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Contributions to Zoology, 81 (3) – 2012

DNA phylogeny of Ryukyus Leucothoidae (Crustacea: Amphipoda)

Kristine N. White1,2, James D. Reimer1

1.  Rising Star Program, Trans-disciplinary Organization for Subtropical Island Studies (TRO-SIS), University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa, Japan 903-0213


Keywords: 18S rDNA,anamixid clade,host specificity,Japan,leucothoid clade.


Commensal leucothoid amphipods collected from sponges, ascidians, and coral rubble from the Ryukyu Archipelago, Japan, were investigated using nuclear 18S ribosomal DNA sequences. Analysis of sequences from 21 species in three genera supported the current morphological species designations and the separation of the family into two clades. Additionally, a possible new generic-level clade was identified and the separation of the genera Anamixis and Paranamixis was not supported. Our results demonstrate that host specificity appears to evolve rapidly in sibling species in this family.


Leucothoid amphipods are commensal peracarid crustaceans, typically associated with sessile invertebrate hosts such as sponges, ascidians, or bivalve mollusks. They are also found in crevices in coral rubble, presumably associated with filter feeding organisms (e.g. sponges) within the rubble. Currently, there are 163 species of Leucothoidae described from coral reefs to the deep sea (White, 2011a; White and Reimer, 2012a, b, c), but it is likely many more undiscovered and undescribed species exist given the cryptic habitats and small sizes of many species in this family (Thomas and Klebba, 2006; Thomas, 2008; White, 2010; Thomas and Krapp-Schickel, 2011).

In 2012, White and Reimer described 24 new species of Leucothoidae with their host associations from the Ryukyu Archipelago in southern Japan (2012a, b, c). Prior to this research only seven species had been reported from mainland Japan, with no reports from the Ryukyu Archipelago, and only one species had associated host information available (discussed in White and Reimer, 2012a).

Despite the progress made in species descriptions of Leucothoidae from some regions of the world, little research has been conducted on their molecular phylogeny, with only two reports in the literature. As of March 3, 2012, GenBank lists approximately 10,000 sequences for Amphipoda, with 921 of these being 18S ribosomal DNA (18S rDNA) sequences (Benson et al., 2005). Molecular sequence data are scarce for Leu­cothoidae, comprising only 94 of the total amphipod sequences. Of these 94 sequences, 30 are 18S rDNA sequences for 10 species in two genera from Florida, Belize, and Indonesia (Anamixis and Leucothoe) (White, 2011b). The remaining 64 are partial COI sequences for three species (Leucothoe ashleyae Thomas and Klebba, 2006; Leucothoe kensleyi Thomas and Klebba, 2006, and one sequence for Anamixis vanga Thomas, 1997) from Florida and Belize (Richards et al., 2007; White, 2011b).

White (2011b) utilized specimens from Florida, Belize, and Indonesia, and documented the utility of 18S rDNA as a species-level molecular marker for the Leucothoidae, while supporting the separation of the family into two large clades. The ‘leucothoid’ clade was shown to contain the genera Leucothoe Leach, 1814 and Paraleucothoe Stebbing, 1899, while the ‘anamixid clade’ contained Anamixis Stebbing, 1897, Nepanamixis Thomas, 1997, and Paranamixis Schellenberg, 1938. The genera Anamixis and Paranamixis are currently distinguished from each other based on the presence of gnathopod 1 and a cleft maxilliped inner plate in Anamixis species. However, in addition to the molecular results of White (2011b), recent morphological analyses also do not support the separation of these two genera based on these characters (White, 2010). Thus, it is clear further phylogenetic analyses are needed to clarify our evolutionary understanding of this unique amphipod family.

In this study, we obtained and phylogenetically analyzed sequences from 21 species of Leucothoidae from the Ryukyu Archipelago. We examine the phylogenetic relationships within the Leucothoidae, and also discuss the potential evolutionary significance of host relationships.

Material and methods

Leucothoid amphipods for this study were collected from marine sponges, ascidians, and coral rubble from throughout the Ryukyu Archipelago, Japan, as reported in White and Reimer (2012a, b, c). These collections included detailed host association and location data (GPS coordinates, depth etc.), as such associations were previously unknown for most of the described species in the Leucothoidae. Specimens, station data, and GenBank accession numbers are listed in Table S1.

