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Evolution of terrestriality in
Hawaiian species of the genus
Ligia (Isopoda, Oniscidea)
ARTICLE JANUARY 2003

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4 AUTHORS, INCLUDING:
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Italian National Research Co
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University of Barcelona
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EVOLUTION OF TERRESTRIALITY IN HAWAIIAN SPECIES OF THE

GENUS LIGIA (ISOPODA, ONISCIDEA)
BY
STEFANO TAITI1,3), MIQUEL A. ARNEDO2,4), STEVE E. LEW2,5)
and GEORGE K. RODERICK2,6)
1
) Centro di Studio per la Faunistica ed Ecologia Tropicali del C.N.R., Via Romana 17,
I-50125 Florence, Italy
2
) Division of Insect Biology, ESPM, 201 Wellman Hall, University of California-Berkeley,
Berkeley, CA 94720-3112, U.S.A.

ABSTRACT
The genus Ligia (Ligiidae) has a worldwide distribution and currently includes more than 30
nominal species. Most of the species are littoral, halophilic, and occur on rocky seashores, but
seven species are strictly terrestrial. Three species of Ligia have been recorded from the Hawaiian
Islands, one introduced, L. exotica, and two edemic, L. hawaiensis and L. perkinsi. Ligia hawaiensis is a littoral species very common along the rocky coasts of the Hawaiian Islands, and Ligia
perkinsi is a montane terrestrial species occurring on Kauai, Oahu, and Hawaii. Morphological and
molecular data of the endemic species of Ligia from the Hawaiian Islands are used to test whether
the adaptation to the terrestrial environment took place only once or, conversely, it evolved on
each island independently. Populations of L. hawaiensis and L. perkinsi from Kauai and Oahu
were examined. Four non-Hawaiian species of Ligia (L. italica, L. pallasii, L. vitiensis, and L. exotica) and the related ligiid Ligidium hypnorum were included in the molecular analysis as an outgroup. All populations of L. hawaiensis were found to be morphologically identical to each other
and distinctly different from L. perkinsi. The Kauai populations of L. perkinsi differ slightly from
the Oahu one in morphology and ecological habitat. Two regions of mitochondrial DNA were sequenced: 675 bp of cytochrome c oxidase subunit I and approximately 490 bp of 16S rRNA. The
cladogram obtained shows that L. hawaiensis and L. perkinsi belong to the same clade and that L.
perkinsi does not constitute a monophyletic unit. The populations of L. hawaiensis show a remarkably high level of geographic structure suggesting that migratory events between the islands
are uncommon. Thus, the independent colonization of terrestrial habitat by an ancestral seashore
population of Ligia is proposed as the most plausible scenario for the origin of the terrestrial populations.

) e-mail: taiti@fi.cnr.it
) e-mail: miquelan@nature.berkeley.edu
5
) e-mail: stevelewalready@yahoo.com
6
) e-mail: roderick@nature.berkeley.edu
4

