Allopatric Divergence and Secondary Contact Without Genetic Admixture

Systematic Entomology (2017), 42, 703–713 DOI: 10.1111/syen.12234

Allopatric divergence and secondary contact without genetic admixture for Arichanna perimelaina (Lepidoptera: Geometridae), an alpine moth endemic to the Hengduan Mountains

XINXIN LI1,2, NAN JIANG1, RUI CHENG1, DAYONG XUE1, YANHUA QU1 andHONGXIANG HAN1

1Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing, China and 2University of Chinese Academy of Sciences, Beijing, China

Abstract. Mountain systems, especially at high altitudes, are an excellent model for determining the mechanisms underlying high species diversity and endemism. Herein, we elucidate the evolutionary history of the alpine moth Arichanna perimelaina (Wehrli), which is endemic to the Hengduan Mountains (HM) region in southwest China, based on three mitochondrial genes and two nuclear genes. Our results revealed six deeply divergent clades that corresponded to populations in different mountain systems in the HM region. Bayesian divergence time estimations suggested a mid- to late Pleistocene genetic divergence. The results also showed that the Mt Yulong (YL) region was a refugium and valley corridors established by glaciation during the Pleistocene allowed populations on the separate mountains to migrate. The reproductive isolation among the different clades on contact zone in the YL region may be associated with the asynchronous mating rhythms and/or the divergent mate recognition caused by the ecological source of divergent selection. Allopatric divergence associated with complex topographies and climatic oscillations, regional dispersal via valley corridors and the suitable refugium of the YL region shaped the genetic divergence and distribution pattern of A. perimelaina in the HM region. These findings highlight the essential role of complex terrain and climatic fluctuations in shaping the unique phylogeographic history of a narrow alpine moth, and provide insights into the mechanisms underlying high species richness and endemism in the HM region.

Introduction cycles (mountain refugia hypothesis) (Lei et al., 2015). In addition, climate fluctuations can induce elevation shifts for montane Mountain systems harbour high levels of biodiversity, and these species and lead to alternating periods of isolation and con- mountain ranges are the hotspots of genetic diversity, species nectivity. Periods of connectivity allow for range expansions, richness and endemism (Taubmann et al., 2011; Fjeldså et al., species dispersal and gene flow, whereas periods of isolation 2012). Various evolutionary and ecological processes have been may lead to genetic divergence and speciation (Hewitt, 2004; proposed to explain such exceptional diversity. Mountainous Waltari & Guralnick, 2009). This pattern of genetic differentia- areas are capable of preventing the dispersal of species, reduc- tion and/or speciation attributed to the Pleistocene climatic fluc- ing gene flow among populations (mountains barrier hypothe- tuations is consistent with the Pleistocene species pump hypoth- sis) and providing suitable habitats during glacial–interglacial esis (Knowles, 2000; Midgley et al., 2001). The Hengduan Mountains (HM) region is characterized by a Correspondence: Hongxiang Han, Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of complex topography, discontinuous landscape and highly vari- Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, able climatic conditions among different subareas, and it is a China. E-mail: [email protected] major global biodiversity hotspot with high levels of endemism

