Molecular phylogeny of shrimps from the genus
Lysmata (Caridea: Hippolytidae): the evolutionary
origins of protandric simultaneous hermaphroditism
and social monogamy
J. ANTONIO BAEZA1,2*, CHRISTOPH D. SCHUBART3, PETRA ZILLNER3,
SOLEDAD FUENTES4 and RAYMOND T. BAUER4
1Smithsonian Tropical Research Institute, Apartado Postal 0843-03092, Balboa, Anc?n, Republic of
Panama
2Smithsonian Marine Station at Fort Pierce, 701 Seaway Drive, Fort Pierce, FL 34949, USA
3Biologie 1, Universit?t Regensburg, D-93040 Regensburg, Germany
4Department of Biology, University of Louisiana at Lafayette, Lafayette, LA 70504-2451, USA
Received 9 May 2008; accepted for publication 20 June 2008
Shrimps from the genus Lysmata are known because of their wide diversity of lifestyles, mating systems, symbiotic
partnerships, and conspicuous coloration. They can occur in crowds (large aggregations), in small groups, or as
socially monogamous pairs. Shrimps from this genus are rare, if not unique among crustaceans, because of their
unusual sexual system. To date, the sexual system of all species investigated comprises a protandric simultaneous
hermaphroditism: shrimps initially mature and reproduce as males and later in life turn into functional
simultaneous hermaphrodites. The evolutionary relationships of the species within the genus are unsettled. A
molecular phylogeny of the group may shed light on the evolutionary origins of the peculiar sexual and social
systems of these shrimps and help resolve standing taxonomic questions long overdue. Using a 647-bp alignment
of the 16S rRNA mitochondrial DNA, we examined the phylogenetic relationship of 21 species of shrimps from the
genus Lysmata from several biogeographical regions; the Atlantic, Pacific, and Indo-Pacific. The resulting phylog-
eny indicates that the genus is paraphyletic and includes the genus Exhippolysmata. The constituent species are
subdivided into three well supported clades: one group exclusively composed of neotropical species; a second clade
comprising the Indo-Pacific and Atlantic symbiotic fish cleaner shrimps; and a third clade including tropical and
temperate species from the Atlantic and Pacific. The molecular phylogeny presented here does not support a
historical contingency hypothesis, previously proposed to explain the origins of protandric simultaneous hermaph-
roditism within the genus. Furthermore, the present study shows that monogamous pair-living is restricted to one
monophyletic group of shrimps and therefore probably evolved only once. ? 2009 The Linnean Society of London,
Biological Journal of the Linnean Society, 2009, 96, 415?424.
ADDITIONAL KEYWORDS: 16s rRNA ? hermaphrodite ? mitochondrial DNA.
INTRODUCTION
Shrimps from the genus Lysmata Risso, 1816 demon-
strate a wide diversity of lifestyles, mating systems,
symbiotic partnerships, and coloration. The 36
described species (Chace, 1997; Rhyne & Lin, 2006;
Baeza & Anker, 2008; Rhyne & Anker, 2008) inhabit
shallow or deep warm temperate and tropical rocky
and coral reefs around the world. Some species live
in crowds (aggregations), others in small groups,
whereas some species are socially monogamous (pair-
living) [e.g. Lysmata grabhami (Gordon, 1935); Wirtz,
1997]. Several species with a somewhat drab colora-
tion dwell freely among rocks in temperate localities,
whereas other more colourful species inhabit tropi-
cal sponges (Lysmata pederseni Rhyne & Lin, 2006;*Corresponding author. E-mail: baezaa@si.edu
Biological Journal of the Linnean Society, 2009, 96, 415?424. With 2 figures
? 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 415?424 415
Rhyne & Lin, 2006). Other strikingly brilliant species
clean fishes [Lysmata amboinensis (De Man, 1888);
Limbaugh, Pederson & Chace, 1961; Fiedler, 1998]
This behavioural and ecological diversity has
attracted the attention of systematists, evolutionary
biologists, and behavioural ecologists (Bauer & Holt,
1998; Baeza, 2006; Baeza & Anker, 2008). Ongoing
studies use various species from this genus as model
systems to test sex allocation and sexual selection
theories and the effect of environmental conditions
in explaining phenotypic plasticity (Baeza & Bauer,
2004; Baeza, 2006, 2007a, b). Due to their diverse
socioecology, shrimps from the genus Lysmata repre-
sent ideal candidates to explore the role of ecological
conditions in explaining evolutionary innovations in
the marine environment.
