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Biol. Lett. (2006) 2, 593–596
doi:10.1098/rsbl.2006.0528
Published online 7 August 2006
A novel obligate cultivation
mutualism between
damselfish and
Polysiphonia algae
Hiroki Hata
†,
*and Makoto Kato
Graduate School of Human and Environmental Studies, Kyoto
University, Yoshida-Nihonmatsu, Sakyo, Kyoto 606-8501, Japan
*Author for correspondence (hata@d01.mbox.media.kyoto-u.ac.jp).
†
Present address: Graduate School of Science, Kyoto University,
Kitashirakawa-Oiwake, Sakyo, Kyoto 606-8502, Japan.
In cultivation mutualisms, farming animals pre-
pare fields for cultivars, enhance their growth
and harvest them. For example, in terrestrial
ecosystems, plant–herbivore cultivation mutual-
isms arose between humans and their crops only
relatively recently. We discovered an obligate
cultivation mutualism between a damselfish and
an alga in a coral reef ecosystem. The damsel-
fish, Stegastes nigricans, manages algal farms
through territorial defence against the invading
grazers and through weeding of unpalatable
algae. As a result, the algal farms of S. nigricans
are dominated by one species, Polysiphonia sp.
We performed an exhaustive survey of algal
assemblages inside and outside the territories of
five damselfish species around the Ryukyu
Islands, Japan, using molecular and morpho-
logical characteristics. Polysiphonia sp. 1 grew
exclusively inside the farms of S. nigricans, and
never elsewhere. Since only Polysiphonia sp. 1 is
harvested and consumed by the damselfish as a
staple food, this interdependent relationship is
an obligate cultivation mutualism. This is the
first record of an obligate plant–herbivore culti-
vation mutualism in a marine ecosystem. Our
data also suggest that three other Polysiphonia
species are facultatively mutual with, commen-
sal with, or parasitic on other damselfish
species.
Keywords: obligate cultivation mutualism;
territorial damselfish; Polysiphonia algae; coral reef
1. INTRODUCTION
Cultivation mutualisms between humans and their
crops have evolved through ‘proto-domestication’ in
which humans use and select plants intruding on
human-disturbed habitats (Rindos 1984). In marine
ecosystems, some herbivorous damselfishes and lim-
pets maintain proto-domesticated algal assemblages
(i.e. algal farms) by excluding grazers and cultivating
distinct crop assemblages (Branch 1981;Ceccarelli
et al. 2001). Some limpets on intertidal rocky shores
in South Africa and the west coast of North
America have evolved facultative cultivation mutual-
isms with species-specific but ubiquitous algae
(Branch 1981). Similarly, the damselfish Stegastes
nigricans has been shown to maintain a monoculture
of a filamentous red-alga, Polysiphonia sp., by
excluding invading herbivores (Hata & Kato 2004).
In addition, they remove less-digestible competitive
algae from their algal farms (Hata & Kato 2002).
This intensive management by S. nigricans results in
selection for fast-growing palatable algae. Cage
experiments that exclude a territory-holding damsel-
fish as well as all herbivores have shown that in the
absence of weeding by the fish, the Polysiphonia sp.
are overgrown by other algal species within a week
(Hata & Kato 2003). When only S. nigricans was
removed, its algal farm was invaded by grazers and
denuded of algae in a few days. Thus, intensive
management and aggressive territorial defence allow
the fish to maintain a monoculture of Polysiphonia
sp. on which it feeds as staple food (Hata & Kato
2002). We investigated whether the dependence of
this alga on the fish is obligatory by determining the
occurrence of the alga outside S. nigricans algal
farms. In addition, we investigated whether other
Polysiphonia spp. algae have species-specific relation-
ships with damselfishes and assessed the phyloge-
netic relationships among Polysiphonia spp. algae
that are cultivated by damselfish.
2. MATERIAL AND METHODS
(a)Sampling
We collected Polysiphonia spp. algae and related algal species from
inside and outside the territories of various damselfish. Whether a
site was inside or outside the territories of damselfish was
determined after 20 min of observation immediately before
sampling. To collect algae exhaustively from outside the territories
of fish, we set line transects from the beach to offshore areas,
perpendicular to the shoreline, at two, four and ten reef flats around
Okinawa Island (26804–52 0N and 1278380–1288190E), Ishigaki
Island (24819–360N and 124804–200E) and Iriomote Island
(24815–250N and 123840–550E) respectively, in 2003 and 2004.
