A new freshwater species of Gnorimosphaeroma (Crustacea, Isopoda, Sphaeromatidae) from Chichi-jima Island, Ogasawara Islands, Japan

This study describes Gnorimosphaeroma rivulare sp. nov. from a stream on Chichi-jima Island, Ogasawara Islands, Japan. This is the second freshwater species of Gnorimosphaeroma and the third Sphaeromatidae from oceanic islands. Gnorimosphaeroma rivulare sp. nov. is morphologically similar to G. boninense Nunomura, 2006, G. naktongense Kwon & Kim, 1987 and G. saijoense Nunomura, 2013. However, G. rivulare sp. nov. differs from these species in various morphological features, such as the shape of pleotelson and pereopod 2, relative length of antennule peduncular articles and pleopod 3 rami, number of setae on maxillula and maxilliped, and seta-tion on pereopod 3. Phylogenetic analyses revealed that G. akanense is sister to G. saijoense , and together they are sister to G. hokuri-kuense . This three taxa clade is sister to G. rivulare sp. nov. with G. iriei basal to them all. Our analysis concludes that G. boninense from Haha-jima Island, Ogasawara Islands is only distantly related to G. rivulare and may represent an independent colonization event.


Introduction
Generally, oceanic islands do not occur on continental shelves.These are islands that have never been connected to a continental landmass.Inland water-dwelling organisms cannot reach oceanic islands without crossing the ocean or speciating from organisms that find themselves in a new habitat to which they have adapted, e.g., marine and then isolated in freshwater.Therefore, the occurrence of freshwater fish and invertebrates on oceanic islands is generally limited (Lévêque et al. 2008;Strong et al. 2008;Väinölä et al. 2008;Wilson 2008).
The Ogasawara Islands, oceanic islands, are a group of approximately 30 islands located in the Pacific Ocean approximately 1000 km southeast of the Japanese archipelago.The Ogasawara Islands have small and well-developed rivers inhabited by freshwater crustaceans, molluscs, and caddisflies.These taxa have limited diversity on oceanic islands (Satake and Cai 2005;Nunomura and Satake 2006;Miura et al. 2008;Tomikawa et al. 2022;Ito et al. 2023).
More than 10,600 species of Isopoda have been described worldwide, occurring in diverse aquatic and terrestrial environments (Boyko et al. 2023).Approximately 950 species of isopods have been recorded from inland waters; however, the number of species occurring in inland waters of oceanic islands is far fewer, with only a few species recorded from Pacific Ocean islands (Jaume and Queinnec 2007;Wilson 2008).The aquatic sphaeromatid genus Gnorimosphaeroma Menzies, 1954 has 26 described species (Boyko et al. 2023).They are restricted to the Japanese Islands, Korean Peninsula, Mainland China, Alaska, and the eastern Pacific coast of North America (Tattersall 1921;Jang and Kwon 1993;Nunomura 2013;Wetzer et al. 2021).Most Sphaeromatidae are marine (Sket and Bruce 2004;Bruce 2005;Jaume and Queinnec 2007).The genus Gnorimosphaeroma is unusual because it includes not only marine species, but also has seven brackish and eight freshwater described species (Fig. 1, Table 1).
Satake and Ueno ( 2013) reported an unidentified species of Gnorimosphaeroma from a stream on Chichi-jima Island, Ogasawara Islands.A recent collection of Gnorimosphaeroma was made on Chichi-jima Island during the author's KT and NN research expedition to the Ogasawara Islands.A colleague provided an additional collection of seven specimens.In this study, we describe this previously undescribed species of Gnorimosphaeroma.To clarify the phylogenetic position of this Gnorimosphaeroma species, we performed a molecular phylogenetic analyses based on nuclear 18S rRNA and mitochondrial 16S rRNA genes.

Samples
Specimens of an unidentified species were collected from under the boulders in the upper stream of Nagatani River, Chichi-jima Island, Ogasawara Islands, Japan (Fig. 2).Our molecular phylogenetic analysis includes nine described species of Gnorimosphaeroma, one unidentified species from Chiba, Japan, one unidentified species from San Francisco Bay, and the new undescribed freshwater species described in this paper (Table 2).The specimens were collected using a fine-mesh hand-net and samples were subsequently fixed in 70% or 99% ethanol in-situ.Specimens fixed in 70% ethanol were transferred to 99% ethanol in the laboratory.

