Phylogeography of the Laniatorid Harvestman Pseudobiantes japonicus and Its Allied Species (Arachnida: Opiliones: Laniatores: Epedanidae)

ABSTRACT 
 To clarify the phylogenetic relationships of Pseudobiantes japonicus Hirst, 1911 and its allied species, Epedanellus tuberculatus Roewer, 1911 and Kilungius insulanus (Hirst, 1911) (Arachnida: Opiliones: Laniatores: Epedanidae), we conducted morphological and nucleotide sequence analyses of this complex. Sequencing of the cytochrome c oxidase subunit I gene of 179 samples recovered six clades comprised of four allopatric lineages within P. japonicus and two monophyletic groups of E. tuberculatus and K. insulanus, respectively, related as follows: ([Clades A + B of P. japonicus] [Clade C of P. japonicus]) (K. insulanus [Clade D of P. japonicus +E. tuberculatus]). This phylogenetic relationship implies that “Pseudobiantes japonicus” is paraphyletic or polyphyletic, unless mitochondrial introgression between species is postulated. Moreover, Clades C and D of P. japonicus were also separable by the combination of a few morphological characters and from Clades A and B in P. japonicus. These results suggest that a thorough taxonomic revision is needed for the classification of P. japonicus and allied species.

Harvestmen (Class Arachnida: Order Opiliones) are a diverse group of arachnids with Ͼ6,000 living species and are commonly found in many terrestrial habitats of every continent except Antarctica (Machado et al. 2007, Kury 2008). Because of low vagility, they often show high geographic differentiation both in external morphology and chromosomes even in a single species (Tsurusaki 2007). Furthermore, because of their general lack of resistance to desiccation, most harvestmen tend to be limited to forests with moderate moisture and several groups, such as Sabacon (Sabaconidae), Caddo (Caddidae), and Triaenonychidae, show typical patterns of disjunct distribution as relicts (Suzuki et al. 1977, Schö nhofer et al. 2013. Their limited occurrence and enormous geographic variability have hampered phylogenetic analyses based on morphological characters because of the paucity of reliable external characters. Recent molecular phylogenetic studies have revealed new aspects for our understanding of the relationships in various groups of harvestmen (Giribet et al. 1999; Thomas and Hedin 2008;Sharma andGiribet 2009, 2011;Derkarabetian et al. 2010Derkarabetian et al. , 2011Giribet et al. 2010;Hedin and Thomas 2010;Schö nhofer and Martens 2010;Burns et al. 2012;Hedin et al. 2012;Sharma 2010;Sharma et al. 2012;Schö nhofer et al. 2013). In this study, as a Þrst trial of the application of molecular phylogenetics for Japanese laniatorids, we focused on the phylogenetic relationships of various populations of the laniatorid Pseudobiantes japonicus Hirst, 1911 (Epedanidae) and its allied species of the same family.
P. japonicus is a common species of harvestman that occurs widely in the western part of the main islands of Japan (Fig. 1). The species is 3.5Ð 4.0 mm in body length (BL) and lives in humid places such as crevices beneath stones and fallen twigs, or in forest ßoor litter. This species shows geographic variation in some external characters such as armaments on the second scutal area (Suzuki 1973a,b). The species also shows male dimorphism in cheliceral size, and the ratio of males with large chelicerae to males with small chelicerae varies geographically (Tsurusaki and Fujikawa 2004).
The distributional range of P. japonica mostly overlaps with that of Epedanellus tuberculatus Roewer, 1911 (Fig. 1). E. tuberculatus is ecologically and morphologically very similar to P. japonica, though E. tuberculatus, whose adult body size reaches Ϸ4.5Ð7.0 mm, is clearly larger than P. japonicus (Fig. 1). These two species have two geo-graphical counterparts that are presumed to be allopatric sister species: Kilungius insulanus (Hirst 1911) in the middle part of the Ryukyu Islands (Island Amami-Ô shima, Island Yoron, Island Okinawa, etc.) and Kilungius bimaculatus Roewer, 1915 in the Sakishima Islands (the southernmost part of the Ryukyu Islands, which includes Island Ishigaki, Island Iriomote, Island Yonaguni, etc.; Fig. 1). Tsurusaki (2006) indicated that these four species constitute a closely related monophyletic group based on their general morphological similarity (Suzuki 1973a,b;Suzuki and Tsurusaki 1999) and their distributional pattern, though they have so far been treated under three different genera in the existing system of classiÞcation (e.g., Roewer 1923).
To resolve phylogenetic relationships of P. japonicus and its allied species, we performed phylogenetic analysis of the group (excluding K. bimaculatus, as samples were unavailable) using the cytochrome c oxidase subunit I (COI) gene.

