Infection, Genetics and Evolution 90 (2021) 104764

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Infection, Genetics and Evolution

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Research paper Shape relatedness between geographic populations of tritaeniorhynchus, the primary vector of Japanese encephalitis virus: A landmark study

R. Morales Vargas a, T. Tsunoda b, J. Noda c, P. Bousses d, T.Y. Nguyen e, F. Hasebe f, J. P. Dujardin g,* a Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, b Laboratory of Medical Entomology, Department of Vector Ecology and Environment, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, c Laboratory of Environmental Health Sciences, Division of Health and Environmental Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan d IRD, UMR MIVEGEC IRD, CNRS, University of Montpellier, F-34398 Montpellier, France e Department of Medical Entomology and Zoology, National Institute of Hygiene and Epidemiology, 1 Yersin street, Hai Ba Trung district, Hanoi, Viet Nam f Research Station, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan g IRD, UMR INTERTRYP IRD, CIRAD, University of Montpellier, F-34398 Montpellier, France

ARTICLE INFO ABSTRACT

Keywords: Background: Japanese encephalitis is a severe disease of acute encephalitis, with children and the elderly pri­ Culex tritaeniorhynchus marily affected, and with mortality rates reaching over 25%. The virus is transmitted mainly by species of the Japan Culex (Culex) vishnui subgroup, primarily the widely spread Cx. tritaeniorhynchus Giles. The latter is known as a Vietnam highly migratory which moves with airflowover large distances. We explored the geometric variation Thailand of the wing venation among distant areas of its geographic distribution. Our working hypothesis was that shape La Reunion Geometric morphometrics variation across geography could reveal known past and present migratory routes. Winds Materials methods: We compared the wing venation geometry of 236 female Culex tritaeniorhynchus from different locations in the Madagascan (La Reunion), Oriental (Thailand, Vietnam) and Paleartic (Japan) regions. To ascertain the taxonomic signal of the wing venation we also used two species as relative outgroups, Cx. whitmorei and Cx. brevipalpis. Results: In spite of an increasing morphometric variation as expected with larger geographic dispersion, our Cx. tritaeniorhynchus samples were clustered as a single species when considered relative to other Culex species. The relationships between geographic sites of Cx. tritaeniorhynchus globally conformed with an isolation by distance model. The shape homogeneity of our Palearctic samples (Japan) contrasted with some heterogeneity observed in the Oriental region (Thailand, Vietnam), and could be related to the different regimes of wind trajectories in these regions. Conclusion: The average shape variation of Culex tritaeniorhynchus disclosed a separation between Madagascan, Oriental and Palearctic regions in accordance with geography. The wing venation not only could reflect geog­ raphy, it also contained a clear taxonomic signal separating three Culex species. Within Cx. tritaeniorhynchus, a contrasting pattern of shape variation between the Palearctic and the Oriental regions is tentatively explained by the influence of wind trajectories.

1. Introduction with children and the elderly primarily affected and with mortality rates reaching over 25% (Foster and Walker, 2009). The JE virus has been Japanese encephalitis (JE) is a severe disease of acute encephalitis, found throughout most of Asia from southeastern Russia in the north to

* Corresponding author at: IRD, UMR INTERTRYP IRD, CIRAD, University of Montpellier, F-34398 Montpellier, France. E-mail addresses: [email protected] (R. Morales Vargas), [email protected] (J. Noda), [email protected] (P. Bousses), [email protected] (F. Hasebe), [email protected] (J.P. Dujardin). https://doi.org/10.1016/j.meegid.2021.104764 Received 21 October 2020; Received in revised form 2 February 2021; Accepted 5 February 2021 Available online 11 February 2021 1567-1348/© 2021 Elsevier B.V. All rights reserved. R. Morales Vargas et al. Infection, Genetics and Evolution 90 (2021) 104764

