Species diversity of corticolous myxomycetes in Tianmu Mountain National Nature Reserve, China.

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ARTICLE Species diversity of corticolous myxomycetes in Tianmu Mountain National Nature Reserve, China Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by UNIV OF MANCHESTER on 12/10/14 For personal use only.

Qi-Sha Liu, Shu-Zhen Yan, Jun-Yong Dai, and Shuang-Lin Chen

Abstract: The species diversity of corticolous myxomycetes on 4 vegetation types in the Tianmu Mountain National Natural Reserve, eastern China, was examined from 2011 to 2012. A total of 1440 moist chamber cultures were prepared with bark samples, which yielded several hundred collections representing 42 species in 20 genera. It was found that 79% of cultures produced some evidence (either plasmodia or fruiting bodies) of myxomycetes. Eight species (Comatricha elegans, Cribraria confusa, Licea pusilla, Cribraria microcarpa, Collaria arcyrionema, Licea biforis, Arcyria cinerea, and Clastoderma debaryanum) were abundant (exceeding 3% of all records), but about a third of all species were classified as rare. Species richness (S = 33) and diversity (exp[H=] = 16.60, S/G = 1.74) of corticolous myxomycetes were the most diverse in the deciduous broadleaf forest. The species recorded from coniferous forest showed the lowest species richness (S = 21) but the highest evenness (J= = 0.91). The cluster analyses were based on the Bray–Curtis similarity matrix, and the results indicated that corticolous myxomycete assemblages were distributed by a seasonal and annual pattern. Canonical correspondence analysis showed that season and pH were key factors in determining species distribution. Key words: corticolous myxomycetes, moist chamber culture, distribution patterns, subtropics, canonical correspondence analysis (CCA). Résumé : On a examiné la diversité des espèces de myxomycètes corticoles propres a` 4 types de végétation, entre 2011 et 2012, dans la réserve naturelle nationale du Mont Tianmu de la Chine orientale. Un ensemble de 1140 cultures en chambre humide ont été préparées a` partir de prélèvements d’écorces, ce qui a permis de constituer plusieurs centaines de collections représentant 42 espèces appartenant a` 20 genres. On a constaté que 79 % des cultures présentaient des signes évidents (plasmodies ou fructifications) de myxomycètes. Huit espèces (Comatricha elegans, Cribraria confusa, Licea pusilla, Cribraria microcarpa, Collaria arcyrionema, Licea biforis, Arcyria cinerea et Clastoderma debaryanum) étaient abondantes (dépassant 3 % des éléments recensés), mais on a estimé qu’environ le tiers des espèces étaient rares. La richesse (S = 33) et la diversité (exp[H=] = 16,60, S/G = 1,74) des myxomycètes corticoles étaient plus élevées dans la forêt de feuillus. Les espèces recensées de la forêt de conifères faisaient preuve de la richesse spécifique la plus basse (S = 21), mais de l’uniformité la plus élevée (J= = 0,91). Les analyses de regroupements s’appuyant sur la matrice de similarité de Bray–Curtis ont révélé que les agencements de myxomycètes corticoles se distribuaient suivant un mode saisonnier et annuel. L’analyse de correspondance canonique a indiqué que la saison et le pH étaient des facteurs clés qui gouvernaient la distribution des espèces. [Traduit par la Rédaction] Mots-clés : myxomycètes corticoles, culture en chambre humide, modes de distribution, zones subtropicales, analyse de correspondance canonique (ACC).

Introduction The myxomycetes (plasmodial slime molds or myxogastrids) are a group of amoeboid protists occurring in terrestrial ecosystems and aquatic habitats. Their life cycle includes 2 trophic stages: one consisting of uninucleate amoeboid or flagellate cells and the other a complex macroscopic multinucleate form (plasmodium), which can achieve macroscopic dimension (Martin and Alexopoulos 1969). Under favorable conditions the plasmodium is mobile, giving rise to one or more fruiting bodies that contain spores. These spores have the potential for long distance dispersal by air currents or animal activities. Reversely, under unfavorable conditions a plasmodium can change into a hardened, resistant structure called a microcyst or sclerotium, able to survive until more favorable conditions occur (Martin and Alexopoulos 1969). Myxomycetes occur throughout the world, with the highest diversity documented in forest ecosystems. Within forests, some myxomycete species inhabit the decaying wood or bark, decaying

leaf litter, and the soil (Stephenson 1988, 1989). The term “corticolous myxomycetes” was first used to describe those species that completed their entire life cycle, from spore to fruiting body formation, on the bark of living trees or vines (Keller and Brooks 1973). There have been numerous studies on corticolous myxomycetes in different terrestrial ecosystems (e.g., Snell and Keller 2003; Everhart and Keller 2008; Everhart et al. 2008). The existence of myxomycetes has been known since the mid17th century. However, studies of this group of species concentrated on their taxonomy with relatively little ecological information collected until ca. 1990 (Stephenson 2011). Although myxomycetes have been recorded in every terrestrial ecosystem, these organisms are not equally abundant in all forest types. For instance, Stephenson et al. (1993) noted that the fruiting bodies of myxomycetes were much less abundant in closed canopy tropical and (or) subtropical regions in southern India than in temperate forests in the United States. Research on myxomycete diversity has focused on temperate and tropical forests (e.g., Stephenson 1988, 1989;