Koninklijke Brill NV, Leiden, 2003

Biology of terrestrial isopods, V: 85-102

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RIASSUNTO
Il genere Ligia (Ligiidae) distribuito in tutto il mondo e comprende pi di 30 specie nominali.
La maggior parte delle specie sono litorali alofile ed abitano le coste rocciose, mentre sette specie
sono strettamente terrestri. Tre specie di Ligia sono segnalate per le Isole Hawaii, una introdotta,
L. exotica, e due endemiche, L. hawaiensis e L. perkinsi. Ligia hawaiensis una specie litorale
molto comune lungo le coste rocciose. Ligia perkinsi una specie terrestre presente nelle isole di
Kauai, Oahu e Hawaii. Viene condotta unanalisi morfologica e molecolare delle specie
endemiche di Ligia delle Isole Hawaii allo scopo di vedere se ladattamento allambiente terrestre
abbia avuto origine una sola volta oppure si sia evoluto indipendentemente su ciascuna isola. Sono
state esaminate diverse popolazioni di L. hawaiensis e L. perkinsi provenienti da Kauai e Oahu.
Nellanalisi molecolare sono state incluse anche altre quattro specie di Ligia (L. italica, L. pallasii,
L. vitiensis e L. exotica) ed una popolazione di Lygidium hypnorum come outgroup. Tutte le
popolazioni di L. hawaiensis esaminate si sono dimostrate morfologicamente identiche, ma
chiaramente distinguibili da quelle di L. perkinsi. In L. perkinsi le popolazioni di Kauai
differiscono da quella di Oahu sia nella morfologia che nellhabitat in cui vivono. Due regioni di
DNA mitocondriale sono state sequenziate: citocromo c ossidasi subunit I e 16S rRNA,
rispettivamente di 675 e circa 490 paia di basi. Il cladogramma ottenuto mostra che L. hawaiensis
e L. perkinsi appartengono allo stesso clade, ma che L. perkinsi non costituisce una unit
monofiletica. Le popolazioni di L. hawaiensis mostrano un alto livello di struttura geografica, che
sembra dimostrare come movimenti migratori fra le diverse isole non siano comuni. La
colonizzazione indipendente dellambiente terrestre da parte di una popolazione litorale di Ligia
rappresenta lo scenario pi probabile per lorigine delle popolazioni terrestri.

INTRODUCTION

Terrestrial isopods (Oniscidea) are derived from marine ancestors and represent a very good example of the evolutionary transition of animals from aquatic
environments to terrestrial environments (Warburg, 1968). The group includes
some 4,000 species occurring in all kinds of terrestrial habitats, from the seashores to high mountains and even deserts. At present the Oniscidea are regarded
as a monophyletic unit (Schmalfuss, 1989; Wgele, 1989; Tabacaru &
Danielopol, 1996a, b) and according to Erhard (1998) the family Ligiidae represents the sister group of all other terrestrial isopods.
The genus Ligia Fabricius, 1798 (Ligiidae) is considered to be transitional between ancestral marine and fully terrestrial forms (Carefoot & Taylor, 1995).
Ligia has a worldwide distribution and currently includes over 30 nominal species. Most of the species are halophilic forms occurring in the supralittoral zone
of rocky seashores, just above the water line, where they feed on seaweed. Seven
species are strictly terrestrial and occur in montane habitats in tropical regions,
mostly cloud forests on island mountain ridges. These species are: Ligia simoni
(Dollfus, 1893) from Colombia and Venezuela, L. perkinsi (Dollfus, 1900) from
the Hawaiian islands, L. platycephala (Van Name, 1925) from Venezuela,

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87

Guiana, and Trinidad, L. latissima (Verhoeff, 1926) from New Caledonia, L.

