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Geological and paleoclimatic events reflected in phylogeographic patterns of intertidal arthropods (Acari, Oribatida, Selenoribatidae) from southern Japanese islands

2021; Wiley; Volume: 59; Issue: 6 Linguagem: Inglês

10.1111/jzs.12480

1439-0469

Tobias Pfingstl, Maximilian Wagner, Shimpei F. Hiruta, Iris Bardel‐Kahr, Wataru Hagino, Satoshi Shimano,

Insect-Plant Interactions and Control

A comprehensive study of the intertidal oribatid mite fauna of southern Japanese islands revealed the presence of the selenoribatid Arotrobates granulatus Luxton, 1992 and two yet undescribed species. The latter are herein described as Indopacifica taiyo n. sp., occurring from the Southern to the Central Ryukyus, and Indopacifica tyida n. sp., which was only found on the most western island of the Ryukyus, namely Yonaguni. A concomitant molecular genetic study using mitochondrial COI and 18S rRNA gene sequences, demonstrated that the phylogeographic pattern of I. taiyo n. sp. reflects recent expansion on the Southern and Central Ryukyus, probably due to existing land bridges during the late Pleistocene. Arotrobates granulatus, on the other hand, shows three distinct lineages, one on Japanese mainland, another on the island of Amami, and the third on part of the Central and Southern Ryukyus. These lineages are most likely the result of the break-up of a large peninsula reaching from China to the Northern Ryukyus about 1.2–1.7 million years ago. Despite emerging land bridges in the late Pleistocene, this species was not able to expand its range again which indicates very low dispersal abilities. Morphometric data of I. taiyo n. sp. show considerable intraspecific variation between island populations correlating with geography. This found variation is suggested to be a result of phenotypic plasticity caused by diverging local environmental factors. From an ecological perspective, all three found species are classified as intertidal rock-dwellers feeding on diverse algae, whereas I. taiyo n. sp. and Arotrobates granulatus occasionally occur in mangrove habitats. Eine umfangreiche Studie der litoralen Hornmilbenfauna der südlichen japanischen Inseln zeigte das Vorkommen von Arotrobates granulatus und zweier bisher unbeschriebener Arten. Diese werden hier beschrieben als Indopacifica tayio n. sp., welche von den südlichen bis zu den zentralen Ryukyus vorkommt, und als Indopacifica tyida n. sp., welche nur auf der Insel Yonaguni gefunden wurde. Eine begleitende molekulargenetische Studie der mitochondrialen COI und 18S rRNA Gensequenzen zeigte, dass das phylogeographische Muster von Indopacifica taiyo n. sp. eine rezente Ausbreitung auf den südlichen und zentralen Ryukyus widerspiegelt, die vermutlich durch, während des späten Pleistozäns bestehender, Landbrücken möglich war. Arotrobates granulatus hingegen zeigt drei klar getrennte Linien, eine auf dem japanischen Festland, eine andere auf der Insel Amami, und eine dritte auf Teilen der zentralen und südlichen Ryukyus. Diese Linien sind vermutlich ein Resultat des Zerfalls einer großen Halbinsel, die vor ca. 1.2-1.7 Millionen Jahren von China bis zu den nördlichen Ryukyus reichte. Trotz der vorübergehenden Landbrücken im späten Pleistozän, war es dieser Art nicht möglich sich wieder auszubreiten, was auf ein geringes Verbreitungspotential hinweist. Morphometrische Daten von Indopacifica taiyo n. sp. zeigen erhebliche intraspezifische Variation zwischen den Populationen verschiedener Inseln, welche stark mit der Geographie korreliert. Vermutlich ist diese Variation eine Folge phänotypischer Plastizität, verursacht durch sich unterscheidende lokale Umweltfaktoren. Vom ökologischen Standpunkt aus, können alle drei gefundenen Arten als litorale Felsbewohner eingestuft werden, die verschiedene Algen fressen, wobei Indopacifica taiyo n. sp. und Arotrobates granulatus gelegentlich auch in Mangroven vorkommen können. The Selenoribatidae represent, together with Ameronothridae, Fortuyniidae, and Podacaridae, an exceptional oribatid mite group as they have adapted to a marine-associated lifestyle instead of living a typical terrestrial life as soil decomposers (e.g., Pfingstl, 2017). While Ameronothridae and Podacaridae are distributed predominantly on polar and temperate coasts, Fortuyniidae