Characterization of ciliate diversity in bromeliad tank waters from the Brazilian Atlantic Forest

Characterization of ciliate diversity in bromeliad tank waters from the Brazilian Atlantic Forest

Accepted Manuscript Title: Characterization of ciliate diversity in bromeliad tank waters from the Brazilian Atlantic Forest Authors: Taiz L.L. Sim˜ao...

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Accepted Manuscript Title: Characterization of ciliate diversity in bromeliad tank waters from the Brazilian Atlantic Forest Authors: Taiz L.L. Sim˜ao, Adriana Giongo Borges, Kelsey A. Gano, Austin G. Davis-Richardson, Christopher T. Brown, Jennie R. Fagen, Eric W. Triplett, Raquel Dias, Claudio A. Mondin, Renata M. da Silva, Eduardo Eizirik, Laura R.P. Utz PII: DOI: Reference:

S0932-4739(17)30024-X EJOP 25506

To appear in: Please cite this article as: Sim˜ao, Taiz L.L., Borges, Adriana Giongo, Gano, Kelsey A., Davis-Richardson, Austin G., Brown, Christopher T., Fagen, Jennie R., Triplett, Eric W., Dias, Raquel, Mondin, Claudio A., da Silva, Renata M., Eizirik, Eduardo, Utz, Laura R.P., Characterization of ciliate diversity in bromeliad tank waters from the Brazilian Atlantic Forest.European Journal of Protistology This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Characterization of ciliate diversity in bromeliad tank waters from the Brazilian Atlantic Forest Taiz L. L. Simãoa, Adriana Giongo Borgesb, Kelsey A. Ganoc, Austin G. DavisRichardsonc, Christopher T. Brownc, Jennie R. Fagenc, Eric W. Triplettc, Raquel Diasd, Claudio A. Mondina, Renata M. da Silvaa; Eduardo Eizirika, Laura R. P. Utza,*


Faculdade de Biociências, PUCRS, Porto Alegre, RS, Brazil


Instituto do Petróleo e dos Recursos Naturais, PUCRS, Porto Alegre, RS, Brazil


Department of Microbiology and Cell Science, University of Florida, FL, USA


Faculdade de Informática, PUCRS, Porto Alegre, RS, Brazil

*Corresponding Author. E-mail address: [email protected] (L.R.P. Utz)

Abstract Bromeliads are a diverse group of plants that includes many species whose individuals are capable of retaining water, forming habitats called phytotelmata. These habitats harbor a diversity of organisms including prokaryotes, unicellular eukaryotes, metazoans, and fungi. Among single-celled eukaryotic organisms, ciliates are generally the most abundant. In the present study, we used Illumina DNA sequencing to survey the eukaryotic communities, especially ciliates, inhabiting the tanks of the bromeliads Aechmea gamosepala and Vriesea platynema in the Atlantic Forest of southern Brazil. Filtered sequences were clustered into distinct OTUs using a 99% identity threshold, and then assigned to phylum and genus using a BLAST-based approach (implemented in QIIME) and the SILVA reference database. Both bromeliad species harbored very diverse eukaryotic communities, with Arthropoda and Ciliophora showing the highest abundance (as estimated by the number of sequence reads). The ciliate genus Tetrahymena was the most abundant among single-celled organisms, followed by apicomplexan gregarines and

the ciliate genus Glaucoma. Another interesting finding was the presence and high abundance of Trypanosoma in these bromeliad tanks, demonstrating their occurrence in this type of environment. The results presented here demonstrate a hidden diversity of eukaryotes in bromeliad tank waters, opening up new avenues for their in-depth characterization.

Keywords: Bromeliads; Ciliates; Metabarcoding

Introduction Bromeliads are a diverse family of plants with rock-dwelling, terrestrial or epiphytic habits, featuring its greatest center of genetic diversity in eastern Brazil, especially in the Atlantic forest (Givnish et al., 2011). Epiphytic bromeliads are intriguing because they have foliar uptake trichomes that foster the absorption of water and nutrients, with the roots, when present, acting mainly as a support organ (Givnish et al., 2011). The genera Aechmea and Vriesea have a special structure formed by foliage arranged in a compact rosette capable of retaining water (phytotelm) that contributes to the energy supply, since the water favors the decomposition of organic matter (microorganisms, leaves, flowers, seeds, small animals, etc.) deposited there (Martinelli et al., 2008). The presence of a specific biota inside bromeliad tanks was first pointed out by Picado (1913) in his work on bromeliads from Costa Rica. Since then, several studies have recorded the presence of metazoans such as rotifers, nematodes, ostracods, and insect larvae in bromeliad phytotelmata (see Kitching 2000 for a review). Regarding unicellular eukaryotes, few studies have included these organisms in their analyses (e.g.; Buosi et al., 2014; Dunthorn et al., 2012; Foissner et al., 2003; Maguire, 1971). The first comprehensive study on ciliates from bromeliad tanks was conducted by Foissner and collaborators (2003) and focused on the morphology of specimens found in bromeliads from Northeastern Brazil, the Dominican Republic, and Ecuador. Subsequent studies (e.g. Foissner 2003a, Foissner 2003b) dealt with the description of new genera and new species of ciliates found in bromeliad tank waters. A total of three new genera and 40 new species of ciliates have been described until now. Dunthorn and co-workers (2012) performed a comprehensive