Genomic DNA was extracted from the urosomes of representative individual ethanol-preserved animals after morphological examination. In total, DNA from one to six specimens each of 24 morphologically determined Leucothoidae species was extracted. Voucher specimens are deposited in The University of the Ryukyus Museum (Fujukan), with the prefix RUMF for museum numbers. Extractions were performed using a guanidine extraction protocol (Sinniger et al., 2010). Partial 18S rDNA sequences were amplified (697-851 base pairs including the V4 hypervariable region) using polymerase chain reaction (PCR) (Saiki et al., 1988). PCR protocols were used following Spears et al. (2005). The following peracarid-specific primers were used for 18S rDNA: 329 (5’-TAATGATCCTTCCGCAGGTT-3’) and Hi- (5’-GTGCATGGCCGTTC TTAG-TTG-3’) (Spears et al., 2005). PCR products were purified using shrimp alkaline phosphatase (SAP) and gel purified with a Qiagen gel extraction kit if non-specific PCR products were apparent after gel electrophoresis.

Sequencing was done by Fasmac (Yokohama, Japan). Sequences were edited, combined, and initially aligned in Bioedit (Hall, 1999) and the alignments were further edited by eye using SeaView 4.3.3 (Guoy et al., 2010) and Se-Al v.2.01 (Rambaut, 2002). Three different alignments were constructed in order to examine the influence of hypervariable regions on resulting phylogenetic trees, as some studies suggest deleting these regions (e.g. Kim and Abele, 1990; Spears et al., 1992; Carmean et al., 1992; Pashley et al., 1993; Campbell et al., 1994; Friedrich and Tautz, 1995; Kim et al., 1996; Chalwatzis et al., 1996; Giribet et al., 1996; Friedrich and Tautz, 1997; Spears and Abele, 2000) while others propose retaining them (Crease and Taylor, 1998; Hwang et al., 2000; White, 2011b). The first alignment contained all variable regions including the V4 hypervariable region (‘long’ alignment). The second alignment retained all variable regions except the V4 hypervariable region (‘medium’). The final alignment retained only conserved regions (‘short’). All three generated alignments contained sequences for 40 taxa (including two outgroup taxa). Representatives of the amphipod family Liljeborgiidae Stebbing, 1899 were chosen as outgroup taxa based on the loss of molar structure and rudimentary charpochelation of gnathopod 1 (more primitive than the fully carpochelate gnathopod 1 in Leucothoidae) in the Liljeborgiidae (Barnard, 1974). The long alignment dataset contained 1205 sites, the medium alignment 751 sites, and the short alignment 426 sites. All three alignments are available from the corresponding author and at the homepage

All three alignments resulted in trees with identical topologies based on preliminary maximum likelihood (ML) and neighbor-joining (NJ) analyses, with the short and medium trees showing generally lower bootstrap support values at most nodes. For these reasons, we therefore utilized the long alignment for all subsequent analyses. Uncorrected measures of percent-sequence divergence were obtained using Mesquite V2.74 (Maddison and Maddison, 2011). The levels of divergence used were 0-0.023 between different populations of a species, 0.090-0.295 between species within a genus, 0.260-0.349 between different genera within the Leucothoidae, and greater than 0.306 between leucothoid genera and the outgroup, Liljeborgiidae (after White, 2011b).

Twenty one ingroup taxa were included in the analyses due to lack of specimens or difficulty of obtaining molecular data from the other four species collected in the Ryukyus. PhyML (Guindon and Gascuel, 2003) was used for ML analyses using an input tree generated by BIONJ (Neighbor Joining) with the Generalized Time Reversible (GTR) model, incorporating invariable sites and a discrete gamma distribution with eight substitution rate categories. Base frequencies were estimated from the dataset. Support for the maximum likelihood tree was measured using the bootstrap method with 1,000 replicates in PhyML.

NJ analysis was accomplished in SeaView 4.3.3 using Jukes Cantor (JC) distance methods. Support was measured using the bootstrap method with 1,000 replicates in SeaView 4.3.3.

Bayesian phylogenetic inference analysis (B) was accomplished using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) under GTR + I + Γ. One cold and three heated Markov chains Monte Carlo (MCMC) with default-chain temperatures were run for 5 million generations, sampling log-likelihoods. Trees at 1000- generation intervals were saved (5000 InLs and trees saved during MCMC). The likelihood plot suggested that MCMC reached a stationary phase after the first 1,000,000 generations, and thus the remaining 4,000 trees were used to obtain clade probabilities and branch-length estimates.