In addition to their assorted lifestyles, shrimps
from the genus Lysmata are unusual among crusta-
ceans for their enigmatic sexual system. All species
studied to date are protandric simultaneous her-
maphrodites (PSH) [L. grabhami: Wirtz, 1997; L.
amboinensis: Fiedler, 1998; Lysmata wurdemanni
(Gibbes, 1850): Bauer & Holt, 1998; Lysmata seti-
caudata Risso, 1816 and Lysmata nilita Dohrn &
Holthuis, 1950: d?Udekem d?Acoz, 2003; Lysmata
californica (Stimpson, 1866): Bauer & Newman, 2004;
Lysmata hochi Baeza & Anker, 2008: Baeza & Anker,
2008; Lysmata bahia Rhyne & Lin, 2006 and Lysmata
intermedia (Kingsley, 1879): Baeza, 2008a; Lysmata
nayaritensis Wicksten, 2000: Baeza, Reitz & Collin,
2008; Lysmata boggessi Rhyne & Lin, 2006 and
Lysmata galapaguensis Wicksten, 2000: J.A. Baeza,
unpubl. observ.]. This unusual sexual system appears
to be a trait shared by all species of the genus (Baeza,
2008a).
In shrimps with PSH, juveniles develop to males
(?male-phase?; Bauer, 2000). The gonads of these males
are ovotestes, with well developed testes but undevel-
oped ovaries, and these shrimps reproduce solely as
males (Bauer & Holt, 1998; Bauer & Newman, 2004;
Baeza & Anker, 2008; Baeza et al., 2008). Later in life,
males mature to functional simultaneous hermaphro-
dites capable of reproducing both as male and female.
These functional simultaneous hermaphrodites have
been termed female-phase individuals or simulta-
neous hermaphrodite phase individuals by Bauer
(2000) and Baeza (2006), respectively. Because most
studies employ the term ?simultaneous hermaphro-
dite? or some variation thereof (Lin & Zhang, 2001;
Baeza, 2006, 2007a, b, c; Calado & Dinis, 2007), the
terms males and hermaphrodites are used hereafter to
describe the two sexual phases.
Protandric simultaneous hermaphroditism appears
to be shared by the related genus Exhippolysmata
Stebbing, 1915 (Kagwade, 1982; Braga et al., 2007;
Laubenheimer & Rhyne, 2008). Other than in the
Crustacea, PSH has been demonstrated for a few
other marine invertebrates (i.e. the polychaete Ophry-
otrocha diadema: Premoli & Sella, 1995; the land
snail Achatina fulica: Tomiyama, 1996; the tunicate
Pyura chilensis: Manr?quez & Castilla, 2005; the bar-
nacle Chelonibia patula: Crisp, 1983). However, PSH
might be more common (Baeza, 2006) than originally
reported among both invertebrates and vertebrates
(Ghiselin, 1974; Policansky, 1982; Crisp, 1983).
Several interesting questions of evolutionary signifi-
cance can be raised about the genus Lysmata, which
has been the focus of an increasing number of studies
focusing on systematics (Rhyne & Lin, 2006; Baeza &
Anker, 2008; Rhyne & Anker, 2008), behavioural
ecology (Baeza & Bauer, 2004; Baeza, 2006, 2007a, b,
2008b; Bauer, 2006), and reproductive biology (Fiedler,
1998; Bauer & Holt, 1998; Bauer & Newman, 2004;
Baeza, 2006, 2008a; Baeza et al., 2008). The evolution-
ary relationships among Lysmata spp. are currently
unknown because no phylogenies, neither morphologi-
cal nor molecular, have been published. This lack of
phylogenetic knowledge is constraining an under-
standing of the evolution of sexual and social systems,
sex allocation patterns, and cleaning behaviours,
among other topics, in marine shrimps.
First, a molecular phylogeny of the Lysmata
should help resolve the historical origins of the rare,
if not unique sexual system from this genus.
Although we now know that the variety of lifestyles
of Lysmata is greater than originally recognized
(Baeza, 2008a; Baeza & Anker, 2008; Baeza et al.,
2008), an emerging dichotomy in social organization
and ecology was noticed in the initial studies. Some
species were noticed to live in crowds (aggregations),
whereas other specialized fish cleaners are socially
monogamous (living in pairs) and live in symbiosis
with sea anemones (Bauer, 2006). Currently, it is not
known whether the symbiotic socially monogamous
condition evolved once or various times indepen-
dently within the genus. Based on this initial
dichotomy, Bauer (2006) suggested that PSH evolved
in the tropics from an ancestral protandric species of
Lysmata that became a specialized fish cleaner.