Received 7 June 2013. Revision received 25 October 2013. Accepted 5 November 2013. Q.-S. Liu, S.-Z. Yan, J.-Y. Dai, and S.-L. Chen. Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, People’s Republic of China. Corresponding author: Shuang-Lin Chen (e-mail: [email protected]). Can. J. Microbiol. 59: 803–813 (2013) dx.doi.org/10.1139/cjm-2013-0360

Published at www.nrcresearchpress.com/cjm on 7 November 2013.

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Tran et al. 2006; Rojas and Stephenson 2007). However, relatively little fieldwork has been conducted on myxomycete diversity in subtropical forests, such as those in Hunan Province, China (Ukkola et al. 2001; Härkönen et al. 2004a, 2004b) and in Big Bend National Park, Texas, USA (Ndiritu et al. 2009). We investigated corticolous myxomycete diversity, including species distribution patterns and the composition of species assemblages, associated with different vegetation types in subtropical forests of China. Novozhilov and Schnittler (2008) noted that the distribution of corticolous myxomycetes may be related to the differences in the bark acidity and texture. Moreover, other environmental factors (e.g., season, elevation, light intensity) interact to influence the occurrence and distribution of corticolous myxomycetes. The primary goal of this survey was to analyze corticolous myxomycete assemblages in response to changes associated with different vegetation types from a subtropical region of China and compare the data with those from other terrestrial ecosystems. In addition, we assessed the relationship between corticolous myxomycetes and some environmental factors such as the bark pH, bark water-holding capacity, and season.

Materials and methods Study area Our study area was in the Tianmu Mountain National Natural Reserve, located in Lin’an City, northwest of Zhejiang Province, China (Fig. 1A). The Tianmu Mountain National Nature Reserve is situated between 30°18=30==–30°24=55==N and 119°23=47==–119°28=27==E, covering an area of approximately 4284 ha (1 ha = 104 m2) (Fig. 1A). This area is strongly influenced by an oceanic climate, with the area acting as a transition from the mid- to north subtropical zone. The mean annual temperature is ca. 15.3 °C (range: –6.8 to 38.1 °C). The mean annual precipitation for this region is 1390– 1870 mm, with the relative humidity ranging from 76% to 81%. The frost-free period is ca. 210 days. The elevation in the reserve ranges from 280 to 1556 m above sea level, with a vertical distribution of the vegetation varying with altitude. The area’s vegetation types consist of evergreen broadleaf forests, mixed evergreen – deciduous broadleaf forests, deciduous broadleaf forests, and shrubs. In addition, coniferous forests dominated by Cryptomeria fortunei occur in the reserve (Ding et al. 2010). This rich natural resource and complex landform offers natural advantages for studying corticolous myxomycete biodiversity. Sampling strategy The study was not intended to provide an exhaustive survey on corticolous myxomycetes in the area. Instead, it represents a limited contribution to the understanding of these organisms. To obtain a representative record of corticolous myxomycetes from the area, 3 surveys were undertaken during 2011–2012. Bark samples, for incubation in moist chambers, were collected in autumn (October) and spring (May). All bark samples were collected from 4 sampling sites (each 0.1 ha) based on the vegetation types (Table 1). At each site, we adopted the opposite angle line 5 spots method to investigate (one plot at the center diagonal position, 4 plots at the equidistance from the center diagonal position), thus 5 different plots were sampled. Four trees were randomly selected from each plot, excluding rotten and small trees (with a height below 15 m). At each plot, a pair of bark samples was taken from each tree, one at 50 cm and the other at 150 cm above the ground. The same trees were resampled during the 3 sampling campaigns (October 2011, May 2012, and October 2012). A total of 480 bark samples were obtained (4 sites × 5 plots per site × 4 trees per plot × 2 heights per tree × 3 times resampling). The method of sampling is shown in Fig. 1. Bark samples were placed in plastic bags separately and stored at –80 °C until needed. A map shows the location of the Tianmu Mountain National Natural Reserve in eastern China and

