Introduction

The Hawaiian Islands are well known for their rich biodiversity and high rate of endemism [1]. Their remoteness, representing the world’s most isolated major archipelago, along with the progressive formation of these islands, are considered crucial for the striking diversification observed in several Hawaiian terrestrial organisms, which include: the Hawaiian Drosophila [2-4], the silversword alliance [5], succineid land snails [6,7], honeycreeper birds [8,9], and others [10-13]. In contrast, endemism in Hawaiian marine invertebrates is strikingly lower than that in Hawaiian terrestrial organisms [14], and with the exception of intertidal Cellana limpets [15], there are no documented marine radiations within the Hawaiian Archipelago. Such disparity suggests that opportunities for allopatric differentiation within the archipelago have been very limited in the marine realm, while abundant in the terrestrial realm. Populations of non-vagile invertebrates endemic to specific coastal patchy habitats located at the interface between sea and land, however, have the potential to be highly isolated and, thus, show elevated levels of allopatric differentiation [16].

The Hawaiian endemic shrimp Halocaridinia rubra, which inhabits anchialine coastal pools, shows evidence of within- and between- island divergence and is comprised of multiple highly divergent lineages [17,18]. An additional interesting case of diversification in a coastal patchy habitat of the archipelago is that of oniscidean isopods in the genus Ligia, which most likely arose from a rocky supralittoral ancestor that arrived to the Hawaiian Archipelago via rafting, and diversified into rocky supralittoral and inland (hereafter, terrestrial) lineages. High levels of genetic differentiation were detected among populations of Hawaiian Ligia from different localities in the islands of Kauaʻi and Oʻahu [19], but populations from other islands have not been studied. Therefore, further phylogeographic analyses of Hawaiian Ligia including populations from previously unsampled islands are needed to better understand the biodiversity and evolution of this group.

The genus Ligia has a worldwide distribution and includes 42 currently recognized species [20-26]; most of which are restricted to rocky supralittoral areas [19,27]. Eight species, however, are strictly terrestrial, inhabiting montane habitats of tropical regions [19,22], and are believed to derive from supralittoral forms [28]. Supralittoral Ligia exhibit morphological, physiological, and behavioral characteristics that are intermediate between ancestral marine and fully terrestrial isopods [29]. Some of the biological characteristics of supralittoral Ligia confer them extremely low vagility [30], such as: direct development (lack a planktonic larval phase, as all peracarids); active avoidance of the open sea (they remain in the area between the exposed intertidal and the supralittoral); extremely low desiccation tolerance (a reason for which they stay close to the water line and are most active at night); limited motility underwater and on sandy shores (rendering them highly vulnerable to predators in these environments).

The biology of supralittoral Ligia severely constrains the movement of these isopods outside the rocky beaches they occupy, effectively isolating populations. This is reflected in the striking radiations of supralittoral Ligia reported in different regions of the world, with extraordinarily high levels of allopatric genetic differentiation, even between localities separated by few kilometers ([19,30-32] and unpublished data). These observations, as well as those by Hurtado et al. [16], challenge earlier suggestions that supralittoral isopods are highly dispersive species, based on their common presence in beaches around the world [33]. Phylogeographic patterns of Ligia in different regions have been shaped by past tectonic events [30], environmental factors, such as sea surface temperature [31], as well as oceanic dispersal events, probably through rafting (unpublished data).

Two endemic species of Ligia are currently recognized in the Hawaiian Islands [34]: the rocky supralittoral Ligia hawaiensis (Dana 1853) and the terrestrial montane Ligia perkinsi (Dollfus 1900). A third species, the cosmopolitan introduced Ligia exotica (Roux 1828), is reported from littoral man-made substrate [19,35]. The supralittoral species L. hawaiensis occurs in rocky intertidal habitats throughout the archipelago [27,34,36], whereas the terrestrial species L. perkinsi is known from high altitude (300–1,500 m above sea level) wet forests on the islands of Kauaʻi, Oʻahu, and Hawaiʻi [34]; although the last report of L. perkinsi in Hawaiʻi was in 1896 [19].