and Selenoribatidae exclusively occur on subtropical and tropical shorelines (e.g., Pfingstl & Schuster, 2014; Schuster, 1989). These mites can tolerate daily tidal flooding by using plastron respiration (Pfingstl & Krisper, 2014; Pugh et al., 1990) and mainly use intertidal algae as substrate and food source (e.g., Pfingstl, 2017). The Selenoribatidae are the most diverse group among these marine-associated oribatid mites, with nine genera and 32 species worldwide. Most members of this family were reported from the Indo-Pacific area (Pfingstl & Schuster, 2014; Procheş & Marshall, 2001); records from neighboring regions, as for example the Japanese Islands, are relatively scarce. Four species, Arotrobates granulatus Luxton, 1992, Schusteria nagisa Karasawa & Aoki, 2005, Schusteria saxea Karasawa & Aoki, 2005 and Rhizophobates shimojanai Karasawa & Aoki, 2005 were found on the southern Ryukyu Islands (Karasawa & Aoki, 2005); the latter three species are only known from this area and so far considered as endemic species for the Japanese Islands. There are yet no records of selenoribatid mites from the close continental coast of China, except for the reports of Arotrobates reticulatus Luxton, 1992, A. granulatus and Psednobates uncunguis Luxton, 1992 from the shore of Hong Kong (Luxton, 1992) and the report of an undetermined species from the littoral of Xiamen (Pfingstl & Schuster, 2014). Therefore, biogeographic connections between those neighboring regions remained mostly unknown. The Ryukyu Islands represent a chain of more than 200 smaller landmasses stretching for about 1200 km from 24° to 31° latitude between Taiwan and Japanese mainland (Kimura, 2002). These islands are divided into three parts, the Northern, Central, and Southern Ryukyus. The Northern and Central parts are separated by the Tokara gap, the Central, and Southern Ryukyus are divided by the Kerama gap (Kojima et al., 2003). These two gaps were formed during the Pliocene (Ota, 1998) and are suggested to be zoogeographical boundaries that might have played important roles in establishing the biogeographical characteristics of the Ryukyus during the Cenozoic (e.g., Ota, 1998). There is a high number of endemic species across invertebrate and vertebrate taxa, and the fauna is largely divided among the above-mentioned three geographic areas which is supposed to be partly caused by these geographic gaps (Muraji et al., 2012). A recent phylogeographic study on intertidal mites from the family Fortuyniidae (Pfingstl, Wagner, et al., 2019) showed a slightly divergent picture. There were no endemic species and while the Tokara gap was strongly reflected in the morphological and molecular genetic sequence data, indications of the Kerama gap acting as an effective barrier to dispersal were completely lacking. This pattern was suggested to be the result of low sea level during the last Pleistocene (Ujiié et al., 2003) which allowed recent expansion and gene flow between several island populations of fortuyniid mites (Pfingstl, Wagner, et al., 2019). Apparently, these tiny wingless organisms show better dispersal abilities than partially volant terrestrial animals, such as insects and birds, and it was suggested that their ability to drift on water may be a key factor (Pfingstl, Wagner, et al., 2019). During the field work for this latter study, we were able to additionally collect numerous populations of selenoribatid mites from various Japanese Islands, which gave us the opportunity to compare their phylogeographic patterns with those of the above-mentioned closely related Fortuyniidae. Therefore, aims of the present study were (a) to determine collected taxa and describe possible new species, (b) to infer phylogeographic scenarios for each found selenoribatid group and compare these to patterns of fortuyniid mites, (c) to interpret the found similarities or divergences, and (d) to update biogeographic information for Japanese intertidal mites. For the present molecular genetic study, 121 selenoribatid specimens were analyzed and for the morphometric study 75 specimens were used. Additionally, we included sequences (COI, 18S) from GenBank from two Indopacifica pantai specimens (accession nrs: MH285652, MH285654, MH285691, MH285692), from one Indopacifica parva specimen (MH285671, MH285690) and from one Thalassozetes