© 2017 The Royal Entomological Society 703 704 X. Li et al. and species richness (Myers et al., 2000; Huang et al., 2010; Materials and methods Lei et al., 2014). This mountainous area experienced complex orogenic and climatic events during the Pliocene and Pleis- Sampling and laboratory methods tocene. Tectonic activities associated with the intense uplift of the Qinghai–Tibetan Plateau (QTP) created a series of par- A total of 128 individuals of A. perimelaina were collected allel, mostly north/south-running high mountain systems (Mt from six mountain ranges (nine sampling sites) (Fig. 1; Table Gaoligong, Mt Nushan, Mt Yunling, Mt Shaluli, Mt Daxue- S1 in Supporting Information) across most of the range of this shan, Mt Qionglai and Mt Minshan), which were separated species. The tissue samples were preserved in anhydrous ethanol ∘ by deep valleys (Shi et al., 1998). The recent rapid uplifts of at −20 C, and the vouchers were deposited in the National the QTP, especially the Kunhuang Movement (1.1–0.6 Ma), Zoological Museum (NZM) at the Institute of Zoology of the uplifted the QTP to 3000 m above sea level (m.a.s.l.) and the Chinese Academy of Sciences (IZCAS) in Beijing, China. associated mountains to over 4000 m.a.s.l. (Li et al., 1996; Li Total genomic DNA was extracted from three legs of differ- & Fang, 1999). This uplift also initiated the following four ent moths using the DNeasy Blood and Tissue Kit (Qiagen, major glaciations on the QTP and adjacent regions during the Beijing, China) following the manufacturer’s instructions. Poly- Pleistocene: the Xixiabangma (1.17–0.80 Ma), Naynayxungla merase chain reaction (PCR) amplification and sequencing were (0.71–0.50 Ma), Guxiang (the penultimate, 0.30–0.13 Ma) and performed for three mtDNA fragments [cytochrome c oxidase Baiyu (the last, 0.07–0.01 Ma) glaciations (Zheng et al., 2002; subunit I (COI), cytochrome b (Cytb) and NADH dehydroge- Zhou et al., 2006). nase subunit 5 (ND5)] and two ncDNA fragments [elongation The unique alpine environment and special Quaternary history factor-1 alpha (EF1𝛼) and glyceraldehyde-3-phosphate dehy- of the HM region have drawn the attention of many researchers; drogenase (GAPDH)]. The primers used in this study are listed however, the mechanisms underlying the genetic diversifica- in Table S2 of Supporting Information. For the PCR amplifica- ∘ tion for endemic alpine insects remain unclear. Previous studies tions, we used the following thermal cycling profile: 94 Cfor ∘ ∘ ∘ focusing on alpine vertebrates have suggested that topographic 4 min; 35 cycles of 94 C for 30 s, 45–62 C for 30 s, 72 Cfor ∘ conditions and Pleistocene climatic changes strongly affected 1 min; and 72 C for 10 min. All of the sequences were deposited the genetic structure of certain mammals (Chen et al., 2015), in GenBank (accessions KY391887–KY392536, Table S3 in reptiles (Guo et al., 2011) and birds (Qu et al., 2011, 2015), and Supporting Information). eco-subregions or subregions in southwest China that exhibit Wolbachia infection may give rise to asymmetrical gene flow island-like characteristics appear to determine this diversifica- (Sasaki et al., 2002), and discordance in mtDNA and ncDNA tion. In addition, these studies emphasized that animals with a phylogenies (Hurst & Jiggins, 2005; Narita et al., 2006; Charlat lower dispersal ability and higher sensitivity to climatic fluctua- et al., 2009). Therefore, to screen for Wolbachia infection, two tions may be particularly suitable for studying the historical pro- gene regions (ftsZ and wsp) were amplified, and the primers are cesses of geographical isolation and genetic divergence caused listed in Table S2 of Supporting Information. A positive control by glacial cycles. by using Arichanna jaguarinaria Oberthür from Mt Cangshan Geometrid moths have low vagility and limited habitat ranges, and a negative control using double-distilled water were both and they are sensitive to climatic changes (Holloway & Bar- included in the PCR reactions. Results showed that except for low, 1992; Dennis, 1993; Hausmann, 2001). Arichanna per- the positive control, DNA bands were not found by agarose gel imelaina (Wehrli), which is an alpine-endemic geometrid moth electrophoresis for any specimens. in the subfamily Ennominae, is mainly distributed in parts of Mt Gaoligong, Mt Yunling, Mt Shaluli and Mt Daxueshan. The habitat altitudes are largely restricted to 2400–3500 m.a.s.l. Phylogenetic and genetic structure analyses Interestingly, some obvious variations in the wing speckle have been found on populations inhabiting different mountain sys- To test if there are significant intraspecific genetic differen- tems (Wehrli, 1939). Furthermore, we also found multiple phe- tiations, the phylogenetic reconstructions were first conducted notypic variations occurred in the Yulong Mountain (YL) region. separately for mitochondrial and nuclear genes via the Bayesian This morphological diversification and allopatric geographic inference (BI) method using mrbayes 3.2.6 (Ronquist et al., distribution of this moth make it an ideal model for exploring 2012) and the maximum likelihood (ML) methods using raxml the mechanisms involved in the diversification, speciation and 8 (Stamatakis, 2014) on the CIPRES Science Gateway (Miller biodiversity of alpine species endemic to the HM region. et al., 2010). The best-fitting substitution model for each gene Here, we examine the evolutionary history of A. perimelaina was selected via mrmodeltest 2.3 (Nylander, 2008). For the BI based on mitochondrial DNA (mtDNA) data and nuclear DNA analyses, the settings were as follows: the Markov chain was run (ncDNA) data with the aim of: (i) testing if there is strong genetic for 10 million generations and sampled every 1000 generations, differentiation across such a restricted mountain area; (ii) if so, the first 25% of each chain was discarded as burn-in, andtwo exploring if the YL region is a contact zone of genetically diver- independent analyses were conducted, both with one cold and gent populations, considering it contains multiple variable phe- three heated chains. tracer 1.5 (Rambaut & Drummond, 2009) notypes; and (iii) if so, investigating the potential mechanisms was used to plot the log-likelihood values and to confirm that underlying the reproductive isolation among the sympatric pop- the Markov chains reached stationarity. The settings for the ML ulations in the YL region. searches were as follows: a random seed value was specified for

MJR N (7) DDDR JSR YLR (6)