philoscoides Jackson, 1938 from the Austral Islands, L. boninensis Nunomura,
1979 from the Bonin Islands, Japan, and L. taiwanensis Lee, 1994 from Taiwan.
The terrestrial species of Ligia are believed to be derived from littoral forms,
which has led to the suggestion that Ligia provides a good model of colonization
of terrestrial biotopes by isopods (Schmalfuss, 1978). On the basis of morphological similarities, a sister species relationship may be hypothesized between some
terrestrial species of Ligia and species occurring on nearby coasts, e.g., L. perkinsi
and L. hawaiensis Dana, 1853 in the Hawaiian Islands, L. taiwanensis and L. exotica Roux, 1828 in Taiwan, and L. philoscoides and L. rugosa Jackson, 1938 in the
Austral Islands (S. Taiti, pers. obs.). However, nothing is known about the phylogenetic relationships of these species. In the present study we analyse the possible phylogenetic relationships of Ligia species occurring in the Hawaiian Islands.
The Hawaiian Archipelago is the most isolated major archipelago in the world,
lying in the middle of the Pacific Ocean 4,000 km from the nearest major land
masses (North America and Japan) and the nearest comparable island groups (the
Marquesas). The islands are the emerged summits of submarine volcanoes and are
progressively younger proceeding from northwest to southeast (Carson & Clague,
1995): Kauai, Waialeale 5.1 Myr; Oahu, Waianae Range 3.7 Myr, Koolau Range
2.6 Myr; Molokai, Puu Nana 1.9 Myr, Kamakaou, 1.76 Myr; Maui, Puu Kukui
1.32 Myr, Haleakala 0.75 Myr; and Hawaii, Kohala 0.43 Myr, Mauna Kea 0.38
Myr, Mauna Loa and Kilauea 0.4 Myr to present.
Three species of Ligia occur on the Hawaiian Islands, two littoral (L. exotica
and L. hawaiensis) and one terrestrial (L. perkinsi). Ligia exotica has a pantropical distribution and has been recorded from the Hawaiian Islands by Dollfus
(1893) and Richardson (1905). This species is certainly introduced to Hawaii
since it occurs only on the docks of the harbours of Honolulu, Oahu, and Hilo,
Hawaii (S. Taiti, pers. obs.) (fig. 1). Ligia hawaiensis is endemic to the Hawaiian Islands, where it is very common along the rocky coasts. It has been recorded from almost all the major islands (fig. 1), and also from the northwestern
Hawaiian Islands (Taiti & Ferrara, 1991; Taiti & Howarth, 1996; Taiti, unpubl.
data). Ligia perkinsi is also endemic to the Hawaiian islands. It was first recorded by Dollfus (1900) from Kauai and Hawaii, and more recently (Taiti &
Howarth, 1996) from Oahu (fig. 1). On Hawaii Island this species seems to be
no longer present, since it has not been reported since 1896, and numerous attempts to recollect it in recent years have been unsuccessful. This is a montane
species living on wet mossy tree trunks, wet vertical rocky cliffs, rheocrenes,
and stream margins.

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Fig. 1. Recorded distribution of Ligia species from the Hawaiian Archipelago (northwestern islands
not included). The numbers refer to the populations used in the present study: 1, Honolulu Harbour;
2, Kauapea Beach; 3, Kapaa; 4, Lihue; 5, Kukuiula; 6, Pupukea; 7, Coconut Island; 8, Pouhala
Marsh; 9, Ala Wai Canal; 10, Makaleha Mts; 11, Mt. Kahili; 12, Haupu Range; 13, Nuuanu Pali.

Fig. 2. Alternative hypotheses of the evolution of terrestrial lifestyle (closed boxes) in Hawaiian Ligia.
A, independent evolution of terrestrial lifestyle; B, single origin of adaptation to terrestrial habitats.

In the present study a morphological analysis of several populations of the two

Hawaiian endemic species of Ligia (L. hawaiensis and L. perkinsi) from the islands
of Kauai and Oahu is undertaken to detect intra- and interspecific differences. Molecular data are used to test whether these species are the result of a single colonization to the archipelago and whether the adaptation to the terrestrial environment
took place independently on the two islands (i.e., whether L. perkinsi is not monophyletic) (fig. 2A) or only once (i.e., L. perkinsi is monophyletic) (fig. 2B).

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89

MATERIALS AND METHODS

Eight populations of Ligia hawaiensis and four of L. perkinsi were sampled

from the islands of Kauai and Oahu (fig. 1, table I) for both morphological and
molecular analyses.
Adult male and female specimens from each population have been considered
in the morphological analysis. The following characters were compared using a
stereomicroscope and a compound microscope for micropreparations: body size,
cephalon structure and eye dimensions, shape of antennule, antenna, buccal
pieces, pleotelson, pereopods, and pleopods.
Four additional Ligia species were included in the molecular analysis (table
I): L. exotica, L. pallasii Brandt, 1833, L. vitiensis Dana, 1853, and L. italica
Fabricius, 1798. The species Ligidium hypnorum (Cuvier, 1792) in the family
Ligiidae was used as an outgroup. Two to four pereopods from live specimens
collected in the field and fixed in 95% ethanol (in a few cases specimens preserved in 75% ethanol from museum collections) were used for DNA extractions, and the rest of the specimen kept as a voucher (stored at Essig Museum of
Entomology at University of California, Berkeley, U.S.A.).
Total genomic DNA was extracted following the phenol/chloroform protocol
of Palumbi et al. (1991) or using QIAGEN DNeasy Tissue Kits. Partial fragTABLE I
Species and populations sampled in the present study
Species