Taiti et al. [19] investigated whether the origin of terrestriality in L. perkinsi populations from the Hawaiian islands of Kauaʻi and Oʻahu occurred as a single event (i.e., L. perkinsi from both islands constitute a monophyletic group sister to a lineage of L. hawaiensis) or as two independent events, one in each island (e.g. two clades each showing reciprocal monophyly of L. hawaiensis and L. perkinsi from the same island). They conducted Maximum Parsimony phylogenetic analyses with sequences of two mitochondrial genes (COI and 16S rDNA), and included individuals of L. perkinsi and L. hawaiensis from Kauaʻi and Oʻahu. They obtained strong support for the monophyly of the Ligia lineages endemic to the Hawaiian Archipelago (also observed in [30]), which were divided into three main clades: one comprised of the L. perkinsi from Kauaʻi; another clade comprised of L. perkinsi from Oʻahu; and a third clade comprised of L. hawaiensis individuals. The L. hawaiensis clade was divided into three lineages: one comprised of the Kauaʻi individuals and the other two comprised of the Oʻahu individuals. The two main L. perkinsi clades were paraphyletic (L. perkinsi from Oʻahu was sister to a clade of L. perkinsi from Kauaʻi + L. hawaiensis), thus, the results were inconclusive as to whether a single or two origins of terrestriality occurred. In addition, they observed high divergences among populations of L. hawaiensis, implying long-standing isolation among them, and with phylogeographic patterns suggesting inter-island dispersal events have been rare throughout the history of the Hawaiian endemic Ligia lineages [19].

Herein, we expanded on Taiti et al.’s [19] previous work by incorporating populations from previously unsampled main Hawaiian Islands (i.e., Maui, Molokaʻi, Lanaʻi, and Hawaiʻi), increasing the number of gene markers, which include nuclear genes, and applying more current phylogenetic approaches. Sampling across all main Hawaiian Islands enables a better understanding on the diversity and evolution of endemic Hawaiian Ligia. Specifically, we asked: (1) whether the younger islands (i.e., Maui, Lanaʻi, and Molokaʻi, all with an age ~ 1.3 My, and Hawaiʻi with an age of 0.4 My; ages from [37]) harbor highly divergent L. hawaiensis lineages, as observed in the older islands (i.e., Kauaʻi [5.1 My] and Oʻahu [3.7 My]); (2) whether evolution of Ligia in the Hawaiian Islands followed a pattern consistent with the progression rule (i.e., lineages from older islands are basal to those from younger islands), a back dispersal pattern (lineages from younger islands colonized the older islands), or an unresolved and/or highly stochastic pattern (indicative of a complex evolutionary history, probably with frequent inter-island dispersal); and (3) whether the new data shed light on the origin of terrestriality in Kauaʻi and Oʻahu (i.e., a single origin or independent origins in each island). Lastly, we incorporated geometric-morphometric analyses to test for differences among three highly divergent supralittoral lineages and determine whether these methods can be used for their discrimination.

Materials and Methods

Sampling

Our molecular dataset included twenty-four L. hawaiensis populations (i.e., localities) from across the main Hawaiian Islands and four L. perkinsi populations from Kauaʻi and Oʻahu (Figure 1A, Table S1). The collection of these specimens did not require a permit from the Department of Land and Natural Resources of Hawaii, and these isopods are not considered endangered or protected species. We also included publicly available sequences for L. hawaiensis and L. perkinsi (Table S1). We were unable to collect L. hawaiensis in the islands of Niʻihau and Kahoʻolawe, because they are private property and a state reserve, respectively. Populations were sampled by hand and preserved in 70–100% Ethanol. As outgroups, we included specimens from Ligia vitiensis, Ligia occidentalis, and Ligia exotica, as previous research [19,30] and preliminary analyses suggest they are the closest extant relatives to Ligia from the Hawaiian Archipelago.

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Figure 1. Sampled localities (A) and inferred phylogeny (B) of Ligia lineages endemic to the Hawaiian Archipelago.