Indonesia in the south, from Japan in the east to the west coast of Table 1 in the west (Rosen, 1986). It is transmitted by the Culex (Culex) vishnui Sp, species; n, number of specimens; H, habitat; RU: rural; SU: Sub-Urban; Est: subgroup, primarily Culex tritaeniorhynchus Giles, as well as by a few Estuary; Rub: rubber plantation; L4: Larva, 4th instar; P: Pupa; A: Adult; N: other Culex species. The primary vector,Cx. tritaeniorhynchus, is North; S:, South; E: East; F, France; J, Japan; V, Vietnam; T, Thailand; t, Cx. distributed throughout the Oriental region, extending west into the tritaeniorhynchus; b, Cx. brevipalpis; w, Cx. whitmorei. Middle East, eastern Mediterranean and large part of the Ethiopian re­ Code Sp. Locality n H Date Stage Geographic gion, the Madagascan region, and is also found in the southeastern and coordinates ◦ eastern Palearctic region (Becker et al., 2010). Culex (Culex) tritaenio­ TO t Toyama, J 23 SU July A 36 36 N, ◦ 2011 137 14 E rhynchus is a member of the Vishnui subgroup, which is one of the five ◦ NA t Nagasaki, J 38 SU June A 32 49 N, subgroups of the Sitiens group (Rattanarithikul et al., 2005). In spite of ◦ 2013 130 03 E ◦ the wide geographic distribution of Cx. tritaeniorhynchus, little is known HA t Hanoi, V 40 SU May A 21 04 N, ◦ about the amount of possible morphological variation (Kanojia et al., 2011 105 66 E ◦ 2010; Karthika et al., 2018). TB t Thai Binh, V 38 RU May A 20 38 N, ◦ 2011 106 16 E We explored this aspect using geometric morphometrics, a modern ◦ AY t Ayutthaya, 60 SU March A 14 36 N, approach to the phenotype allowing the separation of size and shape ◦ T 2011 10 058 E ◦ ◦ properties of most organisms (Rohlf and Marcus, 1993; Zelditch et al., SP t La Reunion, 37 Est April L4, P 20 99 S, 55 28 2004). Its most recent application is based on the use of anatomical F 2011 E ◦ KK1 b Khon Kaen, 25 Rub April A 16 22 N, homologous landmarks allowing valid interindividual comparisons for ◦ ′ T 2011 102 46 E size and for shape (Bookstein, 1991; Rohlf, 1990). The method has ◦ ′ KK2 w Khon Kaen, 21 Rub August A 16 22 N, ◦ ′ proven successful for shape-based species recognition, even of sibling T 2012 102 46 E species, as well as for characterization of within species distribution across geographic areas or ecotopes (Dujardin, 2011; Dujardin and Slice, 2006). Shape (not size) as disclosed by this approach is currently dissection. Those from Toyama (Japan) were dipped in 70% ethanol, recognized as a polygenic character (Klingenberg and Leamy, 2001; then brought to the laboratory and kept until dissection. The material Klingenberg et al., 2004) and most of its variation has been attributed from La Reunion was composed of laboratory emerged imagos that were mainly to genetic drift rather than to environmental conditions kept dry in eppendorfs until dissection. We followed the mounting (Dujardin, 2011; Dujardin and Slice, 2006; Perrard et al., 2014). technique and morphometric data collection as in Morales et al. (2013) Exploring the geographic patterns of shape variation of another (Morales Vargas et al., 2013). Fourteen wing landmarks covering the left medically important mosquito, Aedes albopictus, Morales et al. (2013) wing surface were selected (Fig. 2). concluded that it was best explained by genetic drift, and that apparent departures from expectations based on geography could reveal known 2.3. Morphometric analyses past and present migratory routes (Morales Vargas et al., 2013). Accordingly, the shape of Cx. tritaeniorhynchus was expected to be We examined the metric properties (size and shape) using a similar between close geographic areas, to be divergent between iso­ landmark-based approach, i.e. the coordinates of anatomical landmarks lated or distant areas, and, in the case of some recent invasion of the instead of, traditionally, the linear measurement between them (Rohlf mosquito in a given place, the similarity with the origin was expected to and Marcus, 1993). We compared the global size and the geometric be apparent (Dujardin et al., 2007). shape of the wings of Cx. tritaeniorhynchus between four distant geographic samples, i.e., Japan or the Paleartic region (two samples: 2. Materials and methods Toyama and Nagasaki), the Oriental region composed of Vietnam (two samples: Hanoi and Thai Binh) and Thailand (Ayutthaya), and the La 2.1. and sampling sites Reunion island representing the Madagascan region (Fig. 1). The same wing venation geometry was used to compare Cx tritaeniorhynchus with Culex tritaeniorhynchus female mosquitoes were collected by oral the close Cx. whitmorei and with the more distant Cx. brevipalpis. aspirators in cattle barn and pigsty in Nagasaki (Japan), by light traps Wing size was measured as the centroid size (CS), i.e., the square root from cattle barns in Toyama (Japan), in peridomestic and human of the sum of squared distances of the 14 landmarks to their centroid dwellings in Thailand, and by backpack aspirators from pigsty in Viet­ (Bookstein, 1991). Wing shape was computed as Procrustes residuals, i. nam; in La Reunion, 4th instar larvae and pupae were collected in e., orthogonal projections of aligned specimens on the Euclidean plane wetlands and reared to adult (female) stage (Table 1, Fig. 1). tangent to the consensus form (GPA, or generalized Procrustes analysis, To assess the species homogeneity of the target group (Cx. tritae­ see (Rohlf, 1990)). The principal components of these variables were niorhynchus), we selected two other Culex species as a tentative out­ used as final shape variables. group: Cx. withmorei belonging to another subgroup of the same The contribution of size to the geographic variation of the Cx. tri­ subgenus (Culex) and of the same group (Sitiens) as Cx. tritaeniorhynchus, taeniorhynchus shape was estimated by regressing separately the shape- and Cx. brevipalpis belonging to another subgenus of Culex, the Eume­ derived principal components on size. The search for a common allo­ lanomyia subgenus. Females adult Cx. whitmorei and Cx. brevipalpis were metric component among areas followed the example given in the help collected in rubber plantations in the northeast of Thailand, by BG fileof TPSregr (Rohlf), answering the following question: do the various sentinel traps. Morphological determination was based on Rattanar­ groups have the same relationship with an independent variable (the ithikul et al. (2005) (Rattanarithikul et al., 2005), except for the speci­ centroid size)? Thus, a multivariate analysis of covariance (MANCOVA) mens from La Reunion which were determined according to Hamon with final shape variables as dependent variables, locality as the cate­ (1953) (Hamon, 1953). gorical factor and centroid size as the covariate, was performed to test for homogeneity of regression slopes among geographic origins. 2.2. Wing preparation We verified the conditions underlying the discriminant analysis (Kovarovic et al., 2011), i.e. the covariances matrices equality using a Insects from Thailand were transported in dry ice to the laboratory, simple procedure (Garcia, 2012), and the multivariate normal distri­ then kept at 20 degrees until dissection and mounting of the wings. bution using the multivariate extension of the Shapiro-Wilks W test Specimens collected from Vietnam and from Nagasaki (Japan) were (Royston, 1982). Because these assumptions were rejected, the shape brought alive to the laboratory, then kept at 80 degrees until divergence between the mean shape of each locality / species was