Restricted mobility of individuals due to their asso-
ciation with the host, and hence a reduced probabil-
ity of encountering mating partners, would have
favoured PSH (Bauer, 2000). Under such a scenario,
the crowd warm-temperate species that do not
exhibit specialized cleaning behaviours would have
evolved from tropical species with specialized clean-
ing behaviours and more complex mating systems
(social monogamy) (Bauer, 2006).
A phylogeny should also help answer other long-
standing controversies about various systematic
questions. In the present study, we have specifically
focused in addressing the phylogenetic position of the
416 J. A. BAEZA ET AL.
? 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 415?424
genus Exhippolysmata, the only other known car-
idean genus with PSH (Braga et al., 2007; Laubenhe-
imer & Rhyne, 2008), with respect to members of the
genus Lysmata.
We present a molecular phylogeny of the genus
Lysmata based on the large subunit 16S mitochon-
drial rRNA gene upon examination of 20 available
species from the genus plus outgroups. The signifi-
cance of the phylogeny for answering the questions
posed above is discussed.
MATERIAL AND METHODS
A total of 28 specimens from 21 species of shrimps
from the genus Lysmata and Exhippolysmata oplo-
phoroides were included in the present study
(Table 1). One specimen each of Merguia rhizophorae,
Heptacarpus palpator, Tozeuma carolinense, Hip-
polyte williamsi, and Hippolyte inermis was also
included as the outgroup during the phylogenetic
analyses. Most shrimp species were collected between
Table 1. Lysmata species and other hippolytid shrimps used for the phylogeny reconstruction
Species Collection site (year) CN/GenBank
Lysmata amboinensis (De Man, 1888), Java Aquarium store, Singapore (2000) SMF 32281/EU861487
Lysmata amboinensis, Philippines Aquarium store, FL, USA (2006) UMML 32.9451/EU861488
Lysmata ankeri Rhyne & Lin, 2006 SMEE, Fort Pierce (2006) UMML 32.9452/EU861501
Lysmata bahia Rhyne & Lin, 2006 Bocas del Toro, Panama (2006) UMML 32.9453/EU861503
Lysmata boggessi Rhyne & Lin, 2006 St Petersburg, FL, USA (2006) UMML 32.9454/EU861505
Lysmata californica (Stimpson, 1866) La Jolla, CA, USA (2006) UMML 32.9455/EU861498
Lysmata debelius Bruce, 1983, Indo-Pacific Aquarium store, LA, USA (2001) SMF 32009/EU861491
Lysmata debelius, Java Aquarium store, Singapore (1999) SMF 32280/EU861493
Lysmata debelius, Philippines Aquarium store, FL, USA (2006) UMML32.9456/EU861492
Lysmata galapaguensis Wicksten, 2000 Islas Secas, Panama (2007) UMML32.9457/EU861480
Lysmata gracilirostris Wicksten, 2000 Venao, Panama (2006) UMML32.9458/EU861502
Lysmata grabhami (Gordon, 1935), Haiti Aquarium store, FL, USA (2006) UMML32.9459/EU861489
Lysmata grabhami, Madeira Madeira, Portugal (2001)
(Ricardo Calado)
SMF 32007/EU861490
Lysmata hochi Baeza and Anker, 2008 Long Key, FL (2007) UMML32.9460/EU861507
Lysmata intermedia (Kingsley, 1879) Bocas del Toro, Panama (2007) UMML32.9461/EU861484
Lysmata moorei (Rathbun, 1901) Galeta, Panama (2007) UMML32.9462/EU861481
Lysmata nayaritensis Wicksten, 2000 Chumical, Panama (2007) UMML32.9463/EU861506
Lysmata nilita Dohrn and Holthuis, 1950 Giglio, Italy (2000) (C?dric
d?Udekem d?Acoz)
SMF32005/EU861482
Lysmata olavoi Fransen, 1991 Azores, Portugal (1999) (C?dric
d?Udekem d?Acoz)
SMF 32006/EU861494
Lysmata pederseni Rhyne & Lin, 2006 Carrie Bow, Belize (2007) UMML 32.9464/EU861504
Lysmata rafa Rhyne & Anker, 2008 Aquarium Store, FL, USA UMML 32.9465/EU861495
Lysmata seticaudata (Risso, 1816) Corsica, France (2003) SMF 32004/EU861485
Lysmata seticaudata Cabo Raso, Cascais, Portugal
(C?dric d?Udekem d?Acoz)
SMF 32003/EU861486