(5) N SiS chc uaan Basin ° LCR 30 NJR (4) JZW GG

WNG (3) DXS (2) (1) China Altitude YL Myanmar °N High 5 25

Low >30 11-30 WSG 6-10 1-5 200 km 95°E 1000°0 E 1005°E

Fig. 1. Sampling locations of Arichanna perimelaina in the Hengduan Mountains (HM) region. An image of an adult is shown in the lower-right corner. The dot size corresponds to the sample size of each locality. Different colours represent different clades: cyan, clade I; red, clade II; purple, clade III; green, clade IV; orange, clade V; pink, clade VI. The encircled numbers represent different mountain systems: 1, Mt Gaoligong; 2, Mt Nushan; 3, Mt Yunling; 4, Mt Shaluli; 5, Mt Daxueshan; 6, Mt Qionglai; 7, Mt Minshan. YL, Mt Yulong; DXS, Mt Daxueshan of Zhongdian; JZW, Mt Jianziwan; GG, Mt Gongga; WNG, the western slope of northern Mt Gaoligong; WSG, the western slope of southern Mt Gaoligong. The dash lines represent rivers: NJR, Nujiang River; LCR, Lancangjiang River; JSR, Jinshajiang River; YLR, Yalongjiang River; DDR, Dadu River; MJR, Minjiang River. The base map is based on the altitude layers obtained from the WorldClim-Global Climate Data (http://www.worldclim.org/) at a spatial resolution of 30 arc-seconds, and it was constructed using arcgis 10.2 (http://www.esri.com/software/arcgis/arcgis-for-desktop). [Colour figure can be viewedat wileyonlinelibrary.com]. the initial parsimony search and rapid bootstrapping search for Divergence dating, genetic diversity and history demography the best-scoring ML tree, and GTR + gamma was used for the bootstrapping phase and the final tree (all other parameters were The timing of divergence was estimated in beast 1.8.2 (Drum- default values). We considered those nodes with ≥0.95 Bayesian mond et al., 2012) based on the combined mtDNA genes. Both posterior probabilities and ≥70 bootstrap values as strongly sup- a strict molecular clock model and an uncorrelated lognormal ported (Bryson et al., 2011). Arichanna jaguarinaria Oberthür relaxed molecular clock were attempted, and the model comparison of the Bayes factors by the Akaike information content was used as the outgroup. In addition, median-joining haplo- (AIC) was conducted in tracer 1.5. The evolution model for type networks were also constructed for the individual mtDNA each gene was selected by mrmodeltest 2.3, and the ‘HKY+I’ and ncDNA datasets with the maximum parsimony criterion of model was selected for all three genes. Because of the lack of network 4.6.1.2 (Bandelt et al., 1999). dated fossils or geological events, we used the modified muta- Genetic differentiation within this moth was quantified using tion rate for the insect mitochondrial COI gene of 3.54% per two methods based on two datasets. First, permut 1.1 (Pons million years (1.77% per site per million years) (Papadopoulou & Petit, 1996) was used to calculate the total gene diversity et al., 2010). The mutation rate was modulated by multiply- (HT) and average population gene diversity (HS)toevaluatethe ing the ratio of the average genetic distance for Cytb (or ND5) level of genetic variation, and GST and NST were also calculated versus that for COI to deduce the substitution rate for Cytb (or to estimate the differentiation between populations. Second, an ND5)(Quet al., 2010). Three hundred million generations were analysis of molecular variance (amova) was used to investigate executed with sampling every 30 000 generations, with the ini- the genetic variation within and between populations using tial 10% discarded as burn-in. The stationarity of the chains and Arlequin 3.5 (Excoffier & Lischer, 2010). convergence of the two runs was monitored by tracer 1.5 to

© 2017 The Royal Entomological Society, Systematic Entomology, 42, 703–713 706 X. Li et al. determine whether the effective sample size of each parameter region, the mean COI barcode genetic distances within and was >200 as recommended. treeannotator was used to sum- between populations were firstly calculated with mega 5.2 marize the information from the individual post burn-in trees (Tamura et al., 2011) using the Kimura two-parameter distance onto a single maximum clade credibility (MCC) tree, with the model. Furthermore, to determine whether historical gene flow first 5000 trees discarded as burn-in. Finally, the MCC treewas occurred among the sympatric populations found on the YL visualized using figtree 1.4.2 (Rambaut, 2014). region, IMa2 (Hey, 2010) was performed based on the concate- And then, the number of haplotypes (Hn), haplotype diversity nated mtDNA and ncDNA data. The model parameter functions (Hd) and nucleotide diversity (𝜋) were estimated based on each were estimated in the M-mode based on 1000 000 generations, dataset in dnasp 5.1.0 (Librado & Rozas, 2009). In addition, with the initial 100 000 generations discarded as a burn-in. The two methods based on each dataset were used to infer the demo- Markov chain Monte Carlo run was repeated three times with graphic history. First, Tajima’s D (Tajima, 1989) and Fu’s Fs different random starting seeds to confirm the convergence of (Fu, 1997) were examined with 10 000 coalescent simulations similar values. After M-mode runs, the parameters were esti- in arlequin 3.5. Second, mismatch distribution analyses were mated using likelihood ratio tests implemented in the L-mode. also conducted using arlequin 3.5. Significant values for sum At last, we randomly selected two male–female pairs from each of squared deviations (SSD) or raggedness index (R) indicated a population, and the genitalia was examined following the stan- population-stable model through time. Significance values were dard procedure published by Robinson (1976). calculated with 1000 bootstrap replicates.