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Fig. 1. Sketch map of the geographic locations from where samples of Culex specimens were collected. TO, Toyama (Japan); NA, Nagasaki (Japan); HA, Hanoi (Vietnam); TB, Thai Binh (Vietnam); AY, Ayutthaya (Thailand); KK, Khon Kaen (Thailand); SP, Saint Paul (La Reunion, France). computed as Euclidean distances between their between-group principal 2.4. Geographic statistics components (Mitteroecker and Bookstein, 2011). These distances between group means were used to build an average Because of the known windborne migratory behaviour of Cx. tritae­ linkage hierarchical clustering (UPGMA), and its empirical statistical niorhynchus (Sellers, 1980), we also examined the wind trajectories be­ significance computed on 1000 bootstrapped datasets (Couette et al., tween collecting sites separated by a distance up to around 1000 km, i.e. 2005; Morales Vargas et al., 2013). A non parametric Mantel test was between Thailand and Vietnam in the Oriental region, as well as be­ applied between shape divergence and geographic distances between tween Toyama and Nagasaki in the Palearctic region. The back- areas (9999 runs). trajectories of air masses arriving at the sampling location were calcu­ The Euclidean distances between average individuals were also lated by NOAA’s HYSPLIT atmospheric transport and dispersion computed between each single specimen and the group means, modeling system (Rolph et al., 2017; Stein et al., 2015) using meteo­ including its own group minus itself, allowing to build a detailed vali­ rological data from the Global Data Assimilation System (GDAS). The dated reclassification table. In order to help understand the low accu­ examined locations and analyzed periods for each site are for the month racies observed in the Oriental region, the reclassificationtable presents that the mosquitoes were collected at each sampling site. in detail the wrong assignments instead of the correct ones. Mean monthly temperatures corresponding to the month and year of capture 2.5. Software were obtained from https://www.worldweatheronline.com. A simple parametric linear regression test was used to examine possible We used the freely available CLIC package (Dujardin et al., 2010; relationships between mean shape values and annual average temper­ Dujardin and Slice, 2006) and the online XYOM software (Dujardin and atures of areas. A non parametric Mantel test was also applied between Dujardin, 2019)(https://xyom.io). Also, the R software shape divergence and temperature differences between areas (9999 (https://www.r-project.org/) was used to perform the multi­ runs). variate normality Shapiro-Wilks test as well as the Mantel tests (R mvnormtest package). NOAA’s HYSPLIT atmospheric transport and dispersion modeling system (Rolph et al., 2017; Stein et al., 2015) using