Color-coding and labels correspond between panels and with other figures and tables. Detailed information for each locality is presented in Table S1. A. Sampled localities for supralittoral L. hawaiensis: D1-Kapua'a Beach Park, D2-Kauapea Beach, D3-Kapa'a, D4-Lihu'e, D5-Kukui'ula (Kaua’i Island); E1-Ala Wai Canal, F1-Pupukea, F2-Pouhala Marsh (O’ahu Island); E2-Papohaku Beach Park, E3-North of Puko'o; Lana’i: E4-Manele Bay (Moloka’I Island); A1-Waiopai, E5-Poelua Bay, E6-Spreckelsville, E7-Keanae, F3-Honomanu Bay (Maui Island); A2-Kealakukea Bay, A3-Pu'unalu Beach Park, A4-Isaac Hale Beach Park, F4-Keokea Beach, F5-Onekahakaha Beach Park, F6-Leleiwi Beach, F7-South Point, F8-Kapa'a State Park (Hawai’i Island). Sampled localities for terrestrial L. perkinsi: C1-Mt Kahili, C2-Makaleha Mts, C3-Haupu Range (Kaua’i Island); B1-Nu'uanu Pali (O’ahu Island). Boldfaced labels indicate localities used in the nuclear analyses. * indicates localities examined in the geometric morphometric analyses. B. Majority rule consensus tree (GTR +Γ model in RaxML; TreeBase http://purl.org/phylo/treebase/phylows/study/TB2:S14886) of the concatenated mitochondrial dataset of Ligia samples from the Hawaiian Archipelago and several outgroups. Numbers by nodes indicate the corresponding range of percent Bootstrap Support (BS; top) for Maximum likelihood; and Posterior Probabilities (PP; bottom) for Bayesian inference methods (clade support values for each analysis are shown in Table S4). Nodes receiving 100% for all methods are denoted with an *. NS: less than 50% node support. Samples examined by Taiti et al. [19] are marked with †.

https://doi.org/10.1371/journal.pone.0085199.g001

Geometric-morphometric methods

We captured digital images of the dorsal side of L. hawaiensis specimens using QCapture v3.1.2 and an Olympus QColor3 digital camera attached to an Olympus SZ61 stereomicroscope. We removed all pereopods (i.e., legs) to ensure specimens laid flat. Dissected pereopods and pleopods were not used for morphometric comparisons. The male pereopods and pleopods, which are fundamental in distinguishing Ligia species, did not show distinct differences among the L. hawaiensis populations [19]. During dissections, we determined and noted the sex of each specimen by visually inspecting the endopod of the 2^nd pleopod. Sex was noted as either: male (M); gravid female (F); or juvenile/non-gravid female (J). We characterized body shape by digitizing 27 landmarks (LMs), using TpsDig v2.16 [65], on the periphery of Ligia bodies (Figure 2A). We included landmarks that capture taxonomically informative regions and can be noted unambiguously. For example, we placed landmarks on the medial and the lateral boundaries of the eyes at the body periphery. These landmarks capture the relative size of the eyes and the inter-ocular distance, characters used to distinguish Ligia species [19]. We characterized the relative width of body segments and overall body shape, also important in Ligia taxonomy [19,25,27,66,67], by placing landmarks on the lateral posterior tergite tips. Lastly, we captured the shape of the pleotelson, another trait used in Ligia taxonomy [19,25,68], by placing landmarks at its posterior tip and the lateral posterior points.

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Figure 2. Landmarks (LMs) placement (A) and thin-plate-spline transformations (B) for Ligia hawaiensis (clades D, E, F).

A. LMs 1 and 25 represent the posterior margin of the eyes at the body perimeter; LMs 2-11 and 15–24 are the posterior-most point of each segment; LMs 12 and 14 are the lateral posterior points of the pleotelson, whereas LM 13 is the posterior-most point of the pleotelson; LMs 26 and 27 correspond to the inner-most margin of the eyes. B. Thin-plate-spline transformations of LM positions are shown magnified (10 ×) to reach the natural extremes observed in the clades, which are also indicated with images for specimens with the highest probability of being correctly assigned to each clade.

https://doi.org/10.1371/journal.pone.0085199.g002

As the body plan of Ligia is bilaterally symmetric, all but the pleotelson tip LMs are anatomically homologous and should not be treated as independent in statistical analyses. As suggested by Zelditch et al. [69], we reflected and averaged homologous landmarks across the body midline as defined by the pleotelson tip and the midpoint between the medial eye LMs. Corrected landmarks were centered, scaled and rotated, to best align with the consensus, using the method of generalized least squares, and projected to a flat shape space using tpsRelw v1.49 [70]. We calculated principal components of aligned coordinates to yield orthogonal shape variables, retaining the minimum number of components needed to represent ≥ 95% of the overall variation. These principal components were taken as shape variables to test for differences in shape between lineages, sexes, and sizes. We used the centroid size (the summed square distances of landmarks from the centroid; [71]) as an estimate of body size.