Lysmata cf. trisetacea (Heller, 1861) Chumical, Panama (2007) UMML 32.9466/EU 861483
Lysmata wurdemanni (Gibbes, 1850), TX Port Aransas, TX, USA (2000) SMF 32008/EU861496
Lysmata wurdemanni, West FL St Petersburg, FL, USA (2006) UMML 32.9467/EU861497
Lysmata wurdemanni, East FL Fort Pierce, FL, USA (2006) UMML 32.9468/EU861500
Exhippolysmata ophloporoides (Holthuis, 1948) Ubatuba Bay, Brazil (2006) UMML 32.9469/EU861510
Heptacarpus palpator (Owen, 1939) La Jolla, CA, USA (2001) SMF 32282/EU861509
Hippolyte inermis Leach, 1815 Venice Lagoon, Italy (1997)
(C?dric d?Udekem d?Acoz)
SMF 32283/EU861511
Hippolyte williamsi Schmitt, 1924 Puerto Aldea, Chile (2007) UMML 32.9470/EU861512
Merguia rhizophorae (Rathbun, 1900) Bocas del Toro, Panama (2007) UMML 32.9471/EU861508
Tozeuma carolinense Kingsley, 1878 St Petersburg, FL (2007) UMML 32.9472/EU861513
The sites of collection, dates, museum catalogue number (CN: UMML, University of Miami Marine Laboratories,
Rosenthiel School of Marine Science, University of Miami, SMF Senckenberg Museum Frankfurt, Germany) and the
Genbank accession numbers (GenBank) are shown for each species.
MOLECULAR PHYLOGENY OF LYSMATA SHRIMPS 417
? 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 415?424
2006 and 2007 from different localities in Bocas del
Toro and Islas Secas and other localities close to Naos
Island (Panama), Florida and Texas (USA). Four
species (Lysmata ankeri, Lysmata debelius, L.
amboinensis and L. grabhami) were either purchased
from aquarium stores in Fort Pierce (FL, USA) or
donated from the Smithsonian Marine Ecosystems
Exhibit (Fort Pierce, FL, USA). The remaining
species were from colleague donations (see Acknowl-
edgements). Immediately after collection or purchas-
ing, specimens were preserved in 95?99% ethanol. In
the laboratory, the different species were identified
as previously reported by Bruce (1983), Rhyne & Lin
(2006), Baeza & Anker (2008), Rhyne & Anker (2008)
and using the keys of Chace (1972, 1997) and
Wicksten (2000).
Total genomic DNA was extracted from abdominal
muscle tissue using the Qiagen DNeasy Blood and
Tissue Kit following the manufacturer?s protocol. The
polymerase chain reaction (PCR) was used to amplify
an approximately 550-bp region (excluding primers)
of the 16S rRNA with the primers 16L2 (5?-
TGCCTGTTTATCAAAAACAT-3?), and 1472 (5?-
AGATAGAAACCAACCTGG-3?) (Schubart, Neigel &
Felder, 2000; Schubart, Cuesta & Felder, 2002). Stan-
dard PCR 25-mL reactions [2.5 mL of 10 ? Taq buffer,
2 mL of 50 mM MgCl2, 2.5 mL of 10 mM dNTPs, 2.5 mL
each of the two primers (10 mM), 0.625 U Taq,
1.25 mL of 20 mM BSI and 8.625 mL double distilled
water] were performed on a Peltier Thermal Cycler
(DYAD) under the conditions: initial denaturation
at 96 ?C for 4 min followed by 40 cycles of 94 ?C for
45 s, 48?52 ?C (depending on the species) for 1 min,
and 72 ?C for 1 min, followed by chain extension at
72 ?C for 10 min. PCR products were purified with
ExoSapIT (a mixture of exonuclease and shrimp
alkali phosphatase; Amersham Pharmacia) and
sequenced with the ABI Big Dye Terminator Mix
(Applied Biosystems) at the Laboratory of Analytical
Biology of the National Museum of Natural History
(Washington, DC), which is equipped with an ABI
Prism 3730xl Genetic Analyser (Applied Biosystems).
All sequences were confirmed by sequencing both
strands and a consensus sequence for the two strands
was obtained using the software SEQUENCHER,
version 4.5 (Gene Codes Corp.). A smaller number of
sequences were obtained at the laboratories of the
University of Regensburg using the same primers and
PCR conditions and otherwise the protocol outlined in
Schubart et al. (2002).
