Bi-Hemispheric Distribution and Ecology of the Commensal Amphipod Leucothoe nagatai Ishimaru, 1985 (Crustacea: Leucothoidae)

Abstract

Reports on the taxonomy and distribution of the amphipod crustacean Leucothoe nagatai Ishimaru, 1985, are discussed including significant range extensions from the type locality in the Sea of Japan. This research was based on existing museum specimens, and in situ collections of new material. Originally described from the branchial chamber of the solitary ascidian Styela plicata (Lesueur, 1823), several new ascidian and sponges host species from California and New Zealand are documented for L. nagatai. Molecular studies confirm that northern and southern hemisphere populations are genetically indistinguishable. Color patterns in live and freshly preserved specimens of L. nagatai are diagnostic across its geographic range aiding in identification by nontaxonomists. Feeding habits of leucothoids within their invertebrate hosts are elucidated and discussed. Figures and plates of known invertebrate hosts, color patterns, and taxonomic structures are included.

Leucothoid amphipods commonly occur as inquiline commensals of ascidian, sponge, and bivalve mollusk hosts. The current Leucothoidae combines two previously distinct families, Anamixidae and Leucothoidae, into a single family (Lowry and Myers 2017). Historically, collecting efforts often encountered leucothoids as incidentals in larger benthic samples, and while known as associates inside certain invertebrate hosts, specific host information was lacking from early collections. During the past 20 years, investigators collected and processed invertebrate hosts in situ for leucothoid inhabitants leading to documentation of specific invertebrate hosts and a surge in new leucothoid taxa (Thomas and Barnard 1983, Thomas 1997, 2015, Thomas and Klebba 2006, 2007, Winfield and Alvarez 2009, Winfield et al. 2009, Thomas and Krapp 2011, Ortiz and Winfield 2012, Thomas and Watling 2012, White and Reimer 2012a, b, c, White 2019). In addition to new taxa descriptions, these studies provided detailed ecological information and behavioral observations elucidating functional morphology, especially in feeding studies and observations.

For the purpose of clarity, the authors chose the term "introduced" in lieu of "invasive" as the latter is associated with impact or harm to the environment (Jim Carlton, pers. comm.).

Ishimaru (1985) described L. nagatai (Figure 1) from Japanese waters from the ascidian hosts Styela plicata (Lesueur, 1823) (Figure 2A) and Halocynthia roretzi (Drasche, 1884) (Figure 2B, Table 1). However, there remains a dearth of published taxonomic

Figure 1.

Leucothoe nagatai. (A) Female, Pt. Loma, California. Photo credit: Greg Rouse; (B) Female, Nelson Marine, Nelson, New Zealand. Photo credit: James D. Thomas.

records for L. nagatai in the years since its description. This paper documents occurrences of Leucothoe nagatai (Ishimaru, 1985) (Figure 1) from additional host ascidian and sponge species and documents L. nagatai as introduced in coastal waters of California and New Zealand.

Nagata (1965; p. 159, figs 9–10) collected Leucothoe specimens from the Seto Island Sea, identifying them as Leucothoe alata (Barnard 1955). Subsequently, Ishimaru (1985) revised this material to L. nagatai, noting several distinctions from L. alata. Nagata (1965) did not fully illustrate the mandibular incisors, lacinia mobiles, or raker spine rows of his material, all of which are diagnostic features of L. nagatai. Briefly, L. nagatai is distinguished from L. alata by the following characteristics (corresponding features of L. alata in parenthesis if necessary): antennae 1 and 2 short, stubby (much longer); gnathopod 1, anterior margin of propodus linear (margin inflated); maxilliped, basal article 2 moderately alate (strongly); uropod 3, peduncle, and rami short (much longer).

Observations of in situ feeding of leucothoids show how members of the Leucothoidae utilize host-generated currents to feed inside the hosts, and in some cases may exhibit social structure and defense against conspecifics from other hosts (Thomas 1997). Feeding is accomplished by the amphipod orienting into host generated feeding currents and utilizing setae-covered medial surfaces of the carpus and propodus of the second gnathopods to filter food particles (Figure 3). The antennae, setose medial margins of the smaller first gnathopods, and the maxilliped palp form a setal comb to gather food material from the second gnathopods and bring the food particles to the mouthpart region for ingestion.

Improved digital imaging and stacked focus software programs now allow the capture of color patterns in live and recently collected materials, which is valuable in identifying and documenting species, especially for nontaxonomists and other workers involved in ecological surveys (White and Reimer 2012a,b,c). Once color patterns have faded after fixation and preservation, identification usually requires dissection under a microscope.