Results

All new sequences produced in this study have been deposited in GenBank under accession Numbers KF546528-KF546728 (Table S1). Annotated alignments used in analyses are in Datasets S1–S4, TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S14886), and DataDryad (doi:10.5061/dryad.5k56c).

Nuclear gene patterns

We sequenced two nuclear genes for all Study Area lineages with the exception of the L. perkinsi lineage from Oʻahu (B). Multiple attempts to amplify nuclear genes from this population proved unsuccessful. The patterns inferred from the nuclear genes (Figure 3; colors for clades correspond with those in Figure 1) were, in general, consistent with those inferred from the mitochondrial genes. For the NaK gene (Dataset S4), we only observed six alleles, separated by 1–6 steps. For Clade A members, we detected two alleles separated by a single step. These alleles were separated from the other alleles by 2–6 steps, which is concordant with the high divergence observed in mitochondrial genes between Clade A and all other lineages. The allele observed for the individual of Clade C (L. perkinsi from Kauaʻi) was divergent from the other alleles by 5–6 steps, also consistent with the mitochondrial results. The NaK results show a closer relationship among members of clades D, E, and F, also congruent with the mitochondrial results. Three alleles were observed for members of these clades, which were separated by only 1–2 steps, with Clade E members from Lanaʻi, Molokaʻi and Maui sharing an allele with a Clade F member from Hawaiʻi (F5); whereas another member of the Clade F from Oʻahu (F1) harbored a unique allele. The Clade D individual from Kauaʻi harbored a unique allele.

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Figure 3. Haplotype networks for two nuclear gene fragments for Ligia from the Hawaiian Archipelago.

The nuclear markers examined are the alpha subunit of the Sodium-Potassium ATPase (NaK) and the 28S rDNA gene. Colors correspond with those in Figure 1. Locality ID is indicated next to each allele (locality information is provided in Figure 1 and Table 1). Empty circles represent inferred unsampled (i.e., missing) alleles. Numbers within branches indicate number of mutational steps separating haplotypes. The size of circles is proportional to the frequency at which an allele was recovered. Numbers in parenthesis, indicate the number of individuals observed with the corresponding allele.

https://doi.org/10.1371/journal.pone.0085199.g003

For the 28S rDNA gene, we excluded 60 poorly aligned positions, resulting in a final alignment of 967 characters, 31 of which were parsimony informative (Dataset S3). For this gene, we detected thirteen alleles. Seven of these alleles were recovered from Clade E individuals (Figure 3), with the other lineages harboring one or two alleles. Concordant with mitochondrial phylogenetic findings, alleles from different major lineages appeared highly differentiated, with most lineages separated by 10–21 steps. We note, however, that the single allele found in Clade D (Kauaʻi; D1) was separated by only two steps from one of the two alleles observed in Clade F (from the Oʻahu F1 locality). The F1 and D1 alleles were in turn separated by 11 steps from the other Clade F allele (from the Hawaiʻi F5 locality). This pattern could be the result of incomplete lineage sorting or a past hybridization event. Examination of additional informative nuclear markers in individuals from multiple populations per clade is needed to resolve this question. Indeed, multispecies coalescent analyses of numerous unlinked markers are likely needed to resolve relationships with more certainty [76]. Nevertheless, as shown by Mateos et al. [77], datasets with three loci, including one with strong phylogenetic signal (i.e., the mitochondrial dataset) and one or two with low phylogenetic signal (e.g. NaK) are not well suited for multispecies coalescent approaches, and different methods lead to different results. Therefore, we consider that at the present stage, the mitochondrial phylogeny represents the most plausible hypothesis.