Can. J. Microbiol. Vol. 59, 2013

the positions of 4 sampling sites, which were drawn using the program ESRI ArcGis version 9.3 (Fig. 1A). Vascular plant names followed those described by Ding et al. (2010). We have briefly described each sampling site, geographic location, elevation, and the dominant vegetation in Table 1. Moist chamber cultures In the laboratory, all bark samples were placed in moist chambers, which consisted of Petri dishes (9 cm in diameter) lined with moistened filter paper, as described by Stephenson (1989). For 480 bark samples, we prepared 3 replicates, a total of 1440 chambers. Sterile distilled water adjusted to pH 7 with KOH was added in the chamber around the bark. After approximately 24 h, the pH of each culture was measured using a flat plate pH meter (Fisher Accument Model PHS-3C, JingKe Company, Shanghai, China). After excess water was poured off, all cultures were maintained under indirect natural light at room temperate (23–25 °C). The cultures were observed every 2 or 3 days for approximately 2 months. To maintain the moist environment, water was added to the cultures when required. The cultures were regularly checked for corticolous myxomycete plasmodia and fruiting bodies. The total number of records produced was quantified following Novozhilov et al. (2000). However, the multiple occurrences of a particular species in a moist chamber were considered as one record (Stephenson 1989). The water-holding capacity of bark was determined following the methods of Snell and Keller (2003). The uncultured portions of bark were oven-dried at 60 °C for 3–4 days until a constant mass was reached. To calculate the water-holding capacity of bark (% water per gram of dry bark), samples were placed separately in plastic bags with water added and maintained for 24 h. The water was then drained, with the bark placed on absorbent paper to remove excess water. The bark samples were then weighted. Water-holding capacity of the bark was calculated by the percentage of water to the mass of dry bark. Classification and identification of species Species of corticolous myxomycetes were identified using the morphological characteristics of the fruiting bodies, as explained in known reference works on the subject (Martin and Alexopoulos 1969; Neubert et al. 1993, 1995, 2000). Regarding myxomycete taxonomy, we used the classical morphological species concept and the traditional taxonomy of myxomycetes. Nomenclature followed Hernández-Crespo and Lado (2005). Laboratory specimens were dried at room temperature and then deposited in the Center of Microbial Cultures of Nanjing Normal University. Species richness estimation To estimate the completeness of the survey in terms of the recorded corticolous myxomycetes, species accumulation curves were constructed using the program EstimateS version 8.2 (Colwell 2006). For each site, a total of 20 trees were sampled (4 trees in each of 5 plots). Since the trees were resampled 3 times, we consider a total of 60 samples for the construction of species accumulation curves. Incidence-based coverage estimator (ICE), abundance-based coverage estimator (ACE), and Chao2 were calculated to assess the completeness of the sampling effort for each sampling site, which were recommended by Unterseher et al. (2008). These values were based on the sample size, distribution, and abundance patterns under investigation. The percentage of completeness was calculated using the actual number of species recorded by the mean number of species expected using the 3 estimators (Ndiritu et al. 2009). Data analysis A number of basic diversity parameters were calculated for each sampling site. The relevant data assessed were species diversity, evenness, and dominance using the Hill family of diversity indices (Hill 1973), where Pi is the proportion of i species, S is Published by NRC Research Press

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Fig. 1. (A) Map showing the location of Tianmu Mountain National Natural Reserve in Zhejiang Province. Sampling sites were located within 4 vegetation types at elevations from 400 to 1100 m above sea level. The sampling site codes are explained in Table 1. (B) The sampling strategy: 4 sites × 5 plots per site × 4 trees per plot × 2 heights per tree.

Table 1. Sampling site locations and vegetation characteristics. Elevation (m above sea level)

Key vegetation type

Vegetation

430

Evergreen broadleaf forest

800

Mixed evergreen–deciduous broadleaf forest

Quercus myrsinifolia, Ginkgo biloba, Cryptomeria fortunei Trachycarpus fortunei, Fokienia hodginsii, Liquidambar formosana, Mallotus apelta, Cryptomeria fortunei Carpinus viminea, Carya cathayensis, Pistacia chinensis, Stephanandra chinensis, Fagus engleriana, Trachycarpus fortunei Cryptomeria fortunei, Cephalotaxus fortunei, Pseudolarix amabilis, Pinus massoniana