Populations

Ligidium hypnorum (Cuvier, 1792)

Ligia italica Fabricius, 1798
Ligia vitiensis Dana, 1853
Ligia pallasii Brandt, 1833
Ligia exotica Roux, 1828
Ligia hawaiensis Dana, 1853

Austria: Tirol, Ziller Tal, near Fgen

Italy: Tuscany, Giannutri Island
Madagascar: Nosy-Be
Canada: British Columbia, Sepping Island
U.S.A.: Hawaiian Is, Oahu, Honolulu Harbour
U.S.A.: Hawaiian Is, Kauai, Kauapea Beach
U.S.A.: Hawaiian Is, Kauai, Lihue
U.S.A.: Hawaiian Is, Kauai, Kapaa
U.S.A.: Hawaiian Is, Kauai, Kukuiula
U.S.A.: Hawaiian Is, Oahu, Pouhala Marsh
U.S.A.: Hawaiian Is, Oahu, Coconut Island
U.S.A.: Hawaiian Is, Oahu, Ala Wai Canal
U.S.A.: Hawaiian Is, Oahu, Pupukea
U.S.A.: Hawaiian Is, Kauai, Mt Kahili
U.S.A.: Hawaiian Is, Kauai, Mt Kahili
U.S.A.: Hawaiian Is, Kauai, Makaleha Mts
U.S.A.: Hawaiian Is, Kauai, Haupu Range
U.S.A.: Hawaiian Is, Oahu, Nuuanu Pali
U.S.A.: Hawaiian Is, Oahu, Nuuanu Pali

Ligia perkinsi (Dollfus, 1900)

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ments of the mitochondrial genes cytochrome c oxidase subunit I (CO1) and 16S
rRNA (16S) were amplified using the primer pairs C1-J-1490 and C1-N-2198
(Folmer et al., 1994) (675 bp) and LR-N-13398 (Simon et al., 1994) and LR-J12864 (5-CTCCGGTTTGAACTCAGATCA-3) (Hsiao, pers. comm.) (approx.
490 bp), respectively. In a few cases, a shorter CO1 fragment was amplified with
primer pair C1-J-1751 and C1-N-2191 (Simon et al., 1994) (421 bp). The Perkin
Elmer 9700, Perkin Elmer 9600, or the BioRad i-Cycler were used to perform 25
iterations of the following cycle: 30 s at 95C, 45 s at 42-45C (depending on the
primers) and 45 s at 72C, beginning with an additional single cycle of 2 min at
95C and ending with another one of 10 min at 72C.
The PCR reaction mix contained primers (0.48 M each), dNTPs (0.2 mM
each) and 0.6 U Perkin Elmer AmpliTaq DNA polymerase (for a 50 l reaction) with the supplied buffer and, in some cases, adding an extra amount of
MgCl2 (0.5 to 1.0 mM). PCR products were cleaned using Geneclean II (Bio
101) or QIAGEN QIAquick PCR Purification Kits following the manufacturers
specifications. DNA was directly sequenced in both directions using the dye
terminator cycle sequencing method (Sanger et al., 1977) and the ABI PRISM
BigDyeTM Terminator Cycle Sequencing Ready Reaction with AmpliTaq DNA
Polimerase FS kit. Sequenced products were cleaned using Princeton Separations CentriSep columns and run out on an ABI 377 automated sequencer. Sequences were edited using the Sequencher 3.1.1 software package (Gene Codes
Corporation). Sequences were subsequently exported to the program GDE 2.2
(Smith et al., 1994) running on a Sun Enterprise 5000 Server, and manual alignments were constructed taking into account secondary structure information from
secondary structure models available in the literature. These initial alignments were
used to identify fragments of well-supported homology (i.e., fragments of identical
or well-conserved flanking regions), which were subsequently used as input files
for the analysis after their gaps were removed. All sequences obtained in the present study are available from Genbank under accession numbers: AY051319AY051337 (CO1) and AY051338-AY051356 (16S).
Partial and combined sequence data sets were analysed using the direct optimization method (Wheeler, 1996) as implemented in the computer program POY
(Wheeler & Gladstein, 2000). This method circumvents problems inherent in a
priori alignment by incorporating the search for the most parsimonious optimizations of insertion/deletion events into the evaluation of candidate topologies. The
heuristic search strategy used combined 100 iterations of random addition of taxa,
each of the iterations followed by additional rounds of tree-fusing and tree-drifting
(Goloboff, 1999). Sensitivity of the results to changes in parameter values was investigated by running analyses with different combinations of gap and transver-