study on ciliates from bromeliad tanks based on molecular analyses of cultivated organisms. Their results revealed a high diversity of ciliates, including many undescribed species. Given that previous studies of bromeliad tank ciliates relied on morphological and/or molecular analyses of cultivated organisms, it is likely that the actual diversity present in these habitats is even higher, since not all taxa are expected to be successfully cultured. An alternative approach to survey such diversity is to apply DNA metabarcoding, which has become a standard reference for the identification and classification of all groups of organisms by providing short sequences that can be compared to reference databases (Handley, 2015). Morevover, this approach can reveal low-abundance taxa, since their DNA can be detected even at low concentrations. Here we have applied a DNA metabarcoding approach to characterize the diversity of microbial eukaryotic communities, especially ciliates, from the phytotelmata of two bromeliad species inhabiting a preserved site within the Atlantic Forest of southern Brazil. This is the first study that analyzes the diversity of the microeukaryotic community of bromeliad tank waters using highthroughput DNA sequencing.

Material and Methods Water sample collection We collected water samples from the tanks of bromeliads belonging to two species (Aechmea gamosepala and Vriesea platynema; Figure 1A) seasonally, from January to October 2010, at the Pró-Mata Center for Research and Nature Conservation. This area owned by the Pontifical Catholic University of Rio Grande do Sul, located in São Francisco de Paula municipality, southern Brazil (29o27'- 29o35'S; 50o08' - 50o15'W), comprises approximately 3,100 ha and is located about 900m above sea level. We sampled two epiphytic individuals of each bromeliad species (identified as A1 and A5 for A. gamosepala and V1 and V5 for V. platynema). We sampled two pairs of individuals, each comprising one individual of each species located <100m from each other (Figure 1B). The distance between pair A1/V1 and pair A5/V5 was approximately 450m along a steep forest trail. At each time point, we collected 2 ml of whole water from each individual using a sterile Pasteur pipette. Immediately after collection, we added 2 ml of

the lysis buffer TES (100 mM Tris, 100 mM EDTA, 2% SDS) to each sample, and stored it at -20ºC.

DNA extraction and high throughput DNA sequencing Total DNA was extracted from 250μL of water from each bromeliad using the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA, USA). The blood protocol was slightly adjusted for use with bromeliad water, considering that it was already diluted 1:1 in a lysis buffer, and employing the maximal volume permitted by the manufacturer’s instructions. Partial 18S rRNA gene sequences were amplified using primers Fw and Rv (Nolte et al., 2010) with the addition of a barcoded sequence and the required Illumina sequencing adapters (Fagen et al., 2012). PCR conditions consisted of one initial denaturation step at 94°C for 4 min, 30 cycles including denaturation for 45 s at 94°C, annealing for 30 s at 50°C, and extension for 1 min at 72°C, with one final extension step for 7 min at 72°C. PCR products were purified using the Qiagen PCR purification kit, following the manufacturer’s protocol with the exception of a final elution in sterile water. Sequencing was conducted on an Illumina HiSeq 1000 machine (Illumina Inc., CA, USA) with two 101 base-pair, paired-read cycles. Since all samples were sequenced in a multiplexed Illumina run, barcode sequences were used to extract data from each sample from the total sequencing output. The sequences obtained were first filtered for quality (phred >20) and the primer region was removed using Prinseq software (Schmieder & Edwards 2011). The fastqjoin command implemented in USEARCH 8 (Edgar 2010) was used to join the sequences and to add a gap between them, consisting of 8 unknown bases (called ‘N’). The 8 unknown bases were then replaced by 88 unknown bases with the purpose of approximating the actual fragment size. A total of 110,029 sequences, representing a per-sample average of 8,304 reads for A. gamosepala and 13,701 reads for V. platynema, were then submitted to dereplication and removal of singletons using USEARCH. We also used USEARCH to cluster filtered reads into Operational Taxonomic Units (OTUs), which are roughly equivalent to specieslevel taxa, by applying a 99% identity threshold relative to a centroid sequence. We then assigned the resulting OTUs to phyla and genera using a BLAST-based method

implemented in QIIME (Caporaso et al., 2010), employing the SILVA version 111 database ( as the reference. Bar graphs depicting the number of sequence reads per taxon in each sample were built using de ggplot2 library implemented in R (R Development Core Team, 2015).

