On the brink of extinction: a new freshwater amphipod Jesogammarusacalceolus (Anisogammaridae) from Japan

Abstract Freshwater habitats, especially cold springs, are environments in which the risk of extinction faced by organisms remains high due to human activities. To conserve endangered species, it is important to describe and name them. Here, a new, endangered freshwater anisogammarid amphipod species, Jesogammarus (Jesogammarus) acalceolussp. nov., found in a spring in Aomori Prefecture, Japan, is described which is potentially the sole remaining habitat of this species. Both morphological and molecular phylogenetic results strongly support the nesting of the new species within Jesogammarus. Jesogammarus (J.) acalceolussp. nov. is the first species of genus Jesogammarus that was found to lack a calceolus, a sensory organ located on male antenna 2. Thus, the diagnostic criteria for this genus required amendment. A reconstruction of ancestral calceoli, based on a molecular phylogenetic tree, revealed that the common ancestor of Jesogammarus possessed calceoli, which were secondarily lost in J. (J.) acalceolussp. nov. Our results indicate that this new species, which is key to clarifying the evolution of the calceolus, is of high conservation significance.


Introduction
Fresh water is indispensable to human life. It is also an important habitat for many aquatic organisms. Fresh water accounts for ca. 2.5% of all water on Earth (Lehner remaining habitat of this species (Fig. 1). We describe this species as J. (J.) acalceolus sp. nov. Describing and naming this species, as have been done here, can be considered important first steps leading to its conservation. In addition, we investigated the evolution of calceoli in Jesogammarus species with molecular phylogenetic analyses and ancestral state reconstruction.

Sample collection
Specimens of J. (J.) acalceolus sp. nov. were collected from Haguro Shrine Spring, Hirosaki, Aomori Prefecture, Japan (40. 6153°N, 140.3854°E). Amphipods were collected by a fine-mesh hand net from fallen leaves and mosses. Specimens were fixed in 99% ethanol on the site.

Morphological observation
Appendages of the examined amphipods were dissected using needles under a stereomicroscope (Olympus SZX7) and mounted in gum-chloral medium on glass slides. Prepared specimens were examined by a light microscope (Nikon Eclipse Ni) and illustrated using the aid of a camera lucida attached to the light microscope. The body length was measured from the tip of the rostrum to the base of the telson along the dorsal curvature to the nearest 0.1 mm following Tomikawa et al. (2017). The specimens have been deposited in the Tsukuba Collection Center of the National Museum of Nature and Science, Tokyo (NSMT).

PCR and DNA sequencing
Genomic DNA was extracted from the pleopod muscle of the specimens following procedures detailed by Tomikawa et al. (2014). The primer sets for PCR and cycle sequencing reactions used in this study were as follows: for 28S rRNA (28S), 28SF and 28SR (Tomikawa et al. 2012); for cytochrome c oxidase subunit I (COI), Am-COI-H and Am-COI-T (Tomikawa 2015); and for 16S rRNA (16S), 16STf (Macdonald III 2005) and 16Sbr (Palumbi 1996). PCR and DNA sequencing were performed using the method detailed by Tomikawa (2015). The newly obtained DNA sequence has been deposited in the International Nucleotide Sequence Database Collaboration (IN-SDC) through the DNA Data Bank of Japan (DDBJ) ( Table 1).

Molecular phylogenetic analyses
The phylogenetic analyses were conducted based on sequences of nuclear 28S rRNA and mitochondrial COI and 16S rRNA genes. The alignment of COI was trivial, as no indels were observed. The sequences of 28S and 16S were aligned using the Muscle algorithm implemented in MEGA X (Kumar et al. 2018). Phylogenetic relationships were reconstructed via Maximum Likelihood (ML) and Bayesian Inference (BI). The best evolutionary models were selected based on the corrected Akaike Information Criterion (AIC) for ML and Bayesian Information Criterion (BIC) for BI using MEGA X (Kumar et al. 2018). ML phylogenies were conducted using MEGA X (Kumar et al. 2018) under the substitution model GTR+G+I, and 1,000 bootstrap replications (Felsenstein 1985) were performed to estimate statistical support for branching patterns. BI analyses were estimated using MrBayes v3.2.6 (Ronquist et al. 2012) under the substitution model GTR+G+I, with Markov chains of 10 million generations. Parameter estimates and convergence were checked using Tracer v1.7.1 (Rambaut et al. 2018), and the first 1 million trees were discarded as burn-in. Two gammarid species, Gammarus mukudai Tomikawa, Soh, Kobayashi &Yamaguchi, 2014 andG. nipponensis Uéno, 1966, were included in the analyses as outgroup taxa. Haguro Shrine Spring, Aomori, Japan

