Research Article |
Corresponding author: Yves Basset ( bassety@si.edu ) Academic editor: Carolina de Castilho
© 2019 Raphaële Solé, Sofia Gripenberg, Owen T. Lewis, Lars Markesteijn, Héctor Barrios, Tom Ratz, Richard Ctvrtecka, Philip T. Butterill, Simon T. Segar, Mark A. Metz, Chris Dahl, Marleny Rivera, Karina Viquez, Wessley Ferguson, Maikol Guevara, Yves Basset.
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation:
Solé R, Gripenberg S, Lewis OT, Markesteijn L, Barrios H, Ratz T, Ctvrtecka R, Butterill PT, Segar ST, Metz MA, Dahl C, Rivera M, Viquez K, Ferguson W, Guevara M, Basset Y (2019) The role of herbivorous insects and pathogens in the regeneration dynamics of Guazuma ulmifolia in Panama. Nature Conservation 32: 81-101. https://doi.org/10.3897/natureconservation.32.30108
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A significant proportion of the mortality of rainforest trees occurs during early life stages (seeds and seedlings), but mortality agents are often elusive. Our study investigated the role of herbivorous insects and pathogens in the early regeneration dynamics of Guazuma ulmifolia (Malvaceae), an important tree species in agroforestry in Central America. We reared pre-dispersal insect seed predators from G. ulmifolia seeds in Panama. We also carried out an experiment, controlling insects and pathogens using insecticide and/or fungicide treatments, as well as seed density, and compared survivorship of G. ulmifolia seeds and seedlings among treatments and relative to untreated control plots. We observed (1) high pre-dispersal attack (92%) of the fruits of G. ulmifolia, mostly by anobiine and bruchine beetles; (2) negligible post-dispersal attack of isolated seeds by insects and pathogens; (3) slow growth and high mortality (> 95%) of seedlings after 14 weeks; (4) low insect damage on seedlings; and (5) a strong positive correlation between seedling mortality and rainfall. We conclude that for G. ulmifolia at our study site the pre-dispersal seed stage is by far the most sensitive stage to insects and that their influence on seedling mortality appears to be slight as compared to that of inclement weather. Thus, the regeneration of this important tree species may depend on effective primary dispersal of seeds by vertebrates (before most of the seed crop is lost to insects), conditioned by suitable conditions in which the seedlings can grow.
Anobiidae , fungal pathogen, negative density-dependence, rainforest, seed predation
In tropical rainforests a significant proportion of tree mortality occurs during the early life stages of the plant (seeds and seedlings), and patterns of mortality at these early stages of life may be critical in influencing the diversity of older individuals (
Insects, more than herbivorous mammals, are responsible for most herbivory on seedlings in tropical rainforests (Cruz and Dirzo 1987). In addition, many insects, but very few mammals, preferentially attack seeds or plants near conspecific adults (
Seedling establishment and survival in tropical rainforests may also be influenced by pathogens, especially fungal pathogens (
One way to assess the effect of insect herbivores or fungal pathogens on rainforest seeds or seedlings is to exclude them with (a) insecticide and/or fungicide or (b) mesh exclosures in treatment plots and compare survivorship of seeds or seedlings between treatment plots and control plots (
Several studies have used insecticide and fungicide applications to assess the role of insects and pathogens in maintaining plant diversity and causing conspecific negative density-dependent plant survival as predicted by the Janzen-Connell hypothesis (e.g.