Sampling of P. japonicus and Allied Species.
To obtain a comprehensive phylogenetic sample of P. japonicus and its allied species, 146 P. japonicus, 7 E. tuberculatus, and 15 K. insulanus were sampled from across the ranges of these species ( Fig. 1; Table 1). We failed to obtain samples suitable for DNA extraction for K. bimaculatus. As outgroup taxa, three Proscotolemon sauteri Roewer, 1916 (formerly Phalangodidae but now incertae sedis) and two Metanippononychus daisenensis Suzuki, 1974 (Triaenonychidae) were collected from the wild, and sequence data for Bishopella laciniosa (Crosby & Bishop, 1924) (Phalangodidae) (EU162812: Thomas and Hedin 2008), Metanonychus setulus (Briggs, 1971) (Triaenonychidae) (HM056732: Derkarabetian et al. 2011), Sclerobunus sp. (Triaenonychidae) (HM056742: Derkarabetian et al. 2011), andFumontana deprehendor Shear, 1977 (Triaenonychidae) (EU162773: Thomas and Hedin 2008) were obtained from the DNA Data Bank of Japan. All Dotted lines indicate postulated seashore line during low sea level periods in the early Pleistocene (Ϸ1,500,000 yr ago) with a gap between the southern arcuate peninsula extending from Taiwan and the northern main islands of Japan called the "Tokara Gap" (Ota, 1998). Note that P. japonicus and E. tuberculatus are widely sympatric in the western part of the main islands of Japan and they clearly differ in body size. (Online Þgure in color.)  Fig. 2) were measured for each individual, and the morphology of the second scutal area was recorded (Fig. 2). We measured 146 P. japonicus, 7 E. tuberculatus, and 15 K. insulanus. Measurements of the BL and the ESL were made using a digital caliper.
DNA Extraction, Amplification, and Sequencing. All DNA extractions were performed using QIAGEN DNeasy kits (Qiagen, Valencia, CA), according to the manufacturerÕs protocol for animal tissue samples. The isolated DNA was resuspended in TrisÐEDTA buffer and stored at Ϫ20ЊC until use. For all specimens, we ampliÞed the COI gene, previously shown to be useful to reveal population-level relationships (Crosby and Bishop 1924, Hedin 2008, Derkarabetian et al. 2011), was ampliÞed using the previously pub-  TWG AYA TRG CRT  ARA TTA TMC-3Ј), and Yoshi2Ð2R (5Ј-CCT ACD GTR AAT ATR TGR TG-3Ј). DNA was ampliÞed by incubation at 94ЊC for 10 s followed by 45 cycles of incubation at 94ЊC for 1.5 min, 48ЊC for 2 min, and 72ЊC for 3 min, with a Þnal extension at 72ЊC for 15 min. DNA was ampliÞed by polymerase chain reaction (PCR) in a 50-l reaction volume containing Ϸ50 ng of total DNA, 10 mM TrisÐHCl buffer (pH 8.3) with 50 mM KCl and 1.5 mM MgCl 2 , 0.2 mM of each dNTP, 1.25 U TaqDNA polymerase (TaKaRa, Tokyo, Japan), and 0.5 M of each primer. After ampliÞcation, reaction mixtures were subjected to electrophoresis in 1% low-melting-temperature agarose gels and puriÞed using QIAGEN QuickSpin kits according to the manu-facturerÕs speciÞcations. We sequenced the puriÞed PCR products using a BigDye Terminator Cycle Sequencing Kit (ABI PRISM DNA Sequencing kit, Perkin-Elmer Applied Biosystems, Tokyo, Japan) and ABI PRISM 3100-Avant Genetic Analyzer according to the manufacturersÕ instructions.
Data Analysis. To construct phylogenetic trees for P. japonicus and its allied species, sequences were aligned using ClustalW (Thompson et al. 1994). Moreover, we conÞrmed insertions or deletions (indels) using MEGA5 (Tamura et al. 2011). In the neighborjoining (NJ) method, the model maximum composite likelihood was used. Bootstrap resampling used 1,000 replications for the NJ analysis. In the maximum-likelihood (ML) method was reconstructed by MEGA program, version 5.05 (Tamura et al. 2011). The model test function in MEGA was used to choose models for ML analyses by the Bayes information criterion. The reliability of branching patterns in ML trees was tested by bootstrapping (1,000 samples). The following DNA sequence evolution models were used TN93 ϩ G ϩ I.