Ancestral state reconstruction
The ancestral states of the calceolus on male antenna 2 were reconstructed on the tree (Fig. 2) via the likelihood model using Mesquite v3.61 (Maddison and Maddison 2019). The Markov K-state 1 parameter model was used for likelihood reconstruction at each ancestral node with equal probability for all particular character state changes.

Molecular phylogenetic analyses
The monophyly of Jesogammarus was inferred with maximum (100% bootstrap support [BS]) and relatively low (

Ancestral state reconstruction
The likelihood reconstruction (Fig. 2) demonstrated that the calceolus on male antenna 2 was an ancestral character state of the most recent common ancestor (MRCA) of the Jesogammarus species, with 0.96 proportional likelihood (PL). The character

Taxonomic account
Family Anisogammaridae Bousfield, 1977 Genus Jesogammarus Bousfield, 1979 Type species. Anisogammarus jesoensis Schellenberg, 1937 Diagnosis. Pleonites not carinate dorsally, with slender and robust setae (robust setae often lacking). Dorsal margins of urosomites with 4 (3), 4 (2), 2 (4) clusters of setae or single robust seta; urosomite 2 without prominent median tooth. Antenna 1 longer than antenna 2; article 1 of peduncle subequal to or slightly longer than article 2. Male antenna 2, flagellum with or without calceoli. Maxilla 1, palp article 1 without Filled circles at each species represent states of habitat; pie charts at internal nodes present proportional likelihoods of reconstruction. Key nodes are labelled with the proportional likelihood of the presence or absence of the calceolus on male antenna 2, which was reconstructed as most likely to be at that node. setae. Female gnathopods 1 and 2 strongly dissimilar. Coxal gills on gnathopod 2 and pereopods 3-7, gills 2-5 each with 2 accessory lobes, gills 6 and 7 each with 1 accessory lobe. Uropods 1 and 2, rami extending beyond peduncle of uropod 3. Uropod 3, inner ramus not longer than 0.4 times of that of outer ramus; terminal article distinct. Brood plate 2 of female broadly expanded anteroproximally.
Mouth parts. Upper lip (Fig. 3K) with fine seta on rounded distal margin. Mandibles (Fig. 3L-N), left and right incisors comprising five and four teeth, respectively, left lacinia mobilis comprising four teeth, right lacinia mobilis bifid with many denticles; molar process triturative with plumose seta; left and right mandibles with seven and five blade-like setae on accessory setal rows, respectively; palp comprising 3 articles with length ratio of 1.0 : 3.3 : 2.8; article 1 of palp without setae; article 2 with 25 setae; article 3 bearing pair of setae on inner surface, three clusters of setae and single seta on outer surface. Lower lip (Fig. 3O), outer lobes broad, inner lobes indistinct. Maxilla 1 (Fig. 3P) with medial margin of inner plate bearing 20 plumose setae; eleven serrate robust setae on outer plate apically (Fig. 3Q); palp comprising 2 articles, article 1 marginally bare, apical margin of article 2 with five robust setae and two slender seta. Maxilla 2 (Fig. 4A) with inner plate bearing oblique inner row of 17 plumose setae. Maxilliped (Fig. 4B) with inner plate bearing three apical and two inner marginal robust setae; outer plate, apical margin with plumose setae and inner margin with robust setae; palp comprising four articles, inner margin and submargin of article 2 with rows of setae, article 3 bearing facial setae, slightly curved article 4 with slender nail.
Gnathopods. Gnathopod 1 (Fig. 4C, D) with coxa bearing marginal setae ventrally; basis with long setae on anterior and posterior margins; length of carpus 1.4 × width, with seta on anterior margin; length of propodus 1.3 × carpus and 1.4 × width, bearing two clusters of setae on anterior margin, propodus with oblique and weakly convex palmar margin bearing six medial and ten lateral peg-like robust setae; dactylus weakly curved, as long as palmar margin. Gnathopod 2 (Fig. 4E, F) with coxa bearing marginal setae ventrally; basis with anterior and posterior margins bearing long setae; length of carpus 1.8 × width, bearing setae on anterior margin; length of propodus 1.1 × carpus and 1.6 × width, respectively, with two clusters of setae on anterior margin, propodus with oblique and weakly convex anterior margin bearing eight medial and five lateral peg-like robust setae; dactylus weakly curved, as long as palmar margin.
Coxal gills (Fig. 4J-O) with two accessory lobes on gills 2-5, posterior lobes longer than or equal to anterior ones, one accessory lobe on gills 6 and 7.