Discussion

In this study, we conducted phylogeographic analyses of the Ligia lineages endemic to the Hawaiian Archipelago including a comprehensive sampling of these isopods across the main Hawaiian Islands, which greatly enhances our understanding on the diversity and evolution of this group. Previous genetic work by Taiti et al. [19] on Ligia from the Hawaiian islands of Kauaʻi and Oʻahu reported highly divergent lineages of the terrestrial L. perkinsi and the coastal L. hawaiensis in these islands. By including populations from previously unsampled main Hawaiian Islands, we detected additional lineages of L. hawaiensis and expanded the known range of others. We report the discovery of a new lineage of L. hawaiensis (i.e., Clade A), distributed in Maui and Hawaiʻi, which is highly divergent (> 10% COI K2P) from previously reported Ligia lineages in the Hawaiian Archipelago. We also discovered that the distribution of a lineage (Clade E) previously sampled in a locality in Oʻahu (E1) extends to Molokaʻi, Lanaʻi, and Maui. Similarly, we discovered that Clade F, which was previously identified in Taiti et al. [19] from Oʻahu, contains two additional lineages; one distributed in Maui (F3) and one in Hawaiʻi (F4-F8). Divergence among the three Clade F lineages is > 3% COI K2P. Considering that intra-specific divergences in marine invertebrates are typically < 3% for the same COI fragment used in this study [78], it is likely that some of the divergent Hawaiian Ligia lineages represent cryptic species, thus, the taxonomy of the group needs revision.

High levels of genetic differentiation among populations of the Hawaiian Ligia are congruent with studies of Ligia in other parts of the world ([30-32] and unpublished data). Biological characteristics of these isopods severely restrict their dispersal potential, contributing effectively to the isolation of populations, and, in the long-term, to allopatric genetic differentiation [30]. Nonetheless, phylogeographic patterns indicate that past dispersal events have been important in shaping the evolutionary history of these isopods in the Hawaiian Archipelago.

The monophyly of the endemic Hawaiian Ligia lineages is well supported, suggesting that evolution of this group likely occurred within the archipelago. Lineages identified as the closest relatives of the endemic Hawaiian Ligia are highly divergent and found in other Pacific localities (unpublished data). Large divergences observed among Hawaiian Ligia lineages also suggest a long evolutionary history for this group. In addition, the phylogeographic patterns observed among Hawaiian Ligia, although not fully resolved, do not support simpler patterns of evolution, such as colonization from older to younger islands (i.e., progressive rule), or vice versa (i.e., from younger to older islands), but rather a complex evolutionary history. Illustrating the complexity of the evolutionary history of the Hawaiian Ligia, the three most basal lineages include one found only in the younger islands (Clade A), a terrestrial lineage restricted to Oʻahu (Clade B), and one that is the most diverse. This latter clade (CDEF) includes supralittoral and terrestrial lineages, and divergent lineages restricted either to the older island of Kauaʻi or to the other islands. Dispersal and local extinctions likely contributed to shape this complex evolutionary history.

Despite the complex evolutionary history of Hawaiian Ligia, some phylogeographic patterns emerge that appear congruent with the geological history of the main Hawaiian Islands. First, Kauaʻi, the oldest of the main Hawaiian Islands, harbors only one endemic L. hawaiensis lineage (Clade D), which is highly divergent from the other lineages. This is consistent with the older geological history of this island and its high degree of isolation, as no overland connections are thought to have existed between Kauaʻi and other Hawaiian islands [37]. Second, in contrast to the pattern observed in Kauaʻi, sharing of highly divergent lineages is observed among the other main Hawaiian Islands, suggesting inter-island dispersal among these islands. The geological history of these islands may have provided opportunities for the exchange of colonizers from divergent lineages. Molokaʻi, Maui, Lanaʻi, and Kahoʻolawe, are thought to have existed as a single landmass, the Maui Nui Complex, throughout most of their geological history, first splitting up some ~0.6 million years ago (Mya) and retaining land connections during glacial low sea level stands [79]. The Maui Nui complex is thought to have been connected via a land bridge to Oʻahu between 2.2–1.9 Mya forming the short lived Oʻahu Nui complex [37,79]. These past connections may have facilitated the dispersal of Clade E, restricted to the Maui Nui islands and Oʻahu. The low genetic divergences observed among Clade E populations (< 2.5% COI K2P) may indicate a recent history of isolation among populations of this clade. Dispersal of Clade F across these islands may also have been facilitated by these connections. Third, historically, the oceanic channel separating Maui and Hawaiʻi does not appear to constitute a very effective barrier for inter-island dispersal of coastal organisms that disperse through rafting, as two divergent lineages are shared between Hawaiʻi and Maui (i.e., clades A and F). Remarkably, however, members from Clade E were not found in Hawaiʻi, despite their wide distribution in the Maui Nui islands.