Results
Phylogenetic Analyses of P. japonicus and Its Allied Species. We reconstructed phylogenetic relationships using 179 samples of P. japonicus and its allied species including outgroup species. The length of the P. japonicus COI gene was 1,057 bp, with insertions of 6 bp compared with Pr. sauteri. Gaps in the sequences were eliminated in all of the following analyses, thus 1,051 bp were used in our study. We reconstructed phylogenetic trees based on the NJ method (Figs. 3Ð5). Our phylogenetic trees indicated that P. japonicus was comprised of a paraphyletic assemblage and divided into four subgroups, denoted Clades A to D (Figs. 3Ð5). In addition, we reconstructed another phylogenetic tree based on ML method (Figs. 6 Ð 8). Branching patterns between NJ and ML trees were mainly congruence. It is surprising that both E. tuberculatus and K. insulanus were recovered with one P. japonicus group and the monophyletic K. insulanus was sister to remove Clade D of P. japonicus and E. tuberculatus (Figs. 5 and 8). The monophyly of each subgroup of P. japonicus and both K. insulanus and E. tuberculatus were supported by relatively high bootstrap values (94 Ð100%); however, basal nodes had low support values in NJ and ML trees. Figure 9 shows the geographical distribution of the four clades in P. japonicus. Clade A consisted of 27 individuals distributed in the eastern half of Shikoku and an isolated population (no. 78, Nakanohama in Tosashimizu City) at the southwestern end of Shikoku. In total, 60 individuals from the western half of Shikoku, northern part of Kinki District, and Chu- tuberculatus, these values were 5.64 Ϯ 0.05 (male) and 5.53 Ϯ 0.16 (female), while for K. insulanus they were 4.23 Ϯ 0.46 (male) and 4.01 Ϯ 0.42 mm (female). Males and females of E. tuberculatus were signiÞcantly larger than those of any of other species. However, male K. insulanus were signiÞcantly larger than males in any of the other clades of P. japonicus (Fig. 10).  Fig. 11). Those for males and females of E. tuberculatus were 0.96 Ϯ 0.07 and 1.07 Ϯ 0.08 mm, respectively, and those for males and females of K. insulanus they were 1.30 Ϯ 0.20 and 1.12 Ϯ 0.22 mm, respectively. Of these, male individuals of K. insulanus had the signiÞcantly longest ESL and Clades A and B had the shortest ESL of all examined samples (Fig. 11). There were no signiÞcant differences among male individuals of Clades C and D and K. insulanus (P value Ͼ0.05). Female ESL of Clades A and B were also signiÞcantly shorter than those of the remaining samples (P value Ͻ0.05). No signiÞcant differences were detected between Clades A and B in the ESL of either males and females (P value Ͼ0.05).
Armaments on the second scutal area of P. japonicus and the allied species can be divided into two types: 1) second scutal area unarmed or only with rudimentary spines, and 2) second scutal area with a pair of short spines; and the results of the examination are shown in Table 2. Individuals of Clades A, B, and D had an unarmed second scutal area, except for two males of Clade A. However, Clade C was prominent in having spines on the second scutal area without exception. E. tuberculatus usually had two median granules on the second scutal area or lacked them (nonspined type). K. insulanus had a transverse row of granules on the same position (nonspined type).

Discussion
Phylogeographic Relationship Within P. japonicus and Possible Roles of the Ota and Asahi Rivers as Pleistocene Geographical Barriers. Ikeda et al. (2012) reported that the loss of ßight promotes speciation in coleopteran insects. It is expected that genuinely ßightless animals such as P. japonicus and related species would be bound to show more prominent geo-  graphical population differentiation. In fact, our results indicated that individuals of P. japonicus are divided into four allopatric clades across their distribution.