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91

sion/transition costs. Gap costs of 1, 2, 4, and 8 times the base transformations

were combined with transversion/transition costs of 1, 2, 4, and 8 (only combinations with gap cost equal to or higher than transversion/transition cost were assayed). Congruence across data partitions as measured by the ILD (Mickevich &
Farris, 1981) has been proposed as an objective criterion to choose among different parameter combinations. However, these measurements do not seem to be independent of the actual parameter values considered (Faith & Trueman, 2001).
Equal weighting is usually rendered as just another type of character weighting.
However, equal or uniform weighting differs epistemologically from other weighting schemes, because it neither adds any extra information to the background
knowledge nor reduces the empirical content of the resulting cladograms. Therefore, equal weighting maximizes the explanatory power of the characters (Kluge,
1997; Frost et al., 2001). Results obtained from the analysis under uniform costs
were considered to represent the best current estimate of the phylogenetic relationships of the taxa sampled in the present study.
Pair-wise uncorrected genetic distances (p-values) were calculated from the
implied alignment derived from the preferred tree under equal parameter costs.
Clade support was assessed by means of heuristic calculations of Bremer support
(Bremer, 1988), as implemented in POY, and bootstrap proportions, as calculated with the computer programs Winclada (Nixon, 1999) and NONA
(Goloboff, 1998), using 1000 iterations of an heuristic search of 15 random additions of taxa holding a maximum of 20 trees per iteration and up to 1000 total
trees (calculated from the implied alignment). Support of data for alternative topologies was examined using constrained searches.

RESULTS

Morphological analysis
The comparison of the eight populations of the littoral Ligia hawaiensis from
the islands of Kauai and Oahu showed that they are all morphologically homogeneous and no remarkable differences were observed. The terrestrial Ligia
perkinsi shows clear differences from Ligia hawaiensis, and some minor differences are found also between the populations of L. perkinsi from Kauai and that
from Oahu. No differences were observed among the three populations of L.
perkinsi from Kauai.
Size. The maximum sizes observed are: for Ligia hawaiensis from both
Kauai and Oahu, 22 10 mm, 13 5 mm; for L. perkinsi from Kauai, 13
5 mm, 17 8 mm; for L. perkinsi from Oahu, 15 6 mm, 17 8.5 mm.

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Fig. 3. A-B, Ligia hawaiensis Dana, 1853, 18 mm long from Pouhala Marsh, Oahu: A, cephalon;
B, pleotelson. C-D, L. perkinsi (Dollfus, 1900), 13 mm long from Mt. Kahili, Kauai: C, cephalon;
D, pleotelson. E-F, L. perkinsi, 15 mm long from Nuuanu Pali, Oahu: E, cephalon; F, pleotelson.

Ligia hawaiensis is larger than L. perkinsi, and in L. hawaiensis males are larger
than females while in L. perkinsi females are larger than males.
Cephalon. The distance between the eyes is proportionally smaller in L.
hawaiensis (ratio distance between eyes/eye width = ca. 3/5) (fig. 3A) than in L.
perkinsi (ratio ca. 1/1) (fig. 3C, E).
Pleotelson. The shape of the pleotelson is identical in both L. perkinsi from
Kauai (fig. 3D) and Oahu (fig. 3F). In L. hawaiensis, the pleotelson is very similar to that of L. perkinsi, only the lateral posterior points are slightly more protruding (fig. 3B).
Pereopods. The dactylus of the sixth and seventh pereopod in both sexes has
a tuft of very long thin setae on the tergal margin in Ligia hawaiensis (fig. 4A), in

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93

Fig. 4. A, Ligia hawaiensis Dana, 1853, 18 mm long from Pouhala Marsh, Oahu: pereopod 7 dactylus; B, L. perkinsi (Dollfus, 1900), 13 mm long from Mt. Kahili, Kauai: pereopod 7 dactylus;
C, L. perkinsi, 15 mm long from Nuuanu Pali, Oahu: pereopod 7 dactylus.