The final set of consensus sequences was aligned
with the integrated CLUSTALW and corrected manu-
ally with BIOEDIT, version 7 (Hall, 1999) and then
exported to PAUP* (Swofford, 2002) and MrBayes
(Huelsenbeck, 2000). First, the dataset was analysed
with MODELTEST, version 3.7 (Posada & Crandall,
1998) in PAUP*, which compares different models of
DNA substitution in a hierarchical hypothesis-testing
framework to select a base substitution model that
best fits the data. The optimal model found by MOD-
ELTEST (selected with hierarchical likelihood ratio
tests) was a TVM+I+G evolutionary model
(-ln L = 6189.1997). The calculated parameters were:
assumed nucleotide frequencies A = 0.3441,
G = 0.1839, T = 0.3796, C = 0.0924; substitution rate
matrix with A?C substitution = 1.0, A?G = 6.4199,
A?T = 1.0, C?G = 1.0, C?T = 9.3030, G?T = 1.0; rates
for variable sites assumed to follow a gamma distri-
bution (G) with shape parameter = 0.3711 and a pro-
portion of invariable sites (I) = 0.2193.
Phylogenetic analyses conducted herein were
maximum parsimony (MP) and maximum likelihood
(ML) (in PAUP*) and Bayesian inference (BI; in
MrBayes). MP analysis was performed as a heuristic
search with a starting tree obtained via stepwise
addition, random addition of sequences, random rep-
licates, and tree-bisection-reconnection branch swap-
ping. For ML, the specifications were the same as in
MP. However, branch swapping was performed in the
starting tree and all other parameters used were
those of the default option in PAUP*. For BI, we used
unique random starting trees in the Metropolis?
coupled Markov Monte Carlo Chain (MCMC)
(Huelsenbeck, 2000). During a first preliminary run,
convergence was achieved after 5000 generations at a
likelihood value of -6222.45. A final analysis was
performed for 6 000 000 generations. Every 100th
tree was sampled from the MCMC analysis obtaining
a total of 60 000 trees. From the preliminary run we
determined a burn-in period of 10 000 generations,
calculating a consensus tree with the 50% majority
rule for the last 59 900 sampled trees. We assessed
the robustness of the MP and ML tree topologies by
bootstrap reiterations of the data 2000 and 100 times,
respectively and reconstructing trees using each resa-
mpled data set (Felsenstein, 1985). Support for nodes
in the BI tree topology was obtained by posterior
probability values that represent the frequency with
which each clade occurred within the collection of
trees provided by the analysis.
RESULTS
A total of 647 homologous alignment positions were
used during the present phylogenetic analysis and
257 of these were found to be parsimony informative
positions. It is noteworthy that the species L. bahia
had an insertion of 71 bp in the middle of the 16S
fragment and not shared by any other species. All
phylogenetic trees obtained with the different infer-
ence methods (MP, ML, and BI) resulted in the same
general topology (Fig. 1). Considering our pool of out-
418 J. A. BAEZA ET AL.
? 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 415?424
group species, belonging to three different genera
within the Hippolytidae, the genus Lysmata plus
Exhippolymata oplophoroides represent a monophylo-
genetic clade and Lysmata olavoi is the most basally
positioned species within this clade, which is sup-
ported by a high posterior probability obtained from
the BI analysis. Bootstrap support from the ML and
MP was in general lower. The overall tree topology
suggests that the genus Lysmata can be divided into
three main clades plus a number of unresolved
species. One clade, hereafter named ?Tropical Ameri-
can?, is composed almost entirely by species from the
Caribbean. Within this clade, the only species from
outside the Caribbean is Lysmata gracilirostris from
the eastern tropical Pacific. The basal position of
this species within the clade is not well supported.
Also, two pairs of species are well supported as
sister species: L. pederseni?L. ankeri and L. boggessi?
Lysmata rafa. Interestingly, the specimens of L. wur-
demanni collected from distant localities from the
northern Gulf of Mexico (Texas and western Florida)
are more closely related to each other than to the
specimen collected from the east coast of Florida.
Consequently, the latter specimen holds a basal posi-
tion within this species.
The second so-called ?Cosmopolitan? clade is com-
posed of six species, two from the Mediterranean
(L. seticaudata and L. nilita), two from the Atlantic
(Lysmata moorei and L. intermedia) and two from
the tropical eastern Pacific (L. galapaguensis and
Lysmata cf. trisetacea). Within this clade, the status of
L. intermedia and L. cf. trisetacea as a pair of trans-
isthmian sister species is well supported and may be
used for molecular clock calibrations in the future.