The main objectives of this study were to investigate the impacts of insect herbivores and fungal pathogens on the early regeneration of a tree species of great importance in agroforestry in Central America and Mexico, Guazuma ulmifolia Lam. (
Our protocol involved assessing both the damage on seeds caused by insect predators and the potential joint effects of insect herbivores and fungal pathogens on seed germination and the survival of young seedlings. We then evaluated (1) which stage (seed, seed-to-seedling transition, seedling) is particularly vulnerable to insect herbivores and pathogens; (2) whether the negative impact of insect herbivores at the seedling stage is higher than that of fungal pathogens; and (3) whether seed or seedling density affects the action of insect herbivores and fungal pathogens, as predicted by the Janzen-Connell hypothesis (
Insect seed predators were reared from Guazuma ulmifolia fruits collected on Barro Colorado Island (BCI) and at a nearby site, El Charco (ELC). As the use of insecticide is not allowed on BCI, our experiment assessing the role of insects and pathogens at the seed-to-seedling transition stage was conducted at ELC. Barro Colorado island is a biological reserve (9.15N, 79.85W, elevation 120–160 m) of 1,500 ha covered by lowland rainforest and created by the opening of the Panama Canal in 1914. A permanent ForestGEO vegetation plot (
Guazuma ulmifolia (Lam. 1789, Malvaceae, “Guácimo” in Panama) was selected for this study because of the relatively high availability of seeds at the study sites. It is also an important tree species in agroforestry, being used for firewood, medical treatment, the production of living fences and rehabilitation of degraded soils by fostering forest regeneration (
Of the seven species in the Guazuma genus, G. ulmifolia is the only one to occur in Panama (
Individual Guazuma trees can produce up to 5,000–10,000 fruits per crop. The capsule of the fruit is elliptical or hemispherical, measuring between 18–25mm long, and 14–22mm wide. When mature, it is black (Suppl. material
For rearing insects we collected 320 fruits freshly fallen (without apparent decomposition) from G. ulmifolia on BCI. They were obtained between 2011–2013 from 22 trees (average 14.5 fruits collected per tree). To assess pre-dispersal attack (i.e., while seeds are still on trees) of G. ulmifolia fruits, in 2016 we collected 100 fruits directly from the crown of 5 trees near the 1ha plot in ELC (20 fruits collected per tree). In both cases fruits were stored in individual plastic pots, lined with tissue paper and covered with 1 mm mesh netting for ventilation and to avoid subsequent colonization/contamination of fruits. Rearing pots were checked twice weekly, and any emerging insects were collected, preserved, mounted and then identified with the assistance of taxonomists (see Acknowledgements) and/or with molecular techniques (
Ten experimental blocks (B01 to B10) were set up near the permanent plot at ELC (coordinates in Suppl. material
We used the insecticide ENGEO 247 SC (Syngenta SA, Basel, Switzerland) with active ingredients thiamethoxam (a neonicotinoid) 141 g L-1 and lambda-cyhalothrin (a pyrethroid) 106 g L-1. According to the manufacturer, thiamethoxam has root-, leaf- and stem-systemic activity, while Lambda-cyhalothrin has a strong contact action. We used 0.0025ml m-2 in 50 ml sprayed on each quadrat every week, as recommended by the manufacturer and used in previous studies (e.g.
In total, 1,400 fresh seeds of G. ulmifolia, collected from different trees near ELC, were sown into the experimental blocks in May 2016 at the onset of the rainy season. Prior to sowing, capsules were opened and seeds were checked for insect damage. Only apparently intact seeds were sown. To mimic near natural conditions, seeds were not treated to increase germination rates (
We first ensured that there was no or little spatial pseudoreplication in our data by running simple Mantel tests (1,000 randomizations) between the dependent variables (see below) and the coordinates of the blocks. There was no obvious spatial autocorrelation for any of the response variables: germinated seeds: Mantel’s t = 0.092, p = 0.303; dead seedlings: t = 0.117, p = 0.248; damaged seedlings: t = -0.079, p = 0.654. We used general linear models (GLM) to test the effect of treatment (Control, Fungicide, Insecticide and Insecticide + Fungicide), seedling density (10 or 25 seeds) and the interaction between these two fixed effects on the three dependent variables, at the end of the 14 monitoring weeks: number and percentage of germinated seeds, seedling mortality (number and percentage of dead seedlings) and number and percentage of seedlings damaged. All statistical analyses were conducted using R v 3.3.3 (
The insects reared from the fruits of G. ulmifolia are summarized in Table
Summary of insects reared (total number of individuals per species) from fruits of G. ulmifolia at BCI (320 fruits) and ELC (100 fruits). Barcode Index Number (BIN) indicated when available and is linked to available insect pictures. Host specificity was inferred from a larger study on insect seed predators on BCI (