Morphological observation
All appendages of G. rivulare sp.nov.were dissected in 80% ethanol and mounted in gum-chloral medium.The slides were examined using a stereomicroscope (Olympus SZX7, Japan) and a light microscope (Nikon Eclipse Ni, Japan), and the body and appendages were illustrated using a camera lucida.One male (paratype, NSMT-Cr 31507) was dehydrated through a graded ethanol series, and dried using hexamethyldisilazane (HMDS) (Nation 1983).They were then sputter-coated with gold and observed using scanning electron microscopy (SEM, JSM-6510LV).Body length was measured as a straight-line distance from the rostral point to the posterior margin of pleotelson within the nearest 0.1 mm.Type specimens were deposited at the National Museum of Nature and Science, Tsukuba, Japan (NSMT).
The phylogenetic analyses were conducted based on 16S and 18S sequence data generated for this project and also includes previously published sequences.Ancinus sp.(Ancinidae) and two Chitonosphaera species (Sphaeromatidae), C. lata (Nishimura, 1968) and C. salebrosa (Nishimura, 1969) were used as the outgroup (Wetzer et al. 2013(Wetzer et al. , 2018)).The sequences were aligned using the Muscle algorithm implemented in MEGA XI (Tamura et al. 2021).The aligned lengths of the 16S and 18S were found to be 518 and 559 bp, respectively.Moreover, the concatenated sequences yielded 1077 bp of alignment positions.
Pereopod 1 (Fig. 4A, B) basis with a ventrodistal simple seta, ventral margin with fine setae; merus lobate dorsodistally, dorsodistal corner with 4 setae, ventral margin with robust setae and fine setae; carpus short with 2 robust setae and fine setae on ventral margin; propodus oval, swollen, length 2.5 times as long as wide, with 2 robust setae on ventral margin and 5 setulate setae close to ventral margin; dactylus length 0.6 times as long as propodus.Pereopod 2 (Fig. 4C, D) basis with a ventrodistal simple seta; merus weakly lobate dorsodistally, with 5 setae on dorsodistal corner, ventral margin with fine setae; carpus with 6 robust and 3 slender setae on distal surface, ventral margin with fine setae; propodus subrectangular, not swollen, length 2.8 times as long as wide, with 3 robust setae on ventral margin; dactylus length 0.5 times as long as propodus.Pereopod 3 (Fig. 4E, F) basis with a ventrodistal simple seta; merus lobate dorsodistally, dorsodistal corner with 6 setae, ventral margin with sparse fine setae; carpus with 5 robust and some slender setae on distal margin, ventral margin with sparse fine setae; propodus length 2.8 times as long as wide, with 2 robust setae and a slender single seta on ventral margin; dactylus length 0.5 times as long as propodus.Pereopod 4 (Fig. 4G) basis with a simple seta on ventrodistal corner and some broom setae on dorsal margin; ischium with fine setae on ventral margin; merus lobate dorsodistally with robust setae, dorsal and ventral margins with fine setae; carpus with sparse fine setae on ventral margin; propodus with 3 robust setae on ventral margin, dorsal and ventral margins with sparse fine setae.Pereopod 5 (Fig. 4H) basis with a simple seta on dorsodistal corner; merus and carpus with robust setae on distal margins; propodus with 3 robust setae on dorsal margin.Pereopods 6 and 7 (Fig. 5A, B) basis with a simple seta on dorsodistal corner; merus and carpus with robust setae on distal margins; propodus with 2 robust setae on dorsal margin.
Etymology.The specific name rivulare is derived from a Latin adjective rivularis, which means brook living, referring to the habitat of the new species.
Distribution and habitat.This species is known only from the type locality.The specimens were collected from beneath the cobbles in an upper stream of Nagatani River.

Discussion
Our molecular phylogenetic analyses revealed that marine, brackish, and freshwater taxa appear mixed throughout the tree.Gnorimosphaeroma boninense from a small stream on Haha-jima Island, Ogasawara Islands and G. rivulare sp.nov.are not sister taxa; however, the latter forms a monophyletic clade with G. akanense from Akan River, G. hokurikuense from freshwater streams in Honshu, G. iriei from springs in Kyushu, and G. saijoense from brackish waters at the mouths of rivers in Shikoku.This suggests that G. boninense and G. rivulare sp.nov.may have colonised the Ogasawara Islands independently.However, without including all Gnorimosphaeroma species in the analyses and also carefully reviewing the ecologies of each species it is not possible to definitively determine invasion and evolutionary history.Multiple invasions of freshwater invertebrates into the Ogasawara Islands have been reported for the amphipod Crustacea (Tomikawa et al. 2022).Tomikawa et al. (2022) showed that two species of melitid amphipods, Melita nunomurai Tomikawa &Sasaki, 2022 andM. ogasawaraensis Tomikawa &Sasaki, 2022, which occur in freshwater of Ogasawara Islands, are closely related to the species occurring on Honshu Island and the Nansei Islands, respectively.Possibly, they may originate from different regions.Although the origin of freshwater Gnorimosphaeroma on the Ogasawara Islands cannot been clarified in this study, we suggest that further elucidation of the evolutional history of freshwater isopods on oceanic islands will be possible with enhanced taxon sampling and broad genetic sampling in the future.
The freshwater invertebrate fauna of Chichi-jima Island, Ogasawara Islands, has been relatively well investigated (Satake and Ueno 2013;Satake et al. 2019).However, G. rivulare sp.nov.has only been found at one site in the upper reaches of Nagatani River on Chichi-jima Island (Satake and Ueno 2013).In addition to the low water volume in this species' habitat, the water flow is being dammed up by the roots of the non-native plant Bishop wood, Bischofia javanica.Freshwater Gnorimosphaeroma have been found in springs and mountain streams (e.g.Nunomura 1998) and is considered to prefer oxygenated lotic waters.A stagnant bottom environment may not be favourable for G. rivulare sp.nov., and could severely limit its future persistence.For this newly described species survive, conservation of this restricted habitat is an urgent issue that needs to be addressed.

Figure 1 .
Figure 1.Map showing the type localities of Gnorimosphaeroma species.

Figure 8 .
Figure 8. Maximum likelihood tree for 1077 bp of 16S rRNA and 18S rRNA markers.Numbers on nodes represent ultra-bootstrap values for maximum likelihood and Bayesian posterior probabilities.Symbols beside the species name indicate the habitats shown in Fig. 1.

Table 1 .
The type localities and habitats of Gnorimosphaeroma species.

Table 2 .
Samples used for the phylogenetic analyses.Sequences marked with an asterisk (*) were newly obtained in the present study.ND, no sequence available.