Evolution of terrestriality appears to have occurred early during the diversification of the Hawaiian Ligia, as clades B and C are highly divergent and occupy a relatively basal position in the phylogeny of Hawaiian Ligia. Our results confirm the paraphyly of L. perkinsi, previously observed by Taiti et al. [19]. Therefore, from the phylogeographic patterns it is uncertain (1) whether evolution of terrestriality occurred independently in each island (i.e., Kauaʻi and Oʻahu) or (2) whether terrestriality evolved once. Both of the above hypotheses are equally parsimonious assuming a coastal ancestor for the endemic Hawaiian Ligia clade, which we consider most likely, as the closest lineages are coastal and because of the predominantly coastal nature of the genus. In addition, the remoteness of the Hawaiian archipelago makes colonization by a coastal lineage more likely than by a terrestrial one. Under the observed phylogenetic patterns, an independent origin of terrestriality would have required two steps: the evolution of terrestriality along the branches of clades B and C. Conversely, a single origin of terrestriality may only be explained by invoking the adaptation to terrestrial habitats followed by a reversal to the marine habitat (i.e., 2 steps). Taiti et al. [19] considers the independent adaptation to terrestrial habitats in Hawaiian Ligia as the most plausible explanation for the origin of L. perkinsi populations in Oʻahu and Kauaʻi. They consider a shift from terrestrial to seashore conditions unlikely, given that most of the species in the genus occupy supralittoral habitats, and that terrestrial forms appear to have derived from supralittoral forms [28]. Similar conditions could have acted in Oʻahu and Kauaʻi that facilitated the independent colonization of freshwater habitats. Nonetheless, not enough is known about the biology of L. perkinsi to discard the possibility of a reversal. This species is often found in the rocky shores of streams [34], with populations in Oʻahu occurring less than 3 Km from shore and at low altitude [80]. Furthermore, L. perkinsi exhibits the highest osmoregulatory capabilities observed among Ligia species [81]. Post-hoc analyses from Approximately Unbiased (AU) tests implemented in CONSEL [82] rejected the monophyly of L. perkinsi (p = 1 × 10^-6), which would have supported a single origin of terrestriality. These tests also rejected the monophyly of L. hawaiensis (p = 2 × 10^-5), as well as the monophyly of L. perkinsi from Kauaʻi + L. hawaiensis from Kauaʻi (p = 1 × 10^-5).

Similar to the Hawaiian Ligia, the shrimp Halocaridinia rubra, which inhabits anchialine coastal pools, also shows multiple highly divergent lineages, with allopatric distributions [17,18]. In contrast with Ligia, however, presence of a highly divergent lineage in multiple islands is rare in the shrimp, with only one case reported among the three islands studied (i.e., Oʻahu, Maui, and Hawaiʻi). A shrimp lineage that was found mainly on the west coast of Hawaiʻi was also found in one locality of Oʻahu, represented by a single haplotype that was very close (only one substitution) to some haplotypes present in Hawaiʻi. Therefore, inter-island dispersal appears to have been more limited in this shrimp.

Morphological differences have been previously reported between L. perkinsi lineages (B, C), but not between L. hawaiensis lineages (D, F) [19]. Previous comparisons, however, relied on classic taxonomic characters. Geometric-morphometric approaches have proven useful in identifying differences between otherwise cryptic species in other invertebrate taxa [83-86], including crustaceans [87,88]. They also have been used to test body shape differences at the intraspecific level in isopods [89]. Our geometric-morphometric analyses revealed significant differences in body shape among the three highly genetically divergent supralittoral lineages examined (D, E, and F). Rates of correct assignment of quadratic DFAs after LOOCV were low, however, indicating body shape alone is of limited use as a taxonomic diagnostic tool. On average, lineages differ in traits widely used in the taxonomy of Ligia, including relative body width, distance between the eyes, and the protrusion of the distal-most point of the pleotelson [19,25,27,66,67]. Interestingly, individuals from Clade D exhibit average differences with respect to the other two clades in traits that were previously used to describe Ligia kauaiensis, such as a narrow oblong-ovoid body shape and shorter distances between the eyes [90]. This species, now considered a synonym of L. hawaiensis [20,91], was first described from individuals from the shores of Kalihiwai Bay, Kauaʻi [90], the same location (D1) included in our molecular and morphometric analyses. Therefore, the deep genetic divergence of Clade D (at least 13.57% COI K2P), its discrete geographic distribution, and apparently distinct morphology suggest that L. kauaiensis may be a valid species.