The average length of the 16 line transects was 477 m (1057 m
maximum and 156 m minimum). We set a 1!1 m quadrat outside
the territories at 50 m intervals on each transect and scraped all
algae and seagrass inside the quadrat into a mesh bag by grazing
the entire substratum with a knife. In total, 158 samples were
collected from outside damselfish territories. Five territorial herbi-
vorous damselfishes inhabited these study areas: S. nigricans,
Stegastes lividus,Hemiglyphidodon plagiometopon,Dischistodus prosopo-
taenia and Plectroglyphidodon lacrymatus. Whenever we found these
damselfishes along the line transects, we collected all the algae from
7!7 cm quadrats placed inside the territories. Stegastes nigricans
was found on 14 lines, together with all the other damselfishes
except H. plagiometopon, which was the sole inhabitant of one site
near a river mouth. In total, 53, 18, 9, 19 and 13 samples were
collected from the territories of S. nigr icans,S. lividus,
H. plagiometopon,D. prosopotaenia and P. lacrymatus, respectively.
We also collected algae outside the damselfish territories in
Makaha Beach and Pokay Bay around Oahu Island (21815–420N
and 1578380–1588160W) in Hawaii in October 2003, and inside
and outside territories of H. plagiometopon in a coral reef around
Koh Hae Island (78460N, and 98821 0E) in Thailand in March
2004. The algal samples were immediately preserved in 100%
ethanol. In the laboratory, samples were displaced using distilled
water, and all Polysiphonia algae were sorted under a microscope.
Small thalli of Polysiphonia algae collected from inside (nZ67) and
outside (nZ28) territories were classified into 16 species using
molecular data. The total biomass of algae in samples collected
from damselfish territories and that of Polysiphonia species were
measured in wet weight.
(b)Molecular methods
We extracted total DNA from field-collected, ethanol-preserved
algae. A fragment of the 18S ribosomal RNA gene was amplified by
PCR using the primers 50-ACCTGGTTGATCCTGCCAG-30and
G07 and was directly sequenced using the above two and other
four primers (Saunders & Kraft 1994). All the sequences were
deposited in the NCBI GenBank database (accession nos.
AB219858–AB219930).
(c)Phylogenetic analyses
Maximum-parsimony (MP) and maximum-likelihood (ML)
analyses were conducted using PAUP
v. 4.0b10; Bayesian
Received 13 July 2006
Accepted 15 July 2006
593 q2006 The Royal Society
inference (BI) was conducted using MRBAYES v. 3.0b4. The MP
analyses employed the heuristic search option with TBR (tree
bisection and reconnection) branch swapping and 1000 random-
taxon-addition replicates, identifying the 60 most parsimonious
trees of length 468 steps, C.I.Z0.607 and R.I.Z0.804. Heuristic
MP bootstrap analysis consisted of 1000 pseudoreplicates with 10
random-taxon-addition replicates per pseudoreplicate. The
likelihood ratio test implemented in MODELTEST v. 3.06 found that
the TrNCGCI model best fits the sequence data, and this model
was employed in a heuristic ML analysis. A heuristic search with
10 random-taxon-addition sequences and TBR branch swapping
was performed. BI was carried out based on the model of GTRC
GCI with 1 000 000 generations, sampling every 100 generations.
The first 100 samples were discarded as burn-in.