barbara specimen (MW289085, MW298484). For more details see Appendix 1. Samples of littoral algae were scraped off rocks, concrete walls, and mangrove roots with a small shovel and then put in Berlese-Tullgren funnels for 12–24 h to extract mites. Afterward, specimens were fixed in ethanol (100%) for morphological and molecular genetic investigation. The majority of samples was taken by Shimano, Hiruta, and Pfingstl and if not, the name of the collector is provided. Each sample location was given a code; these codes are presented in parentheses and will be used throughout the manuscript to allow easier linking of geographic information. The suffixes "-jima/-shima" and "-gawa" are Romanized Japanese meaning "island" and "river," respectively; in the graphs and tables showing results, these suffixes are not given due to a shortage of space. Japanese mainland Kyushu, pref. Kagoshima: Iriki, Fukiage-cho, Hioki City (JP_86) algae on mussels and barnacles on concrete seawall near the mouth of the river; 31°30′14.70″N 130°19′10.56″E; 10 Jun. 2018. Southern Ryukyus Ishigaki-jima, pref. Okinawa: Ohama Beach (JP_36) Bostrychia on rocks on sandy beach; 24°20′40.61″N 124°12′5.58″E; 15 Mar. 2019. Kabira Bay (JP_62) Bostrychia on large rocks on sandy beach; 24°27′49.14″N 124° 8′39.09″E; 20 Mar. 2019. Yamabare (JP_65) thick cushions of Bostrychia on large rocks; 24°26′56.11″N 124°10′46.41″E; 20 Mar. 2019. Fukido-gawa estuary (JP_66) diverse algae growing on mangrove roots; 24°29′10.48″N 124°13′48.29″E; 20 Mar. 2019. Kabira (JP_90) unknown algae on rocks; 24°28′25.7″N 124°08′10.5″E; 31 Mar. 2019; leg. H. Uchida. Central Ryukyus For this study, 121 specimens of the family Selenoribatidae (67 of genus Indopacifica, 53 of the genus Arotrobates and one outgroup specimen of T. barbara) were analyzed. Whole genomic DNA was extracted from ethanol-fixed individuals using Chelex resin according to the adjusted protocols in Pfingstl, Lienhard, et al. (2019). We amplified a partial sequence of the mitochondrial DNA cytochrome c oxidase subunit I (COI) gene (600 bp amplicon length) and the complete 18S ribosomal RNA gene (ca. 1900 bp amplicon length) of major mitochondrial lineages, following the protocols of Pfingstl, Lienhard, et al. (2019). PCR conditions and primers as well as sources are summarized for each locus in Table S1. Subsequent DNA purification steps included enzymatic ExoSAPIT (Affymetrix) and Sephadex G-50 resin (GE Healthcare). Cycle sequencing, using BigDye Sequence Terminator v3.1 kit (Applied Biosystems), was conducted according to Schäffer et al. (2008). Automatic capillary sequencing and sequence visualization was operated on an ABI3500XL (Applied Biosystems) device. Furthermore, already published sequences of I. parva (18S rRNA: MH285690 and COI: MH285671) and I. pantai (18S rRNA: MH285691, MH285692 and COI: MH285654, MH285652) (Pfingstl, Lienhard, et al., 2019) were downloaded from GenBank and included in the alignment. MUSCLE (Edgar, 2004), integrated in the software MEGA 7.0 (Kumar et al., 2016), was used to align sequences. Concatenation of the single locus datasets and indel coding of the 18S alignment was performed in the perl-based program 2matrix (Salinas & Little, 2014). Finally, three alignments were created and analyzed (a) COI (564 bp), (b) 18S (1964 bp; incl. indels and binary coded gaps), and (c) COI + 18S (2528 bp; incl. indels and binary coded gaps). For more detailed information, see alignments in Data S1S–3. All generated sequences have been deposited at GenBank and are accessible at following numbers, MW289085–MW289203 (COI) and MW298484–MW298513 (18S). The best fitting model of molecular evolution was determined based on Bayesian Information Criterion (BIC) in PartitionFinder v2.1.1 (Lanfear et al., 2017). Additionally, for the COI dataset, based on all three codon positions as starting scheme, a "greedy" partition search (Lanfear et al., 2012) in PartitionFinder v2.1.1 was conducted. The results of these analyses are summarized in Table S1. Phylogenetic inference was conducted for each single locus and the concatenated dataset in RaxML HPC v.8.0. (Stamatakis, 2014) and MrBayes v3.2 (Ronquist et al., 2012). Node support for the maximum likelihood framework was assessed using 10.000 bootstrap replicates and GTR-GAMMA model of evolution. Posterior