L. perkinsi from Kauai there are some short setae (fig. 4B), and in L. perkinsi
from Oahu (fig. 4C) there is an intermediate condition with some setae shorter
and thicker than in L. hawaiensis but longer than in L. perkinsi from Kauai.
Male characters. The major differences are found in the male characters.
Ligia species usually possess fields of papillae on the male first three pereopods
which function as anti-slide structures during copulation (Schmalfuss, 2003). In
Ligia hawaiensis, the papillar fields on carpus and merus of the first pereopod are
large (fig. 5A), while they are reduced in both populations of L. perkinsi from
Kauai (fig. 5C) and Oahu (fig. 5E). The propodus of the first pereopod is proportionally shorter and thicker in L. hawaiensis than in L. perkinsi and shows a protruding triangular process on the distal part. This process is typical of the IndoPacific species belonging to the exotica-group, and can be considered as a synapomorphic character of the species belonging to this group. This process is missing in both the two island populations of L. perkinsi. In the second and third pereopods, the papillar field is large on the carpus and small on the merus of L.
hawaiensis (fig. 5B), reduced on carpus and lacking on merus of both L. perkinsi
from Kauai (fig. 5D) and Oahu (fig. 5F). Distinct differences are found also in the
endopod of the male second pleopod: in L. hawaiensis it is enlarged in the distal
part with an obliquely truncate apex (fig. 6A), in L. perkinsi from Kauai the apical
part is not enlarged and the apex is rounded, slightly bilobed (fig. 6B), while in L.
perkinsi from Oahu the distal part is not enlarged and apically truncate (fig. 6C).

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Fig. 5. A-B, Ligia hawaiensis Dana, 1853, 18 mm long from Pouhala Marsh, Oahu: A, pereopod 1;
B, pereopod 2. C-D, L. perkinsi (Dollfus, 1900), 13 mm long from Mt. Kahili, Kauai: C, pereopod 1;
D, pereopod 2. E-F, L. perkinsi, 15 mm long from Nuuanu Pali, Oahu: E, pereopod 1; F, pereopod 2.

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Fig. 6. A, Ligia hawaiensis Dana, 1853, 18 mm long from Pouhala Marsh, Oahu: pleopod 2; B, L.
perkinsi (Dollfus, 1900), 13 mm long from Mt. Kahili, Kauai: pleopod 2; C, L. perkinsi, 15 mm
long from Nuuanu Pali, Oahu: pleopod 2.

Moreover, the two island populations of L. perkinsi differ also in the habitat
where they occur: on Kauai the species is common in wet moss on indigenous
trees of montane rain forests above 600 m, while on Oahu it occurs on a windward wet rocky cliff at approximately 300 m on Koolau Range.
Molecular analysis
Partitioned analyses of the two gene fragments under equal weights resulted
in two trees of 632 steps for the CO1 and two trees of 592 steps for the 16S (not
shown). The simultaneous analysis of the two genes yielded one tree of length
1238 (fig. 7). Results from the partitioned as well as the combined analyses were
very similar, and differences were mostly restricted to the non-Hawaiian species
(16S) and the relationships of the L. hawaiensis haplotypes from Oahu. Partitioned and combined analyses agree in supporting the monophyly of the Hawaiian species, the monophyly of the sampled haplotypes of L. hawaiensis, and the
monophyly of populations of L. perkinsi within each island. However, L.
perkinsi is shown to be paraphyletic with respect to L. hawaiensis, with the Oahu
population splitting first and the Kauaian haplotypes forming the sister clade of
L. hawaiensis. The Oahu populations of L. hawaiensis are shown to be paraphyletic with respect to the monophyletic Kauaian lineage.
The effect of different parameter combinations analysed on specific topological hypotheses is summarized in fig. 8. The monophyly of the Hawaiian species,
the monophyly of the L. hawaiensis haplotypes, and the paraphyly of L. perkinsi

