Edaphologia, No. 104: 19–24, March 29, 2019 19 Possible occurrence of reproductive isolation between two geographical clades of a laniatorid harvestman Pseudobiantes japonicus (Arachnida: Opiliones: Epedanidae) in Shikoku Yoshimasa KUMEKAWA1,2, Osamu MIURA2, Haruka FUJIMOTO3, Katsura ITO2, Ryo ARAKAWA2, Jun YOKOYAMA4, Tatsuya FUKUDA5 1 The United Graduate School of Agricultural Sciences, Ehime University, Monobe 200, Nankoku, Kochi, 783-8502 Japan 2 Faculty of Agriculture and Marine Science, Kochi University, Monobe 200, Nankoku, Kochi, 783-8502 Japan 3 The Graduate School of Integrated Arts and Sciences, Kochi University, Monobe 200, Nankoku, Kochi,783-8502 Japan 4 Faculty of Science, Yamagata University, 1-4-12, Kojirakawa-machi, Yamagata, 990-8560 Japan 5 Faculty of Knowledge Engineering, Tokyo City University, 1-28-1, Tamazutsumi, Setagaya, Tokyo, 158-8557 Japan Corresponding author: Yoshimasa Kumekawa ([email protected]) Received 19 February 2018; Accepted 7 September 2018 Abstract Phylogeographic analyses of a laniatorid harvestman Pseudobiantes japonicus Hirst, 1911 (Arachnida: Opiliones: Laniatores: Epedanidae) in Japan have provided evidence for long-term isolation among allopatric clades from different refugia, and there was a possible narrow hybrid zone between two different phylogroups, Clade A and Clade B, in central part of Shikoku. We performed PCR-RFLP analyses of mitochondrial DNA (mtDNA) and nuclear DNA (nrDNA) to clarify whether hybridization or introgression between them occur or not in the area where ranges of Clade A and Clade B meet. We were able to detect neither heterozytes for nrDNA genes nor the incongruence between mtDNA and nrDNA. These results indicated that P. japonicus have not experienced hybridization or introgression be- tween Clade A and Clade B in the contact zone, suggesting that they could be considered as cryptic species. Key words: Central Shikoku, cryptic species, phylogeny, Pseudobiantes japonicus Widespread and non-vagile taxa that currently occupy Introduction both historically glaciated and unglaciated areas are ideal It is widely accepted that geographical isolation can result systems to examine the generality of intra- and interspecific in speciation, and it has also long been proposed that incidental phylogenetic patterns caused by allopatric fragmentation and genetic divergence in allopatry can result in substantial postzy- range shifts (Hewitt, 1996, 2000). Harvestman (Arachnida: gotic isolation (Dobzhansky, 1937). The recent studies on Opiliones) is one of such taxa and they often show high geo- mitochondrial DNA (mtDNA) phylogeography have revealed graphic differentiation both in external morphology and chro- a lot of cases of apparently deep historical subdivision within mosomes even in a single species (Tsurusaki, 2007). Of these, species, many of which were morphologically cryptic or even Pseudobiantes japonicus Hirst, 1911 (Laniatores: Epedanidae), discordant with subspecies boundaries (Avise, 2000). Many which is a common species of harvestmen that occurs widely studies have revealed cryptic species with little to no obvious in the western part of the main islands of Japan, is 3.5–4.0 morphological differentiation, but levels of neutral genetic mm in body length and lives in humid places such as crevices divergence equal to or greater than those between morphologi- beneath stones and fallen twigs, or in forest floor litter. Suzuki cally diagnosable species (e.g., Zamudio and Savage, 2003; (1973a, b) reported that P. japonicus shows geographic varia- Elmer et al., 2007), and some studies revealed that these pheno- tion in some external characters, and Tsurusaki and Fujikawa typically cryptic lineages may have evolved independently for (2004) indicated the male dimorphism of cheliceral size in this millions of years (Hoskin et al., 2005; Elmer et al., 2013). species. Recent morphological and phylogenetic study using 20 Yoshimasa Kumekawa, Osamu Miura, Haruka Fujimoto, Katsura Ito, Ryo Arakawa, Jun Yokoyama, Tatsuya FukudaFig.1 E132 E133 E134 E135 E136 Ⅰ N35 E130 E135 E140 E145 N45 N34 Shikoku N40 Japan N35 Ⅰ Fig. 1. Distribution of individuals of Clade A, B, C and D of P. japonicus in Shikoku and adjacent areas (Kume- kawa et al., 2014). Open and solid circles indicate Clade A and Clade B, respectively. Open and solid tri- angles indicate Clade C and Clade D, respectively. this species and its allied species, Epedanellus tuberculatus light on the extent and nature of reproductive isolation (Barton Roewer, 1911 and Kilungius insulanus (Hirst, 1911), revealed and Hewitt, 1985, 1989). Considering these studies, it is very that P. japonicus is divided into four phylogenetic groups interesting to detect hybridization and/or introgression be- (Clades A–D), and one of which (Clade D) forms a monophy- tween clades of A and B of P. japonicus in possible secondary letic group with E. tuberculatus and K. insulanus, and each contact zones. clade of