Uropods. Uropod 1 (Fig. 4R) with peduncle bearing basofacial robust seta, two robust setae on inner and outer margins, one and two robust setae on inner and outer distal corners, respectively; length of inner ramus 0.8 × that of peduncle, inner margin of inner ramus with two robust setae; length of outer ramus 0.9 × that of inner ramus, inner margin of outer ramus with robust seta. Uropod 2 ( Fig. 4S) with peduncle bearing two robust setae on inner and outer margins, respectively, and robust seta on inner and outer distal corners; length of inner ramus 0.9 × that of peduncle, inner margin of inner ramus with two robust seta; length of outer ramus 0.8 × that of inner ramus, inner margin of outer ramus with robust seta. Uropod 3 (Fig. 4T, U) with peduncle length 0.3 × that of outer ramus; length of inner ramus 0.3 × that of outer ramus, inner ramus with slender setae on inner margin and setae apically; outer ramus comprising two articles, proximal article with two clusters of setae on inner and outer margins, some of which robust, lacking plumose setae, length of terminal article 0.2 × that of proximal article, apical part of terminal article with simple setae. Telson (Fig. 4V) 0.8 times as long as wide, cleft for 67% of length, with robust seta and slender setae on each lobe. Gnathopods. Gnathopod 1 (Fig. 5C, D) with carpus bearing cluster of setae on anterior margin; length of propodus 1.2 × that of carpus and 1.5 × width; propodus with eight medial and two lateral robust setae on palmar margin. Gnathopod 2 (Fig.  5E, F) with carpus bearing cluster of setae on anterior margin; propodus and carpus approximately the same length, propodus with three medial and two lateral robust setae and one medial and one lateral pectinate robust setae on palmar margin.
Pereopods 5-7 with more expanded posterior margin of bases than those of male ( Fig. 5G-I).
Variations. Although almost all specimens have a pleonite 1 with a pair of setae on the dorsal margin, a few specimens have three setae. Some specimens have a urosomite 1 with a pair of lateral robust setae and a pair of clusters of robust setae on its dorsal margin. The numbers of setal clusters on the posterior margins of the peduncular articles 1-3 of antenna 1 ranged from two to four, six or seven, and two to four, respectively. The number of setal clusters on the posterior margins of the peduncular articles 4 and 5 ranged from five or six and four or five, respectively. Some specimens have robust setae on the outer margin of the outer ramus of uropod 1 and lack robust setae on the inner margin of the outer ramus of uropod 2. Some specimens have a telson with 2 robust setae on each lobe. The number of eggs is up to 9.
Etymology. The new specific name derived from the absence of calceolus. Assessment of conservation status. Jesogammarus (J.) acalceolus sp. nov. was found in a spring located 120 m above sea level, on the slope of the volcanic Mt. Iwaki, Aomori Prefecture, Japan. Although we conducted an intensive survey of inland waters at more than 400 sites in the Aomori Prefecture, this new species was present only in this one spring described above and not found in any others (unpublished data). In most of the freshwater habitats that were investigated, J. (J.) jesoensis Schellenberg, 1937, which is distributed in Hokkaido and northern Honshu, was present. Because J. (J.) acalceolus sp. nov. and J. (J.) jesoensis are not closely related (Fig. 2), it is expected that the current distributions of both species are a result of different evolutionary processes. As a positive aspect, the type locality of J. (J.) acalceolus sp. nov. is in the precincts of the Iwaki Haguro Shrine, built in AD 807, as a result of which this type locality has been treated with care by locals for more than 1,000 years (Sasaki 1995). Therefore, the environment of this spring has been preserved in good condition, allowing the present J. (J.) acalceolus sp. nov. population to survive. At present, this spring has an abundance of water (60 m 3 /day) (Yamamoto 1994), and its environment is stable. However, amphipods are known to be highly sensitive to chemicals, such as pesticides (Schulz 2003;Nyman et al 2013). This species inhabits only a few meters of a spring brooklet surrounded by apple plantations. Therefore, the deterioration of its habitat due to an inflow of agricultural chemicals into spring water may lead to its extinction.