3. RESULTS AND DISCUSSION
Our field collections revealed four Polysiphonia,
specialized to specific damselfish species (figure 1;
Fisher’s exact test, all p!0.001; figure 2). These four
Polysiphonia species were morphologically distin-
guished from 21 species known from Japan (Yoshida
1998) in having four pericentral cells, ecorticated
fronds and rarely branched erect axes (figure 1). This
indicates that these Polysiphonia species have never
been found as free-living forms, and thus, we called
the algal species, Polysiphonia spp. 1–4. Polysiphonia
sp. 1, which was always dominant in the algal farms
of S. nigricans, was encountered only inside the farms
of S. nigricans and never outside them, irrespective of
intense sampling (figure 1). This suggests that only
S. nigricans can provide Polysiphonia sp. 1 with the
exposed sunny habitat, where grazing pressure is
moderate and competitive algae are weeded out. In
this way, Polysiphonia sp. 1 is obligately dependent on
S. nigricans. The damselfish manages its algal farm
dominated by Polysiphonia sp. 1 and feeds exclusively
in the farm (Hata & Kato 2002,2004), suggesting
that the fish depends on Polysiphonia sp. 1 for staple
food. Therefore, this interdependent relationship
between S. nigricans and Polysiphonia sp. 1 is an
obligate cultivation mutualism (table 1). We found
that another damselfish, H. plagiometopon,hada
‘semicultivated’ (Harris & Hillman 1989)Polysiphonia
species. Algal farms of this fish species were always
dominated by Polysiphonia sp. 3 (figure 1). However,
Polysiphonia sp. 3 also inhabited the algal farms of
other damselfishes and was found to occur outside
damselfish farms. This association represents a facul-
tative cultivation mutualism, in which the fish
depends on the alga, but the alga does not necessarily
depend on the fish (table 1).
Polysiphonia species that correspond to ‘weeds’
(Harlan 1992) in terms of human cultivation were
also encountered. Polysiphonia sp. 2 and 4 were
found inside the algal farms of P. lacr ymatus and
D. prosopotaenia, respectively. These algae were rare
outside the territories of damselfish, but did not
dominate the farms (figure 1). These algae are
obligately associated with specific fish, whereas the
fish do not necessarily depend on the algae for staple
food. Damselfishes manage their farms in a range
of intensities (table 1), as both monocultures and
mixed cultures (Hata & Kato 2004). Only in
intensive farming systems, damselfish seem to have
evolved obligate cultivation mutualisms, such as
for S. nigricans.Plectroglyphidodon lacrymatus and
D. prosopotaenia, which maintain mixed-culture farms
by management without weeding, appear to engage
only in facultative cultivation mutualism. Stegastes
lividus did not have any species-specific algae in
its mixed-culture farm. On the other hand, the
0
100
50
(b) S. lividus (n = 18)
0
100
50
0
100
50
0
100
50
(d) H. plagiometopon (n = 9)
(e) D. prosopotaenia (n = 19)
(c) P. lacrymatus (n = 13)
P
olysiphonia
sp.2 (n = 3)
***
P
olysiphonia
sp.1 (n = 53)
***
P
olysiphonia
sp.3 (n = 51)
***
P
olysiphonia
sp.4 (n = 9)
***
0
100
50
0
100
50
(f) outside fish territories (n = 158)
per cent occurrence of each alga inside and outside damselfish territories (%)
1 mm
(a) S. nigricans (n = 53)
Figure 1. Percent occurrence of four Polysiphonia spp. algae
inside and outside the territories of the damselfishes (a)
Stegastes nigricans,(b)S. lividus,(c)Plectroglyphidodon
lacrymatus,(d)Hemiglyphidodon plagiometopon and (e)
Dischistodus prosopotaenia. The probability of occurrence of
each algal species among these sites was analysed using
Fisher’s exact test.
p!0.001.
594 H. Hata & M. Kato Obligate cultivation mutualism
Biol. Lett. (2006)
Polysiphonia species that are found exclusively symbio-
tically with specific damselfishes are not monophyletic
(figure 2), suggesting that the adaptations of these
algae to damselfishes originated independently.