Maximum likelihood (ML) analysis of partial 18S rDNA sequences of all available Ryukyus species’ sequences resulted in a tree with generally high bootstrap support values for most nodes. Neighbor Joining (NJ) analysis resulted in an identical distance tree with high bootstrap support values. Bayesian analysis (B) resulted in a phylogenetic tree identical to the ML tree with high posterior probabilities.

The resulting 18S rDNA tree depicted two major clades (Fig. 1), one composed of Anamixis and Para­namixis species (ML = 100%, NJ = 100%, B = 1.00) and the other containing Leucothoe species (ML = 92%, NJ = 72%, B = 1.00). The anamixid clade did not have prominent subclades, grouping Anamixis sentan White and Reimer, 2012c and Paranamixis thomasi White and Reimer, 2012a into one clade. The 18S rDNA sequence data also supported the connection of leucomorphs and anamorphs of P. thomasi (ML = 69%, NJ = 99%, B = 1.00). The connection of anamorphs and leucomorphs was not possible for A. sentan as only leucomorphs were sequenced. The leucothoid clade depicted two distinct subclades within the genus Leucothoe. A new generic-level subclade grouped morphologically similar species (Leucothoe toribe White and Reimer, 2012b, Leucothoe obuchii White and Reimer, 2012a, Leucothoe enko White and Reimer, 2012c, and Leucothoe kebukai White and Reimer, 2012c) together (ML = 100%, NJ = 100%, B = 1.00). Within the larger Leucothoe subclade (ML = 86%, NJ = 98%, B = 1.00), multiple specimens of each species formed subclades.


Fig. 1. Maximum likelihood (ML) phylogenetic tree of ‘long’ partial 18S ribosomal DNA gene sequences from replicates of 21 Leucothoid taxa collected from depths of 1-33 meters, rooted with an outgroup of two Liljeborgiid species. Numbers at branches represent ML/Neighbor joining (NJ) bootstrap values. Thick lines represent Bayesian posterior probabilities >0.95.

Four morphologically similar species (Leucothoe vulgaris White and Reimer, 2012a, Leucothoe ama­miensis White and Reimer, 2012a, Leucothoe akuma White and Reimer, 2012c, and Leucothoe akaisen White and Reimer, 2012c) formed a subclade separate from all other Leucothoe species within the leucothoid subclade (ML = 92%, NJ = 100%, B = 1.00). Five specimens of Leucothoe elegans White and Reimer, 2012a formed a subclade with Leucothoe hashi White and Reimer, 2012b, Leucothoe zanpa White and Reimer, 2012b and Leucothoe lecroyae White and Reimer, 2012b, depicting minor population differences between Okinawa–jima, Amami–oshima, and Hiroshima populations of L. elegans (ML = 90%, NJ = 67%, B = 1.00). The final subclade in the Leucothoe subclade was comprised of morphologically similar species (Leucothoe trulla White and Reimer, 2012a, Leucothoe bise White and Reimer, 2012b, Leucothoe nurunuru White and Reimer, 2012b, Leucothoe daisukei White and Reimer, 2012b, and Leucothoe akaoni White and Reimer, 2012b) (ML = 68%, NJ = <50%, B = 1.00).


After many unsuccessful attempts by the first author at amplifying and sequencing multiple molecular markers (cytochrome oxidase subunit 1, 12S ribosomal DNA, 16S ribosomal DNA) for Leucothoidae, this study focused on nuclear 18S rDNA. Several studies have found that the hypervariable regions of 18S are taxon specific and useful as a species-level marker (Hwang et al., 2000; White, 2011b) and several amphipod molecular phylogenies have been based at least partially on 18S rDNA, including gammaridean Lake Baikal amphipods (Sherbakov et al., 1999; Macdonald et al., 2005), subterranean gammaridean amphipods (Englisch and Koenemann, 2001), Gammarus species (Hou et al., 2007), and caprellid and corophioid taxa (Ito et al., 2008).

While all three alignments in this study grouped the taxa into the same clades, the long alignment, containing the V4 hypervariable region, showed the strongest bootstrap support for species, suggesting that full length 18S rDNA sequences are an excellent marker at the species-level and potentially useful for population studies. Removing the variable regions in the shorter 18S rDNA alignments resulted in the loss of some resolution at the species level, but still supported the major clades with lower support and shorter distances.