Results Ecological niche modelling Phylogenetic and genetic structure analyses Ecological niche modelling (ENM) was used to evaluate the suitable habitat continuity during the Last Glacial Maximum In total, we obtained 2981 bp from mitochondrial genes (COI, (LGM) and the Last Interglacial (LIG) periods. A total of 675 bp; Cytb, 1005 bp; ND5, 1301 bp) and it contained 126 13 occurrence records were obtained for the niche modelling, polymorphic sites, 116 of which were parsimony-informative, including nine records from the fresh specimens, one from the defining 47 haplotypes. We also acquired 1607 bp from nuclear literature (Wehrli, 1939) and three records from pinned spec- genes (EF1𝛼, 905 bp; GAPDH, 702 bp) and it contained in 24 imens at the NZM, IZCAS (Table S1 in Supporting Informa- polymorphic sites, 20 of which were parsimony-informative, tion). The environmental layer variables of the current period, defining 37 haplotypes. Both the BI and ML phylogenies based LGM (the Community Climate System Model; CCSM) and the on the mtDNA data supported six clades (I–VI; Fig. 2a), LIG were downloaded from WorldClim-Global Climate Data although the bootstrap values for two nodes in the ML tree (version 1.4; http://www.worldclim.org/). To reduce the model were low. Clade I & II was the sister group of all other clades. over-fitting caused by environmental variables redundancy, we Clade I consisted of individuals from the YL region and the Mt first ran maxent 3.3 using 19 environmental variables and Jianziwan (JZW) region. Clade II consisted of individuals from then calculated Pearson’s correlation coefficientr ( ) between the western slope of northern Mt Gaoligong (WNG) region. each pair of variables using spss (IBM SPSS Inc., Chicago, IL, Clade III, the sister group of clade IV & V & VI, consisted of U.S.A.). Variables with r > 0.9 were considered as highly cor- individuals from the western slope of southern Mt Gaoligong related, and eight variables (annual mean temperature, mean (WSG) region. Clade IV consisted of individuals from the Mt diurnal range, isothermality, temperature seasonality, maximum Daxueshan of Zhongdian (DXS) region and the YL region. temperature of warmest month, annual precipitation, precipi- Clade V consisted of individuals from Mt Gongga (GG), and tation of the driest month and precipitation seasonality) were clade VI consisted of individuals from the YL region. The selected for the final analyses. haplotype network of mtDNA data (Fig. 2b) also presented six maxent was used to construct the models using a random clusters corresponding to six clades. The star-like shapes in the selection of 75% of the occurrence data, and the remaining 25% network of clades I and IV both indicate at least one population were used to test the models. The default convergence threshold expansion event. The ancestral haplotype occurred in both the (10−5), 2000 maximum iterations and 10 replicates were used. JZW and YL regions for clade I, and the DXS and YL regions The assessment index of the ENM prediction included the area for clade IV. under the receiving operator characteristics curve (AUC) and Unlike the mitochondrial phylogeny, the phylogeny based the binomial probabilities. An AUC >0.9 indicated that the on ncDNA dataset (Fig. 3a) did not present the six clades sup- predicted results of the model were better than those predicted ported by the mtDNA dataset completely. However, a shallow stochastically. divergence pattern was observed. Four major clade groups (I & II, III, IV, V & VI) were recognized in the nuclear phylogeny (Fig. 3b). Clade II was nested within clade I, and clade VI Genetic distance, historical gene flow and genitalia dissections was embedded into clades V. The sympatric populations were located in different clade groups. Moreover, the four major clade To explore the potential mechanisms that underlie the repro- groups were also observed in the nuclear haplotype network. ductive isolation among the sympatric populations in the YL There was one shared haplotype for populations GG and YL,

14 WSG III 4 11 10 1/100 3 4

JZW & YL I 1/93 4 0.001 >17 9–11 12–16 Outgroup WNG II 5–8 1–4

Fig. 2. (a) Phylogenetic tree of Arichanna perimelaina based on the mitochondrial DNA (mtDNA) dataset. Numbers near branches are Bayesian posterior probabilities and bootstrap values. YL, Mt Yulong; DXS, Mt Daxueshan of Zhongdian; JZW, Mt Jianziwan; GG, Mt Gongga; WNG, the western slope of northern Mt Gaoligong; WSG, the western slope of southern Mt Gaoligong. (b) Median-joining haplotype network constructed based on mtDNA genes using network. The haplotype circle size denotes the number of sampled individuals, and the number of base pair changes (no number = 1 bp) is indicated. Solid circles represent haplotypes that were either not sampled or extinct. (c) Adults with coloured circles correspond to the different clades. Scale bar = 1 cm. [Colour figure can be viewed at wileyonlinelibrary.com].