probabilities for the Bayesian inference were obtained from Metropolis-coupled Markov chain Monte Carlo (MCMC) simulations using two independent runs, eight chains, a sampling frequency of 10,000 and the respective model of evolution. The analyses were run for 20e−6, 15e−6, and 10e−6 generations for the concatenated, COI and 18S dataset, respectively. To ensure parameter convergence and stationarity of chains, split deviation frequencies lower than 0.01 were assured and effective sample sizes (ESS) were investigated in Tracer 1.6 (Rambaut et al., 2014) (available at http://beast.bio.ed.ac.uk/Tracer). The first 25% of obtained trees of the MrBayes run were discarded and the rest summarized in terms of a 50% majority rule consensus tree. All trees were visualized in FigTree v1.4.2 (Rambaut, 2014) (available at http://tree.bio.ed.ac.uk/software/figtree). Net interspecific p-distances between groups, using 1000 bootstraps replications, were calculated in MEGA 7.0. Minimum interspecific and the maximum intraspecific divergence (in %) were calculated in R vs. 3.6.1 (R Core Team, 2017) using the functions "nonConDist" and "maxInDist" from the R-package SPIDER v.1.5 (Brown et al., 2012). Raw intraspecific genetic distances were obtained for both genera by the function "dist.dna" implemented in the R-package APE v.5.4 (Paradis et al., 2004) and visualized using the package PHEATMAP v.1.0.8 (Kolde & Kolde, 2015). Furthermore, to infer the phylogeographic relationships among genera, TCS networks (Clement et al., 2002) were calculated in PopART (Leigh & Bryant, 2015; available at http://popart.otago.ac.nz). Specimens were embedded in lactic acid for temporary slides, and measurements were made using a compound light microscope (Olympus BH-2) and ocular micrometer. A set of 15 continuous variables (bl—body length, dPtI—distance between pedotecta I, dbi—distance between inner borders of bothridia, dbo—distance between outer borders of bothridia, nwda—notogastral width on level of seta da, nwdm—notogastral width on level of seta dm, nwdp—notogastral width on level of seta dp; cl—camerostome length, cw—camerostome width, dcg—distance between camerostome and genital orifice, dac3—distance between acetabula 3, gl—genital orifice length, gw—genital orifice width, al—anal opening length, aw—anal opening width, see Figure S1) were measured in 75 Indopacifica specimens from six different populations originating from four different Ryukyu islands (Yonaguni-jima, Iriomote-jima, Ishigaki-jima, and Okinawa-jima). For species discrimination, all populations from a species were pooled; for the comparison of populations in the wider distributed Indopacifica taiyo n. sp., 61 specimens from five different locations from above given four islands were used for analysis. Specimens used for morphometric comparison were not the same as used for molecular genetic analyses, but they belonged to the exact same populations (same sample, approx. 10 cm2 patch of alga). Specimens were sexed based on internal structures (spermatopositor, ovipositor), and sexes were separated for morphometric analyses. For univariate statistics of Indopacifica, minimum, maximum, mean, and standard deviation for each variable were calculated. Multivariate analyses investigating differences between putative Indopacifica species included a Principal Component Analysis (PCA; using a variance-covariance matrix) and a Non-metric Multidimensional Scaling (NMDS; based on Euclidian distances, two-dimensional); both analyses were performed on log10-transformed size-corrected data. No rotation was applied to the multivariate data. Size correction was done by dividing each variable through the geometric mean of the respective specimen. For the investigation of divergence among different populations of Indopacifica, a Kruskal–Wallis and a Mann–Whitney U-test (Bonferroni-corrected p-values) were used for comparing the means of variables for pairwise comparisons to clarify whether single variables differ significantly between the populations. Additionally, a Discriminant Analysis (LDA) was performed on log10-transformed raw data to show size and shape differences between populations. All analyses were performed with PAST version 3.11 (Hammer et al., 2001). Due to the relatively low number of A. granulatus specimens available, we were not able to