchid species might display asexual reproduction triggered under certain climatic

366conditions.

367

368Effect of spatial configuration

369The effect of habitat configuration in the surrounding landscape at increasing radii, varied

370associated with the spatial scale. At local scale (i.e. 1 km radius), we found a significant and

371positive effect of edge on orchid species and abundance. This can be related to the small

372spatial scale of the surrounding area considered, and to the fact that within a forest matrix, the

373edges can sustain equal or even more orchid species than the forest core (Parra Sánchez et al.

3742016). Edges are in general related to ecotone habitats, with considerably lower levels of

375competition for edaphic and light resources (i.e. low abundance of dominant plant species and

376low cover of trees and shrubs); these open niches can be colonized by pioneer species with

15 377weak competitive ability or high light requirements such as orchids (Arditti & Ghani, 2000;

378Fekete et al. 2020). In this sense, the surveyed areas are close to urban nuclei, showing

379anthropogenic habitats that provide refuge for orchid species, such as cemeteries, orchards

380and roadsides; thus, the edges can provide refugial conditions (Fekete et al. 2017) and act like

381ecological corridors by facilitating pollination and seed dispersal between suitable habitats on

382therefore spread of the orchid taxa (Van Rossum & Triest, 2012).

383 The positive effect of habitat cohesion at intermediate spatial scales (i.e. surrounding

384areas of radii 2 km and 5 km) is supported by previous findings. Greater habitat cohesion

385implies reduced fragmentation and consequently associated disturbance. It is well known that

386the dynamics of pollinator bees, and therefore the orchid-pollinator relationship, as well as the

387fruit set are positively associated with both the density and the condition of the surrounding

388vegetation matrix (Parra-Tabla et al. 2011; Akhalkatsi et al. 2014).

389 At larger spatial scales (i.e. surrounding landscape of 5 km and 10 km radii), we found

390an a priori unexpected negative effect of the total area of remnant vegetation on the diversity

391of orchid communities at patch-scale. It has been previously stated that peripheral orchid

392populations can perform better than populations living at the optimal conditions, despite being

393under more stressful conditions (García et al. 2010). In this sense, larger areas of remnant

394habitat could diminish orchids’ potential to find edges or semi-disturbed conditions in which

395they might perform better. In addition, it has been observed that habitat fragments of modest

396size can be ideal to maintain functional pollinator populations (Cane 2001). Considering the

397spatial scale pollinators can cover, it makes sense that habitat fragmentation negatively

398impacts orchids at smaller spatial scales while a trend that would seem opposite prevails at

399larger spatial scales.

400

401Effect of ecological disturbance

16 402High cover of exotic species had a negative effect on all orchid biodiversity metrics, whereas

403low cover seemed to not negatively affect orchid communities (Fig. 2). In addition, cover of

404exotic plant species had a significant effect on the orchid community composition, most likely

405related to the ability of the different orchid species to compete for resources (e.g. light, water,

406pollinators) with generalist exotic plants. Exotic plant species pose a threat to 65% of the

407orchid species present in Australia (Nevill 2010; Wraith & Pickering, 2019). Exotic plants are

408often pollination generalists (Bartomeus et al. 2008), dominating the diet of pollinators

409(Morales & Traveset 2009; Pyšek et al. 2011), and achieving high reproductive success

410(Thompson & Knight, 2018). Moreover, greater presence of exotic species is often associated

411with greater grazing intensity (Wraith & Pickering, 2019); in this sense, the orchid species that

412are able to survive in more invaded places could be benefiting from grazing being mainly

413directed to palatable exotic grasses. In addition, in cases where exotic plants also form

414mycorrhizal associations, native orchids can become displaced due to overlapping webs of

415fungi (Bonnardeaux et al. 2007).

416 Ecological condition positively influenced orchid individual abundance, although

417surprisingly it did not have an effect in the models for species richness and Shannon diversity

418index. Previous studies showed that good ecological condition (e.g. greater native vegetation)

419is related to diminished proportion of orchids at risk of extinction (Wraith & Pickering, 2019).

420The lack of effect of this factor in our models can be due to the fact that poor ecological

421condition is linked to habitat modification and fragmentation and increased levels of grazing

422and exotic cover (Wraith & Pickering, 2019). In this sense, the fact that we separated each

423effect showed that the main risk for orchids in disturbed habitats is the one posed by exotic

424plant species. However, we observed a threshold of ecological condition below which orchid

425species were not able to survive (Fig. 2).

426 It has been previously stated that orchids are negatively affected by high levels of

427irradiation and intensive grazing pressure (Akhalkatsi et al. 2014; Prowse et al. 2019); however,

17 428most of the orchid species restricted to Mediterranean climate ecosystems prefer habitats that

429appear disturbed to a certain degree (Martin-Fores, 2017; Tsiftsis & Antonopoulos, 2017).