The third group, hereafter named ?Cleaner? clade, is
composed by the fish-cleaning shrimps L. debelius,
L. amboinensis and L. grabhami. In the tree, the
phylogenetic positions of L. bahia, L. californica and
L. nayaritensis are not well supported by any of our
phylogenetic analyses. Most interesting is that the
Caribbean L. hochi and the Brazilian Exhippolysmata
ophlophoroides form a monophyletic clade in all recon-
struction methods, which is highly supported by BI.
However, the position of this clade among the other
representatives of Lysmata is not well supported.
Interestingly, all of the species whose sexual system
have been examined (all of them feature PSH) belong
to all three natural clades here revealed (Fig. 1).
The distributions of the different socioecologies (social
monogamy versus crowds) and lifestyles (symbiosis
versus free-living) were not interspersed in the tree
(Fig. 2). Social monogamy was restricted to the
?Cleaner? clade, whereas crowd species were found in
other clades either more or less derived than the
?Cleaner? clade. Some species recently reported as
living in small groups with local abundances much
lower than that reported for crowd species (L. bahia,
L hochi, and L. intermedia; Baeza et al., 2008) are
also present in two natural groups other than the
?Cleaner? clade (Fig. 2). In addition, symbiotic species
are almost invariably restricted to the ?Cleaner?
clade. The only exception is L. pederseni from the
?American-Tropical? clade known from tube sponges in
the Caribbean.
DISCUSSION
We present, for the first time, a molecular phylogeny
of shrimps from the genus Lysmata based on a
segment of the 16S rRNA mitochondrial gene.
Although not all of the 36 species described for the
genus could be included, our analyses with three
different phylogenetic reconstruction methods support
the monophyly of this genus together with the genus
Exhippolysmata. Therefore, the genus Lysmata is cur-
rently paraphyletic. Below, we discuss our findings
with respect to the evolutionary origins of PSH and
the different social structures observed in this genus.
EVOLUTIONARY ORIGINS OF PSH AND
LIFESTYLES IN LYSMATA
In our molecular phylogeny, the symbiotic fish cleaner
shrimps L. amboinensis, L. grabhami, and L. debelius
cluster together within a well-supported clade. This
?Cleaner? clade holds a derived phylogenetic position
with respect to the ?Cosmopolitan? clade and L. olavoi.
By contrast to the ?Cleaner? clade, composed solely by
socially monogamous species, several species in the
?Cosmopolitan? clade as well as in the other natural
clades live in crowds and do not engage in any symbi-
otic partnership with sessile macroinvertebrates
(L. galapaguensis, L. cf. trisetacea; J.A. Baeza, unpubl.
data). Furthermore, all of the species belonging to the
?Cosmopolitan? and ?American Tropical? clades whose
sexual system have been examined, were found to be
protandric simultaneous hermaphrodites (Rhyne &
Lin, 2006; Baeza, 2008a; J. A. Baeza, unpubl. data).
Indeed, it appears that PSH represents a conserved
trait within the genus Lysmata (Baeza, 2008a) and
Exhippolysmata (Kagwade, 1982; Braga et al., 2007).
The fact that a basally positioned group of species
pertaining to the ?Cosmopolitan? clade features PSH
and most commonly lives freely in the intertidal as
aggregations (crowds) does not support Bauer?s (2000)
view about the historical origins of the sexual system.
According to the historical contingency hypothesis of
Bauer (2000), PSH originated in an ancestral protan-
dric species of Lysmata that became a symbiotic
specialized fish cleaner. The crowd warm-temperate
species that do not exhibit specialized cleaning behav-
iours would have invariably evolved from tropical
MOLECULAR PHYLOGENY OF LYSMATA SHRIMPS 419
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420 J. A. BAEZA ET AL.
? 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 415?424
species with specialized cleaning behaviours and more
complex mating systems (social monogamy) (Bauer,
2006). Thus, we a priori expected that all of the species
with a free-living life style and not featuring special-
ized cleaning behaviours (originally termed as ?crowd?