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Fig. 2. Landmarks (white circles) collected on the wings of Culex sp., ordered according to the numbers from 1 to 14. meteorological data from the Global Data Assimilation System (GDAS) Simple metric distances are not necessarily reflectingthe complexity was used to calculate the air masses back trajectories. of shape divergence between forms (Rohlf, 1990). To improve the un­ derstanding of shape relatedness, figures representing superposed con­ 3. Results figurationsof landmarks after the GPA were computed for the following samples: the three species (Fig. 5, top), the three large regions, i.e., We examined a total of 282 female individuals subdivided according Paleartic, Oriental and Madagascan regions (Fig. 5, bottom), the pair to three species, Cx. tritaeniorhynchus (n = 236), Cx. whitmorei (n = 21) Toyama-Nagasaki from the Palearctic region (Fig. 5, bottom) and the and Cx. brevipalpis (n = 25). Culex tritaeniorhynchus was collected in two collecting sites of Vietnam (Fig. 6, top) and the pair Vietnam- three zoogeographic regions: the Palearctic, the Oriental and the Thailand from the Oriental region (Fig. 7). Madagascan regions, where a total of seven collecting sites were visited Between species, relatively large spacing between many homologous between 2011 and 2013, with an average sampling of 39 insects by lo­ landmarks was observed, especially in the central area of the wing cality (Table 1). (Fig. 5, top). Between the two subgenera Cx. (Culex) sp. and Cx. Between the three species there was no significant difference be­ (Eumelanomyia) sp., no landmark could be correctly or closely super­ tween sizes. Within Cx. tritaeniorhynchus, an important size variation posed. It is worth noting that the complete curve of the anterior border was observed between samples, with the largest wings observed in of the wing did not fit between subgenera, and that the central area of Japan and the smallest ones in Vietnam. the wing showed large distances between homologous landmarks The covariance matrices of the Cx. tritaeniorhynchus shape variables without needing artificialamplification to be clearly visible (Fig. 5, top). within localities were generally compatible with equality, but multi­ The central area of the wing was also the place of changes between variate normality was strongly rejected (P <10 6), preventing us to large geographic regions (Oriental, Paleartic, Madagascan) of Cx. tri­ apply a multiple linear discriminant analysis and its metric, the Maha­ taeniorhynchus, with smaller landmarks displacements, visible at less lanobis distance (Kovarovic et al., 2011). Therefore, the between groups landmarks (see Fig. 5, bottom). Between the two close collecting sites of principal components analysis (gPCA) was used, as generally recom­ Vietnam (Fig. 6, top), as well as the two collecting sites of the Palearctic mended by (Mitteroecker and Bookstein, 2011). region (Fig. 6, bottom), the displacement of landmarks was modest, Despite striking differences between some areas (Fig. 3 bottom), the barely visible, giving the impression of a perfect fit.Finally, between the global size of Cx. tritaeniorhynchus poorly but significantly (P = 0.011) two neighboring countries of the Oriental region, Vietnam and Thailand, contributed to the geographic variation of shape: the coefficient of we observed an intermediate situation with small but visible displace­ determination was 5% for the first principal component (gPC1, repre­ ments at almost all the landmarks (Fig. 7), consistent with the inter­ senting 66% of the total variance) and 4% for the second gPC2 (34%). mediate metric distances separating them (Table 2). The hypothesis of a common allometric component among areas was According to the validated reclassificationbased on the gPCA, there rejected (P < 0.002), preventing us to completely remove the residual was some confusion between Cx. whitmorei and Cx. tritaeniorhynchus allometric component of shape. reducing the total correct classificationscore to 88% (Table 3, top). The The Mantel test examining the relation between geographic and same validated reclassification based on the gPCA performed on Cx. metric distances was significant, although not highly significant (P = tritaeniorhynchus, correctly reclassified distant countries like Japan and 0.02), supporting the model of isolation by distance. A global vision of La Reunion (at around 85% or more), but could not clearly distinguish shape relatedness between samples of Cx. tritaeniorhynchus was brought neighboring countries like Vietnam and Thailand (Table 3, bottom). by the between-group PCA (Fig. 4, top) and by the hierarchical clus­ Unexpectedly, the poor reclassification of Vietnam and Thailand (55% tering (Fig. 4, bottom). Both statistical techniques disclosed a separation and 73%, respectively) did not arise from a large confusion between the of the Madagascan sample of Cx. tritaeniorhynchus from the Paleartic and two neighboring countries, but because of some confusion with the the Oriental ones, as well as a further subdivision between Palearctic distant Japan. 20% of the Vietnamese specimens were wrongly assigned and Oriental regions. to Japan, versus 14% to Thailand, and 15% of the Thailand mosquitoes

