Antonie van Leeuwenhoek (2009) 96:71–78
DOI 10.1007/s10482-009-9337-4
ORIGINAL PAPER
Study of the diversity of culturable actinomycetes
in the North Pacific and Caribbean coasts of Costa Rica
Godofredo Solano Æ Keilor Rojas-Jiménez Æ
Marcel Jaspars Æ Giselle Tamayo-Castillo
Received: 9 September 2008 / Accepted: 25 March 2009 / Published online: 14 April 2009
 Springer Science+Business Media B.V. 2009
Abstract In this study, 137 actinomycetes were The observed pattern of species distribution might be
isolated from subtidal marine sediments in the North the result of several factors including the character-
Pacific and Caribbean coasts of Costa Rica. Bioin- istics of the ecosystems, presence of endemic species
formatics analysis of the 16S rRNA gene sequences and the influence of terrestrial runoff.
assigned the isolates to 15 families and 21 genera.
Streptomyces was the dominant genus while the Keywords Actinomycetes  Microbial Ecology 
remaining 20 genera were poorly represented. Nearly Cahuita  Gandoca-Manzanillo  Las Baulas 
70% of the phylotypes presented a coastal-restricted Cabo Blanco
distribution whereas the other 30% were common
inhabitants of both shores. The coastal tropical waters
of Costa Rica showed a high diversity of actinomy-
cetes, both in terms of the number of species and Introduction
phylogenetic composition, although significant dif-
ferences were observed between and within shores. The actinomycetes comprise a large and diverse
group of Gram-positive bacteria that are extensively
distributed in several environments. These bacteria
are primarily saprophytic and are known to contribute
Electronic supplementary material The online version of in nutrient turnover, using many available nutrient
this article (doi:10.1007/s10482-009-9337-4) contains
supplementary material, which is available to authorized users. sources for their development (Williams et al. 1983;
Jensen et al. 1991; Stackenbrandt et al. 1997; Mincer
G. Solano  M. Jaspars et al. 2002). Many actinomycetes are recognized for
Chemistry Department, University of Aberdeen,
their metabolic versatility that frequently is accom-
Aberdeen AB24 3UE, UK
panied by the production of secondary metabolites of
G. Solano  K. Rojas-Jiménez  G. Tamayo-Castillo (&) economic importance (Lazzarini et al. 2000; Bull
Unidad Estratégica de Bioprospección, et al. 2000; Bull and Stach 2007). During the last few
Instituto Nacional de Biodiversidad,
P.O. Box 22-3100, Santo Domingo de Heredia, decades, isolation of actinomycetes has been mainly
Heredia, Costa Rica performed as part of drug discovery programs and has
e-mail: gtamayo@inbio.ac.cr focused on terrestrial sources, but recent efforts
turned to marine environments because of the
G. Tamayo-Castillo
Escuela de Quı́mica, Universidad de Costa Rica, increasing discovery rate of new isolates and prom-
San Jose, Costa Rica ising bioactive compounds from these ecosystems
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72 Antonie van Leeuwenhoek (2009) 96:71–78
(Fenical 1993; Bull et al. 2000; Donadio et al. 2002; Materials and methods
Lam 2006). Some actinomycetes have been studied in
detail due to their biotechnological importance; Sample collection
however, more comprehensive studies are needed to
understand the diversity, distribution and ecology of This study was conducted in four marine protected
the large majority of actinomycetes in marine habitats areas of Costa Rica under permit number R-004-
(Stach et al. 2003; Maldonado et al. 2005b; Bull and 2004-OT-MINAE of the National Ministry of Envi-
Stach 2007). ronment. Two sampling trips were performed in
Several statistical tools are currently being devel- December 2004 to the Caribbean Coast, specifically
oped for describing and comparing microbial com- to Cahuita National Park and Gandoca-Manzanillo
munities. For example 16S rRNA gene sequences can National Wildlife Refuge and two further trips in
be easily assigned to taxonomical hierarchies based January 2005 to Cabo Blanco Absolute Natural
on naı̈ve Bayesian classifiers (Wang et al. 2007) or Reserve and Las Baulas Marine National Park in
used to perform quantitative analyses of the commu- the Pacific. A total of 26 samples were collected,
nities (Martin 2002). Existing tools assign sequences where factors such as distance from the coast, depth
to operational taxonomic units (OTUs) based on and sampling site uniqueness were considered for the
genetic distances between sequences which also collection. The sampling of sea bottom sediments
allow the estimation of ecological parameters such was carried out with a grab sampler while shoreline
as richness, diversity and degree of similarity samples were collected using a small hand-held
between microbial communities (Schloss and Han- stainless steel shovel. The samples were put in
delsman 2005, 2006). The application of this bioin- polyethylene ziplock bags and kept at room temper-
formatics-based approach is very promising in ature in the field, then on ice until transported to
microbial ecology, particularly for the understanding laboratory where they were stored at 4C. A
of organisms such as Actinobacteria in marine description of collecting sites is given in Table 1.