The current distribution of biological diversity cannot be understood without information about how organisms responded to historical changes in geological and climatic conditions. There is no doubt that the glacialÐinterglacial cycles of the Quaternary period would have played an important role in shaping the distribution of biodiversity among current populations, even in warm temperate zones where the land was not covered with ice sheets (Minato and Ijiri 1976). During the Last Glacial Maximum, the mean annual temperature was Ϸ5Ð9ЊC cooler and the precipitation was lower than present levels (Tsukada 1984). Therefore, climatic cooling caused southward shifts and shifts to lower altitude in the geographical ranges of various species (Tsukada 1974). Palynological evidence indicates that broadleaved deciduous and evergreen forests in Japan were subjected to cold periods at least four times during the Quaternary (Tsukada 1974). Aoki et al. (2004) hypothesized that the pollen record indicates that refugial populations were sparsely distributed along the coasts of the PaciÞc Ocean, while refugia of broadleaved evergreen forests were limited to southern areas mainly at the southern end of Kyushu. Moreover, fossil evidence also indi-

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ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 107,no. 4 cates that these populations migrated northward from refugia after the Last Glacial Maximum (Tsukada 1984, Matsuoka andMiyoshi 1998). These studies suggest that populations of some forest-dependent insects and other arthropods responded to the environmental changes associated with the glacial cycles in a similar fashion to that of their associated forests. Our studies indicated that P. japonicus consists of four clades occupying different geographical areas; Clade A (eastern Shikoku), Clade B (northern Kyushu, western Shikoku, Chugoku, Kinki, and the western part of Hokuriku Districts), Clade C (from southern Kinki to Tokai Districts), and Clade D (southern and middle part of Kyushu; Fig. 9). P. japonicus is widely distributed in western Japan from evergreen forests near sea level to Japanese beech Fagus crenata Blume forests, which usually grow in mountainous areas above Ϸ800 m in altitude in western Japan. Palynological evidence shows distributional ranges of F. crenata moved southward and only grew in southern Kyushu, southern Shikoku, and the Kii Peninsula of the Kinki District as refugia during the last ice age 18,000 Ð20,000 yr ago (Fujii et al. 2002). It is presumed that the Ota River that ßows down from the western part of the Chugoku Mountains of Honshu ran southward through the present day Bungo Channel that separates Kyushu Island and Shikoku Island when the Seto Inland Sea regressed at that time. However, the Asahi River that originates from the Mt. DaisenÐHiruzen area in the eastern part of the Chugoku Mountains had run through the Kii Channel (Ota et al. 2004) until the Seto Inland Sea was reformed Ϸ10,000 yr ago. Thus, it is plausible that these two rivers (and the present day Bungo and Kii Channels) have acted as barriers to gene ßow for land animals with very low vagility, such as P. japonicus. The distributional patterns of the four clades of P. japonicus strongly suggest that these clades had been restricted to evergreen forests along the coasts of the PaciÞc Ocean in the last glacial age and were differentiated under the inßuence of the ancient Ota River and ancient Asahi River, and have recently expanded their distribution independently from each refugium.
The comparison of intraspeciÞc phylogeographic patterns with other taxa over the same area and the search for congruent geographic patterns of genetic variation play important roles in indicating the inßuence of common historical factors (Arbogast andKenagy 2001, Hewitt 2004 (Suzuki and Tsurusaki 1981). A phylogeographic study based on the mitochondrial COI gene for the metallic blue earthworm Metaphire sieboldii (Horst, 1883) showed that the Group III occurring in the eastern part of Shikoku was replaced by Group I in the Kii Peninsula (Minamiya et al. 2009). In Japanese beech, F. crenata, the Clade III chloroplast DNA haplotype that occurs in Kyushu, the western half of Chugoku District, and Shikoku is substituted by Clade II in the Kii Peninsula (Fujii et al. 2002). Furthermore, recent phylogeographical analyses of mammals such as the Japanese monkey Macaca fuscata and Japanese deer Cervus nippon have also shown that there are separate mitochondrial  clades between Shikoku and the Kii Peninsula (Kawamoto 2007, Nunome et al. 2010. The arrangement of analyzed morphological characters of P. japonicus on our phylogenetic tree could reveal that each clade of this species was recognized by the combination of BL, ESL, and the spine type of the second scutal area (Fig. 12). For example, individuals of Clade C have the longest ESL and the spine type of second scutal area, but Clade D lacks of them despite having the longest ESL of all P. japonicus. Individuals of Clades A and B have a relatively short ESL and the granule and the loss types of second scutal area, except for two male individuals with the spine type in Clade A. Unfortunately, individuals of Clade A could not be distinguished with those of Clade B by morphological characters we examined. A remaining problem is the occurrence of two clades (Clade A and Clade B in eastern and western Shikoku, respectively) of P. japonicus in Shikoku. Additional molecular markers may lend support to the scenario inferred here, and multiple markers should be surveyed to Þnd further variation within this species. Such research would help to validate existing COI data sets and facilitate new interpretations.
Historical hybridization between different genotypes could be most commonly identiÞed by the heterogeneity of nuclear DNA (nrDNA) and the incongruence between mitochondrial DNA (mtDNA) and nrDNA phylogenies that may indicate different parental contributions to the hybrid genome (e.g., Funk and Omland 2003). In this study, it allows for the discussion about hybridization, introgression, and incomplete linage sorting between phylogenetic groups of P. japonicus to add the nrDNA information. Previous studies indicated that polymorphisms in 28S rRNA in nrDNA had proven to be effective tools to clarify the relationship of closely related taxa in harvestmen (Wheeler and Hayashi 1998;Giribet et al. 1999;Sharma andGiribet 2009, 2011;Derkarabetian et al. 2010;Giribet et al. 2010;Hedin and Thomas 2010;Sharma 2010;Sharma et al. 2012). Therefore, the phy-  logenetic study using both mtDNA and an additional nrDNA could provide an answer not only to reticulated evolution but also to cryptic speciation between genotypes in P. japonicus.