430

431Fruiting success

432According to our results, fruiting success for D. pardina and G. major is mainly stochastic and

433not obviously controlled by local floral resources and surrounding vegetation, contradicting a

434priori predictions. Fruiting plants of both species were often observed in clusters, which

435suggests chance encounters with pollinators that visited neighbouring plants. It is also possible

436that developing fruits could have been grazed by kangaroos, although grazing pressure in the

437area did not seem to be high and many spent flowers were observed during fruiting surveys,

438confirming that they had not been pollinated. Overall, the success rate of the studied species

439appeared to be low; however, for food deceptive orchids, it is common that the reproductive

440success rate does not reach 20% (Gill 1989; Jacquemyn & Brys 2010). In this sense, populations

441of these species might rely on a small number of individuals producing seed to replenish the

442population or even long-lived individuals that are rarely pollinated, contrarily to other species,

443such as orchids from the genus Thelymitra for which developing fruits were observed

444frequently in the same plots. Contrary to our expectation, fruiting success of Diuris pardina

445was not significantly correlated with the native pea it mimics, Pultenaea largiflorens, despite

446bees being observed visiting P. largiflorens individuals during sampling. However, fruiting

447success of D. pardina positively correlated with the abundance of Lavandula stoechas, an

448introduced labiate woody species which strongly attracts honeybees (Kantsa et al. 2018). This

449trend was also observed in Australia for Diuris magnifica, which did not increase its

450reproductive success associated with the presence of native pea plants but appeared to be

451influenced by another non-model plant (Scaccabarozzi et al. 2019). The fact that G. major was

452positively correlated with the abundance of Wurmbea dioica might be due to the fact that

453male individuals of this dioecious lily attract two species of Australian native bees, and, to a

18 454lesser extent, honeybees (Vaughton & Ramsey 1998). Previous studies focusing on the

455pollination and fruiting success of the Australian orchid versicolor also observed in

456their plots of study co-flowering plants of Wurmbea dioica and two species from the genus

457Drosera (Reiter et al. 2019).

458

459Orchids as indicators

460The combined effects of factors on orchid communities is complex to interpret and our results

461suggest that orchids are not ideal as a general or early-warning ecological indicators. Despite

462being sensitive to ecological conditions, orchids also appear to be favoured by edges at local

463scale, and small size habitat patches at larger scales. Preventing degradation of ecosystems

464and promoting high ecological condition and low exotic species cover is crucial to preserve

465orchids. However, the recommendations towards habitat configuration appear unclear, and

466need to be carefully considered at different spatial scales in order to inform management

467strategies. Previous literature suggested that the effect of habitat configuration in the

468surrounding landscape can differentially influence orchid population viability depending on the

469pollination syndrome displayed by the different species and on their specificity with the

470pollinator involved (Newman et al. 2013). Further studies should aim to elucidate the effect of

471the habitat configuration on orchid diversity controlling for the species’ pollination syndrome,

472clonal ability, and tolerance to disturbance. Therefore, unravelling responses of different

473functional groups within the orchid family to environmental factors such as the ones

474considered in this study would be necessary as a preliminary step to be able to use orchids as

475reliable indicators. This will also help to design improved conservation strategies adjusted to

476the configuration of the area to be managed.

477

478ACKNOWLEDGEMENTS

19 479We thank the TERN Ecosystem Surveillance team, Rosalie Lawrence, Robert Lawrence, Penny

480McLachlan, Katie Irvine, Michael Starkey, Sally O’Neill, Candy Guerin, Anthelia Bond, the

481District Councils of Mount Barker and Alexandrina and the South Australian Department for

482Environment and Water. This work has been funded by the grant 327-2018 – Australian Orchid

483Foundation - Climate and habitat condition controls on orchid populations – research

484outcomes associated with a citizen science program. Likewise, it has been possible thanks to

485the grant from the Department of Industry, Innovation and Science – Inspiring Australia,

486Citizen Science Grants – Wild Orchid Watch. We have no conflicts of interest to declare.

487

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28 FIGURES

Fig. 1. Representation of the effect of habitat parameters on orchids diversity at different spatial scales and the sign of the effect. See supplementary material S4 for the best subsets of models, and S5 for estimates, statistics and significance for fixed effects of the best models.

29 Fig. 2. Orchid species richness and individual abundance at different levels of disturbance, with regards to cover of exotic species, ecological condition and grazing intensity.

30 Fig. 3. Non-metric multidimensional scaling, in which the differences in orchids communities among sites can be differentiated by the colours of the polygons. Vectors show the disturbance variables that were significantly correlated to the ordination space.

31 Fig. 4. Number of flowers and fruiting success for Diruis pardina and Glossodia major. Photos of both species are original from the authors and were taken at Spring Gully Conservation

Park, South Australia.

32