environments (Bull et al. 2005).
Costa Rica is a narrow strip of land that connected Isolation of actinomycetes
South America with North America in relatively
recent times (approximately 3 million years ago) and Upon arrival at the laboratory, about 1 g of each
thus acts as a natural barrier to the transit of marine sample was taken for actinomycete isolation. Sam-
forms between the Caribbean and the Pacific Ocean ples were suspended in sterile saline solution (0.85%
(Rich and Rich 1983). The Caribbean littoral length NaCl), treated by thermal shock at 60C for 6 min
of the country is comprised of 1,228 km and is and serial diluted to 10
-3. Aliquots (0.1 ml) of each
characterized by a regular shape where sandy beaches dilution were spread by duplicate on M1 medium
alternate with rocky promontories. The Pacific littoral plates (Jensen et al. 2005a; prepared with artificial sea
is 1,016 km long and presents several irregularities water) and glucose-yeast extract medium (Godon and
such as peninsulas, gulfs and bays that contain a Mihm 1962; prepared with distilled water) amended
number of sandy beaches formed by the accumulation with nalidixic acid (10 mg/ml) and nystatin (50 mg/
of sediments and distributed by the tides. In general, ml). Plates were incubated at room temperature up to
beaches of the Caribbean and North Pacific show six weeks and checked regularly for the emergence of
high concentrations of calcium in sediments, which is presumptive actinobacteria. New actinomycetes were
indicative of the abundant reef structures near these subcultured onto new M1 or ISP2 (Shirling and
beaches (Cortes 2002; Salazar et al. 2004). In this Gottlieb 1966) plates supplemented with the above
study a culture-dependent approach was used for antibiotics. Isolates were compared in their colony
isolating actinomycetes from the North Pacific and and morphological characteristics to select no more
Caribbean coasts of Costa Rica. The 16S rDNA gene than two representatives of the redundant colonies
sequences were processed by a series of bioinfor- present in each sediment. A total of 137 presumptive
matic tools with the aim of describing the composi- actinomycete strains were obtained, for a mean
tion, diversity and similarity of the actinomycete isolation rate of 5.26 strains per sample. Each
microbial communities. microorganism was included into a database with
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Antonie van Leeuwenhoek (2009) 96:71–78 73
Table 1 Description of the sampling sites
Protected Coast Collecting site description Sampling Location
area depth (m)
Gandoca- Caribbean Open sea shallow waters located nearby fringing reefs, -10 to -6 93701000N 823605900W
Manzanillo patch reefs, and carbonate banks and algal ridges.