We also detected significant differences in the average overall body shape between sexes, with females exhibiting wider anterior segments than males and juveniles. These differences may be caused by the development of the ventral marsupium (i.e., brood pouch) in females. This structure forms from thoracic sterna and overlapping oostegites prior to copulation in mature females, and was present in all samples identified as females. As we did not observe obvious differences between males and juveniles, and females were classified based on the presence of the marsupium, a temporary structure [92], differences between the sexes in L. hawaiensis may be temporary, and thus, not relevant to the taxonomy of Ligia in the Hawaiian Archipelago. Lastly, we detected an effect of body size on body shape for L. hawaiensis, with larger individuals exhibiting a more elongated body with relatively smaller head than smaller individuals. Allometry has been reported also for Ligia pallasii, where in contrast to our results, larger individuals (usually males) tend to be disproportionality broader than smaller ones [93,94], a pattern that we have also observed in other Ligia from the northern East Pacific (unpublished data).

Conclusions

Our study revealed new lineages and expanded the distribution range of several lineages of supralittoral Ligia in the Hawaiian Archipelago. A previously unknown highly divergent lineage (Clade A) of supralittoral Ligia was found in the islands of Hawaiʻi and Maui. We found that Clade E, a lineage previously reported only from Oʻahu, is widely distributed in the Maui-Nui islands. Clade F, a lineage also previously reported only from Oʻahu, contains two additional moderately divergent lineages with geographically restricted distributions; one in Maui and one in Hawaiʻi. Our results supported the monophyly of all Ligia lineages endemic to the Hawaiian Archipelago, consistent with a single colonization event. Our results, however, did not support the monophyly of all the supralittoral lineages (currently grouped into L. hawaiensis), and/or the monophyly of the terrestrial lineages (currently grouped into L. perkinsi), implying more than one evolutionary transition between coastal and inland forms. The lack of monophyly of currently recognized species and the relatively high genetic divergences of several lineages argues for a taxonomic revision. The phylogeographic patterns of Ligia in the study area are complex, inconsistent with simple evolutionary patterns proposed for the Hawaiian Islands, such as the progression rule. Evidence for multiple historical dispersal events between islands is observed among Hawaiʻi, Oʻahu, and the Maui-Nui islands. In contrast, the oldest and most geographically distant of the major islands (Kauaʻi) shares no lineages with the other islands. Although multiple lineages inhabit several islands, no instances of sympatry at the locality level were detected. Significant body shape differences were observed among three L. hawaiensis clades examined (D, E, and F), one of which was consistent with previous recognition of a separate coastal species in Kauaʻi (i.e., L. kauaiensis).

Finally, our results are relevant for the protection of unique and highly localized biodiversity found in an environment subject to high pressure from human disturbances in the Hawaiian Islands. Destruction and modification of rocky intertidal beaches through activities such as construction and conversion to sandy beaches (e.g. by sand nourishing) can destroy the natural habitat of Ligia and, thus, jeopardize the survival of unique lineages endemic to the Hawaiian Islands.

Supporting Information

Acknowledgments

Dr. Miquel A. Arnedo, Dr. Francis G. Howarth, Alejandro Hurtado, and Shuang Liu helped in the collection of Ligia specimens used in this study. Several Texas A&M undergraduate students provided laboratory assistance, including: Jessica Fleming, Ellen Giddens, and Lauren Lescure. Dr. Mary Wicksten provided helpful comments. The Texas A&M University Brazos HPC cluster was used for phylogenetic analyses <brazos.tamu.edu>. This is contribution No. 234 of the Center for Biosystematics and Biodiversity.