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Fig. 4. Top: Factor map of the two first principal components illustrating the spatial proximity of shape between Cx. tritaeniorhynchus from La Reunion, Japan, Vietnam and Thailand. Squares are the mean values. Bottom: Hierar­ Fig. 3. Quantile boxes illustrating centroid size variation. Top: size variation chical classification of our various samples: Cx. (Eumelanomyia) brevipalpis and among three Culex species: Cx. (Culex) tritaeniorhynchus (left), Cx. (Culex) Cx. (Culex) whitmorei, both samples collected at Khon Kaen in Thailand, and Cx. whitmorei (mid) and Cx. (Eumelanomyia) brevipalpis (right). Bottom: siz varia­ (Culex) tritaeniorhynchus coming from distant areas: two localities of Japan tion within Cx. tritaeniorhynchus among four countries: Japan (two collecting (Toyama, TO and Nagasaki, NA), two localities of Vietnam (Hanoi, HA and Thai sites), Vietnam (two collecting sites), Thailand and the French island La Binh, TB), one of Thailand (Ayutthaya, AY) and one of La Reunion (Saint Paul, Reunion. Values are millimeters as converted from pixels. SP). The tree algorithm is the UPGMA (Unweighted Pair Group Method with Arithmetic Mean); the input variables are shape variables, excluding isometric were wrongly assigned to Japan, versus only 5% to Vietnam (Table 3, change of size. bottom). The simple parametric linear regression test used to examine possible 4.2. Shape divergence and hierarchical clustering relationships between shape and temperature among areas (Table 4) was not significant( P = 0.69), nor was the Mantel test between matrices As shown by shape variation (Fig. 4, top) and by the hierarchical of shape divergence and temperature differences (simulated P = 0.19). clustering (Fig. 4, bottom), the classificationexclusively based on shape The air mass trajectories between collecting sites showed connection variation could reproduce the known taxonomic classification, nicely in the Palearctic region only, i.e., between Nagasaki and Toyama (Fig. 8, separating the two subgenera Cx. Culex and Cx. Eumelanomyia, and, panels A & B). On the contrary, in the Oriental region no air mass within the Cx. Culex subgenus, the two subgroups Sitiens (Cx. whitmorei) appeared to connect Vietnam (either Hanoi or Thai Binh) and Thailand and Vishnui (Cx. tritaeniorhynchus). This congruence with systematics is (detailed results not shown). one more example of the phylogenetic signal often embedded in the geometry of the wings (Dujardin, 2011; Dujardin and Slice, 2006). 4. Discussion Furthermore, the same classificationtree also could organise the various samples according to their geographic proximity (Fig. 4, bottom), a 4.1. Size pattern in accordance with the Mantel test supporting an isolation by distance model, and, as a consequence, in agreement with the idea of Size variation of Cx. tritaeniorhynchus had apparently no relation genetic drift affecting shape more than the environment (Dujardin, with either geography (Fig. 3 bottom) or taxonomy (Fig. 3, top), and it 2011; Morales Vargas et al., 2013; Perrard et al., 2014). had a poor impact on passive shape divergence between groups (less than 5% on gPC1 or on gPC2). Among the Cx. tritaeniorhynchus sub­ populations, the low size effect on shape variables gave shape-based 4.3. Procrustes superposition differences more credibility as pure shape divergence (Dujardin and Slice, 2006). In contrast with the Procrustes superposition of wings as disclosed between species (Fig. 5, top), we observed only small changes within Cx.

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Fig. 6. Top: Superimposition of aligned Hanoi (HA) and Thai Binh (TB) mean configurations onto the consensus of Vietnam. Bottom: Superimposition of aligned Toyama (TO) and Nagasaki (NA) mean configurations onto the consensus of Japan.