Sediments were dark and sandy
Cahuita Caribbean The largest fringing reef in Costa Rica consisting of -17 to 0 94504400N 82490300W
three barriers also presents patch reefs and carbonate
banks. Sediments were coralline sandy. Fine sands
from the beach were sampled as well
Cabo Blanco North Pacific Open sea shallow waters located neary patch reefs with -20 to 0 93304800N 8550400W
coarse sandy sediments of coralline origin. Coarse
sands from the beach were sampled as well
Las Baulas North Pacific Open sea shallow waters located in front of the mouth -20 to 0 10180500N 855101600W
of the Tamarindo estuary. Fine sandy sediments
associated information such as morphological Genbank using the BLAST tool (Altschul et al.
descriptions, date of collection and date of isolation. 1997) and against the Ribosomal Database Project II
using the Classifier tool (http://rdp.cme.msu.edu). The
DNA manipulations latter assigned the 16S rRNA gene sequences to a
taxonomical hierarchy based on a naı̈ve Bayesian
The Actinobacteria were grown in liquid ISP2 rRNA classifier with a 95% confidence interval (Wang
medium (Atlas 1993) at 25C for 8–15 days with et al. 2007). The number of sequences belonging to the
constant shaking at 150 rpm. Genomic DNA from major phylogenetic groups was recorded.
actinomycete strains was isolated using the Gen
EluteTM Bacterial DNA Extraction Kit (Sigma– Ecological analyses
Aldrich Co., MO, USA) following the manufacturer’s
instructions. The 16S rRNA gene was PCR amplified The sequence set was aligned using the Infernal
from the total DNA extracts using primers 27f and secondary-structure based aligner implemented in the
1,525r (Lane 1991). The reactions were performed on RDPII page (Cole et al. 2009) and optimized by hand
a GeneAmp PCR system 9700 (Applied Biosystems, with BioEdit. The alignment created was processed
CA, USA) at the following temperature program: with DNADIST program of PHYLIP (http://evolution.
95C for 10 min followed by 35 cycles of 94C for genetics.washington.edu) to generate a distance
1 min, 52C for 1 min and 72C for 1 min and 10 min matrix, which was used as an input file for DOTUR
extension at 72C. The PCR products were purified (http://www.plantpath.wisc.edu). This program grouped
using the Gen EluteTM PCR Clean-up Kit (Sigma– the sequences into OTUs according to different
Aldrich Co., MO, USA) according to manufacturer’s sequence similarity cutoffs, which allow the estima-
protocols. Sequencing was performed at the sequenc- tion of the community composition regardless of the
ing facility of the Dana Farber Cancer Institute at the abundance effect (Schloss and Handelsman 2005). In
University of Harvard, Boston, Massachusetts, using this study, we used the standard 97% sequence simi-
the same PCR primers. The primer sequences of the larity to distinguish between different bacterial species
molecular marker were carefully compared to the where two sequences with an identity greater than 97%
corresponding chromatogram and then contiged with were assigned to the same OTU and thus to the same
the program BioEdit (Hall 1999). species (Stackebrandt and Goebel 1994). Once
DOTUR assigned sequences to OTUs, it was possible
Phylogenetic classification to calculate the number of OTUs observed in the col-
lecting sites (relative richness) and the predicted
The set of 137 sequences, 64 from the Pacific and 73 number of OTUs that might exist in each ecosystem
from the Caribbean, was compared against the (expected richness), based on the bias-corrected
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Chao1 estimator (Chao 1984; Chao and Lee 1992). North Pacific Caribbean
DOTUR was also used to calculated the Shannon
index, a very useful tool for comparing the diversity
Actinomadura Nocardiopsis
between communities, and the Simpson index, that Amycolatopsis Streptomyces Microbacterium
shows the likelihood that two individuals taken at Mycobacterium Nocardia Brachybacterium
random from a community belong to the same species. Curtobacterium Nonomuraea Glycomyces
Jiangella Saccharopolyspora
We constructed a phylogenetic tree with all the Leucobacter
Kribbella Rhodococcus Pseudonocardia
sequences from the two actinobacterial communities Saccharomonospora Gordonia
Terrabacter
with the program MEGA 4.0 (Kumar et al. 2008) using Salinospora
the neighbor joining method and applying a Tamura-
Nei distance-correction (Saitou and Nei 1984). The
generated phylogenetic tree and an additional file Fig. 1 Distribution of genera per site of isolation for the 137
describing each sequence with the corresponding coast actinomycetes analyzed in this study
of origin were used as inputs for running the UniFrac
web tool (http://bmf2.colorado.edu/unifrac/index.psp) analyzing the diversity within the specific sites, Las
in order to determine statistical differences between Baulas and Cabo Blanco located at the North Pacific
communities according to the UniFrac significance contained 10 and 8 actinomycete genera, respectively.