low (mtDNA, H = 0.439; ncDNA, H = 0.553). Further- (a) (b) IV S S more, the NST value was significantly higher than the GST IV > < value (mtDNA, NST = 0.975 GST = 0.561, P 0.01; ncDNA, > < NST = 0.737 GST = 0.447, P 0.01). The amova results (Table 1) revealed similar results for the two datasets. The F among populations indicated high levels of genetic II I & II ST I differentiation (mtDNA, FST = 0.955 83, P = 0.00; ncDNA, FST = 0.610 22, P = 0.00). And most of the variation (mtDNA, 95.58%; ncDNA, 61.02%) occurred among populations, whereas the variation within populations was low (mtDNA, 0.0001 III V & VI 4.42%; ncDNA, 38.98%). 1/97 VI V III 9–12 >12 Outgroup 1–4 5–8

Fig. 3. (a) Phylogenetic tree of Arichanna perimelaina based on the Divergence dating, genetic diversity and history demography nuclear DNA (ncDNA) dataset. Numbers near branches are Bayesian posterior probabilities and bootstrap proportion from a maximum The results of the model comparison showed that the uncor- likelihood analysis. (b) Median-joining haplotype network constructed related lognormal relaxed molecular clock is superior to the based on ncDNA genes using network. The haplotype circle size others for COI (lognormal relaxed vs strict, 57.925; lognor- denotes the number of sampled individuals. Solid circles represent mal relaxed vs exponential relaxed, 4.176) and the exponential haplotypes that were either not sampled or extinct. [Colour figure can relaxed molecular clock is more appropriate for Cytb (exponen- be viewed at wileyonlinelibrary.com]. tial relaxed vs lognormal relaxed, 713.629; exponential relaxed vs strict, 37248.88) and ND5 (exponential relaxed vs lognormal relaxed, 290.627; exponential relaxed vs strict, 1234.926). indicating a shallow population structure, whereas no haplotype The time to the most recent common ancestor for A. perime- was shared by the sympatric populations in the YL region. laina and outgroups was approximately 1.343 Ma [95% high- The result of the permut analyses showed that the total gene est posterior density (HPD): 1.095–1.598 Ma]. The divergence diversity was high (mtDNA, HT = 1.000; ncDNA, HT = 1.000), time results (Fig. 4) showed that the intraspecific divergence whereas the average intra-population gene diversity was occurred during the mid- to late Pleistocene. The first split

Table 1. Analysis of molecular variance (amova) results separately based on mitochondrial and nuclear datasets.

Source of variation d.f. Sum of squares Variance components Percentage variation Fixation index

Mitochondrial DNA Among populations 5 1383.81 17.112 94 Va 95.58 0.955 83*** Within populations 117 92.517 0.790 74 Vb 4.42 Total 122 1476.358 17.90368 Nuclear DNA Among populations 5 113.637 1.273 38 Va 61.02 0.610 22*** Within populations 122 99.230 0.813 36 Vb 38.98 Total 127 212.867 2.086 74

***P < 0.001. Va and Vb indicate the difference level. Probabilities were calculated by 1023 random permutations of individuals across populations.

0.171 (0.099–0.236) 𝜋 = 0.000 71; ncDNA, Hd = 0.703, 𝜋 = 0.00072) and GG VI (mtDNA, Hd = 0.679, 𝜋 = 0.00026; ncDNA, Hd = 0.893, 0.362 (0.259–0.466) V 𝜋 = 0.001 29) showed high genetic diversity, whereas populations WNG (mtDNA, Hd = 0.000, 𝜋 = 0.000; ncDNA, 𝜋 0.413 (0.313–0.509) Hd = 0.000, = 0.000 00), WSG (mtDNA, Hd = 0.154, IV 𝜋 = 0.00005; ncDNA, Hd = 0.410, 𝜋 = 0.000 27) and JZW (mtDNA, Hd = 0.526, 𝜋 = 0.00039; ncDNA, Hd = 0.870, 𝜋 = 0.001 34) showed low genetic diversity. III The neutrality test results (Table 2) showed that both Tajima’s 0.463 (0.357–0.567) D (−1.75) and Fu’s Fs (−3.98) were significantly negative for population JZW, indicating historical population expansion. However, the results were not significant for the other populations. Because genetic variation was not observed among the mtDNA sequences of population WNG, the mismatch distribu- 1.343 (1.095–1.598) I tion could not be calculated. The mismatch distribution results for the other populations, especially for populations JZW and 0.093 (0.055–0.130) DXS (Table 2), indicated nonsignificant lower SSD and R values, suggesting population expansion.