Site code

Name

Location

EF

Chan Yuan Temple

EDF

Seven Mile Pavilion

30°19=14.30==N, 119°26=51.27==E 30°20=1.22==N, 119°26=25.95==E

DF

Parking Plot

30°20=37.56==N, 119°26=25.77==E

1100

Deciduous broadleaf forest

CF

Hong Miao

30°19=23.94==N, 119°27=43.23==E

500

Coniferous forest

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the total number of species recorded, for species richness; N1 = −exp[冱Piln(Pi)] = −exp[H=], the expectation of Shannon diversity index; N2 = (冱Pi2)−1 = reciprocal of Simpson’s index; Ninf = (Pi)−1 = reciprocal of Pi index, for the commonest species; and J= = ln(N1)/ ln(S), for Pielou’s evenness (Pielou 1975). These diversity parameters were calculated using the program PRIMER version 5.0. Hill family indices have been used for the comparison of myxomycete communities (Ndiritu et al. 2009). In addition, values of the overall taxonomic diversity were assessed using the mean number of species per genus ratio (S/G) (Stephenson et al. 1993). According to S/G values, a community where species have diversified among many genera is considered more diverse than one where most of the species belong to a few genera. We used the relative abundance index, described by Stephenson et al. (1993), where the indices A is abundance (>3% of all records), C is common (1.5%–3%), O is occasional (0.5%–1.5%), and R is rare (2 (Ter Braak 1987; Braak and Smilauer 2002). Each moist chamber representing one microhabitat was coded in the environment dimension according to the states classified for each environmental parameter. Randomized 499 Monte-Carlo permutation tests were performed to determine which environmental variables exerted significantly more influence on the distribution of corticolous myxomycetes at P < 0.05. The significance of the first ordinance axis was tested with all 4 axes together. To avoid introducing noise by rare species, species with a relative frequency of 3% of all records). However, 15 of the 42 species were classified as rare for this region. The occurrence of Physarales was occasional or rare, for instance: Physarum pusillum (0.3%), Didymium iridis (0.4%), Didymium difforme (0.8%), and Physarum nutans (1.3%; Table 2). The primary data are shown in Supplementary Table S11. Here, for the first time, we stored the bark samples at –80 °C until the cultures were prepared. Interestingly, myxomycetes had survived upon this treatment, but it cannot be ruled out that some species may be lost due to this treatment. Thus, the impact of this treatment on the assemblages of myxomycetes should be evaluated deeply in future studies. Completeness of the survey Our species accumulation curves were generated using the data based on the same number of the moist chambers (Fig. 2). Because the actual recorded number was larger than the estimate number by ACE index (21 > 19, Table 2), ACE underestimated richness and thus was not used to determine the percent sampling effort. The sampling effort was calculated using the actual number divided by the mean value of 2 indices (ICE and Chao2 species richness estimators). We found that the sampling effort of the present survey was 77%–89% complete (Table 2). The sampling effort for deciduous broadleaf forest had the most complete result (89%). Reversely, the minimum sampling effort for mixed evergreen – deciduous broadleaf forest was 77%. Species diversity The corticolous myxomycetes obtained from the moist chamber cultures are shown in Table 2 and Fig. 3. Relative abundance data (N1, N2, and Ninf) were calculated for various dataset based on the Hill family of diversity indices. The species richness, diversity, and evenness varied considerably in association with the vegetation types (Table 2; Fig. 3). The corticolous myxomycetes obtained from the deciduous broadleaf forest were more diverse than species associated with other vegetation types according to the N1 index (exp [H=] = 16.60, Table 2) and species per genus ratio (S/G = 1.74, Table 2). The values of 4 indices (S = 21, N2 = 12.95, J= = 0.91, and

Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/cjm-2013-0360. Published by NRC Research Press

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Table 2. Occurrence of myxomycetes associated with 4 vegetation types, including a number of species recorded, statistical data, and sampling effort.


Recordb Species name

Acronym

Abundance indexa

Arcyria cinerea Arcyria denudata Arcyria leiocarpa Arcyria pusillum Ceratiomyxa fruticulosa Clastoderma debaryanum Clastoderma pachypus Collaria arcyrionema Comatricha aequalis Comatricha elegans Comatricha laxa Comatricha nigra Craterium minutum Cribraria confusa Cribraria languescens Cribraria microcarpa Diderma effusum Didymium difforme Didymium iridis Echinostelium minutum Hemitrichia clavata Hemitrichia serpula Lamproderma columbinum Lamproderma scintillans Licea biforis Licea floriformis Licea kleistobolus Licea operculata Licea pusilla Metatrichia vesparium Paradiacheopsis erythropodia Paradiacheopsis solitaria Perichaena corticalis Perichaena depressa Perichaena liceoides Physarum nutans Physarum pusillum Stemonitis fusca Stemonitis herbatica Trichia botrytis Trichia persimilis Willkommlangea reticulata

ARCcin ARYden ARClei ARCpus CERfru CLAdeb CLApac COLarc COMaeq COMele COMlax COMnig CRAmin CRIcon CRIlan CRImic DIDeff DIDdif DIDiri ECHmin HEMcla HEMser LAMcol LAMsci LICbio LICflo LICkle LICoper LICpus METves PARery PARsol PERcor PERdep PERlic PHYnut PHYpus STEfus STEher TRIbot TRIper WILret

A O O R C A R A R A R C R A R A C O R O R O R O A O R O A R O C C O O O R O R R R O

Species diversityc Records made % Positive of moist cultures Species richness (S) Genus recorded (G) Species/genus ratio (S/G) Exponential Shannon (N1) Reciprocal of Simpson (N2) Evenness (J=) Commonness (Ninf) Species estimated ACE ICE Chao2 Estimated species % Sampling effortd