Material and methods

In the field, potential amphipod host species were collected in situ either by SCUBA or by sampling biofouling communities (ropes, floats, dock pilings) in harbors and marinas. In the lab, host specimens were dissected to extract the commensal amphipods. Material was fixed in seawater buffered 2% formalin for morphological investigations and in 99.5% ethanol for molecular analysis.

Genomic DNA was extracted from urosomes of individual ethanol-preserved L. nagatai specimens from three populations

Figure 2.

Host species for Leucothoe nagatai. (A) Styela plicata, Irukabana, Etajima, Hiroshima Bay, Japan. Photo credit: Daisuke Uyeno; (B) Halocynthia roretzi, Irukabana, Etajima, Hiroshima Bay, Japan. Photo credit: Daisuke Uyeno; (C) Pyura vittata, Matoba, Takehara, Seto Inland Sea, Japan. Photo credit: Daisuke Uyeno; (D) Leucandra heathi, Copyright Neil McDaniel, World Porifera Database; (E) Ciona robusta. Photo credit: Melissa Frey, Burke Museum; (F) Leucoselenia eleanor, Copyright Diver.net, House of Scuba; (G) Styela clava. Photo credit: Richard Taylor.

Table 1.

List of Locations and Hosts for Leucothoe nagatai Ishimaru, 1985

Figure 3.

Carpal feeding net, Leucothoe gnathopod 2. Photo credit: James D. Thomas.

(California, New Zealand, and Japan) after morphological examination (Table 1). Voucher specimens are deposited at the National Museum of Nature and Science in Tokyo, Japan (NMST) (Table 2). Extractions and amplification of 18S ribosomal DNA (18S rDNA) and cytochrome oxidase subunit I mitochondrial DNA (COI mtDNA) were performed following the protocols of White and Reimer (2012d) and White et al. (2016). Invertebrate-specific primers were used for 18S rDNA (Spears et al. 2005) and for COI mtDNA (Geller et al. 2013).

PCR products were sequenced in both directions by Eurofins MWG Operon (U.S. A.). Consensus sequences were made, edited, and aligned in Bioedit 7.0.5.3 (Hall 1999). The 18S rDNA alignment contained sequences of 771 bp for 21 taxa (two outgroup, eight California, six New Zealand, and five Japan sequences) and COI mtDNA alignment contained sequences of 586 base pairs (bp) for eight of the same taxa (two outgroup, three California, one New Zealand, and two Japan sequences). 18S rDNA sequences included the V4 hypervariable region (indels), which is a known species-level marker for Leucothoidae (White 2011, White et al. 2016). Outgroup sequences were from Leucothoe tunica White, 2019 from Florida, U.S.A. Both alignments are available from the corresponding author and sequences are available from GenBank (Table 2).

Maximum likelihood analyses were completed using PhyML (Guindon and Gascuel 2003) with an input tree generated by BIONJ (Neighbor Joining) with the Generalized Time Reversible (GTR) model, invariable sites, and a discrete gamma distribution with eight substitution rate categories. Base frequencies were estimated from the dataset. The bootstrap method was used to measure support for the maximum likelihood tree with 1,000 replicates in PhyML. The datasets

Table 2.

List of Specimens Used for DNA Sequence Data, Including Museum and GenBank Accession Numbers

generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Results

Amphipods collected from Japan, California, and New Zealand are identified as L. nagatai based on morphological and molecular analyses (Table 1). The levels of 18S rDNA sequence divergence were 0–0.003 among L. nagatai sequences from all populations and 0.177–0.203 between L. nagatai and outgroup sequences. The levels of COI mtDNA sequence divergence were 0–0.232 among L. nagatai sequences from all populations and 1.594–1.751 between L. nagatai sequences and outgroup sequences. Maximum likelihood (ML) analysis of 19 18S rDNA sequences of L. nagatai from California, New Zealand, and Japan resulted in a tree depicting a monophyletic clade with a 100% bootstrap support value. The same results were evident in the resulting tree from analysis of the COI mtDNA sequences. Sequences from all populations were scattered throughout both trees (Figure 4).

Expanded information on host data for Leucothoe nagatai was collected from three solitary ascidians in Japan: Styela plicata (Figure 2A), Halocynthia roretzi (Figure 2B), and Pyura vittata (Stimpson, 1852) (Figure 2C). In California coastal waters, L. nagatai was found most frequently in the ascidian Ciona robusta Hoshino and Tokioka 1967 (Figure 2E), and from regional endemic sponges Leucandra heathi Urban, 1906, (Figure 2D) and Leucoselenia eleanor Urban 1906 (Figure 2F). In New Zealand, specimens were collected from ports and marinas from the ascidian S. plicata and Styela clava Herdman, 1881 (Figure 2G).