II Outgroups Ecological niche modelling 0.5 0.4 0.3 0.2 0.10 Ma mid-Pleistocene late-Pleistocene Holocene The ENMs for A. perimelaina exhibited good predictive ability, with an average training AUC of 0.991. Our analysis Fig. 4. Estimation of the divergence times for Arichanna perimelaina. indicated that more extensive and continuously suitable moun- The number at each node represents the mean value of the most recent tainous habitats across the HM region occurred during the LGM common ancestor with a 95% confidence interval. [Colour figure can be (Fig. 5). For this period, mountainous habitats occurring in the viewed at wileyonlinelibrary.com]. Mt Qionglai, Mt Daxueshan, Mt Shaluli, Mt Yunling and Mt Gaoligong regions were connected with each other. In sharp occurred at 0.463 Ma (95% HPD: 0.357–0.567 Ma), and it sepa- contrast, the suitable habitat range was significantly restricted rated clades I & II from the other clades. The second divergence during the LIG, suggesting that this period would have provided occurred between clades III and IV & V & VI at approximately the least favourable climatic conditions for this species. Fur- 0.413 Ma (95% HPD: 0.313–0.509 Ma). The divergence time thermore, the suitable habitats of different mountains remained between clades IV and V & VI was approximately 0.362 Ma almost isolated during this period. Moreover, Lijiang (indicated (95% HPD: 0.259–0.466 Ma) and between clades V and VI by the green line) and adjacent areas were in the core of the most was approximately 0.171 Ma (95% HPD: 0.099–0.236 Ma). suitable habitats in the HM region during both the LGM and LIG Clade II diverged from clade I at 0.093 Ma (95% HPD: periods, whereas other alpine habitats were in the periphery. 0.055–0.130 Ma). These divergence times (0.463–0.093 Ma) correspond to the last three glaciations (0.71–0.01 Ma) and get later than the Kunhuang Movement (1.1–0.6 Ma). Genetic distance, historical gene flow and genitalia dissections The genetic polymorphisms are shown in Table 2. The genetic diversity for whole population was high (mtDNA, The genetic distance results revealed intra-population variabil- Hd = 0.934, 𝜋 = 0.008 31; ncDNA, Hd = 0.952, 𝜋 = 0.002 09). ity in the COI barcode region for distances within population Populations YL (mtDNA, Hd = 0.943, 𝜋 = 0.00744; ncDNA, ranging from 0.00% to 0.11%, and between populations rang- Hd = 0.936, 𝜋 = 0.001 71), DXS (mtDNA, Hd = 0.879, ing from 0.38% to 1.81% (Table S4 in Supporting Information).

Table 2. Nucleotide polymorphisms, neutral test and mismatch distribution results for each population and the whole population.

Population Sample size Hn Hd 𝜋 Tajima’s D Fu’s Fs SSD P-values RP-values

Mitochondrial DNA JZW 23 8 0.526 0.000 39 −1.75* −3.98* 0.0043 0.457 0.0352 0.687 WNG 4 1 0.000 0.000 00 – – – – – – WSG 13 2 0.154 0.000 05 −1.15 −0.54 0.0281 0.208 0.5030 0.417 DXS 14 7 0.879 0.000 71 −1.53 −1.91 0.0055 0.348 0.0534 0.529 GG 8 3 0.679 0.000 26 0.07 −0.22 0.0495 0.235 0.2896 0.245 YL 66 31 0.943 0.007 44 1.09 0.20 0.0104 0.669 0.1365 0.874 Total 128 47 0.934 0.008 31 0.79 −0.35 0.0397 0.012 0.0133 0.124 Nuclear DNA JZW 23 11 0.870 0.001 34 −0.38 −4.93** 0.0011 0.557 0.0455 0.278 WNG 4 1 0.000 0.000 00 – – – – – – WSG 13 3 0.410 0.000 27 −0.91 −0.79 0.0085 0.430 0.1716 0.483 DXS 14 4 0.703 0.000 72 0.72 −0.09 0.0049 0.422 0.0710 0.469 GG 8 5 0.893 0.001 29 0.34 1.08 0.0195 0.540 0.1110 0.477 YL 66 21 0.936 0.001 71 −0.38 −10.32 0.0101 0.377 0.2041 0.593 Total 128 37 0.952 0.002 09 −0.70 −24.74*** 0.0027 0.083 0.0250 0.371

*P < 0.05; **P < 0.01; ***P < 0.001. Hn, the number of haplotypes; Hd, haplotype diversity; 𝜋, nucleotide diversity; SSD, sum of squared deviations; R, raggedness index; JZW, Mt Jianziwan; WNG, the western slope of northern Mt Gaoligong; WSG, the western slope of southern Mt Gaoligong; DXS, Mt Daxueshan of Zhongdian; GG, Mt Gongga; YL, Mt Yulong.