Fig. 5. Superimposition of aligned mean configurations onto their general consensus. Top: three Culex species: Cx. tritaeniorhynchus and Cx. whitmorei (Culex subgenus), and Cx. brevipalpis (Eumelanomyia subgenus). Bottom: three taxonomic regions of Cx. tritaeniorhynchus: the Palearctic region (Japan), the Oriental region (Vietnam and Thailand), and the Madagascan region (La Reunion). tritaeniorhynchus, between taxonomic regions: shifts in the central area of the wing were discrete and there was no discrepancy at the anterior border of the wing (Fig. 5, bottom). Between closer collecting sites, in Japan (TO and NA) or in Vietnam (HA and TB), almost no visible change Fig. 7. Superimposition of aligned mean configurationsof Cx. tritaeniorhynchus could be detected between collecting sites (Fig. 6). These patterns were from Vietnam and Thailand onto the general consensus of the Oriental region. also in agreement with an isolation by distance model, although they Arrows indicate the disagreements between configurations. could not be clearly confirmed in the Oriental region (Vietnam and Thailand). Landmark displacements between Vietnam and Thailand were were not exclusively located at the central part of the wing, but Table 2 dispersed at many landmarks, including some landmarks at the anterior Above diagonal, the geographic distances in kilometers. Under diagonal, the border of the wing (Fig. 7). Accordingly, Vietnam and Thailand, sepa­ Euclidean distances between group means (after between group PCA). TO, rated by 1280 km, showed a much larger metric distance (from 0.019 to Toyama (Japan); NA, Nagasaki (Japan); HA, Hanoi (Vietnam); TB, Thai Binh 0.022) than the one between Toyama and Nagasaki (0.009), separated (Vietnam); AY, Ayutthaya (Thailand); SP, Saint Paul (La Reunion, France). by 1085 km (Table 2). TO NA HA TB AY SP

TO 0 1085 2738 2720 3614 9894 4.4. Individual reclassification NA 0.00919 0 3491 3489 4389 10,684 HA 0.02229 0.0212 0 75 1277 7193 TB 0.02826 0.02837 0.01216 0 1281 7275 The above comparisons provided information about the average AY 0.03607 0.03111 0.01938 0.02167 0 6301 wings, they represented the main trends of shape relatedness between SP 0.02858 0.02919 0.02751 0.03635 0.03644 0 groups. The closer view brought by the individual shape variation pro­ duced new information. Considering the shape similarity of single in­ of resolution of the morphometric traits as used here (Table 3). dividuals with these average wings, i.e., performing validated Within Cx. tritaeniorhynchus, such individual reclassification raised reclassification, some discrepancies with the main trends were dis­ new questions, especially about the neighbor countries Vietnam and closed. For instance, the individual reclassification to species did not Thailand. Contrary to the individuals belonging to more distant areas reach a perfect score as when comparing average values (except for Cx. like Japan (85%) or La Reunion (86%), individuals from Vietnam and brevipalpis). Some confusion between Cx. whitmorei and Cx. tritaenio­ from Thailand were poorly reclassified to their geographic origin (55% rhynchus highlighted their higher phylogenetic proximity, or some lack

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Table 3 Individual reclassification.Errors in the validated reclassificationafter a between group PCA. Wrong assignments: number of incorrectly assigned specimens / group sample; percentages between squared brackets. Detailed wrong assignments: the partition of wrongly assigned specimens according to species (top) or main localities (bottom). t, Culex tritaeniorhynchus; w, Culex whitmorei; b, Culex brevipalpis. The 31 specimens of “t” that were confused with “w” were composed of specimens from Thailand (18), from Vietnam (11), and from Japan (2). J, Japan; V, Vietnam; T, Thailand; LR, La Reunion.

