Description of a novel Ligia species from Nihoa, a remote island in the Papahānaumokuākea Marine National Monument

Introduction

The Hawaiian Islands (hereafter HI) are a series of islands, atolls, islets, and rocky outcroppings of volcanic origin spanning ∼2,400-km of the northern Pacific Ocean. Islands in the archipelago are arranged in a relatively linear manner, with younger islands located towards the eastern end of the archipelago and older islands found in its western end. Younger islands include eight major islands, all of which have formed in the past 5 million years (in decreasing age: Ni‘ ihau, Kaua‘ i, O‘ ahu, Moloka‘ i, Maui, Lanai, Kaho‘ olawe, and Hawai‘ i). The older islands, found west of Kaua‘ i, include ten island groups ranging widely in size and elevation (in decreasing age: Kure Atoll, Midway Atoll, Pearl & Hermes Atoll, Lisianski, Laysan, Maro Reef, Gardner Pinnacles, French Frigate Shoals, Necker, and Nihoa). These islands are part of the Papahānaumokuākea Marine National Monument (hereafter “PMNM”), a protected area of the United States of America established by presidential decree on June 15, 2006 to protect natural and cultural resources from the region. The Monument initially protected 362,073 km² of marine habitats; however, it was extended by President Barack H. Obama in 2016 to encompass 1,508,870 km² of the Pacific Ocean. This makes the PMNM the largest contiguous fully protected conservation in the United States of America and one of the largest marine preserves in the world. The habitats of the PMNM support an incredible diversity of coral, fish, birds, marine mammals and other flora and fauna, many of which are endemic to the PMNM (Starr & Martz, 1999; Starr & Starr, 2008; Kane, Kosaki & Wagner, 2014). Nonetheless, recent descriptions of new species from the PMNM suggest additional new species may exist in this region (Stein & Drazen, 2014; Pyle, Greene & Kosaki, 2016; Sherwood et al., 2020; Alvarado et al., 2022; Sherwood et al., 2022).

Intertidal habitats of the PMNM are known to harbor Ligia isopods, a genus of poorly dispersing isopods shown to hold high levels of cryptic diversity (Taiti et al., 2003; Hurtado, Mateos & Santamaria, 2010; Eberl et al., 2013; Santamaria et al., 2013; Raupach et al., 2014; Santamaria, Mateos & Hurtado, 2014; Santamaria et al., 2017; Greenan, Griffiths & Santamaria, 2018; Santamaria, 2019). Currently, nine Ligia species are thought to be endemic to the HI: eight coastal species that inhabit rocky intertidal habitats, and a terrestrial species that inhabit terrestrial habitats at elevation in the islands of Kaua‘ i, O‘ ahu and Hawai‘ i. The eight coastal species were formerly recognized as L. hawaiensis Dana 1853; however, they were split into these species on the basis of molecular, morphological, and geographic distributional data (Santamaria, 2019). Despite reports of “L. hawaiensis” from the islands of Nihoa, Necker, and La Perouse Pinnacle in the PMNM (Taiti & Howarth, 1996), which are located 190 to 675-km from Kaua‘ i, no specimens from these islands were included in any of the molecular studies characterizing Ligia from the HI to date (Taiti et al., 2003; Santamaria et al., 2013; Santamaria, 2019). Given the limited dispersal potential exhibited of Ligia isopods and the long-term isolation of these oceanic islands, molecular characterizations of these populations are likely to uncover additional cryptic species of Ligia in the region.

In this study, we use molecular approaches to characterize Ligia isopods from the island of Nihoa, the easternmost island in the PMNM. Doing so, we aim to determine: (a) whether Ligia individuals from this highly remote island harbor any unique genetic lineages, (b) if so, what are the phylogenetic relationships of these lineages to other Ligia species previously reported from the HI, (c) whether these lineages are divergent enough to be considered a novel species, and if so (d) describe said lineages as a new species. We do so by incorporating phylogenetic reconstructions, and distance- and phylogeny-based molecular species delimitation methods on a multi-locus dataset comprised of all extant Ligia species from the Hawaiian Islands and newly collected specimens from Nihoa. Our results indicate Ligia from Nihoa represent a highly divergent genetic lineage that is reciprocally monophyletic with all other Ligia species from the HI. Given its genetic uniqueness and geographic isolation, we describe this lineage as Ligia barack sp. nov. on the basis of molecular characters. The formal description of this cryptic species adds to our understanding of the biodiversity of the PMNM.