test (Lozupone and Knight 2005) and the P test of At the Caribbean, Cahuita and Gandoca-Manzanillo
significance (Martin 2002). This program computes harbored 7 and 9 actinomycete genera. A remarkable
the phylogenetic distance between the sequence sets as feature of this data set was that Streptomyces was the
the fraction of the branch length of the tree that leads to dominant genus recovered from both shores, compris-
descendants from either one site or the other (Lozu- ing nearly two-thirds (64.5%) of the total of isolates.
pone and Knight 2005; Lozupone et al. 2006). The The other 20 genera were poorly represented, account-
similarity in the actinomycete composition between ing for the remaining 35.5% of the isolates.
communities was estimated with SONS (http://www.
plantpath.wisc.edu/fac/joh/sons.html). This program Species richness and diversity
used as primary input the ‘‘*.list’’ output file from
DOTUR and a file containing each sequence identifier The analysis of the ecological estimators showed that
with its assigned community of origin. It was possible 44 phylotypes of actinomycetes were recovered from
to estimate the fraction of shared sequences between the sampling sites at the North Pacific and the
communities, the fraction of shared OTUs and the Caribbean of Costa Rica (Table 2). Also, the estima-
overall community similarity (Jaccard’s estimator), by tion of the number of species predicted according to
accounting for the abundance distributions of OTUs the Chao1 estimator indicated that both communities
that were either endemic to one site or shared by the combined might harbor a mean of 67 species (95%
two communities (Schloss and Handelsman 2006). CI: 52–111). When calculated separately by coast,
the program determined that each of the sites
presented 22 OTUs, although not necessarily corre-
Results sponding to the same species. The total number of
species expected for the North Pacific community
Taxonomic composition was 37 species (95% CI: 25–86) and 33 species for
the Caribbean (95% CI: 24–63). The diversity of the
Results of the comparison of the 16S rRNA gene actinomycete collection, according to the Simpson
sequences against the RDP II database assigned the index, was 0.09. This represents a probability of 9%
isolates to 15 families of actinomycetes and 21 genera that two individuals taken at random from the
(Fig. 1). From these, six genera were isolated from both collection belong to the same species (Table 2).
the Pacific and the Caribbean (Streptomyces, Nocardia, However, when Simpson index was estimated for
Nonomuraea, Rhodococcus, Saccharopolyspora and each coast, results showed a value of 0.07 for the
Gordonia) while 8 genera were found exclusively in the Pacific and 0.15 for the Caribbean, indicating that the
Pacific and 7 exclusively in the Caribbean. When likelihood of finding identical species in the Pacific
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Antonie van Leeuwenhoek (2009) 96:71–78 75
Table 2 Main ecological estimators calculated for the overall the number of species in each community to the total
collection, the North Pacific and the Caribbean samples number of species. When comparing within Carib-
Overall North Pacific Caribbean bean sites, results showed that 62% of the isolates in
collection samples samples Gandoca-Manzanillo and 57% in Cahuita were
common to both places. However, the number of
OTUs observed 44 22 22
OTUs shared between these sites was close to 31%.
OTUs expected 67 37 33
Furthermore, the Jaccard’s estimation of the similar-
Simpson 0.09 0.07 0.15
ity between these communities was 18%. The com-
Shannon 3.33 2.75 2.39
parison between Cabo Blanco and Las Baulas in the
Data were estimated by DOTUR program using a 3% distance North Pacific showed that a high percentage of the
cutoff sequences and OTUs were common to both ecosys-
tems (Table 3).
samples is lower than in the Caribbean samples and
hence, an indirect measurement of higher diversity in
the Pacific. The estimation of the diversity using the Discussion
Shannon index, for the overall collection was 3.33.
