Molecular Phylogenetics and Evolution 94 (2016) 47–54 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Phylogenetic analysis of the winter geometrid genus Inurois reveals repeated reproductive season shifts q ⇑ Satoshi Yamamoto a, , Eugene A. Beljaev b, Teiji Sota c a Graduate School of Human Development and Environment, Kobe University, 3-11 Tsurukabuto, Nada, Kobe 657-8501, Japan b Institute of Biology and Soil Science, Prospect 100 Let Vladivostoku 159, Vladivostok 690022, Russia c Department of Zoology, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502, Japan article info abstract Article history: Winter geometrid moths belonging to the genus Inurois comprise nine species that reproduce during Received 12 March 2015 early winter, three species that reproduce in late winter, and polymorphic species with genetically Revised 15 July 2015 diverged early and late winter populations that co-occur widely across the species’ range. In our previous Accepted 17 August 2015 studies, we demonstrated that differences in reproductive timing resulted in allochronic reproductive Available online 22 August 2015 isolation between sympatric populations. In the present study, to assess the evolutionary pattern of reproductive timing within the genus, we determined the phylogenetic relationships among species Keywords: using nuclear and mitochondrial gene sequences. Nuclear gene tree showed that reproductive season Allochronic speciation shifts occurred independently in four of 13 divergence events. In two divergence events, allochronic sister Introgressive hybridization Parallel divergence lineages were formed in sympatry, suggesting that the segregation of the reproductive season was asso- Temporal isolation ciated with diversification in the genus Inurois. We also found that the mitochondrial gene tree was quite different from the nuclear gene tree and that mitochondrial introgression may have occurred in a few cases. Although it remains unclear whether early and late winter species actually have hybridized with each other and how strong or stable is the reproductive isolation provided by the reproductive season segregation, our study illuminates the potential importance of allochronic isolation in the diversification process of the genus Inurois. Ó 2015 Elsevier Inc. All rights reserved. 1. Introduction tophagous insects (Filchak et al., 2000; Berlocher and Feder, 2002; Drès and Mallet, 2002; Thomas et al., 2003; Savolainen Temporal disjunction of reproductive activities functions as a et al., 2006; Franks and Weis, 2009; Shimono et al., 2009; hybridization barrier between closely related species (Lamont et al., Matsubayashi and Katakura, 2009; Papadopulos et al., 2013; 2003; Danley et al., 2007; Sachet et al., 2009; Thomassen et al., Richter-Boix et al., 2013; Thomassen et al., 2013; Powell et al., 2013) as well as facilitates genetic differentiation among populations 2014). In addition, climate can be a major factor that causes allo- (Hall and Willis, 2006; Smith and Friesen, 2007; Sota et al., 2013)and chronic isolation. For example, a seabird species has sympatric sexual isolation among individuals within populations (Hirao and populations that breed in different seasons (hot and cool seasons) Kudo, 2008). Reproductive isolation owing to differences in repro- on separate islands (Monteiro and Furness, 1998; Friesen et al., ductive timing is known as allochronic isolation. This isolation should 2007; Bolton et al., 2008). Another example is the winter geome- be important for understanding the mechanism of diversification in a trid moth Inurois punctigera showing a reproductive season that specific lineage, because in some cases, allochronic isolation can be is separated into early and late winter in cool habitats owing to an initial step of speciation (Alexander and Bigelow, 1960; Marshall harsh midwinter conditions; however, its reproductive season is and Cooley, 2000; Abbot and Withgott, 2004; Friesen et al., 2007; not separated in warm habitats (Nakajima, 1998). Genetic differen- Yamamoto and Sota, 2009; Santos et al., 2007). tiation between sympatric early and late winter populations has Allochronic isolation is often strongly associated with diver- been demonstrated in habitats with severe midwinter conditions gence in ecological traits, such as host preferences in phy- (Yamamoto and Sota, 2009). In these studies, the segregation of reproductive seasons was shown to have occurred in parallel in dif- ferent localities with similar climatic conditions (Friesen et al., q This paper was edited by the Associate Editor Francesco Frati. 