Materials and Methods

Results

We successfully produced sequences for four mitochondrial and three nuclear genes for six Ligia specimens from Nihoa (hereafter L. barack sp. nov). Unique haplotypes have been deposited in GenBank and BOLD (see Table 1 for GenBank Accession Numbers). The addition of these sequences to the alignment produced by Santamaria (2019) produced a concatenated dataset 3,996-bp long prior to the removal of poorly aligned positions for the 16S, 12S, and 28S rDNA gene. This final alignment included 196 individuals from 40 localities in the HI including Nihoa (Fig. 1, Table 1). Removal of poorly aligned sites (43, 17, and 49 for the 16S, 12S, and 28S rDNA genes respectively) produced a final alignment 3,887-bp long containing 543 parsimony informative sites (COI = 185; Cyt-b: 120; 12S rDNA = 99; 16S rDNA = 91; 28S rDNA = 39; NaK = 6; H3A = 3). An annotated alignment is provided as Dataset S1.

All phylogenetic reconstructions completed in this study were similar to those reported by Santamaria et al. (2013) and Santamaria (2019), with the exception of L. barack sp. nov individuals (Fig. 2). These individuals were placed in a well-supported clade (BS = 100; PP = 100, Fig. 2) that excluded individuals from all other Ligia species endemic to the HI. Our phylogenetic reconstructions identified four highly divergent and reciprocally monophyletic lineages consisting solely of coastal Ligia endemic to the Hawaiian Islands: (a) Clade A (lavenders and purples in all figures; BS = 100; posterior probability (PP) = 100) which consisted of all L. dante (A2, A5 in Hawai‘ i), L. honu (A3–4 in Hawai‘ i) and L. eleluensis (A1, A6–7 in Maui) individuals; (b) Clade D (green in all figures; BS =100; PP =100) which included all L. hawaiensis individuals from Kaua‘ i (D1–2, D6) as well as L. barack sp. nov. (D7); (c) Clade E (oranges and yellows in all figures; BS = 100; PP = 100) consisting of all L. mauinuiensis individuals from O‘ ahu (E10), Moloka‘ i (E2, E3), Lana‘ i (E4), and Maui (E5–E9); and lastly (d) Clade F (reds in all figures; BS = 100; PP = 100) which included all L. kamehameha (F4–F11 in Hawai‘ i), L. rolliensis (F1–2 and F13–16 in O‘ ahu), and L. pele (F3, F12 in Maui) individuals. We also observed two lineages consisting of individuals of the terrestrial L. perkinsi: (a) Clade B (black in all figures) from O‘ ahu (B1), and (b) Clade C (blue in all figures; BS = 100; PP = 100) from Kaua‘ i (C1–3).

Clades D, E, and F were placed in a well-supported monophyletic group (BS = 100; PP = 100) with clades E and F identified as each other’s sister clade (BS = 81–100; PP = 91–97). The sister to the “D + E + F” clade was Clade C (BS = 99–100; PP = 100), which consisted of the terrestrial L. perkinsi from Kaua‘ i. Clade B, consisting of the terrestrial L. perkinsi from O‘ ahu, was identified as the sister clade to the large monophyletic group consisting of clades C, D, E, and F (BS = 100; PP = 100). The most basal group was Clade A, which consisted of coastal Ligia species from the islands of Maui and Hawai‘ i.

COI K2P distances between Ligia species from HI ranged between 3.0–17.8%, with comparisons between L. barack sp. nov and other species in the region ranging between 3.0–17.8% (Table 2). Genetic diversities produced when comparing L. barack sp. nov were low, ranging between 0.0–0.3% (Table 2).

Molecular species delimitations consistently identified L. barack sp. nov as a separate and distinct species from other Ligia species endemic to the HI. ASAP analyses of the COI dataset placed all Nihoa specimens in a separate putative species containing no Ligia from other localities in nine of the ten best partitions produced by ASAP, with only the ninth best supported partition (p-value rank = 4; W rank = 18; threshold distance = 0.040249) grouping Nihoa Ligia with L. hawaiensis individuals (Kaua‘ i). All tree-based MSDAs carried out in PTP, bPTP, and GMYC recognized L. barack sp. nov as a separate species. Lastly, comparisons between this newly described species and L. hawaienesis, its sister taxon, in KoT produced a K/θ ratio of 9.912.

Taxonomy

Based on the long-term and geographical isolation for Nihoa, morphological comparisons, results of phylogenetic reconstructions and MSDAs, COI K2P pairwise distances reported herein, and K/θ ratio between it and its sister taxon, we describe Ligia barack sp. nov., a new species of Ligia from Nihoa. A holotype and three paratypes were deposited at the Florida Museum of Natural History (FLMNH) in Gainesville, FL, USA. We describe L. barack sp. nov. primarily using molecular characters and some diagnostic morphological characters. We include a broad description of the holotype that covers traits evaluated by previous authors (Taiti et al., 2003; Santamaria et al., 2013; Santamaria, 2019), a photograph of the holotype of L. barack (Fig. 3), and illustrations of diagnostic features of this species (Fig. 4). Other traits not mentioned below (e.g., pereopods 2–6) are as described and/or illustrated by Taiti et al. (2003), Taiti, Ferrara & Kwon (1992), and Jackson (1933).