When estimated separately the North Pacific coast In this study we obtained 137 actinomycete isolates
showed a value of 2.75 and the collection from the from four sampling sites located in the North Pacific
Caribbean a value of 2.39. This index is the most and Caribbean waters of Costa Rica. Several bioin-
widely used in ecology, where values typically fall formatics tools were used to analyze 16S rRNA gene
between 1.5 and 4, with higher values representing sequences and to determine species composition,
higher diversity. diversity and other ecological parameters. Taxonomic
composition analysis of the collection shows that the
Comparison of the actinomycete communities number of genera isolated in this study is comparable
or even higher to that found in other culture-
The comparison of the actinomycete composition dependent studies (Mincer et al. 2002; Maldonado
between the North Pacific and Caribbean coast was et al. 2005a, b; Jensen et al. 2005b; Bredholdt et al.
computed with UniFrac, based on phylogenetic 2007). In addition, it was remarkable that 15 out of
information. Results of this comparison using both the 30 families described for the order Actinomyce-
the UniFrac and the P significance tests revealed that tales were represented in the collection (Stacken-
species composition of either coast differed signifi- brandt et al. 1997). This suggests that, despite the
cantly from the other (P \ 0.01; n = 137). This was modest number of isolates, the coastal sediments
consistent with results of the similarity analyses sampled in these tropical waters might be considered
determined with SONS showing that the percentage as rich sources of Actinobacteria, both in terms of the
of phylotypes shared between the communities was number of species and the phylogenetic composition.
33–35%, at a distance level of 3%. The overall The most abundant genus in this collection was
Jaccard’s similarity between the North Pacific and Streptomyces, comprising nearly two-thirds of the
Caribbean collections was 0.20, which is the ratio in isolates, while the other 20 genera remained less well
Table 3 Main statistical estimators of the comparisons between the communities of marine actinomycete isolates
Communities compared n Shared sequences Shared OTUs Jaccard’s similarity
Comm. 1 Comm. 2 Comm. 1 Comm. 2
Caribbean-North Pacific 137 0.63 0.31 0.35 0.33 0.20
Gandoca-Cahuita 73 0.62 0.57 0.31 0.31 0.18
Cabo Blanco-Baulas 64 0.82 0.67 0.64 0.53 0.41
Data were calculated with SONS program using a 3% distance cutoff
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76 Antonie van Leeuwenhoek (2009) 96:71–78
represented. Interestingly, genera such as Micromo- differ from the other marine sites sampled. Another
nospora and Salinispora that have been reported as physical factor that could have an effect on species
dominant in other marine ecosystems were scarcely distribution, particularly in the North Pacific, was the
detected in this study (Magarvey et al. 2004: upwelling, which is a wind-driven effect that replaces
Maldonado et al. 2005a: Bull et al. 2005: Jensen the warmer surface waters with subsurface nutrient-
et al. 2005a, b). This outcome might be explained by rich waters. This effect occurred precisely during the
differences in the sample pre-treatment used and sampling period (Brenes et al. 2002), and could
media isolation employed, which may account for the provoke the homogenization of sediment-living bac-
high presence of Streptomyces and the absence of teria and the circulation between locations as judged
Micronospora and Salinospora. When analyzing the by the higher percentage of sequences and OTUs
geographic species distribution using both phyloge- shared between the two sites sampled in this coast.
netic-based and distance-based approaches, it was The second major influence could be the presence
observed that nearly 70% of the genera presented an of endemic species. The amount of singletons detected
apparent coastal-restricted distribution while the in all the sites suggests that functional changes could
remaining 30% were detected in both the North be occurring in these bacteria in order to adapt to the
Pacific and Caribbean. However, these results should specific ecological niches, which might be accompa-
be treated with caution since lack of recovery cannot nied with the development of speciation processes.