species; Bauer, 2000), but shown as belonging to the
?Cosmopolitan? and ?Tropical-American? clades in the
present study, would have clustered in one or more
clades invariably separated on their own branch from
the ?Cleaner? socially monogamous clade. Recent
studies have demonstrated that shrimps from the
genus Lysmata feature a diversity of lifestyles, wider
than initially recognized (Baeza, 2008a; Baeza &
Anker, 2008; Baeza et al., 2008). Further detailed
studies on the lifestyle and sexual system of the species
within this genus and others related (Lysmatella,
Mimocaris, Exhippolysmata) and the development of a
new more robust phylogeny with additional molecular
Figure 1. Phylogenetic tree obtained from Bayesian inference (BI) analysis of the partial 16S rRNA gene for shrimps
from the genus Lysmata, and other selected taxa from the family Hippolytidae. Numbers above or below the branches
represent the posterior probabilities from the BI analysis and bootstrap values obtained either from maximum likelihood
(ML) or maximum parsinomy (MP) analyses in PAUP* (BI/ML/MP). The general topology of the trees obtained from MP
and ML analyses was the same. PSH indicates that protandric simultaneous hermaphroditism has been confirmed as the
sexual system of a particular species (for details, see text). The images of the shrimps (from top to bottom) represent
Lysmata boggessi, Lysmata debelius, and Lysmata galapaguensis.

Figure 2. Sociobiology and lifestyle in shrimps from the genus Lysmata. On the left, the white, gray and black squares
represent the socioecology (social monogamy, small groups and crowds, respectively) of each species. On the right, the
white and black squares represent the adoption of a symbiotic or free-living lifestyle by a particular species, respectively.
The absence of a square indicates that the socioecology or lifestyle of a particular species is not known. In the text, ?social
monogamy? and ?crowds? are occasionally used as terms for ?pair-living? and ?aggregations?, respectively.
MOLECULAR PHYLOGENY OF LYSMATA SHRIMPS 421
? 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 415?424
markers should help us elucidate the actual historical
origins of PSH among shrimps in the near future.
The fish cleaner shrimps L. amboinensis, L. grab-
hami, and L. debelius clustered together within a well
supported clade that is derived compared to the ?Cos-
mopolitan? clade and L. olavoi. The information above
suggests that social monogamy and the adoption of a
symbiotic lifestyle (e.g. associated with sea anemones)
are derived conditions within the genus and that
these traits evolved once in the ancestor of the
?Cleaner? clade. A second independent origin of a sym-
biotic lifestyle occurred in the ?Tropical American?
clade because L. pederseni is known only from tube
sponges (Rhyne & Lin, 2006; J. A. Baeza, unpubl.
data). Unfortunately, the host use pattern and popu-
lation distribution of this species in their sponge host
is presently not known. Most importantly, for the
?Cleaner? clade, the origins of pair living and the
adoption of a symbiotic lifestyle appear to be linked in
some manner with the origins of cleaning behaviour;
all three traits are present in the ?Cleaner? clade but
absent in the remaining natural clades, except for
L. pederseni. However, the reasons for the association
among these three traits are not evident at first sight.
Social monogamy is known to occur in several other
free-living and symbiotic crustaceans, including
shrimps (Baeza, 1999, 2008b; Baeza & Thiel, 2007).
Various hypotheses have been proposed to explain the
origins and adaptive value of social monogamy (?mate
guarding? hypothesis: Parker, 1970; Grafen & Ridley,
1983; ?territorial cooperation? hypothesis: Wickler &
Seibt, 1981; ?environmental constraints? hypothesis:
Baeza & Thiel, 2007). Because food obtained from
client fish might be the most important food source
for these cleaner shrimps, and a cleaning station
therefore would be a highly valuable resource (as
suggested for cleaner fish; Sikkel, Cheney & C?t?,
2004), it might be possible that a pair of shrimps will
be more successful in establishing and defending a
cleaning spot (i.e. a territory) than a single individual.
The benefits derived from the shared defence of a
cleaning station might be driving territoriality and
social monogamy in these shrimps once cleaning
behaviour has evolved. Alternatively, cleaner shrimps
might live in pairs but not necessarily in long-term
monogamy; shrimps might be moving rather fre-
quently among cleaning stations shifting mating
partners serially during their lifetime, as suggested
for other socially monogamous shrimp Hymenocera
picta (see Wickler & Seibt, 1981). The adaptive value
of social monogamy for Lysmata cleaner shrimps
needs to be clarified. Shrimps in the ?Cleaner? clade
might be interesting examples with which to experi-
mentally test hypotheses about social monogamy and
the adoption of symbiotic lifestyles in the marine
environment.
Interestingly, although social monogamy in the
Cleaner clade appears to represent a conserved trait,
the lifestyle in the other clades is variable, with
closely related species occurring as small groups or
aggregations within the same clade. Some species of
Lysmata inhabit environments in which persistence
might be difficult. For example, L. pederseni occur at
very low frequencies in tube sponges that harbour a
diverse assemblage of fish and other crustaceans
(Rhyne & Lin, 2006; JAB, unpublished observations).