Results and Discussion The eukaryotic community in the bromeliad tank waters sampled here exhibited very high diversity, with a total of 33 different identified phyla (Figure 2). Among these phyla, Ciliophora was the second most abundant, reaching 30% of the DNA sequence reads in one individual of V. platynema collected during the winter (Figure 2). The other eukaryotic phyla among the top-20 during the study period included Arthropoda, Annelida, and Apicomplexa. Goffredi et al. (2015) had found a similar result for bromeliads from Costa Rica. In their study, metatranscriptome analyses revealed a high abundance of annelids and insect larvae. Morphological studies focusing on the invertebrate community present in bromeliad waters had also pointed out the dominance of arthropods and annelids (e.g. Picado, 1913; Richardson, 1999; Zots and Traunspurger, 2016). The high frequency of annelids and arthropods reported in bromeliad tank waters could explain the high abundance of apicomplexans found in the present study, since apicomplexans are unicellular endosymbionts and are generally found in association with aquatic oligochaetes and arthropods (Hausmann et al., 2003). A highly frequent group of unclassified eukaryotes was also observed among the 20 most abundant eukaryotic groups (Figure 2). This indicates that an important portion of the eukaryotes that inhabit bromeliad phytotelmata is still unknown and/or unrepresented in DNA sequence databases. Within phylum Ciliophora, nine different classes were detected in the sampled bromeliads, with class Oligohymenophorea having the largest number of genera and OTUs (Figure 3). Dunthorn and collaborators (2012) detected seven ciliate classes in their analyses of bromeliad phytotelmata biota, based on cultured organisms. Ciliate classes Plagiopylea and Prostomatea were not found in their study, but were identified at our study site using the DNA metabarcoding approach. The former was represented by the genus Trimyema, while the latter was represented by the genus Coleps, a very common organism in freshwaters. Future analyses should address the issue of whether these classes are more

difficult to detect using a culture approach, relative to DNA metabarcoding, or whether their presence is indeed variable at different study sites. The two remaining ciliate classes, Karyorelictea, (which is predominantely marine with genus Loxodes the only representative in freshwater) and Armophorea, remain undocumented in bromeliad phytotelmata. The most abundant ciliate genera found in V. platynema and A. gamosepala in the present study were Glaucoma, Obertrumia, Phacodinium, and Tetrahymena (Figure 4). The genus Glaucoma was present during the whole study period and presented an abundance peak (~10%) in August in one individual of V. platynema. In A. gamosepala, its abundance peak was observed in a sample collected in May. Glaucoma is a very common limnetic genus found in freshwater environments, and was mentioned in the past as an example of genera that had never been found in bromeliad waters (Dunthorn et al., 2012). Obertrumia presented a high frequency in A. gamosepala, and its peak of abundance was observed in a sample collected in August. In V. platynema its highest abundance was observed in October, reaching ca. 3%. The genus Obertrumia is considered cosmopolitan, and is generally found in the plankton or benthos of freshwater environments, especially when cyanobacteria are abundant (Foissner et al., 1999). The genus Phacodinium was present in both sampled individuals of A. gamosepala and in one individual of V. platynema (V5) in May and August. This is a limnetic genus generally found in freshwater, and is reported here in bromeliad waters for the first time. The genus Tetrahymena was one of the most abundant genera of eukaryotes found in the present study (Figure 4) with a peak of biomass reaching 15% in one V. platynema sampled in August. Tetrahymena is a cosmopolitan genus commonly found in freshwater environments. Most species in the genus are bacterivores feeding also on organic matter, but some species are histophagous and are found associated with fishes or invertebrates (Lynn, 2008). Bacterivores play an important role as grazers in aquatic environments, especially when bacteria concentration is high (Gerhardt et al., 2010). Other genera of unicellular eukaryotes that were abundant during the study period were the apicomplexans Babesia and Leydiana. Babesia was present during the whole study period and recorded from both A. gamosepala and V. platynema (Figure 4). Leydiana was recorded from one individual of V. platymena (V1) in October and from the other