2007; Yamamoto and Sota, 2012). Given that specific climatic con- ⇑ Corresponding author. ditions can facilitate allochronic isolation at different localities in a E-mail address: [email protected] (S. Yamamoto). http://dx.doi.org/10.1016/j.ympev.2015.08.016 1055-7903/Ó 2015 Elsevier Inc. All rights reserved. 48 S. Yamamoto et al. / Molecular Phylogenetics and Evolution 94 (2016) 47–54 similar manner to these cases, the species diversity of a lineage gation factor 1 alpha (EF-1a), tektin, and arginine kinase (ArgK), that occupies a wide area, including habitats with similar climatic were polymerase chain reaction (PCR) amplified using the primers conditions, may be associated with repeated evolutionary shifts in 50-TGCGGTGGTATCGACAAGAG-30 (Yamamoto and Sota, 2009) and the reproductive season. 50-GATTTACCRGWACGACGRTC-30 (Kawakita et al., 2004) for EF- In East Asia, the winter moth genus Inurois, including I. punctig- 1a,50-ACCAGTGGRGAYATYCTWGG-30 and 50-CGCAGTTTYTGATRC era, provides an opportunity to examine the hypothesis of repeated TYT-30 (Mallarino et al., 2005) for tektin, and 50-GACAG reproductive season shifts. This genus exhibits variations in its CAARTCTCTGCTGAAGAA-30 and 50-GGTYTTGGCATCGTTGTGGTAGA reproductive season, where some species reproduce in early winter TAC-30 (Kawakita et al., 2003) for ArgK. In addition to these nuclear and others in late winter if their habitats have severe midwinter sequences, a partial sequence of the mitochondrial cytochrome oxi- conditions (Fig. 1), suggesting that the separation of reproductive dase subunit I (COI) gene was also PCR amplified using the primers: season may have occurred in other lineages of this genus, in addi- 50-TTATTTTTGGAATTTGAGC-30 and 50-CCTGTTAATCCTACTGT-30 tion to I. punctigera. Furthermore, given that the divergence of the (Yamamoto and Sota, 2009). A BigDye Cycle Sequencing Kit reproductive season can occur repeatedly in I. punctigera, the (Applied Biosystems, Foster City, CA) was used to analyze the PCR reproductive season may also have diverged independently in products with an ABI3130xl sequencer (Applied Biosystems) and the entire Inurois lineage. Because Inurois species occupy almost the same primers. We found that the ArgK sequence could not be the same geographic range as I. punctigera, this genus should have determined for some samples due to length polymorphisms, so also been affected by the same climatic environment; thus, harsh we conducted additional sequencing with the inner forward primer climatic conditions may have promoted repeated reproductive sea- ARGinF 50-TTCGAGAACTTGGACTCCGG-30 using the initial PCR son shifts in the genus Inurois. products of ArgK. Finally, we obtained a 926 bp sequence of EF-1a, In this study, we addressed the evolution of reproductive season a 702 bp sequence of tektin, a 415 bp sequence of ArgK, and a in the winter moth genus Inurois and the importance of the sepa- 900 bp sequence of COI. The sequence data were deposited in Gen- rated reproductive season in diversification (i.e. speciation and Bank (accession numbers: AB552989–AB553279; AB980810– maintenance of reproductive isolation between species). To this AB980945). In the molecular phylogenetic analyses, we also used end, we studied the phylogenetic relationships among all Inurois 21 EF-1a sequences, which were reported in our previous study species using both nuclear and mitochondrial genes. Introgression (GenBank accession numbers: AB467986–88, AB467990, of mitochondrial gene among Inurois species was also examined. AB468003–04, AB468006–07, AB468015, AB468028, AB468031, We hypothesized that reproductive season shift has occurred AB468033–35, AB468037–39, AB468044, AB468047–48, repeatedly in Inurois and the separation of reproductive season AB468050; Yamamoto and Sota, 2009). Genital character states between species and lineages would play an important role in were determined for all the Inurois species, as well as A. japonensis diversification of Inurois, as suggested in I. punctigera. and C. zabolne (Table S2). We could not obtain morphological and nuclear sequence data for A. acroama, so the outgroup species were not included in the analyses based on nuclear sequences and mor- 2. Materials and methods phological data. Molecular phylogenetic analyses were performed with the 2.1. Sampling and phylogenetic analyses three datasets: nuclear sequences (three regions combined), COI sequences, and combined nuclear + morphological data. The Inurois species are distributed around the Sea of Japan (southern sequences of each gene were separated based on the codon posi- Russian Far East, China, Korean Peninsula, and Japan; Fig. 2). We tions, and general time reversible (GTR) + G substitution models collected fresh specimens to extract DNA from all Inurois species were applied to each of them. GTR + G substitution models were in Russia (around