Between species

Species Wrong assignments Detailed wrong assignments

t w b

Cx. tritaeniorhynchus 31/236 [13%] / 31 0 Cx. whitmorei 2/21 [10%] 2 / 0 Cx. brevipalpis 0/25 [0%] 0 0 / Total 33/282 [12%] 2 [1%] 31 [11%] 0

Between Cx. tritaeniorhynchus subpopulations Locality Wrong assignments Detailed wrong assignments J V T LR Japan (J) 9/61[15%] / 3[5%] 2[4%] 4[7%] Vietnam (V) 35/78[45%] 16[20%] / 11[14%] 8[10%] Thailand (T) 16/60[27%] 9[15%] 3[5%] / 4[7%] La Reunion (LR) 5/37[14%] 3[8%] 2[5%] 0[0%] / Total 65/236[28%] 28[12%] 8[3%] 13 [6%] 16[7%]

reclassification of Vietnam and Thailand was due to a consistent pro­ Table 4 portion of individuals closer to Japan (20% and 15%, respectively) and Temperatures (from https://www.worldweatheronline.com). Values are much less to some confusion between nearby areas (11% and 3%) given in Celsius degrees. (Table 3). The conformational affinity of many Oriental specimens Locality Month Year Mean monthly temperature (18%) to the distant Japan instead of to the closest country, deserves Toyama July 2011 23.00 some discussion. Was it explained by some artifacts, or could it be the Nagasaki June 2013 22.00 result of either environmental or genetic causes? Hanoi May/June 2011 26.00 Thai Binh May/June 2011 26.00 Ayutthaya March 2011 27.00 4.4.1. Vietnam and Thailand differences as artifacts? La Reunion April 2013 24.00 The most obvious non-biological causes possibly incriminated in morphometric variation could be related to either the capture tech­ niques, the capture dates, the sample transportation and preservation, or and 73%, respectively). The intuitive explanation, in agreement with the both. The capture techniques, as well as the transportation and con­ isolation by distance model, would be that confusions are more likely servation conditions, were indeed different between Thailand and between neighboring countries (Vietnam, Thailand) than between Vietnam, but also between each collecting site. Although non-biological distant areas (Japan, La Reunion). Unexpectedly, the poor influencecan not readily be excluded from the observed morphometric

Fig. 8. Air mass backward trajectories to the collection places of Toyama (TO), Japan. The analyzed height above ground level (AGL) = 0 m, duration = 72 h, and start time = 12:00 (UTC) of each day, from May 30 (A) through June 26 of 2011 (B). Each line represents the trajectory of each air mass, while the color of each line corresponds to the starting date according to the description appearing below each panel. Air mass trajectories connecting Nagasaki (NA) and Toyama (TO) are pointed with a red arrow. Such a connection between collection sites did not appear in the Oriental region.

7 R. Morales Vargas et al. Infection, Genetics and Evolution 90 (2021) 104764 variation, we consider that this possible effect was, at least, not deter­ properly Cx. tritaeniorhynchus from some other members of the subgroup minant. We observed indeed that the two collecting sites showing the (Karthika et al., 2018). Could the presence of cryptic species - or of highest morphometric similarity in our sample were Toyama and ongoing cryptic speciation - explain the unexpected individual reclas­ Nagasaki for which capture method, capture dates, transportation and sification patterns in the Oriental Cx. tritaeniorhynchus (Table 3, bot­ preservation conditions were different. tom)? The geometry of the wing is a powerful characterizing tool, but its accuracy is generally not reaching 100%, as already observed in the 4.4.2. Vietnam and Thailand differences as different environments? Culex genus (Dujardin, 2011). Its resolution in our study was perfect For mosquitoes, different breeding sites might have an influence on between subgenera, but showed some weakness in distinguishing the development of size differences, and, as a passive effect, an influence morphologically close species. A total of 31 Cx. tritaeniorhynchus speci­ on shape differences also. However, breeding sites were of the same kind mens (13% of 236), most of them coming from Thailand (18/31), were throughout the sampling territory: open wetlands, such as rice fields in confused with Cx. withmorei. Thus, an unknown proportion of a truly Japan, Vietnam and Thailand, or flooded meadows in La Reunion cryptic species could have gone unnoticed in the sample, affecting the (Hamon, 1953). It is commonly admitted that the climatic impact on homogeneity of the Oriental samples. Even if this hypothesis is not the metric properties is acting on size much more than on shape (Morales most likely one, it is worth noting that the species homogeneity of the Vargas et al., 2010; Phanitchat et al., 2019). In this regard, it is worth Oriental population of Cx. tritaeniorhynchus has been questioned various noting that in spite of Japan and Vietnam having the most different sizes times. In Thailand, a morphological variant collected in Chiang Mai (Fig. 3, bottom), the shape of 20% of the Vietnamese specimens was (500 km North of Ayutthaya, our collecting site) was named as var. confused with the one of Japan (Table 3). The relationship of size with siamensis (Barraud and Christophers, 1931). This variant, later recog­ temperature showed a negative trend, as expected from Bergmann’s rule nized as a subspecies, i.e. Cx. tritaeniorhynchus summorosus (Sir­ (Atkinson, 1994), but it was not significant(details not shown), nor was ivanakarn, 1976), was not reported anymore in Thailand by the relationship of shape with temperature. Unfortunately, the number (Rattanarithikul et al., 2005). Two genetic studies published the same of our collection sites (Table 4) was too low to expect enough power year (2015) on Indian samples provided opposite conclusions: a specific from statistical tests, so that the climatic influencecould not be explored status for Cx. tritaeniorhynchus summorosus (Airi and Kaur, 2015), and further. Cx. tritaeniorhynchus as a simple taxon (Rajavel et al., 2015).