perform a morphometric analysis of this species. Preserved specimens were embedded in Berlese mountant for microscopic analysis in transmitted light. Drawings were performed with an Olympus BH-2 Microscope equipped with a drawing attachment, and then they were scanned and afterwards processed and digitized with the free and open-source vector graphics editor Inkscape (https://inkscape.org). For photographic documentation, specimens were air-dried and photographed with a Keyence VHX-5000 digital microscope. Morphological terminology used in this paper follows that of Grandjean (1953) and Norton and Behan-Pelletier (2009). Family Selenoribatidae Schuster, 1963 Genus Indopacifica Pfingstl, Shimano & Hiruta LSID: urn:lsid:zoobank.org:act:A71290A4-6B56-473F-9ABF-4C02992AF37B Type material. Holotype: female, Japan, Ryukyus, Iriomote-jima, pref. Okinawa, Nadara-gawa estuary (JP_47) Bostrychia and other algae from boulder; 16 Mar. 2019. Four paratypes: two males and two females, same location and date as holotype. All deposited at the Collection of Arachnida, Department of Zoology, National Museum of Nature and Science, Tokyo (NMST). Two additional paratypes from Yonaguni-jima, pref. Okinawa, Kataburu Beach (JP_56); 18 Mar. 2019, deposited in the collection of the Naturhistorisches Museum Wien/NHM Vienna. Etymology. The sound of specific epithet "taiyo" has the meaning of both "big ocean" and "the sun" in the general Japanese and "big ocean" refers to the wider coastal distribution from the Southern to the Central Ryukyus. It is given as noun in apposition. Diagnosis. Prodorsal setae short; bothridial seta clavate and barbed; gastronotic region round in dorsal view; transversal depression on anterior part of notogaster; median longitudinal, hourglass-shaped depression on epimeron I; two pairs of adanal setae; proximoventral tooth on claws present. Description of adult. Measurements. Females (n = 26), length: 289–325 µm (mean 304 µm), width: 185–203 µm (mean 192 µm); males (n = 33), length: 277–319 µm (mean 296 µm), width: 175–197 µm (mean 189 µm). Integument. Color brown. Cerotegument of prodorsum, ventral region and legs granular. Notogastral cerotegument densely granulated with larger granules irregularly surrounded by smaller granules. Prodorsum (Figure 1a). Rostrum rounded in dorsal view, clearly demarcated from remainder of prodorsum by transverse ridge. Pair of anteriorly converging faint prodorsal ridges, difficult to observe. Rostral (ro) and lamellar seta (le) simple and short. Interlamellar seta (in) minute, exobothridial seta (ex) minute. Bothridium large cup with lateral incision. Bothridial seta clavate, distally barbed. Gnathosoma. Chelicera chelate, with two teeth on each digit. Setae cha and chb of approximately same length, both dorsally slightly pectinate. Palp setal formula 0–2–1–3–8 (+solenidion ω). Distal part of rutellum developed as thin, triangular, slightly inwardly curved membrane with longitudinal incision. Setae a and m long, smooth. Mentum regular, finely granular, seta h simple, long. Notogastral region (Figure 1a). Notogaster rounded, nearly circular in dorsal view. Pair of indistinct humeral projection. A median transversal depression present between lyrifissure ia. Fourteen pairs of thin, short setiform notogastral setae (length 10–16 µm), c1, c3, da, dm, dp, la, lm, lp, h1–3, p1–3; c3 absent. Orifice of opisthonotal gland gla anterior to seta la. Lyrifissure im between setae la and lm. Lateral aspect (Figure S2). A broad lateral furrow reaching from dorsal to ventral sejugal scissure, caudally delimited by conspicuous ridge. Pedotectum I present, round, small. Lateral enantiophysis consisting of two opposite projections; the anterior rounded, the posterior pointed. Discidium di developed as prominent conical bulge. Podosoma and venter (Figure 1b). Median longitudinal, hourglass-shaped depression on epimere I covered with granules and inconspicuous semicircular depression on posterior border of epimere III. Three pairs of short, fine genital setae present. Preanal organ triangular in ventral view, interior part anchor-shaped. Two pairs of short adanal setae ad1–2 present. Lyrifissure iad slightly oblique, next to anterior border of anal opening. Legs (Figure 2). Long hook-like claw with one small proximoventral tooth. Cerotegument granular. No