EF 14 1 3 1 1 26 3 25 1 29 0 13 0 61 1 15 4 2 0 3 0 1 1 5 11 0 0 1 42 0 0 10 5 0 4 0 0 1 0 0 1 0

EDF 40 7 3 1 2 10 0 43 0 101 1 12 0 64 1 19 5 2 1 6 0 3 0 2 22 9 1 4 39 0 6 23 3 1 1 0 2 10 2 0 0 4

DF 13 5 0 0 14 5 0 9 1 2 3 2 2 10 2 53 7 2 3 6 2 2 2 0 45 1 0 1 39 1 0 0 14 4 7 15 1 1 0 4 0 9

CF 4 0 0 0 5 6 2 8 0 14 0 2 0 11 1 19 5 3 1 0 0 5 2 7 3 0 0 0 5 0 0 0 8 3 3 0 0 0 0 0 0 0

285 84% 28 16 1.75 13.49 9.49 0.78 4.67

450 95% 33 17 1.94 14.45 9.51 0.76 4.46

287 94% 33 19 1.74 16.6 10.73 0.80 5.42

117 44% 21 11 1.91 16.11 12.95 0.91 6.16

31 38 31 35 80%

33 42 44 43 77%

34 37 36 37 89%

19 23 26 25 84%

aRelative abundance of records for a particular species (Stephenson et al. 1993). A, abundance (>3% of all records); C, common (1.5%–3%); O, occasional (0.5%–1.5%); R, rare ( 19).


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Fig. 2. Species accumulation curves of samples versus cumulated species numbers for the myxomycete assemblages associated with 4 vegetation types investigated. The site codes are explained in Table 1. The figures in parentheses are the estimated number of species recorded.

Ninf = 6.61; Table 2) indicated that corticolous species from the coniferous forest were the most even and common, with the corticolous myxomycetes from the mixed evergreen – deciduous broadleaf forest the most uneven (S = 33, J= = 0.76, and Ninf = 4.46; Table 2). Except for species richness (S) and Pielou’s evenness (J=) indices being statistically significant (P < 0.05); all other indices showed nonsignificant differences. Community analysis There were differences in the corticolous myxomycete assemblages of the 6 major taxonomic groups (Ceratiomyxales, Echinosteliales, Liceales, Trichiales, Physarales, and Stemonitales) related to the different vegetation types. Members of the orders Stemonitales, Trichiales, and Liceales were the majority of specimens obtained in this survey (Fig. 4). Interestingly, the order Physarales contained less than a quarter of all recorded corticolous myxomycetes from each vegetation type. For instance, only 2 species were represented in Physarales in the evergreen broadleaf forest. A comparison based on the CC index (Table 3) indicated that corticolous myxomycete assemblages showed a high similarity among the 4 sampling sites (means CC = 0.70, between 20 to 27 species shared; Table 3). This demonstrated a core assemblage shared by all vegetation types where corticolous myxomycetes were recorded (e.g., Cribraria confusa, Comatricha elegans, Hemitrichia serpula, Diderma effusum; Table 2). The results showed that different trends in the CC index and shared species were related to the same vegetation types (Table 3). For instance, the myxomycete biota in the deciduous broadleaf forest showed the lowest similarity with the mixed evergreen – deciduous broadleaf forest (CC = 0.48, Table 3). However, these 2 forest types showed the highest number of shared species (27, Table 3). The highest similarity occurred between mixed evergreen – deciduous broadleaf forest and evergreen broadleaf forest (CC = 0.87, Table 3). The corticolous myxomycetes obtained from moist chamber cultures using the bark samples collected from October 2011, October 2012, and May 2012 were compared using the CC index, without regard to the differences among sampling sites. The mean value for all combinations of the survey periods was CC = 0.81, sharing 20 common species (Table 4). The similarity index showed that autumn of 2011 and spring of 2012 were the most similar (CC = 0.86, Table 4), whereas autumn of 2011 and 2012 were the least similar (CC = 0.77, Table 4).


Site dynamics and seasonal patterns were calculated using cluster analysis (Figs. 5 and 6). In the dendrogram analysis (Fig. 5), the assemblages of corticolous myxomycetes were classified using a similarity index of 0.3 into 2 groups and then arranged by season. This analysis included data from coniferous forest taken in the spring of 2012. The assemblages in autumn of 2011 and 2012 aggregated into seasonal groups. The same pattern was found in the MDS (Fig. 6). Since the MDS stress was >0.1, cluster analysis results were superimposed onto the 2-multidimensional ordination. The 3 groups were separated according to the seasonal and annual pattern. Groups 1 and 2 clustered species were derived from 2012, while group 3 comprised species obtained from 2011 (Fig. 6). In addition, comparisons of species assemblage were obtained from a paired bark sample (one at 50 cm, one at 150 cm height), but there were no significant height differences in myxomycete community composition of all sites according to the ANOSIM analysis (P > 0.05, Table 5). The primary data of ANOSIM are shown in Supplementary Table S31. Analysis of the pooled data set for corticolous myxomycetes (27 species, excluding rare species) using CCA revealed the relationship between species assemblages and environmental variables (Fig. 7). The relative importance of the 5 environmental parameters (bark pH, bark water-holding capacity, elevation, height above the ground, and season) was recorded. Conditional automatic unrestricted Monte-Carlo permutation tests and randomizations indicated that myxomycetes were strongly influenced by pH, water-holding capacity, elevation, and season (P = 0.002 in all cases), while height above the ground was less significant (P = 0.01). The 4 extracted axes accounted for 42%, 35%, 13%, and 11% of species variance for environmental factors, respectively.