Cultivation mutualisms have also evolved between
fungi and terrestrial invertebrates, i.e. ants, termites
and bark beetles (Vega & Blackwell 2005), and a salt
marsh snail (Littoraria irrorata;Silliman & Newell
2003). However, only high-attine ants, termites and
ambrosia beetles occur in obligate cultivation mutual-
isms with an obligate cultivar (Mueller et al. 2005). In
these obligate mutualisms, most farming insects
transplant inocula of fungi from their natal gardens to
new colonies, and thus cultivars are transmitted
vertically (Mueller et al. 2005). In contrast, the
marine cultivation mutualism is analogous to the
ancestral fungus–termite mutualism in which termites
acquire cultivars horizontally via wind-dispersed
spores from other colonies (Aanen et al. 2002;
Korb & Aanen 2003). In the alga–damselfish
mutualism, algal farms of Polysiphonia sp. 1 are mostly
transmitted by S. nigricans from generation to gener-
ation (Lee & Barlow 2001). When colonizing a new
habitat, S. nigricans may use water-borne spores and/
or fragments of Polysiphonia sp. 1 dispersed from
other algal farms. In fact, some Polysiphonia species
have a high capacity for dispersal by spores (Rindi &
Cinelli 2000) or fragments (Eriksson & Johansson
2005), and inside algal farms, both sexual and asexual
spores of Polysiphonia sp. 1 were observed. Addition-
ally, inside artificial cages that excluded all herbivores,
Polysiphonia sp. 1 newly colonized even outside
S. nigricans territories, although they were soon over-
grown by competitive macroalgae. This experiment
showed a high supply of recruits of Polysiphonia sp. 1
in reefs inhabited by S. nigricans (Hata & Kato 2003).
In the terrestrial cultivation mutualisms mentioned
earlier, farming insects harvest decomposition
products that originate from plant remains. In the
damselfish–Polysiphonia cultivation mutualism,
*Snig Ryukyu (6)
*Snig Ryukyu (6)
P. pacifica
Plac Ryukyu (1)
out Ryukyu (1)
P. howei
*Hpla Ryukyu (1)
*Hpla Thailand (2)
Snig Ryukyu (6)
Sliv Ryukyu (1)
Plac Ryukyu (1)
out Ryukyu (4)
out Hawaii (2)
Womersleyella setacea
100/100
100/100
83/100
64/84
97/100
88/60
P. senticulosa
P. stricta
P. morrowii
out Hawaii (1)
Dpro Ryukyu (3)
other Polysiphonia
Murrayella periclados
Laurencia filiformis
0.005 substitutions/site
51/52
99/98
98/100
Stegastes lividus (Sliv)
Dischistodus prosopotaenia (Dpro)
Stegastes nigricans (Snig)
Plectroglyphidodon lacrymatus (Plac)
Hemiglyphidodon plagiometopon (Hpla)
outside fish territory (out)
Polysiphonia sp.1
Polysiphonia sp.2
Polysiphonia sp.3
Polysiphonia sp.4
D. prosopotaenia
H. plagiometopon
P. lacrymatus
S. nigricans
Figure 2. Phylogeny of Polysiphonia spp. algae found inside and outside the territories of the damselfishes Stegastes nigricans,
S. lividus,Plectroglyphidodon lacrymatus,Hemiglyphidodon plagiometopon and Dischistodus prosopotaenia. The association of each
alga is denoted by the abbreviation and colour of its damselfish host species and by collection site. An asterisk denotes the
dominance of the alga in samples (representing more than 50% of the biomass). Numbers in parentheses indicate the
number of DNA samples. Data for unshaded species denote citations from the NCBI GenBank. The tree was obtained
using ML method, with a log-likelihood score of 4971.063. Branches that collapse in MP, ML and/or BI trees are presented
as dotted lines. Nodal support is assessed by bootstrap values of MP and posterior probabilities of BI (above branches,
MP/BI, respectively). Solid and broken arrows indicate obligate and facultative associations, respectively.
Table 1. Algae that inhabited the algal farms of damselfishes and their relationships with damselfishes.
attributes Polysiphonia sp. 1 Polysiphonia sp. 3 Polysiphonia sp. 2 and 4
habitat only algal farms of
S. nigricans
mainly algal farms of
H. plagiometopon
only algal farms of
P. lacrymatus (sp. 2) or
D. prosopotaenia (sp. 4)
dependence of algae on fish obligate facultative obligate
intensity of farming by fish intensive intensive extensive
dependence of fish on algae obligate obligate partial
type of interaction obligate cultivation
mutualism
facultative cultivation
mutualism
commensalism
status of algae cultivated semicultivated weed
Obligate cultivation mutualism H. Hata & M. Kato 595
Biol. Lett. (2006)
however, the damselfishes harvest photosynthate from
algae cultivated on a sunlit substratum. Thus, this is
the second example of an obligate cultivation mutual-
ism between plant and herbivore, preceded by the
crop–human cultivation mutualism, and the first
example in a marine ecosystem.