porose areas detectable. Setation and solenidia: Leg I (0–3–2–3–18) (1–2–2), leg II (0–3–2–3–15) (1–1–1), leg III (1–2–1–2–13) (1–1–0), leg IV (1–2–1–3–12) (0–1–0) (for details see Table 1). Distribution. The distribution of this species ranges from the Southern Ryukyus (Yonaguni-jima, Iriomote-jima, Ishigaki-jima) to the Central Ryukyus, whereas Okinawa-jima represents its northern limit (Figure 3). Remarks. Indopacifica taiyo n. sp. can be distinguished from Indopacifica iohanna Resch & Pfingstl, 2019 by the differing notogastral depressions (one transverse vs. two oblique) and the lack of a proximoventral tooth on the claws in the latter. Indopacifica mauritiana Pfingstl & Baumann, 2019 and I. parva Pfingstl, Shimano & Lienhard, 2019 show a circular median depression on epimeron I instead of an hourglass-shaped depression in the present species, and I. pantai Pfingstl, Shimano & Lienhard, 2019 exhibits three pairs of adanal setae instead of only two and is significantly larger than I. taiyo n. sp. LSID urn:lsid:zoobank.org:act:11CAAFEE-1B1C-44D0-B1A9-832439D1F01F Type material. Holotype: female, Japan, Ryukyus, Yonaguni-jima, pref. Okinawa, Sonai (JP_59) dark black filamentous algae on rocks; 19 Mar. 2019. Four paratypes: two males and two females, same location and date as holotype. All deposited at the Collection of Arachnida, Department of Zoology, National Museum of Nature and Science, Tokyo (NMST). Two additional paratypes from the same location deposited in the collection of the Naturhistorisches Museum Wien/NHM Vienna. Etymology. The specific epithet "tyida" refers to the Okinawan dialect word for "the sun" and is given as noun in apposition. We choose this term because the type locality Yonaguni-jima is the westernmost point of Japan where one can observe the most beautiful sunsets. Diagnosis. Prodorsal setae short; pair of anteriorly converging prodorsal ridges; bothridial seta lanceolate and barbed; gastronotic region round in dorsal view; irregular oblique depressions in humeral areas of notogaster; median longitudinal, hourglass-shaped depression on epimeron I; two pairs of adanal setae; proximoventral tooth on claws present. Description of adult. Measurements. Females (n = 9), length: 308–319 µm (mean 312 µm), width: 188–197 µm (mean 193 µm); males (n = 7), length: 277–313 µm (mean 296 µm), width: 179–191 µm (mean 183 µm). Integument. Color brown. Cerotegument of prodorsum, ventral region and legs granular. Notogastral cerotegument densely granulated with larger granules irregularly surrounded by smaller granules, resulting in a weak reticulate pattern. Prodorsum (Figure 1c). Rostrum rounded in dorsal view, clearly demarcated by from remainder of prodorsum by transversal ridge. Pair of anteriorly converging prodorsal ridges. Rostral (ro) and lamellar seta (le) simple and short. Interlamellar seta (in) minute, exobothridial seta (ex) minute. Converging bothridium large cup with lateral incision. Bothridial seta lanceolate, distally barbed. Gnathosoma. Chelicera chelate, with two teeth on each digit. Setae cha and chb of approximately same length, both dorsally slightly pectinate. Palp setal formula 0–2–1–3–8 (+solenidion ω). Distal part of rutellum developed as thin, triangular, slightly inwardly curved membrane with longitudinal incision. Setae a and m long, smooth. Mentum regular, finely granular, seta h simple, long. Notogastral region (Figure 1c). Notogaster rounded, oval in dorsal view. Oblique irregular depression in humeral areas. Fourteen pairs of thin, short setiform notogastral setae (length 10–15 µm), c1, c3, da, dm, dp, la, lm, lp, h1–3, p1–3; c3 absent. Orifice of opisthonotal gland gla anterior to seta la. Lyrifissure im between setae la and lm. Lateral aspect (Figure S2). A broad lateral furrow reaching from dorsal to ventral sejugal scissure, caudally delimited by conspicuous ridge. Pedotectum I present, round, small. Lateral enantiophysis consisting of two opposite projections, both pointed. Discidium di developed as prominent conical bulge. Podosoma and venter (Figure 1d). Median longitudinal, hourglass-shaped depression on epimeron I covered with granules and inconspicuous semicircular depression on posterior border of epimeron III. Three pairs of short, fine genital setae. Preanal organ triangular in ventral view, interior part