Taxa | BIN | BCI | ELC | Notes |
---|---|---|---|---|
Coleoptera | ||||
Chrysomelidae – Bruchinae | ||||
Amblycerus cistelinus (Gyllenhal, 1833) | BOLD:ACG0463 | 72 | 0 | Seed predator, host specific on G. ulmifolia |
Amblycerus sp. bru39SG | BOLD:ACJ3956 | 39 | 3 | Seed predator, host specific on G. ulmifolia |
Amblycerus sp. bru30SG | BOLD:ABW8381 | 15 | 0 | Seed predator, host specific on G. ulmifolia |
Mimosestes sp. 1YB | – | 0 | 1 | Seed predator |
Ptinidae – Anobiinae | ||||
Lasioderma sp. ano2SG | BOLD:ACF0770 | 242 | 291 | Seed predator, host specific on G. ulmifolia |
Lasioderma sp. ano4SG | – | 1 | 0 | Seed predator |
Bostrichidae | ||||
Lyctus sp. | – | 0 | 2 | Wood borer |
Silvanidae | ||||
Ahasverus advena (Waltl, 1832) | – | 1 | 0 | Fungal-feeder, cosmopolitan pest of stored products |
Cathartus sp. 1SG | – | 115 | 0 | Probably fungal-feeder |
Unidentified ELC sp. 1 | – | 0 | 1 | Probably fungal-feeder |
Unidentified ELC sp. 2 | – | 0 | 1 | Probably fungal-feeder |
Cucujidae | ||||
Unidentified – cuj1SG | – | 63 | 0 | Ecology unknown |
Nitidulidae | ||||
Stelidota sp. 5SG | – | 2 | 0 | Sapromycetophagous?, generalist, reared from several hosts |
Unidentified larva | BOLD:ACL7065 | 29 | 0 | Sapromycetophagous?, may be larvae of Stelidota sp. 5SG |
Stelidota sp. 6SG | – | 0 | 1 | Sapromycetophagous?, generalist, reared from several hosts |
Curculionidae – Scolytinae | ||||
Xyleborus sp. 1YB | – | 0 | 1 | Wood borer |
Corylophidae | ||||
Unidentified Corylophidae | – | 0 | 11 | Fungal-feeder |
Lepidoptera | ||||
Autostichidae | ||||
Deoclona xanthoselene (Walsingham, 1911) | BOLD:ACF0463 | 51 | 0 | Seed predator, generalist, reared from several hosts |
Blastobasidae | ||||
Blas lep37SG | BOLD:ABV2151 | 2 | 1 | Scavenger, generalist, reared from several hosts |
Blas lep31SG | BOLD:ABV2158 | 1 | 2 | Scavenger, generalist, reared from several hosts |
Hymenoptera | ||||
Braconidae | ||||
Unidentified Braconidae | – | 0 | 1 | Parasitoid |
Pteromalidae | ||||
Unidentified Pteromalidae | – | 1 | 7 | Parasitoid |
TOTAL | 634 | 323 |
After 14 weeks of monitoring, 36.1% of the 1,400 sown seeds had germinated (Suppl. material
Overall, seedling mortality was very high, and in total 95.7% of germinated seeds were dead by Week 14 (Suppl. material
Average percentage of dead seedlings per block, detailed by treatment (C, I, F, I+F, coded as in Fig.
Number of seedlings growing plotted against time for each treatment (coded as in Fig.
The highest seedling mortality was in weeks 4 and 5 (weeks of the 27 May and 3 June 2016), with a total of 77 and 86 dead seedlings, respectively. During week 4, only one week after the highest rate of seed germination (Fig.
After 14 weeks of monitoring, only 24 seedlings were alive, including 9 damaged seedlings, spread only over 4 blocks. The rational models estimated that after one year of monitoring, a total of 9 seedlings for the whole experiment would still be alive. This was not the case: the last two seedlings died on 5 May 2017, one year after the beginning of the experiment, in block 07, quadrat I+F25. The growth and biomass of surviving seedlings in week 14 were also rather slow and low. They measured on average 30.0 ± 1.91 mm and the two last seedlings to die were 125 and 140 mm tall. Since the sample size of surviving seedlings in week 14 was low, we performed a simple ANOVA that indicated that neither treatment (F3,1= 0.896, p = 0.48) nor seed density (F3,1= 0.713, p = 0.42) had any effect on seedling height.
Overall, 100 out of 506 seedlings that germinated were damaged (19.8%, Suppl. material
Regarding this question, we observed that: (a) pre-dispersal attack of the fruits of Guazuma ulmifolia is very high (up to 92% of fruits attacked) and the result of the action of several species of insect seed predators, mainly anobiine and bruchine beetles (Lasioderma spp. and Amblycerus spp.). (b) In Panama, and contrary to what was observed in Costa Rica (
Because of very high pre-dispersal attack of G. ulmifolia fruits at ELC, the number of viable seeds that can germinate on the ground is probably considerably reduced.
In contrast, we believe that post-dispersal attack of G. ulmifolia seeds by insects is infrequent. Amblycerus spp. do not oviposit on fallen fruits even if there are many on the ground (
In relation to this question we further observed that: (a) seedling survival over time was weakly and positively influenced by the fungicide treatment. (b) There was a strong positive correlation between weekly cumulative seedling mortality and cumulative rainfall. (c) About 20% of seeds that germinated were damaged, mostly by leaf-chewing insects, but neither the treatments nor seed density affected the number of damaged seedlings. (d) Insect herbivores were not observed feeding on seedlings during day-time. (e) Seedling growth was slow (3cm tall after 14 weeks), and all 506 seedlings that germinated out of 1,400 seeds sown were dead after one year.