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CRM 002 Sfenthourakis et al. (eds.), BIOLOGY TERRESTRIAL ISOPODS, V

Fig. 7. Single most parsimonious tree obtained by simultaneous direct optimization of the CO1 and
16S fragments, with heuristic Bremer supports (below branches) and bootstrap proportions (above
branches) of the Ligia populations examined in this study.

island populations are recovered regardless of the gap cost and transversion
weighting considered. The monophyly of the Oahu haplotypes of L. hawaiensis
is only supported under particular parameter cost schemes (fig. 8). The branching pattern of the island populations of L. perkinsi is also sensitive to
gap/transformation costs, although most of the parameter combinations suggest
that the Oahu populations of L. perkinsi were the first offshoots of Hawaiian
Ligia, in contrast to what we might expect due to the older age of Kauai. Forcing
monophyly of L. perkinsi under equal costs increases tree length by 13 steps,

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97

Fig. 8. Summary of the results of parameter sensitivity analyses. Ten different parameter combinations
were analysed: gap cost equal, twice, four times, or eight times base transformations (indicated in the
x-axis) combined with transversion (TV) cost equal, twice, four times, or eight times transitions (TS)
(indicated in y-axis). Only combinations with gaps equal to or higher than transversions were assayed.
Black boxes denote support of all trees obtained under this particular parameter combination, grey
boxes indicate partial support (only some of the obtained trees support the specified group) and white
boxes indicate complete lack of support (none of the obtained trees supports the specified group).

while constraining monophyly of the island populations of L. perkinsi and L.

hawaiensis results in 21 extra steps.
Uncorrected genetic distances across taxa are shown in table II. Divergence
values between the island population of L. perkinsi average 0.12 0.003 and between L. perkinsi and L. hawaiensis 0.11 0.006. The larger divergences observed across L. hawaiensis haplotypes are 0.10 0.002, between haplotypes of
Oahu (Ala Wai Canal excluded) and Kauai, and 0.08 0.001 and 0.09 0.002,
between the haplotype from Ala Wai and the rest of Oahu haplotypes and the
Kauai ones, respectively.

DISCUSSION

The cladistic patterns obtained from the sampled populations of Ligia perkinsi
and L. hawaiensis are compatible with both of the proposed hypotheses (fig. 2)

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Fig. 9. Alternative scenarios of the evolution of terrestrial lifestyle in Hawaiian Ligia. A, independent evolution of terrestrial lifestyle (closed boxes), with extinction of sister island populations
of Ligia hawaiensis Dana, 1853 (crossed branches); B, single origin of adaptation to terrestrial
habitats (closed box) and subsequent reversal to primitive littoral, halophilic habitat (open box).

for the adaptation to terrestrial habitats. However, the reconciliation of the preferred topology with either of these hypotheses requires different sets of assumptions. The plausibility of the two alternative scenarios can thus be evaluated
through the comparison of the required assumptions. On the one hand, the independent evolution towards a terrestrial lifestyle would involve the extinction of
both island lineages of L. hawaiensis sister to the respective L. perkinsi island
population (fig. 9A). However, if the Oahu populations of L. hawaiensis are monophyletic, as suggested by some of the parameter combinations, it would be
sufficient to invoke only the extinction of the sister lineage of L. hawaiensis
from Oahu and a subsequent recolonization of the island by a L. hawaiensis
lineage from Kauai. On the other hand, the single origin of adaptation to the terrestrial habitat would require a reversal event to the primitive seashore dwelling
condition (fig. 9B) in the ancestor of the current lineages of L. hawaiensis.
Therefore, selection of one scenario over the other requires deciding what is
more plausible, the extinction of haplotype lineages or the back adaptation to a
seashore habitat from a terrestrial dwelling state. Extinction of haplotype lineages or replacements of old lineages by more recently evolved lineages are regular processes of the dynamics of a population (Avise, 2000). The shift from a
seashore habitat to a mist forest (or the other way around) involves important
physiological changes, mostly related to osmotic regulation (Tsai et al., 1997).
Although adaptation to hypotonic, terrestrial freshwater environments from a
marine littoral environment has been hypothesized in Ligia (cf. Schmalfuss,
1978), there is no evidence to date of a shift in the opposite direction. Therefore,
at this point we consider the independent adaptation to terrestrial habitats in Hawaiian Ligia as the most plausible explanation for the origin of L. perkinsi populations in Oahu and Kauai.
If this hypothesis is correct, then some morphological characters present in
both Oahu and Kauai populations of L. perkinsi must be due to convergence, i.e.,