We thank E. Toby Kiers, Carl Smith and an anonymous
reviewer for helpful comments on the manuscript, and
Atsushi Kawakita and Yudai Okuyama for their help with
molecular experiments. This study is supported by JSPS
Research Fellowships for Young Scientists.
Aanen, D. K., Eggleton, P., Rouland-Lefe
`vre, C.,
Guldberg-Frøslev, T., Rosendahl, S. & Boomsma, J. J.
2002 The evolution of fungus-growing termites and their
mutualistic fungal symbionts. Proc. Natl Acad. Sci. USA
99, 14 887–14 892. (doi:10.1073/pnas.222313099)
Branch, G. M. 1981 The biology of limpets: physical
factors, energy flow, and ecological interactions. Ocea-
nogr. Mar. Biol. Ann. Rev. 19, 235–380.
Ceccarelli, D. M., Jones, G. P. & McCook, L. J. 2001
Territorial damselfishes as determinants of the structure
of benthic communities on coral reefs. Oceanogr. Mar.
Biol. Ann. Rev. 39, 355–389.
Eriksson, B. K. & Johansson, G. 2005 Effects of sedimen-
tation on macroalgae: species-specific responses are
related to reproductive traits. Oecologia 143, 438–448.
(doi:10.1007/s00442-004-1810-1)
Harlan, J. R. 1992 Crops and man, 2nd edn Madison, WI:
American Society of Agronomy.
Harris, D. R. & Hillman, G. C. 1989 Foraging and farming:
the evolution of plant exploitation. London, UK: Unwin
Hyman.
Hata, H. & Kato, M. 2002 Weeding by the herbivorous
damselfish Stegastes nigricans in nearly monocultural
algae farms. Mar. Ecol. Prog. Ser. 237, 227–231.
Hata, H. & Kato, M. 2003 Demise of monocultural algal-
farms by exclusion of territorial damselfish. Mar. Ecol.
Prog. Ser. 263, 159–167.
Hata, H. & Kato, M. 2004 Monoculture and mixed-species
algal farms on a coral reef are maintained through
intensive and extensive management by damselfishes.
J. Exp. Mar. Biol. Ecol. 313, 285–296. (doi:10.1016/
j.jembe.2004.08.009)
Korb, J. & Aanen, D. K. 2003 The evolution of uniparental
transmission of fungal symbionts in fungus-growing
termites (Macrotermitinae). Behav. Ecol. Sociobiol. 53,
65–71.
Lee, J. S. F. & Barlow, G. W. 2001 Recruiting juvenile
damselfish: the process of recruiting into adult colonies
in the damselfish Stegastes nigricans.Acta Ethol. 4, 23–29.
(doi:10.1007/s102110100040)
Mueller, U. G., Gerardo, N. M., Aanen, D. K., Six, D. L.
& Schultz, T. R. 2005 The evolution of agriculture in
insects. Annu. Rev. Ecol. Evol. Syst. 36, 563–595.
(doi:10.1146/annurev.ecolsys.36.102003.152626)
Rindi, F. & Cinelli, F. 2000 Phenology and small-scale
distribution of some rhodomelacean red algae on a
western Mediterranean rocky shore. Eur. J. Phycol. 35,
115–125. (doi:10.1080/09670260010001735701)
Rindos, D. 1984 The origin of agriculture: an evolutionary
perspective. San Diego, CA: Academic Press.
Saunders, G. W. & Kraft, G. T. 1994 Small-subunit rRNA
gene sequences from representatives of selected families
of the Gigartinales and Rhodymeniales (Rhodophyta). 1.
Evidence for the Plocamiales ord. nov. Can. J. Bot. 72,
1250–1263.
Silliman, B. R. & Newell, S. Y. 2003 Fungal farming in a
snail. Proc. Natl Acad. Sci. USA 100, 15 643–15 648.
(doi:10.1073/pnas.2535227100)
Vega, F. E. & Blackwell, M. 2005 Insect-fungal associations:
ecology and evolution. Oxford, UK: Oxford University
Press.
Yoshida, T. 1998 Marine algae of Japan. Tokyo, Japan:
Uchida Rokakuho Publishing.
596 H. Hata & M. Kato Obligate cultivation mutualism
Biol. Lett. (2006)
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