Figure 4.

Maximum likelihood phylogenetic trees of (A) COI mitochondrial DNA and (B) 18S ribosomal DNA gene sequences from specimens of L. nagatai collected from Japan (J), California, USA (U), and New Zealand (N). Trees were rooted with Leucothoe tunica White, 2019 as the outgroup (O). Numbers above branches represent maximum likelihood bootstrap values. Sequences are represented by GenBank accession numbers. Image created in Adobe Illustrator.

Discussion

In California samples of C. robusta, the first author documented an average of 12–50+ specimens per tunicate. When interior cavities of C. robusta became overcrowded in late summer, the amphipods would excavate exterior slits in the tunic material and inhabit these "taco-like" domiciles (R. Velarde, pers. comm.). In mouthpart structure, L. nagatai exhibits significantly larger than normal mandibular incisors, laciniae mobiles, and broad, sharp raker spines (Figure 5) than most leucothoids possibly assisting in forming these excavations. In New Zealand, Styela plicata habitation numbers were comparatively less than found in California, with an average of 2–12 specimens encountered per tunicate. In California, L. nagatai was collected from two regional sponge hosts along with L. alata. This suggests that L. nagatai may displace the native L. alata, at least in the case of these two sponge hosts.

Ciona robusta is a host for L. nagatai; however, Ciona intestinalis Linnaeus (1767) is widely reported as introduced and has been confused with C. robusta. Brunetti et al. (2015) applied molecular tools investigating this matter to elucidate two distinct cryptic species complexes within the C. intestinalis group. The C. intestinalis species group, a North Atlantic species, was recognized as distinct from the C. robusta group found in Japanese and NW Pacific waters.

Leucothoe nagatai exhibits a consistent and distinct color pattern throughout its range in both northern and southern hemispheres. This pattern is represented by a number of

Figure 5.

Leucothoe nagatai, left mandible, 1000×. Photo credit: James D. Thomas.

pinkish to red lateral circles on pereonite and coxal segments, and a distinctive "spread eagle" dorsal coloration on each pereonite segment (Figure 1). In New Zealand coastal regions, the first author collected a currently undescribed leucothoid resembling L. nagatai from the endemic ascidians, Asterocarpa coerulea Quoy and Gaimard (1834), Corella eumyota Trausedt 1882, and Cnemidocarpa bicornuta Sluiter, 1900. This unidentified leucothoid differs from L. nagatai in having a distinct coloration pattern in live and freshly preserved materials (Figure 6) and a uniarticulate (versus 2-articulate) palp on the first maxilla. Both species share the short form dactyl of the first gnathopod. To date, this undescribed species is limited to shallow coastal habitats and has not yet been recorded in New Zealand harbors and marinas, which is dominated by L. nagatai.

In reviewing museum collections and taxonomic literature for occurrences of L. nagatai, the authors failed to discover references or collections prior to the 1980s. The comprehensive works of J.L. Barnard based on museum materials and collections in California and New Zealand from the 1950s to the late 1970s did not encounter L. nagatai (Barnard 1952, 1955, 1959, 1962, 1964a,b, 1966, 1969, 1972a,b, 1979). Thus, it appears that L. nagatai was absent from New Zealand and California coastal waters in the late 1970s and early 1980s. Distribution of L. nagatai between northern and southern hemisphere locales remains unreported, and is expected to be limited to cool temperate, and not tropical and subtropical waters.

These data demonstrate the importance of taxonomically accurate historical collections in evaluating movement and introductions of marine species. They also herein provide a rough timeline for when L. nagatai became established in these two geographically separate areas (Figure 7). The most likely introduction scenario is transport by marine vessel traffic as biofouling on freighter and container ships. A similar pattern was documented for Leucothoe eltoni (Thomas, 2015),

Figure 6.

Undescribed Leucothoe sp. from New Zealand. Photo credit: James D. Thomas.

Figure 7.

Map representing the geographic distribution of Leucothoe nagatai

which has a trans-Pacific distribution (White et al. 2019). Along the western coastline of the United States, L. nagatai appears to have spread progressively northward from marinas and harbors in and around San Diego to San Francisco Bay in the south to San Francisco in the north. Nonhost-associated specimens were taken during California Department of Fish and Game Introduced Species Surveys of bays and harbors. None were taken in similar surveys of the open coast.

In New Zealand, L. nagatai is currently restricted to marinas and harbors, not having spread outside these habitats to wider coastal environs. Future research on L. nagatai distribution should include examination of materials from other museum collections in the southern hemisphere, e.g., Australian Museum in Sydney; the Melbourne Museum in Victoria; and the Western Australian Museum in Perth.