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the reduction of papillar fields on the carpus and merus of the male first three
pereopods and the reduction of a triangular process on the propodus of the male
first pereopod. According to Schmalfuss (2003), large papillar fields in male anterior pereopods and the size of males larger than females indicate a mateguarding behaviour prior to copulation. This behaviour is probably present in L.
hawaiensis and lost in L. perkinsi from both Kauai and Oahu.
Because of the morphological uniformity, the close proximity of the islands,
and the coastal habitat of L. hawaiensis, we expected to find that L. hawaiensis
represented a single, archipelago-wide, panmictic population. However, our results show deep genetic divergences both between and within (Oahus Ala Wai
haplotype) island populations of L. hawaiensis, well above the mitochondrial divergences reported between species, or even genera, of other Hawaiian invertebrates (Gillespie et al., 1994; Shaw, 1996; Thacker & Hadfield, 2000). These results could point towards a long-time presence of Ligia in the Hawaiian Islands.
However, high levels of molecular divergence in 16S have been reported in
other oniscidean species (Michel-Salzat & Bouchon, 2000), which may be indicative of an accelerated rate of evolution in the mitochondrial genes of these
organisms. Ligia hawaiensis also displays a remarkable geographic structure,
with most of the island populations forming exclusive clades, suggesting that inter-island dispersal events have been rare throughout the history of the lineage.
This seems to contradict the hypothesis that littoral species have a great facility
of dispersal as commonly supposed (Vandel, 1960: 63).

CONCLUSIONS

In conclusion, the genus Ligia provides an excellent model to test hypotheses

regarding the evolution of ecological shifts. Although the obtained population
cladogram is compatible with both proposed hypotheses, we conclude that the
independent colonization of the terrestrial habitats of Kauai and Oahu by an ancestral seashore population of Ligia is the most plausible scenario for the origin
of terrestrial populations. Thus, considering also their morphological differences,
the two terrestrial populations of Ligia from Kauai and Oahu should be regarded
as belonging to distinct species. Ligia hawaiensis populations display a remarkable genetic divergence and geographic structure, suggesting that inter-island
colonization events are rare. More work is required to clarify the evolutionary
significance of the patterns found in the present study. More specifically, a more
thorough sampling that includes all the Hawaiian Islands inhabited by Ligia is
necessary for testing further the independent origin of the terrestrial populations

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101

and to look for the presence of the phylogeographic patterns observed in Kauai
and Oahu populations of L. hawaiensis on the remaining islands.

ACKNOWLEDGEMENTS

We would like to express our most sincere thanks to Dr. F.G. Howarth and Mr
D.J. Preston, Bernice P. Bishop Museum, Honolulu, and Dr. A. Asquith, U.S.
Fish and Wildlife Service, Kauai, for their invaluable help in collecting part of
the material examined. The whole staff of the Entomology Department of the
Bishop Museum is particularly acknowledged for their kind help and hospitality
during S.T.s visits to the Hawaiian Islands. We wish to thank Leo Shapiro and
two anonymous reviewers who provided valuable comments on the manuscript.
M.A. was supported by a fellowship from the Spanish Ministerio de Educacin y
Cultura (EX-99-46630819).

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