Our 18S rDNA sequence data strongly support the separation of the anamixid and leucothoid clades, and grouped morphologically similar species into highly supported subclades within the genus Leucothoe. The unexpected new generic-level clade (‘new’ clade hereafter) groups Leucothoe toribe, L. obuchii, L. kebukai , and L. enko together. These species all have a one-articulate palp of maxilla 1, gnathopod 1 basis widened or inflated, gnathopod 1 propodus curved or proximally inflated (not straight), and gnathopod 1 dactylus reaching less than 0.2 × propodus length. Further investigation will clarify whether these ‘new’ clade species belong in a new genus or if they belong in the genus Paraleucothoe, whose members share the diagnostic characters listed above in addition to having moderate sexual dimorphism and an elongate maxilliped inner plate. If this clade is indeed Paraleucothoe, the diagnostic morphological characters of Paraleucothoe will need to be revised. However, Leucothoe togatta White and Reimer, 2012b and L. ouraensis White and Reimer, 2012b do not share the morphological characters of this new generic level clade, suggesting that they may be placed differently in a tree containing sequences from more species; this is further supported by the relatively weak support for these species as members of the new clade (ML = 89%, NJ = 78%, B = 0.92) and their basal position in the grouping.

The three large subclades in the genus Leucothoe group morphologically similar species. One subclade contains Leucothoe vulgaris, L. amamiensis, L. akuma and L. akaisen, which share a rounded anterodistal head margin, gnathopod 1 dactylus reaching greater than 0.2 × propodus length, and wide pereopod 5-7 bases. Additionally, these four species also all have unique color patterns. L. vulgaris has a red ‘saddle’, a red dot on coxa 4, and yellow antennae; L. amamiensis has a thick pink striped pattern; L. akuma has a thin pink striped pattern; and L. akaisen has a yellow ‘saddle’, a red dot on coxa 4, and red striped antennae.

Another subclade contains Leucothoe elegans, L. hashi, L. zanpa, and L. lecroyae, and these species also share some unique morphological characters, including a thin gnathopod 1 carpus and propodus and gnathopod 1 dactylus reaching less than 0.3 × propodus length. Finally, the subclade of L. trulla, L. bise, L. nurunuru, L. daisukei, and L. akaoni share gnathopod 1 dactylus reaching greater than 0.2 × propodus length and gnathopod 2 propodus mediofacial setal row displaced to midline.

The 18S rDNA tree depicts P. thomasi and A. sentan as sister species and; therefore, does not support the separation of the genera Anamixis and Paranamixis. Examination of both morphological and molecular data of all species in the anamixid clade is necessary to determine the status of these genera. The connection of a P. thomasi anamorph and leucomorphs is also confirmed by DNA sequences with levels of divergence between 0 and 0.0175. It is apparent that these two genera will need further future revision, but we do not formally merge them here due to lack of available data for more species from each genus. Once such data are available, we feel such a revision will be necessary.

As shown in White (2011b), molecular identification and analyses of leucothoid taxa supports traditional morphological species definitions. The available molecular data clusters morphologically similar species together, suggesting that the diagnostic morphological characters are not due to convergent adaptation to similar hosts, and do in fact reflect true evolutionary relationships between leucothoid species. However, higher level clades and relationships within Leucothoidae need to be further investigated using both morphological data and additional molecular markers to clarify species groupings within the different genera. Taxonomic revisions are not proposed here due to the lack of morphological and molecular data available for more of the 163 species in Leucothoidae. Further analyses including more species from more regions of the world will help us clarify our understanding of the evolutionary relationships within this family, and may lead to the formal taxonomic revisions that appear needed from this preliminary study.

Finally, from the phylogenetic analyses it is apparent that several closely related species inhabit different hosts, suggesting that host specificity can evolve and change rapidly in sister species. Sponge and ascidian habitation appears to have evolved more than once in the Leucothoidae, as species collected from sponges or ascidians occur in nearly every clade on the tree.


Thanks to Nathan S. White for exceptional collection assistance and to Yuka Irei for assistance with DNA extraction and amplification. Members of the University of the Ryukyus MISE Lab provided support in many forms. The Rising Star Program at the University of the Ryukyus provided laboratory space and logistical support. Reviews from Anne-Nina Loerz and two anonymous reviewers significantly improved the manuscript. Funding was provided by a Japanese Society for the Promotion of Science (JSPS) postdoctoral fellowship awarded to the first author (#P10711). The second author was supported in part by the International Research Hub Project for Climate Change and Coral Reef/Island Dynamics at the University of the Ryukyus.

Received: 19 March 2012

Revised and accepted: 12 June 2012

Published online: 4 July 2012

Editor: R. Vonk


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On-line supplementary material

S1. Leucothoid specimens (from White and Reimer, 2012a, b, c) examined in this study and corresponding GenBank Accession Numbers.