sampled individual (V5) in May and August. In October, it had an abundance peak of more than 10%. In addition, the apicomplexan group Gregarina was present during the whole study period, showing relative abundances of ca. 15% (Figure 4). Among unicellular eukaryotes, the presence of the genus Trypanosoma was a very intriguing result. A total of 40 OTUs defined from our data matched Trypanosoma reference sequences, including 1 OTU matching T. congolense, 1 OTU matching T. cruzi, 30 OTUs matching T. rotatorium and 2 OTUs matching T. scelopori. Although the precise species-level identification of these OTUs would require additional comparisons with reference sequences from the same geographic region (to control for phylogenetic diversity within species, as well as taxonomic uncertainties pertaining to this group), our results provide strong support to the genus-level identification of Trypanosoma in these bromeliad tank waters. Interestingly, Trypanosoma was present in tanks from both bromeliad species, reaching an abundance of more than 10% in A5 sampled in January and in V5 sampled in May (Figure 4). The same V. platynema individual sampled in October had a relative abundance of Trypanosoma of more than 20% (Figure 4). It is noteworthy that Bacigalupo et al. (2006) reported high infestation rates by T. cruzi of triatomine insects associated with terrestrial bromeliads. However, the actual presence of Trypanosoma in the tank waters had so far not been documented. Therefore, our results demonstrate for the first time the presence (and high abundance) of Trypanosoma in bromeliad tanks, indicating that these organisms and their vectors use phytotelmata as a common habitat. The most frequent metazoan genus recorded was the harpacticoid copepod Paramphiascella (Figure 4). This genus is commonly found in marine or brackish habitats and has never been recorded from freshwater. The presence of harpacticoid copepods in the sampled bromeliads was observed during in vivo analyses of the samples (not shown), which corroborates this finding, but the assignment of the sequences to this marine genus may have been caused by lack of sequences in the reference database to this species of the genus found in freshwater habitats. Overall, the results we obtained here for the two surveyed bromeliad species corroborates previous findings reporting that these habitats harbor a hidden diversity of single-celled as well as multicellular organisms. In relation to ciliates, until now 40 new species and three new genera have been described from different species of bromeliads

(Foissner, 2003a; Foissner, 2003b; Foissner et al., 2003), all based on morphological observations. Our data have shown that the diversity of ciliates and other unicellular eukaryotes is much higher than it has been reported by morphological studies. In addition, the composition of these communities is very complex, and varies considerably across species, between individuals of the same species, and also between different time-points for the same bromeliad. By detecting remarkable levels of eukaryotic diversity at phylum and genus level, an unprecedented richness of ciliate classes, and complex variation in community composition, we show that these habitats have only begun to be explored, and will require intensive efforts to be fully characterized. We also demonstrate the usefulness of the DNA metabarcoding approach to conduct large-scale surveys of these ecosystems, allowing fine-scale comparisons within and across study-sites. Although many analyses in community ecology can be performed using sequence-based OTUs, the more desirable goal of attaining species-level (or even genus-level) identification of all ciliates (and eukaryotes in general) present in these habitats remains distant. Addressing this limitation will require not only improvements in present-day reference databases (e.g. Berney et al., 2017) to allow better identification of known taxa, but also intensive taxonomic work (using morphological and molecular studies of live, preserved and cultured specimens) targeting the organisms that occur in these habitats, many of which are likely to be undescribed. Such taxonomic advances will not only significantly improve our understanding of bromeliad water biodiversity, but also foster the development of in-depth studies of the functional ecology of these complex communities and their host plant.

Acknowledgements This work was supported by National Council of Research and Development (CNPq).

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Figure Legends Figure 1: A. Aechmea gamosepala and Vriesea platynema individuals sampled in this study. B. Spatial location of the bromeliad individuals (A1 and A5 for A. gamosepala and V1 and V5 for V. platynema) sampled at the Pró-Mata Center for Research and Nature Conservation, São Francisco de Paula municipality, southern Brazil.

Figure 2: Relative abundance (estimated from the number of sequence reads) of the 20 most abundant eukaryotic phyla identified in the present study. A1 and A5 refer to sampled individuals of Aechmaea gamosepala; V1 and V5 refer to individuals of Vriesea platynema. Numbers 05,08,10 refer to May, August, and October, respectively.

Figure 3: Total number of Operational Taxonomic Units (OTUs – defined using a 99% identity criterion [see Materials and Methods] and roughly equivalent to species) and genera detected in this study and assigned to each ciliate class.

Figure 4: Relative abundance (estimated from the number of sequence reads) of the 20 most abundant eukaryotic genera identified in the present study.