Leaves of G. ulmifolia on mature trees are readily attacked by a variety of insect herbivores (
Apart from a weak positive effect of fungicide treatment on seedling demography (survival in time; as reported in
July 2016 should have been a period of active growth for G. ulmifolia seedlings that germinated in May 2016. July 2016 was the second rainiest July (monthly sum 486.8mm) on BCI since records began in 1925, only surpassed by July 1956 (496.6mm; http://biogeodb.stri.si.edu/physical_monitoring/research/barrocolorado). In these conditions, high rainfall may have caused soil anoxia and some rot in seeds and seedlings, as suggested by the strong correlation between cumulative rainfall and seedling mortality during the experiment. Further, G. ulmifolia seedlings have a relatively low tolerance to flooding (
For the densities considered in this study, post-dispersal seed and seedling density did not greatly affect the negative action of insect herbivores and fungal pathogens. The lack of difference in seedling survival between control quadrats sown at two different seed densities also suggests that intra-specific competition of seedlings is not responsible for their high mortality (
Since post-dispersal attack of G. ulmifolia seeds and seedlings by insects at ELC was infrequent, this suggests that Janzen-Connell effects due to insect herbivores specifically, if they exist, may be rather weak for this tree species.
Since Amblycerus spp. only oviposit in fruits on trees (
First, as indicated by further monitoring of blocks after 14 weeks, seeds were unlikely to germinate after the end of our experiment. This suggests that the length of the study and the germination rate of seeds were appropriate for our experiment (and see
Second, the overall germination rate of G. ulmifolia seeds (36%) was higher at ELC than that reported in the literature for Panama (natural conditions, 20%:
Third, an unknown proportion of seeds was also probably lost in our experiment before germination. We observed damage due to vertebrates crossing the blocks (i.e., excavated soil, seed labels chewed), most likely caused by agoutis, Dasyprocta punctata, and coatis, Nasua narica. We also suspect that ants, particularly Atta spp., removed some of the seeds used in the experiment. In Colombia, several ant species, such as Atta cephalotes, are known to remove preferentially in open pastures small tree seeds including G. ulmifolia (
Our factorial design insured that all treatment groups were exposed to the same experimental conditions. All experimental blocks were thus likely to be affected in the same way by additional environmental factors (such as light conditions, seed removal by vertebrates and rainfall) which were not controlled in our study.
Our study generated new information on the role of natural enemies in the survival of Guazuma ulmifolia at the early stages of the life cycle. We conclude that the pre-dispersal seed stage is by far the stage most vulnerable to insect enemies. Also that the main agent of seedling mortality during the experiment was probably inclement rainfall, and that Janzen-Connell effects that may be specifically due to insect herbivores are probably rather weak for this tree species. The regeneration of G. ulmifolia in the forest of ELC appears to be difficult to account for but not to be the outcome of the action of insect herbivores after seeds are dispersed. This illustrates that each tree species may respond differently to pathogens and herbivores (
What are the implications of our findings for the regeneration of this important tree species in agroforestry? Without addressing what may be suitable conditions for seedling growth, the regeneration of this species may further depend on effective primary dispersal of seeds by vertebrates before most of the seed crop is lost to insects. In this situation, local conservation of dispersal agents may be crucial to enhance escape of seed predation by insects. For example, artificial roosters for frugivorous bats, which disperse seeds of G. ulmifolia (
We thank the Smithsonian Tropical Research Institute in Panama for logistical support. D. Catalina Fernandez, Indira Simon Chaves and Marjorie Cedeño collected most of the insect material on BCI, which was identified by H.B., John Heppner, Josef Jelinek, M.M., Geoffrey Morse, M.R. and Henry Stockwell. Stephany Arizala helped with fieldwork. This work was supported by the Czech Science Foundation [16-20825S to Y.B.]. Field work on BCI was largely funded by a postdoctoral grant from the Academy of Finland to S.G. Grants from the Smithsonian Institution Barcoding Opportunity FY013 and FY014 (to Y.B.) and in-kind help from the Canadian Centre for DNA Barcoding allowed sequencing of insect specimens. Y.B. and H.B. are members of the Sistema Nacional de Investigación, SENACYT, Panama. S.G. holds a Royal Society University Research Fellowship. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA; USDA is an equal opportunity provider and employer.
Supplementary tables S1-S4, Appendix S1, Plate S1