The results from IMa2 were almost identical for independent phylogeny and haplotype network also reveal four major clade runs, and all of these results indicated that significant histori- groups (I & II, III, IV, V & VI) (Fig. 3). Multiple analyses indi- cal gene flow did not occur between each population pair (Table cate that significant genetic differentiation has occurred among S5 in Supporting Information). Further morphological analyses the different populations. The lower within-population genetic of the adults provide diagnostic differences for these six pop- distance and average gene diversity together with the higher ulations. Certain distinct and stable variations in genitalia are between-population genetic distance and total gene diversity observed among the different clades (Fig. 6), especially for the indicate that differentiation is high among populations and low sympatric populations (clades I, IV and VI). The costa of the within populations. The amova results also demonstrate that valve in the male genitalia clade IV is longer and produced an the split among populations is strong but almost homogenous obvious extension of the terminal margin of the valve (Fig. 6a). within each population. Besides, the considerable morphologi- The ductus bursae is strongly sclerotized in the female of clade cal variations occurred in the genitalia of both males and females IV,followed by that of clade VI, and that of clade I is the weakest provides further evidence of significant intraspecific divergence. (Fig. 6b). In addition, the ductus bursae of clade IV is broader The significant genetic divergence is attributed to the diverse anteriorly than the other clades. topography of the HM region caused by the uplift of the QTP, especially the recent Kunhuang Movement events, which have offered ample opportunities for allopatric isolation. Multiple Discussion particular geographic barriers can be easily identified, such as Mt Gaoligong, Mt Nushan, Mt Shaluli and Mt Daxueshan. Given Significant allopatric divergence in the HM region the origin time (1.343 Ma) and the intraspecific divergence Recent studies suggest that species with narrow geographi- time (0.463–0.093 Ma) of this moth, together with the time of cal distributions may present lower genetic variation for traits Kunhuang Movement (1.1–0.6 Ma), vicariance events among related to environmental tolerance (Kellermann et al., 2009). these populations can be pinpointed. The genetic divergence was Weak or lower genetic divergence has been observed for further affected by the Quaternary climatic oscillations, espe- alpine endemic vertebrate animals with strong mobile abil- cially the most severe glaciations during 0.71–0.01 Ma, which ity. For example, weak phylogeographical structure of a bird occurred in the HM region (Zheng et al., 2002). The glacial and Garrulax elliotii (Verreaux) (Qu et al., 2011) and the Chi- interglacial cycles caused considerable fragmentation of subni- nese white-bellied rat Niviventer confucianus (Milne-Edwards) val habitats, which in turn triggered the near-simultaneous onset (Chen et al., 2011) both occurred in eco-subregions/subregions of intraspecific allopatric diversification during the mid- tolate in southwest China. Although A. perimelaina might be expected Pleistocene (0.463–0.093 Ma; Fig. 4). These results suggest that to show less genetic differentiation in such a small mountain the complex topography associated with the tectonic events and region, our results indicate that considerable genetic fragmen- glaciation cycles during the mid- to late Pleistocene had a signif- tation has occurred in six different mountain systems, and the icant role in shaping the current genetic structure of A. perime- mitochondrial phylogeny and the haplotype network analyses laina, and they also provide evidence supporting the hypothesis recovered six major clades (I–VI) (Figs 1, 2a,b) with differ- that mountain systems are the cradle for generating arthropod ent levels of phenotypic variation (Fig. 2c), and the nuclear intraspecific and interspecific diversity (Garrick, 2011).

(a) Current

I II III

IV V VI (b)

LGM

I II III

IV V VI

Fig. 6. Male (a) and female (b) genitalia of different populations of Arichanna perimelaina. The arrows indicate the costa of the valve for male genitalia and ductus bursae for female genitalia. Scale bar = 1 mm. [Colour figure can be viewed at wileyonlinelibrary.com].

Although the nuclear data phylogeny does not present the LIG distinct genetic structure identified by the mtDNA dataset, a shallow divergence pattern is still observed. Excluding Wol- bachia infection, we inferred that incomplete lineage sorting was probably the driver of incongruence between the mtDNA and ncDNA phylogenies.

Secondary contact in the YL region

Both the phylogenies and the haplotype networks inferred separately from the mtDNA data and the ncDNA data reveal strong differentiation among populations from the DSX, JZW and YL regions, suggesting long-term geographical isolation triggered by the Kunhuang Movement. It should be noted that some individuals sampled from the YL region clustered with populations DXS and JZW, which provides evidence for secondary contact of the populations DXS, JZW and YL. Species Fig. 5. Palaeodistributions and the current distribution of Arichanna perimelaina as predicted by ecological niche modelling. LGM, Last at high altitudes can migrate vertically during climatic fluctu- Glacial Maximum; LIG, Last Interglacial. The black spots repre- ations (Hewitt, 1996). During glacial periods, montane vege- sent sampling localities and the occurrence records in the Hengduan tation in different high mountain areas were driven to lower Mountains region. Different colours correspond to different fitting elevations, where they migrated and colonized new mountain indices, with low in blue and high in red. The area outlined with the areas (Schoville & Roderick, 2010). This vegetation migration green line denotes Lijiang region. [Colour figure can be viewed at provided ecological corridors along the valleys between moun- wileyonlinelibrary.com]. tain systems that enabled alpine phytophagous species to come