anchor-shaped. Two pairs of short adanal setae ad1–2. Lyrifissure iad slightly oblique, next to anterior border of anal orifice. Legs (Figure 4). Long hook-like claw with one small proximoventral tooth. Cerotegument granular. No porose areas detectable. Setation and solenidia: Leg I (0–3–2–3–18) (1–2–2), leg II (0–3–2–3–15) (1–1–1), leg III (1–2–1–2–13) (1–1–0), leg IV (1–2–1–3–12) (0–1–0) (for details see Table 1). Distribution. This species was only found in a single location on Yonaguni the south westernmost island of Japan (Figure 3). Remarks. Indopacifica tyida n. sp. can be distinguished from I. taiyo n. sp. by its faint prodorsal ridges (vs. absent in the latter), the differing notogastral depressions (two oblique vs. one transversal) and a significantly larger distance between camerostome and genital opening (as demonstrated later in the morphometry section). Indopacifica iohanna lacks the proximoventral tooth on the claws, and I. parva and I. mauritiana show a circular median depression on epimeron I (instead of an hourglass-shaped depression). Indopacifica pantai is considerably larger and shows three pairs of adanal setae instead of only two. The populations of A. granulatus from Japanese mainland (Figure 5) and the Central and Southern Ryukyus show conformity in their morphology and no distinct differences could be detected (Table 2). A slight deviation is shown only in the specimens from Kagoshima (JP_86) which possess very fine adanal setae instead of all being reduced to their alveoli. The specimens from Hong Kong described by Luxton (1992) differ from the herein investigated Japanese individuals by lacking lamellar setae (le) and one pair of notogastral seta and by having only alveolar interlamellar (in) and epimeral setae (3a, 4a). The specimens from the Japanese Ryukyus described by Karasawa and Aoki (2005) also lack the lamellar setae (le) and show only alveolar interlamellar setae (in), but they lack two pairs of notogastral setae and show variable adanal setation (Table 2). However, most of these differences are subtle and may be results of either intraspecific variation or inaccurate observation. In the case of adanal setae, Karasawa and Aoki (2005) already mentioned that they can be present or absent and this variation was also found in the present study which confirms intraspecific variation at least in some aspects. The lamellar setae which were not detected by the other authors (Karasawa & Aoki, 2005; Luxton, 1992) are very delicate structures and partly covered by cerotegument in most of the presently investigated populations and we also had initial difficulties to find them. Consequently, it is possible that these setae were present in the other studies as well but were overlooked due to their very fine nature. Moreover, the interlamellar setae in our specimens are very short and may also be easily mistaken for simple alveoli. The differences in notogastral setation, on the other hand, may represent real divergences though there are certain discrepancies. Seta c3 of the herein studied specimens is located in a very lateral position so that it is difficult to detect from a dorsal view. However, Karasawa and Aoki (2005) depicted a seta in the exact same position but labeled it as la. So, their lack of a seta of the c-row as well as the different position of the lyrifissure im could be explained by a mislabeling, but it still does not explain the lack of notogastral seta h1. Unfortunately, Luxton (1992) did not label any setae in the figure or the text; therefore, it is impossible to make clear assessments. To conclude, the diagnostic value of the existing morphological differences between specimens from the different studies is not clear and needs further investigation. The topology of the Bayesian Inference (BI) tree based on concatenated data (COI and 18S) (Figure 6) is well resolved with each species showing a distinct clade. Despite differing in certain aspects of the topology, the single gene trees are basically consistent in exhibiting the same distinct clades for each species (Figures S3–S7). Although A. granulatus is represented as a monophyletic clade in the BI tree of the concatenated data, it shows a distinct deep structure with more or less three obvious groups. One group consists of specimens from Japanese mainland (JP_33), another group