Ligia barack nov. sp.

LSID: urn:lsid:zoobank.org:act:558494AB-37D7-47BA-BA54-4E532D7585C6.

BOLD BINs: AFQ9578.

Materials examined: six individuals from the island of Nihoa (D7). Both males and females were included. The holotype (♂; UFID 72496; Fig. 3), and three paratypes (♀♀♀; UFID 72497-72499) from the type locality have been deposited at the Florida Museum of Natural History (FLMNH) in Gainesville, FL, USA.

Type locality: Hanaka‘ ie‘ ie (Adam’s Bay), Nihoa, Hawai‘ i, USA. (D7; 23°03′30.3″N 161° 55′27.6″W).

Description of holotype: male individual that is 17.8 mm long and 6.7 mm wide at the widest point of the pereionite 4 (body length to width ratio of ∼2.7; Fig. 2). Eyes are large (eye length is ∼0.5 greatest width of cephalon) and closely spaced (inter-eye distance ∼0.5 times eye length) (Fig. 4A). Posterolateral processes of the pereionite 7 extend ∼$\frac{1}{3}$ length of the pleonite 3. Antennae extends just past midbody, being about ∼0.6 times the total body length. Article 3 of the peduncle of the antenna is about 1.5X length of the peduncle article 2, with robust setae on either side of the distal end (Fig. 4B). Pleotelson shape similar to that of other Ligia in the Hawaiian Islands; however, the lateral posterior points are nearly parallel to the interior lateral posterior points (Fig. 4C). First three pereopods have papillar fields present, with those in the carpus and merus of the first pereopod present in over half of pereopod segment (e.g., pereopod 1 illustrated in Fig. 4D). The dactylus of the sixth and seventh pereopod have four small robust setae on the tergal margin (Fig. 4F). Apex of the appendix masculina in the endopod of the second pleopod is enlarged and slightly bilobed with distal end rounded (Fig. 4G).

The holotype is deposited in the FLMNH under UFID 72496. GenBank Accession numbers for sequences obtained from the holotype are: PP851829 (COI); PP852382 (16S rDNA); PP852387 (12S rDNA); PP856001 (Cyt-b); PP852394; (28S rDNA); PP856007 (NaK); and PP861092 (H3A).

Remarks: the herein described L. barack sp. nov can be distinguished from other coastal Ligia species endemic to the Hawaiian Islands by the absence of a tuft of very long thin setae on the tergal margin of the dactylus of the sixth and seventh pereopods (Figs. 4E–4F; see Fig. 4 in Taiti et al., 2003). The appendix masculina of the endopod of the second pleopod also differs between L. barack sp. nov and other species in the region (Taiti et al., 2003). The appendix masculina of L. barack is slightly bilobed with the distal end being rounded, which contrasts with the obliquely truncate morphology seen in coastal Ligia from the Hawaiian Islands (see Fig. 4G; contrast with Fig. 6 in Taiti et al., 2003). Lastly, L. barack exhibits slight differences in eye shape and size. This species has large eyes (eye length is ∼0.5 greatest width of cephalon) which is similar to other coastal Ligia species endemic to the Hawaiian Islands; however, the eyes appear to be the most distantly spaced of all these species. The inter-eye distance of L. barack is about ∼0.5 times the eye length, while the smallest ratio for other nearby species is ∼0.7 (seen in L. honu, L. hawaiiensis, L. kamehameha, L, mauiniensis, L. rolliensis; Santamaria, 2019). Lastly L. barack can also be distinguished using molecular characters listed below.

Diagnostic molecular characters:

COI: 1-C; 31-A; 94-C; 526-C

16S: 288(316)-T.

Cyt-b: 181-G; 223-C; 262-G; 265-G; 354-G

12S: 380(398)-G.

Distribution: rocky intertidal habitats of Nihoa.

Hawaiian common name: Pokipoki o Hanaka‘ ie‘ ie. Pokipoki is the Hawaiian name for terrestrial isopods and similar creatures inhabiting aquatic and terrestrial habitats. Meanwhile, Hanaka‘ ie‘ ie refers to the traditional name for Adam’s Bay of Nihoa Island. Thus, this name broadly translates to “the isopod from Adam’s Bay of Nihoa Island.”

Etymology: this species is named after Barack H. Obama, the former President of the United States of America, who was born in the island of O‘ ahu and who is responsible for the expansion of the Papahānaumokuākea Marine National Monument to its current size.