Discussion The transportation of myxomycete spores is reliant on the wind and animals, allowing spores to reach a suitable habitat and germinate (Kamono et al. 2009). Field surveys on myxomycetes have been undertaken using field collections and moist chamber cultures techniques. These approaches confirmed that favorable conditions (e.g., moisture, temperature, food) allow myxomycetes to colonize and complete their life cycle in natural and artificial habitats. This allows myxomycetes to occur in all terrestrial ecosystems. The knowledge of myxomycete assemblages and their association with a particular type of terrestrial ecosystems originated from studies undertaken in temperate forests (e.g., Stephenson et al. 2001). Recent studies have emphasized myxomycetes distribution and ecology in tropical forests (e.g., Lado et al. 2003; Tran et al. 2006; Wrigley de Basanta et al. 2008), with the available data indicating that species assemblages in the subtropics are different from those in temperate forests (Stephenson et al. 1993). Our study focused on the corticolous myxomycetes from 4 different vegetation types in the Tianmu Mountain National Nature Reserve, eastern China. The collection of bark samples was repeated seasonally during the 2-year study (autumn of 2011, spring and autumn of 2012). The number of species expected, based on an exhaustive sampling effort, originated by using the values generated from ICE, ACE, and Chao2 indices. However, the values with equal intensity (with the same number of prepared moist cultures) for all vegetation types indicated 83%–95% completeness, suggesting that sampling effort was insufficient for some vegetation types (e.g., mixed evergreen – deciduous broadleaf forest indicated only 77% completeness; Table 2). Some species were possibly missed because (i) of logistical problems associated with visiting the sampling sites throughout the year, the bark samples were not collected during late spring and summer; and (ii) some corticolous myxomycetes were sporadic or inconspicuous in their occurrence and Published by NRC Research Press

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Fig. 3. Statistical data were calculated for the 4 vegetation types. S, species recorded; N1, expectation of Shannon diversity index; N2, reciprocal of Simpson’s diversity index; J=, species evenness; Ninf, species dominance; S/G, species per genus ratio. Site codes are explained in Table 1. The asterisk (*) indicates a statistically significant relationship at P < 0.05.

Fig. 4. Pattern assemblages of corticolous myxomycetes of the 6 major taxonomic groups for the different vegetation types. The numbers next to the order name is the number of species recorded.

went unobserved to see using the moisture chamber culture technique. These sources of error should be addressed with future investigations. The N1 (exponential Shannon diversity index) and S/G (species per genus) indices were used to analyze the corticolous myxomycete diversity. The value of N1 index associated with the deciduous broadleaf forest was much higher compared with other vegetation types (N1 = 16.6, Table 2; Fig. 3). The S/G index indicated that corticolous myxomycetes in the deciduous broadleaf forest were more diverse than those associated with other vegetation types (S/G = 1.74, Table 2; Fig. 3), although there was no significant difference (P > 0.05, Fig. 3). The coniferous forest showed the

highest N2 value, followed by the deciduous broadleaf forest (N2 = 10.73, Table 2; Fig. 3). The S (recorded species), N2 (Reciprocal of Simpson), J= (Evenness), and Ninf (commonest) indices indicated that species from conifer forest were the most even. Ukkola and Rikkinen (2000) found that wet coniferous forests exhibited lower values of myxomycete richness and productivity in Oregon. In our study, the number of corticolous myxomycetes recorded was lowest in the coniferous forest (S = 21, P < 0.05, Table 2; Fig. 3). Importantly, the elevation of the deciduous broadleaf forest (1100 m) was greater than for other vegetation types (Fig. 1; Table 2) in our study. Our results indicated that the corticolous myxomycete assemblages were more diverse at higher altitude. However, Published by NRC Research Press

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Table 3. Comparison of myxomycete assemblages from 4 sampling sites. EF


EF EDF DF CF

EDF

DF

0.87 25 23 20

CF

0.57 0.48

27 20

0.77 0.77 0.76

Fig. 5. Dendrogram analysis showing the similarity coefficient of corticolous myxomycetes associated with different years and seasons based on the Bray–Curtis similarity matrix. 1 refers to the year 2011, 2 refers to 2012, S represents spring (May), A is autumn (October). The site abbreviations are explained in Table 1.

20

Note: Coefficient of community indices are presented in upper right, and the numbers of species shared in common are presented in the lower left. The site codes are explained in Table 1.

Table 4. Comparison of myxomycete assemblages from 3 periods (October 2011, October 2012, May 2012) excluding the sampling sites. Oct. 2011 Oct. 2011 May 2012 Oct. 2012

May 2012 0.86

20 20

Oct. 2012 0.77 0.80

20

Note: Coefficient of community indices are presented in the upper right, and numbers of species shared in common are presented in the lower left. The site codes are explained in Table 1.