Similarly, L. rafa occur at low frequency in an envi-
ronment where predation pressure seems to be high
(Rhyne & Anker, 2008). PSH in the genus Lysmata
might represent a key innovation favouring the radia-
tion of shrimps into environments in which it might
be difficult to persist for species with separate sexes.
SYSTEMATICS OF THE GENUS LYSMATA
The apparently paraphyletic status of the genus
Lysmata is caused by the position of E. oplophoroides,
from Brazil, that clusters together with the recently
described L. hochi from the Caribbean. Although the
position of these species among the other representa-
tives of Lysmata is not clear, their relatedness was
supported by all tree construction methods. Inclusion
of a shorter unpublished sequence of a second species
of Exhippolysmata made available by Dr. Xinzheng
Li (Institute of Oceanology, Chinese Academy of
Sciences, China), confirmed the position of a second
member of this genus next to L. hochi (not shown).
The differences in morphology between these two
genera are obvious. Shrimps from the genus Ex-
hippolysmata are characterized by slender legs, an
extremely long rostrum (longer than the carapace),
and a dorsal basal crest of teeth (Holthuis, 1948). In
turn, in L. hochi, as well as in other Lysmata spp., the
pereiopods are comparatively robust, the rostrum is
short (usually much shorter than the carapace), and
the dorsal teeth of the carapace are equidistantly
spaced, never forming a crest (Holthuis, 1948; Baeza
& Anker, 2008). This degree of morphological differ-
entiation between closely related species may not be
uncommon within the genus. Because the topology of
our phylogenetic trees does not support the position of
the genus Exhippolysmata as a natural sister clade to
Lysmata, the latter might represent a derived clade
of Lysmata shrimps that colonized deeper waters,
with the presence of a long rostrum and slender long
pereiopods being common among several deep water
shrimp taxa (Bauer, 2004). Because this relationship
was supported by all reconstruction methods, regard-
less whether based on parsimony, likelihood, or dis-
tances (results of simpler distance models not shown),
it appears highly unlikely that the clustering of E.
oplophoroides and L. hochi into a single clade is due
422 J. A. BAEZA ET AL.
? 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 415?424
to long-branch attraction. Future phylogenies includ-
ing more species from both genera as well as other
related ones (i.e. Lysmatella, Merhippolyte, Parhip-
polyte, Mimocaris), and with more and independently
segregating genetic markers, are necessary to confirm
the actual phylogenetic position of Exhippolysmata.
ACKNOWLEDGEMENTS
J.A.B. thanks Bill Hoffman from the Smithsonian
Marine Ecosystems Exhibit (SMEE), Valerie Paul,
Raphael Ritson-Williams, Koty Sharp, and Sherry
Reed from the Smithsonian Marine Station at Fort
Pierce (SMSFP); ORA at Harbor Branch Oceano-
graphic Institution (HBOI); Darryl Felder from the
University of Louisiana at Lafayette (ULL); and
Arthur Anker, Rachel Collin, Arcadio Ortiz, and
Maricela Salazar from the Smithsonian Tropical
Research Institute (STRI), Panama, for their help
during the different steps of specimens collection,
fixation, transportation and sequencing. Arcadio Ortiz
from STRI gently worked out local collecting permits
for shrimps in Kunayala, Panama. Drs C?dric
d?Udekem d?Acoz, Fernando Mantelatto, and Ricardo
Calado kindly made available some species from their
collections. J.A.B. thanks Dr Rachel Collin for invit-
ing him to participate on the research cruise to Islas
Secas, Isla de la Coiba, and other islands off the
tropical eastern Pacific onboard of the R/V Urraca,
STRI, Panama, where various shrimp species were
collected. Many thanks to Jeff Hunt and Lee Weigt at
the Laboratory of Analytical Biology (NMNH, Wash-
ington, DC) for their logistical support. Dr. Xinzheng
Li (Institute of Oceanology, Chinese Academy of Sci-
ences, China) kindly made available an unpublished
sequence of a species of Exhippolysmata. J.A.B.
thanks the support from a STRI Marine Postdoctoral
Fellowship and a SMSFP Postdoctoral Fellowship.
C.D.S. and P.Z. thank J?rgen Heinze for making
available facilities at the Department of Biology of the
University of Regensburg to carry out parts of this
study. This is contribution number 122 of the ULL
Laboratory for Crustacean Research. This is contri-
bution number 753 of the Smithsonian Marine
Station of Fort Pierce, Florida.
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