Discussion

The Hawaiian Islands (HI) were previously thought to harbor a single endemic coastal Ligia species: Ligia hawaiensis. This species, first described by Dana (1853), was determined to represent a cryptic species complex composed of allopatric species with distributional ranges largely limited to rift zones within a single island, single islands, or previously connected islands (Santamaria, 2019). Despite previous reports of L. hawaiensis from the remote and older islands found in the Papahānaumokuākea Marine National Monument (Taiti & Howarth, 1996), none of the molecular studies conducted on Hawaiian Ligia to date have included populations from these islands. This has left unanswered whether Ligia populations from the older and highly remote islands in the PMNM harbor highly divergent genetic lineages and/or novel species in need of description. By using similar molecular approaches to those used by Santamaria (2019) to describe highly genetically divergent yet largely morphologically cryptic lineages of Ligia in the HI as new species, we herein describe L. barack sp. nov from Nihoa.

Our molecular characterizations of Ligia individuals collected in Nihoa show this population to be highly divergent and isolated from other Ligia lineages and species found in the HI. We observed no sharing of haplotypes between L. barack sp. nov individuals and other Ligia populations in the HI at any of the four mitochondrial genes studied (e.g., COI, Cyt-b, 16S and 12S rDNA). Instead, Nihoa specimens harbored unique and private haplotypes that form a well-supported monophyletic group that excludes all other Ligia species from the HI and that are highly divergent from other ones found to date in Hawaiian Ligia. COI K2P divergences between Nihoa Ligia and other Ligia species from Hawaii ranged from 3.0–17.8%, values that are similar to other amongst species comparisons (Table 2). Meanwhile, within species COI K2P divergences amongst L. barack sp. nov individuals ranged from 0.0–0.7%. Not surprisingly, the K/θ ratio between L. barack sp. nov and its sister taxon (L. hawaiensis; K/θ = 9.912), greatly exceeds the K/θ ratio of 4 at which there is a 95% probability that two separate species are being compared (Rosenberg, 2003).

The phylogenetic placement of L. barack sp. nov is of interest, as our analyses recovered with high support both the monophyly of this newly described species and its sister relationship to L. hawaiensis (Fig. 4). The latter is a coastal species of Ligia whose distributional range is thought to be limited to the island of Kaua‘ i, the closest island to Nihoa. These islands are separated by ∼240 km of open ocean and have never been connected. This suggests that oceanic dispersal led to the colonization of these islands by Ligia. Nihoa’s older age (7.5 My) suggests the ancestor to L. hawaiensis in Kaua‘ i may have originated from Nihoa; however, additional work is necessary to establish the origins of these species as back-dispersals appear to have occurred in Ligia from the HI.

Despite consisting of the Ligia from the two oldest islands in our analyses (Kaua‘ i: 5 My, Nihoa 7.5 My), the monophyletic group consisting of L. hawaiensis + L. barack sp. nov clade was not found in a basal position in any of our analyses (Fig. 3). Instead, the most basal clade in all analyses was one comprised of Ligia species found in Maui and Hawai‘ i, the two youngest islands in the archipelago (<1.5 My). These findings are consistent with previous studies of Ligia from the HI (Santamaria et al., 2013; Santamaria, 2019) and suggest that the evolution of Ligia in the region have been shaped by colonization, extinction, and back-dispersal events.

Our description of L. barack sp. nov from the island of Nihoa underscores the importance of molecular approaches in conservation efforts in the PNMN. Future studies of Ligia from other islands in the PNMN are likely to uncover additional highly divergent genetic lineages likely representing new species in need of description. These studies may also help further elucidate the evolutionary history of Ligia in the HI. Meanwhile, molecular characterizations of other poorly dispersing organisms may similarly uncover new species or genetic lineages in other taxa and thus increase our understanding of the biodiversity of these highly remote and isolated islands. Molecular tools may also aid in the monitoring of the spread of alien species, a critical threat to the fauna and flora of the PMNM (DeFelice et al., 1998; Selkoe, Halpern & Toonen, 2008). Ligia exotica has been shown to occur in Midway Atoll, an island within the PMNM (Santamaria et al., 2022). This species of Asian origin known to have been introduced to manmade coastal habitats around the world and is a potential competitor to endemic coastal Ligia (Hurtado et al., 2018). The use of genetic tools such as COI barcoding and eDNA may be useful to monitor the presence of this species in other regions of the PMNM without extensive field-work.

Conclusion

The use of both mitochondrial and nuclear gene fragments to characterize Ligia isopods from Nihoa uncovered a highly divergent lineage of Hawaiian Ligia not previously reported from other localities in the HI. Phylogenetic and species delimitation approaches provide evidence that this lineage represents a new species of Ligia, which we describe as Ligia barack sp. nov. To our knowledge, this species is the first intertidal crustacean that is described from and likely solely endemic to the island of Nihoa. This discovery underscores the unique biodiversity of the PMNM and the need for additional studies of poorly dispersing taxa within it. Our findings also further provide evidence of Ligia isopods as an example of in-situ speciation of a Hawaiian marine animal.

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