Possible Occurrence of Reproductive Isolation Between Two Geographical

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 between 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 different 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. (2013). After amplification, reaction mixtures were LC176242 for Clade B). subjected to electrophoresis in 1% low-melting-temperature agarose gels and purified using QIAGEN QIAquick PCR Pu- Results and Discussion rification Kit according to the manufacturer’s specifications. We determined sequences of COI in mtDNA and 28S We sequenced the purified PCR products using a BigDye Ter- rRNA in nrDNA in P. japonicus, and their lengths were 930 minator Cycle Sequencing Kit (ABI PRISM DNA Sequencing bp and 1038 bp, respectively. Based on the sequence data, kit, Perkin- Elmer Applied Biosystems, Tokyo, Japan) and we found that the amplified COI and 28S rRNA sequences of ABI PRISM 3100-Avant Genetic Analyzer according to the Clade A have Kpn I and Bss II restriction sites, respectively, manufacturer’s instructions. while Clade B has no sites digested by these restriction en- We carried out PCR-RFLP (restriction fragment length zymes (Fig. 3). Moreover, comparison of sequences using polymorphism) analysis, because autapomorphic character- some samples from each clade indicated that these apomor- istics of the COI and 28S rRNA are the restriction sites Kpn phic characteristics of restriction sites are preserved in each I (GGTACC) (Fig. 3a) and Bss II (GCGCGC) (Fig. 3b), re- clade. Thus, we were able to anticipate presence of the above spectively. After designating the restriction sites, the amplified digestion sites for the samples used in this study. In the hybrid products were digested by Mse I and Bss II at 37℃ for more between Clade A and Clade B in P. japonicus, in particular, it than an hour. The digested DNAs were separated on 1.5% is difficult to detect the heterogeneity at the polymorphic site agarose gel and the size of each band was determined. These of 28S rRNA in nrDNA using the sequence results processed sequences have been also deposited in DDBJ/ EMBL/ Gen- by an autosequencer. Therefore, we conducted PCR-RFLP Possible of reproductive isolation of P. japonicus 23 analysis to obtain further evidence of hybridization and introgression between Clades A and B of P. japonicus. Author Contributions We analyzed eight populations in a secondary contact Y. K., K. I., J. Y., R. A. and T. F. designed the study. Y. K. zone between Clade A and Clade B of P. japonicus based on and H. F. collected samples, analysed the data and wrote the previously published study by Kumekawa et al. (2014). Al- manuscript. O. M. analysed the data. Y. K. O. M. J. Y. and T. F. though most of the populations consisted of only individuals wrote the manuscript. of either Clade A or Clade B of P. japonicus, a single population (site d) contained individuals of both clades. The latter 摘要 population (site d) was comprised only by 26 specimens of 粂川義雅 1, 2・三浦収 2・藤本悠 3・伊藤桂 2・荒川良 2・横山潤 4・ Clade A and 27 of Clade B, and there were no specimens that 福田達哉 5（1 愛媛大学大学院連合農学研究科〒 783-8502 can be identified as hybrids between the two clades. The result 高知県南国市物部乙 200・2 高知大学農林海洋科学部〒 783- of digestion pattern indicated that neither hybridization nor in- 8502 高知県南国市物部乙 200・3 高知大学大学院総合人間自 trogression occurred between Clades A and B of P. japonicus 然科学研究科〒 783-8502 高知県南国市物部乙 200・4 山形 in the area studied, and our results also suggested that Kagami 大学理学部〒 990-8560 山形市小白川町一丁目 4-12・5 東京 and Yoshino rivers and their tributaries are the boundary be- 都市大学知識工学部〒 158-8557 東京都世田谷区玉堤 1-28- tween Clade A and Clade B of P. japonicus (Fig. 2). It is very 1）：四国のニホンアカザトウムシ（クモ綱 : ザトウムシ目 : カ interesting that only either Clade A or Clade B was detected in マアカザトウムシ科）における 2 つの地理的系統群間での生 most populations of the area (Fig. 2). 殖的隔離の可能性 As possibilities that can explain this fact, two scenarios ニホンアカザトウムシ Pseudobiantes japonicus の 2 つの異 may be conceivable: 1) only a relatively short time has passed なる系統群（クレード A とクレード B）の分布域が接触す since ranges of Clade A and Clade B meet; 2) Clade A and る四国中央部において両系統群間の交雑や遺伝子移入の有無 Clade B cannot live together due to possible competitive ex- を明らかにするために，ミトコンドリア DNA と核 DNA の clusion or reproductive interference between the two clades. PCR-RFLP 解析を行った．両クレードの混棲が確認されたの Previous study focusing on phylogeographic analysis revealed は 1 地点のみであったが，その地点を含め，この地域内で， the existence of two monophyletic clades of P. japonicus in 核 DNA の遺伝子におけるヘテロ接合と判定される個体やミ Shikoku, Clade A and Clade B, derived from ancestral popu- トコンドリア DNA と核 DNA 間における不一致は発見されな lations, hypothesizing that these arose because of isolation in かった．これは，クレード A とクレード B が接触地域におい independent refugia in the southeastern (Clade A) and south- て交雑や遺伝子移入を経験していないことを示唆し，これら western Shikoku (Clade B), and this explains surprisingly のニホンアカザトウムシは隠蔽種であると考えられた． high intraspecific genetic variation in P. japonicus (Kumekawa キーワード：隠蔽種，系統学，ニホンアカザトウムシ，四国 et al., 2014). Our results support this hypothesis because no 中央部 hybridization and introgression seen to occur between Clade A and Clade B of P. japonicus at least in the contact zone stud- References ied. Thus, our results suggest that these phylogroups of Clade Avise, J. C., 2000. 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