P. japonicus can be recognized by combination of a Hybridization or introgression between different clades few morphological characteristics (Kumekawa et al., 2014). have been most commonly identified by the heterogeneity of Moreover, phylogeographic analyses of P. japonicus have pro- nuclear DNA (nrDNA) and the incongruence between mtDNA vided evidence for long-term isolation of lineages among dif- and nuclear DNA (nrDNA) phylogenies that may indicate dif- ferent refugia: southern Kyushu (Clade D), southwestern Shi- ferent parental contributions to the hybrid genome (Funk and koku (Clade B), southeastern Shikoku (Clade A) and southern Omland, 2003). Although previous studies indicated that the Kii peninsula (Clade C) (Kumekawa et al., 2014). Among cytochrome c oxidase subunit I (COI) gene in mtDNA plays these clades, external characteristics of Clade A is very similar the powerful tool to clarify the relationship among clades of to those of Clade B, and the distributional range of Clade A is P. japonicus (Kumekawa et al., 2014, 2015), it is unknown for parapatric to that of Clade B in central area of Shikoku (Fig. 1; the available markers of nrDNA in this species. Some stud- Kumekawa et al., 2014), indicating occurrence of secondary ies indicated that polymorphisms in 28S rRNA in nrDNA are contact zone between clades of A and B. However, it remains effective to analyze the relationship among closely related unclear whether hybridization and/or introgression between taxa in harvestmen (Giribet et al., 1999; Giribet et al., 2010). these clades have occurred in this area. In general, analysis of These results suggest that the analyses using COI in mtDNA secondary contact zones between lineages or clades can shed and 28S rRNA can detect hybridization and introgression Possible of reproductive isolation of P. japonicus Fig. 2 21 Ⅰ Yoshino river 13 h a d f 2 2 27 26 1 g Kagami river e 7 10 b 12 Ⅰ c Shikoku Fig. 2. Distribution of Clade A and Clade B of P. japonicus in and around the contact zone, characterized by PCR-RFLP. Open and solid sectors indicate Clade A and Clade B, respectively. Numerals in circles indicate numbers of individuals examined. Alphabets in squares indicate population of Table 1. Scale bar = 10 km. Table 1. Sampling areas of P. japonicus in this study. Population corresponds to Fig. 5. Location Population Prefecture City Town Sampling date The number of individual a Kochi Kochi Ino 2 Oct. 2012 2 b Kochi Takaoka Hidaka 18 May 2013 7 c Kochi Kochi Koishigi 15 May 2013 12 d Kochi Kochi Tosayama 21 June–9 Oug. 2013 53 e Kochi Kochi Niida 14 May 2013 10 f Kochi Kochi Tosayamada 15 Oct. 2012 2 g Kochi Kochi Tosayamada 8 Nov. 2012 1 h Tokushima Miyoshi Higashiiya 14 July 2013 13 between clades of P. japonicus. The aim of this study is to the area spanning across a possible boundary between Clades clarify hybridization and/or introgression between clades of A A and B (Fig. 2). All the samples were stored at -30 ℃ until and B of P. japonicus in contact zones. DNA extraction. All DNA extractions were performed using QIAGEN DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA), accord- Materials and Methods ing to the manufacturer’s protocol for animal tissue samples. To detect possible contact zones of the ranges of Clades The isolated DNA was resuspended in Tris-EDTA buffer and A and B of P. japonicus in Kumekawa et al. (2014), we sam- stored at -20℃ until use. For all specimens, we amplified the pled materials from the central part of Shikoku (Fig. 1). A to- COI gene in mtDNA and 28S rRNA gene in nrDNA. We used tal of 100 individuals were collected from eight populations in previously published primers of Kumekawa et al. (2014) for 22 Yoshimasa Kumekawa, Osamu Miura, Haruka Fujimoto, Katsura Ito, Ryo Arakawa, Jun Yokoyama, TatsuyaFig. Fukuda 3 (a) (b) M Clade A Clade B M Clade A Clade B Agarose gel electrophoresis DNA sequence of Kp Bs Clade A DNA sequence of Clade B Fig. 3. Expected restriction sites for molecular characteristics and PCR-RFLP profiles of (a) COI in mtDNA and (b) 28S rRNA in nrDNA. Arrowheads indicate the difference of DNA sequence of Clade B. Kp: restriction site of Kpn I; Bs: restriction site of Bss II; M: size marker. Note that two bands (digested fragments ca. 600 bp and ca. 400 bp long, respectively) are visible in Clade A, while Clade B shows a non-digested single band (ca. 1000 bp) on a slab gel of mtDNA (upper left, a). Clade A shows only a single band (ca. 900 bp) on a slab gel for nuclear genes 28S rDNA, because small fragments (ca. 100 bp long) were washed out. Also note that no heterozygotes between Clade A and Clade B, that should display two bands comprised by ca. 900 and ca. 800 bp long fragments, were found. COI gene and primers used by Mallatt and Sullivan (1998) for Bank International DNA databases (COI: LC176239 for Clade 28S rRNA. DNA amplification follows the method of Kume- A, LC176240 for Clade B; 28S rRNA: LC176241 for Clade A, kawa et al.