Table 5. Results of one-way analysis of similarity (ANOSIM) (R values and significance levels) were for the height differences on the distribution of myxomycete species (one sample at 50 cm, the other at 150 cm above the ground, for each tree).

EF-50 vs. EF-150 EDF-50 vs. EDF-150 DF-50 vs. DF-150 CF-50 vs. CF-150

Sample statistic R

Significance level

−0.370 −0.556 −0.519 −0.481

P > 0.05a P > 0.05a P > 0.05a P > 0.05a

Note: The site codes are explained in Table 1. aNo significant differences.

Stephenson et al. (2004) found a negative association between myxomycete diversity and elevation (elevation from 1200 to 2700 m): increasing elevation resulted in decreasing diversity in the Maquipucuna Cloud Forest Reserve, southern America. This paradoxical result is perhaps due to the differences in the altitudinal range of the 2 surveys. Our study’s altitudinal range was 280–1100 m. The myxomycete spores at lower latitude with a lower incidence may be a result of their dispersal method by wind; branches and foliage could block spores from establishing in the forest location, compared with higher altitudes. It has been previously demonstrated that most myxomycete species do not randomly occur (e.g., Stephenson 1989; Everhart et al. 2008; Novozhilov and Schnittler 2008). In the present study, the occurrence of particular species was associated with certain environmental factors, such as substrate pH, water-holding capacity of the substrate, and height above the ground. Stephenson et al. (2004) noted that microhabitat parameters determine myxomycete distribution to a higher degree than do climate factors. Our results indicated that the corticolous myxomycete assemblages were based on the seasonal influence from environment features. In the CCA biplot, the left axis consisted of corticolous species that usually occurred in autumn (October) and the right axis consisted of species that usually occurred in spring (May) (Fig. 7). The assemblages of corticolous myxomycetes related to a seasonal and annual pattern were compared using cluster analyses (Figs. 5 and 6). The corticolous myxomycetes obtained from the same year were divided into one clade and then separated by a seasonal pattern (Fig. 5). The same pattern has

been reported in the Yassugatake Mountains of central Japan (Takahashi and Harakon 2012). Kamono et al. (2009) found that myxomycete compositions were characterized by a seasonal pattern using molecular methods. In addition, myxomycete occurrence in the spruce forests seemed to follow a strict seasonality from Xinjiang Province of western China (Schnittler et al. 2012). It was probably because corticolous myxomycetes have different development time. The optimum temperature for most myxomycete spores to germinate is 22–30 °C and wet conditions (Stephenson 2011). Under unfavorable conditions (e.g., reduced temperature, desiccation, starvation), plasmodium may change into the sclerotium or microcyst stage, which can quickly revert to the active stage upon the return of favorable conditions. All of these resistant forms can remain in a dormant stage for months to years (≤61 years), depending on the species (Martin and Alexopoulos 1969). The fruit of some species emerge early in the spring and cease sporulation by the middle of summer; others begin in the summer and continue until fall. In the present study, the period from May to October saw corticolous myxomycetes experience a hot and wet summer, where most species could germinate to achieve their life cycle. However, from October to May of the next year, the climate was cold and dry (relatively unfavorable conditions) where some of the species survived in a resistant form (microcyst or sclerotium). No new spores or microcysts germinated during this time. In Table 4, the results indicated that corticolous myxomycete assemblages obtained during autumn of 2011 were remarkably similar to the species from spring of 2012 (CC = 0.86, Table 4), and more dissimilar to those from autumn of 2012 (CC = 0.77, Table 4). In summary, the season was a major factor determining assemblages of corticolous myxomycetes, and species distributed by a seasonal pattern. There were no significant differences in species diversity indices associated with the 4 vegetation types. However, the corticolous myxomycete assemblages were significantly different with the order of dominance being Liceales, Stemonitales, and Trichiales (Fig. 4). In previous studies, Physarales was the dominant order among species. Stephenson (1989) indicated that Stemonitales developed under more acidic conditions compared with Physarales and Trichiales. Estrada-Torres et al. (2009) suggested that Physarales was probably closely associated with high pH (ranging from 7.5 to 10). Novozhilov et al. (2006) reported members of the families Trichiaceae and Cribrariaceae mostly occupied substrate with low pH (usually 3.5–5.5) in the arid regions of the Lower Volga River Basin. The bark pH appears to be an important factor in determinPublished by NRC Research Press

Liu et al.

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Fig. 6. Ordination in 2 dimensions using multidimensional scaling based on the Bray–Curtis similarity matrix. Groups 1 and 2 clustered species were derived from 2012, while group 3 comprised species obtained from 2011. The site codes are explained in Table 1 and caption of Fig. 5.

Fig. 7. Biplot of the canonical correspondence analysis revealed the relationship between 27 common species (>0.5% of all records) and environmental parameters (arrows indicate factors for bark pH, bark water-holding capacity, elevation, height above the ground, and season). The asterisk (*) indicates a statistically significant relationship at P = 0.002. The double asterisk (**) indicates a statistically significant relationship at P = 0.01. Acronyms for species names are defined in Table 2.