© 2017 The Royal Entomological Society, Systematic Entomology, 42, 703–713 Phylogeography of Arichanna perimelaina 711 into contact. In our analyses of ENM, the suitable habitats were et al., 2003; Hausmann et al., 2011). Although distinct varia- continuous in the south-central HM region, and the neighbour- tions were observed in the male and female genitals of different ing mountain systems in the northern HM region were con- populations (Fig. 6), confirming that the lack of gene flow was nected via suitable valleys during the LGM (Fig. 5); both of caused by genital incompatibility is difficult, because consid- these conditions provided migration corridors for populations erable variability in genitalia at the intraspecific level has been inhabiting different mountain systems. For example, the popu- observed for other moth species (Mutanen, 2005; Mutanen & lations of JZW and DXS expanded and colonized the YL region Kaitala, 2006; Mutanen et al., 2006, 2007). Therefore, we spec- along the Yalongjiang River and the Jinshajiang River during ulate that the lack of gene flow among different populations of the LGM, respectively. the YL region may have been caused by an asynchronous mating The reason why the YL region could be the sympatric rhythm or invalid mate recognition. The former prevents indi- area of the divergent populations of A. perimelaina lies in its viduals belonging to different populations from encountering special climatic history and unique vegetation. Three glacia- each other at the same time, and the latter causes them to per- tions, the Yunshanping glaciation (0.600 Ma), Lijiang glacia- ceive each other as incompatible mates. Mating rhythm (Harde- tion (0.316–0.259 Ma) and Dali glaciation (0.240 Ma) (Zheng, land, 1972; Sakai & Ishida, 2001) and mate recognition (Saveer 2000), occurred in Lijiang during the mid-Pleistocene. After the et al., 2014) play critical roles in reproductive isolation among Lijiang glaciation, Lijiang became warmer and even muggy dur- divergent lineages or species. Natural selection for adaptation to ing the late Pleistocene, thereby providing suitable habitats for different ecological conditions has become increasingly recog- many species (Li, 1961; Zheng, 2000). Furthermore, the YL nized as an important factor in the formation and maintenance region has a prominent and complete vertical natural landscape of species (Schluter, 2009; Prada & Hellberg, 2013). Differ- because of the elevation gradient and presents a mid-subtropics ent ecological environments may cause adaptive divergence in zone, temperate zone and even a cold zone, and there are signif- mating rhythm and mate recognition, thereby restricting gene icant differences in the vertical natural landscapes of the east flow between divergent populations. Future research coupling and west slopes of the YL region. Thus, we assume that the behaviour and physiology will provide insights into the mecha- milder climate environment in Lijiang and the complex ecolog- nisms underlying the reproductive isolation that occurs in different divergent populations of this alpine moth and provide a more ical environment of the YL region provided suitable refugia for complete understanding of the species diversity and endemism. species inhabiting adjacent mountain systems during the mid- to late Pleistocene climatic fluctuations. The dispersal between the JZW and YL regions, and between the DXS and YL regions Supporting Information under the influence of the climatic fluctuations, led to thesec- ondary contact in Lijiang. Our findings provide evidence that Additional Supporting Information may be found in the online Lijiang was an important mid- to late Pleistocene refugium and version of this article under the DOI reference: a contact zone hotspot, and they help to explain the extraordinary 10.1111/syen.12234 biodiversity of Lijiang, especially the YL region. Furthermore, Table S1. Sampling information for A. perimelaina. our results provide further evidence that Lijiang should be a high-priority area for biodiversity conservation. Table S2. All primers used in the PCR amplifications. Table S3. GenBank accession numbers for all sequences.

Reproductive incompatibility for different populations in the YL Table S4. The COI barcode genetic distance within and region between different populations. Table S5. Gene flow among the three sympatric populations. Barriers to gene migration might accumulate during periods when gene flow is blocked because of spatial isolation or physical obstacles to migration. However, populations that have Acknowledgements developed incomplete reproductive barriers frequently come into contact at different stages of divergence as habitat ranges We sincerely thank all collectors for sampling assistance in the expand, which provides the opportunity for gene flow (Abbott field. This work was supported by the National Science Foun- et al., 2013). Yet in our analyses, the genetic separation of pop- dation of China (grant nos 31372176, 31572301, 31272288, ulations JZW, DXS and YL revealed by the phylogenies and 31402004), the National Science Fund for Fostering Talents in the haplotype networks, as well as the absence of the histori- Basic Research (grant no. NSFC-J1210002) and by a grant from the Key Laboratory of the Zoological Systematics and Evolution cal gene flow between the sympatric populations demonstrate of the Chinese Academy of Sciences (no. O529YX5105). The that genetic admixture did not occur among the three sympatric authors declare that they have no conflict of interest. populations of the YL region, suggesting reproductive isolation. The genetic distance between each pair of populations (1.20% References for I and IV, 1.53% for I and VI, and 1.52% for IV and VI) was less than the criterion of 2% maximum intraspecific bar- Abbott, R., Albach, D., Ansell, S. et al. (2013) Hybridization and code divergence, thus suggesting intraspecific variations (Hebert speciation. Journal of Evolutionary Biology, 26, 229–246.

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