be assumed as absence of the organism in one site. Finally, the influence of terrestrial runoff may be
Our results raise the question of why some species are important: the sampling sites are directly influenced by
found on both coasts and others just on one. Although rivers that bring sediments and consequently terrestrial
we cannot propose a clear explanation for this actinomycetes into the coasts, i.e. those in the Carib-
observation, one possible reason could be that species bean, Cahuita and Manzanillo are affected by sediment
that are shared in Pacific and Caribbean shores runoff from the La Estrella and Sixaola watersheds,
existed before the Central American Isthmus closed respectively. In the Pacific, Las Baulas is influenced by
the connection between the oceanic waters ca. 3 the Tamarindo mangrove estuary. Thus, at this point of
million years ago (Rich and Rich 1983). Another the study is difficult to establish whether the isolates
possible explanation is that these species might are truly ‘‘marine’’ or have terrestrial origin with
present a ubiquitous distribution (Maldonado et al. possible halo tolerance characteristics.
2005b; Lam 2006). The reasons for the differences in The ecological estimators determined in this study
the actinomycetes composition between populations were consistent in suggesting that these tropical
in the North Pacific and the Caribbean are complex waters showed a high diversity of actinomycetes,
and might be the result of the interaction of several both in terms of the number of species and the species
factors. Firstly, ecosystem characteristics: the inher- composition. However, significant differences in the
ent physical, chemical and biological characteristics phylogenetic distribution were observed between and
of the Caribbean Sea and Pacific Ocean could within the North Pacific and the Caribbean shores.
influence the life forms that developed on their When comparing the Jaccard’s similarity between
respective shores. The ecosystems at the sampling sites, the estimation was consistent with previous
sites in the Caribbean were quite different from the results at showing that nearly one-third of the total
sites in the North Pacific. For example, in the number of OTUs was found in both shores. When
Caribbean, both Cahuita and Gandoca-Manzanillo analyzed separately within shores, the results suggest
are characterized by the presence of fringing reefs, that species composition in Cahuita and Gandoca-
patch reefs and carbonate banks although they differ Manzanillo is quite different, most likely due to the
in size, shape, diversity of species, and terrestrial strong influence of the La Estrella and Sixaola rivers
runoff influence (Cortes 2002). In the Pacific, the and the existence of a local current, perpendicular to
sampling sites in Cabo Blanco presented mainly the coast, that might limit microorganism exchange
patch reefs with other coral species and Las Baulas between these sites. In the Pacific, Las Baulas and
comprised a particular ecosystem located at the Cabo Blanco presented a more homogeneous com-
mouth of the Tamarindo estuary, where salinity, position that could be the result of the mixing effect
organic matter content and the species developed of the wind-driven upwelling as mentioned above.
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Antonie van Leeuwenhoek (2009) 96:71–78 77
To our knowledge, this is the first report of the Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ,
isolation and ecological characterization of actino- Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T,
Garrity GM, Tiedje JM (2009) The ribosomal database
mycetes from the North Pacific and Caribbean coasts project: improved alignments and new tools for rRNA
of Costa Rica. We determined that these tropical analysis. Nucleic Acids Res 37:141–145. doi:10.1093/nar/
waters are rich in terms of composition and that gkn879
important differences can be observed between and Cortes J (2002) The coral reefs of Costa Rica’s Caribbean
Coast. Instituto Nacional de Biodiversidad, Heredia,
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the marine or terrestrial origin of the isolates and the Donadio S, Monciardini P, Alduina R, Mazza P, Chiocchini C,
role they play in their respective ecosystems. Cavaletti L, Sosio M, Puglia AM (2002) Microbial tech-
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Acknowledgments We would like to thank Luis Acosta from J Biotechnol 99:187–198. doi:10.1016/S0168-1656(02)
INBio, the Center of Marine Research (CIMAR) of the 00209-2
University of Costa Rica and Michael Goodfellow from the Fenical W (1993) Chemical studies of marine bacteria:
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