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ing the distribution of particular myxomycete species. However, many myxomycete species appear to have a relatively wide pH tolerance (Everhart et al. 2008), but this is not the case for all species. For instance, some species tolerate acidic substrates (acidophilic species), whereas others never develop under low pH condition. We found that the pH of all the bark samples were low (mean value of pH = 5.64 ± 0.89, see Supplementary Table S21) in the present study. However, some myxomycete species (members from Physarales) were rare or occurred infrequently (Table 2). Species in the Physarales order occurred most often when the substrate had a high pH (Fig. 7). For instance, Didymium effusum (DIDeff), Didymium difforme (DIDdif), and Physarum nutans (PHYnut) supported this trend (Fig. 7). It seemed that some changes in the environment (or climate) resulted in a low bark pH value, affecting the corticolous myxomycete distribution in this area. This suggested that myxomycete assemblages could be used as relatively sensitive biological indicators corresponding to some environmental changes. Various studies have examined the relationship between myxomycete assemblages and bark water-holding capacity. Snell and Keller (2003) found no significant differences in myxomycete species abundance and the bark water-holding capacity. However, Stephenson (1989) and Schnittler et al. (2006) found that the water-holding capacity of bark was positively related with myxomycete occurrence. These conflicting results probably occurred from the varying amount of epiphyte cover on the host substrate or differences in sampling methodologies. In the CCA biplot, corticolous myxomycetes were best explained by the bark waterholding capacity with the relationship being strongly significant (P = 0.002). Interestingly, bark water-holding capacity was inversely related to the pH and elevation (Fig. 7). For example, Perichaena depressa (PERdep) and Ceratiomyxa fruticulosa (CERfru) inhabited higher altitudinal environments on dryer substrates with higher pH. In contrast, Cribraria confusa (CRIcon) preferred a relatively moister and lower altitude with a low pH condition. Spore production is a single most important mechanism for myxomycetes dispersal, largely by wind (Martin and Alexopoulos 1969). Rojas and Stephenson (2007) found that myxomycete spores had the ability to reach a considerable height (>1 km) when transported by wind, and species diversity was higher at lower altitudinal locations. Perhaps, in forests, myxomycete spores are hindered by leaves and branches when they fall, thus myxomycete assemblages are apparently related to the height above the ground. We found that while myxomycete distribution was significantly influenced by the height above the ground (P = 0.01, Fig. 7), other environmental variables, such as season, bark pH, waterholding capacity of bark, had a stronger association (P = 0.002, Fig. 7). However, the significant difference in the height above the ground occurred on a relatively small scale, with the maximum difference being 1 m in our study. Future studies should investigate a higher location of myxomycete assemblages on the trees. The traditional view suggests that myxomycetes occur mainly in humid, warm environments. However, Stephenson (1989) found that myxomycete assemblages appear more diverse in temperate forests than in tropical or subtropical forests. Furthermore, Stephenson (2011) indicated that the corticolous myxomycete biodiversity was much lower in tropical forests than most trees in temperate forests. We recorded 42 species of corticolous myxomycetes in this region, which is less compared with those in temperate forests (Stephenson 1989; Ndiritu et al. 2009). Many reasons may account for the difference in abundance for this region: (i) the pH of bark samples was strongly acidic (pH = 5.64 ± 0.89, see Supplementary Table S21), with some myxomycetes unable to tolerate these acidic conditions; (ii) Schnittler and Stephenson (2000) and Stephenson et al. (2004) noted that the higher humidity, which is caused by rainfall, may lead to the lower frequency and diversity of corticolous myxomycetes. These regions see daily torrential rainfall in summer (or early summer), which potentially washed most corti-


colous myxomycete spores off the bark, and promote conditions ideal for filamentous fungi that contaminated the corticolous myxomycetes. We analyzed the corticolous myxomycete assemblages associated with 4 vegetation types from a subtropical region of China and compared the data with those from other terrestrial ecosystems. The predominant species representing the orders of Liceales, Stemonitales, and Trichiales in this area were different from previous studies. In addition, the corticolous myxomycetes from the deciduous broadleaf forest were the most diverse, and those from the coniferous forest were the most common and most evenly distributed. The results indicated that the assemblages of corticolous myxomycetes were distributed by seasonal and annual pattern. Our findings were consistent with previous studies showing bark pH was an important factor influencing corticolous myxomycete assemblages. Thus, the change of circumstances assumes that myxomycetes alter their distribution pattern according to environmental restrictions.

Acknowledgements We gratefully thank Tian-Peng Song, Ming-Quan Guo, Bin Wei, Wei Tao, and Qian Li for their input and assistance during the fieldwork. We appreciate Dr. Xiang-Ming Tang (Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, China) for helping us use the software. This study was supported a fund from the National Nature Science Foundation of China (31170014) and a fund from the Key Program of Natural Science of Jiangsu Higher Education Institutions of China (12KJA180004).

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