Discussion
Among freshwater habitats, springs have an especially high risk of extinction of species (Fluker et al. 2010). The highly diverse genus Jesogammarus, which is found in spring water habitats of the Japanese Archipelago, has a sensory organ termed the calceolus on male antenna 2. We described a new endangered freshwater amphipod species, Jesogammarus (Jesogammarus) acalceolus sp. nov., found in a spring in Aomori Prefecture, Japan, which is potentially the sole remaining habitat of this species. Although the calceolus is thought to be a sensory organ, its function and evolution are not well understood (Lincoln and Hurley 1981;Godfrey et al. 1988;Read and Williams 1990;Dunn 1998). Therefore, the discovery of J. (J.) acalceolus sp. nov., which lacks calceoli, provides important clues regarding the function and evolution of calceoli. An ancestral reconstruction of calceoli via the molecular phylogenetic tree generated during this study revealed that the common ancestor of Jesogammarus possessed calceoli, which were secondarily lost in J. (J.) acalceolus sp. nov. (Fig. 2). Since Jesogammarus carried calceoli only on the flagellum of antenna 2 of males, it is considered that calceoli have a reproductive function (Bousfield and Shih 1994). Females of amphipods lay eggs just after moulting when the exoskeleton is soft. Therefore, some species display a reproductive behaviour termed "precopula", in which a male holds and guards a female for a couple of days till the female's moulting and subsequent laying eggs. Dunn (1998) reported that calceoli can be used to evaluate the moulting interval of females to find suitable females for mate guarding. In this study, J. (J.) acalceolus sp. nov., the males of which lack calceoli, was also found to practice precopulatory guarding, which suggested that calceoli are not always necessary for precopulatory guarding in J. (J.) acalceolus sp. nov.
The calceolus is a typically club-or paddle-shaped structure found on the antennae of amphipods (Schmitz 1992). Although structures similar to the calceolus are also found in the antennal articles of Anaspidacea and Mysida, these are not considered to be homologous to amphipod calceoli (Bousfield and Shih 1994). Calceoli are used mainly as a taxonomic character in the higher taxa of amphipods (Lincoln and Hurley 1981;Holsinger 1992;Bousfield and Shih 1994). In Anisogammaridae, the presence or absence of calceoli is used as a genus-level taxonomic feature (Bousfield 1979). However, the molecular phylogenetic tree generated in this study confirmed that the non-calceolate species, J. (J.) acalceolus sp. nov., is nested in Jesogammarus, and not in Anisogammarus, Ramellogammarus or Spasskogammarus, the other Anisogammaridae with non-calceolate species (Fig. 2). These results indicated that the calceolus should no longer be used as a diagnostic feature of Jesogammarus and the genus needs to be redefined. Therefore, in this study, we have amended the diagnosis of Jesogammarus. In Gammarus, the seasonal variation of the presence or absence of calceoli was known (Karaman and Pinkster 1977), but J. (J.) acalceolus sp. nov., lacks calceoli year-round, suggesting that male antenna 2 lacking calceoli is a stable taxonomic feature.
Freshwater amphipods have low dispersal ability, and there thus exists a high tendency for endemic species to be distributed throughout each region (Tomikawa 2017). In addition, our taxonomic studies revealed a considerable presence of Jesogammarus fauna in the Japanese Archipelago Tomikawa et al. , 2017Tomikawa 2015). For these reasons, it is unlikely that J. (J.) acalceolus sp. nov. will be found outside type localities, thereby limiting the current habitat of this species to a great extent. In the past, there have been many cold springs in Hirosaki with the type locality of this new species. However, recent, rapid urbanization has led to a depletion of such springs (Sasaki 1995). Besides, the habitat of this species may have been lost due to the disappearance of springs and/or environmental pollution caused by the use of agrochemicals, both of which were associated with apple plantations that flourished in this region. Thus, to conserve what is possibly the only remaining population of J. (J.) acalceolus sp. nov., it will be necessary to conduct further investigations into risk factors and develop a conservation plan with the cooperation of local communities and policymakers. In conclusion, our results indicate that this new species, which is key to clarifying the evolution of the calceolus, is of high conservation significance.