4.4.3. Vietnam and Thailand separated because of genetic drift? 5. Conclusion Genetic drift has been frequently claimed as being the main causes of geometric shape variation among geographic populations (Dujardin, The hierarchical clustering and various statistical tests based on 2011; Morales Vargas et al., 2013; Perrard et al., 2014). According to the average shape variation of Culex tritaeniorhynchus disclosed a separation genetic drift hypothesis, the reclassification of 18% of the Oriental Cx. between Oriental, Paleartic and Madagascan regions, in global agree­ tritaeniorhynchus specimens with Japan could be the reflection of a ment with geographic distances. The pattern of differences in the common origin, and the poor reclassification rates of individuals be­ average landmarks configurations showed either (i) almost perfect su­ tween Vietnam and Thailand could suggest some lack of exchanges be­ perposition, as between localities of the same country, (ii) modest dif­ tween these neighboring countries. ferences at most landmarks, as between Vietnam and Thailand, or (iii) Culex tritaeniorhynchus is known as a highly migratory species which large distances at a few landmarks, as between the Oriental, Paleartic moves northward in east Asia with the southwest airflow in spring and Madagascan regions, and between species. The shape relatedness as (Sellers, 1993). However, true exchanges between Oriental and Paleartic illustrated by average wings were consistent with both taxonomy and regions, because of their large geographic separation, are not likely (but geography. The individual reclassificationshowed less conformity with see (Nabeshima et al., 2009)). Rejecting the idea of such exchanges, we these general patterns, suggesting more dissimilarity between Vietnam would consider the morphological affinity of some Oriental specimens and Thailand than expected according to their geographic proximity. In with Japan as a side effect of the Oriental heterogeneity itself. In such a our sampling, these two countries were separated by approximately the hypothesis, the question restrains to explaining the Oriental heteroge­ same geographic distance as the two collecting sites of Japan (Nagasaki neity of shape; alternatively, it is also about explaining the Palearctic and Toyama). In spite of this, Vietnam and Thailand showed consistently homogeneity of shape. more shape divergence. This led us to suggest some Oriental heteroge­ Wind-borne migration has repeatedly been observed in Culex tritae­ neity of shape within Cx. tritaeniorhynchus, as opposed to high homo­ niorhynchus (Asahina, 1970; Hayashi, 1978; Ji-Guang et al., 1993; geneity of shape in the Palearctic region. Considering the hypothesis of Reynolds et al., 1996). Considering the reported windborne migration passive transportation of the by winds, we showed that in our capacity of this species, the observed wind trajectories connecting sample there was no wind connection between collecting sites of the Toyama and Nagasaki (see Fig. 8) could add some possible clues to their Oriental region, contrary to the one observed between the Palearctic high morphometric similarity. From Thailand to Northern Vietnam sites. Adding to this hypothesis, we suggested that the Annamese however, in spite of the geographic distance being among the shortest mountains could represent a natural obstacle to passive gene flow be­ one of our sample, no connecting wind trajectory could be found tween Vietnam and Thailand. Thus, the Oriental heterogeneity of shape (detailed results not shown). Furthermore, the Annamese Mountains could be explained mainly by geographic isolation, i.e., by genetic drift. could represent a natural obstacle limiting the possibility of true ex­ It could even revive an already engaged taxonomic discussion about the change by wind transportation. Supposing that there is an obstacle to the presence in the Oriental region of another, cryptic species hidden in the passive migration of Cx. tritaeniorhynchus between Thailand and Viet­ Cx. tritaeniorhynchus collections, not detected by the current morpho­ nam, it is not unlikely to see some genetic drift affecting separate sub­ logical criteria as used in the present study. populations. Even if this was not reflected in the hierarchical classification tree (Fig. 4, bottom), our validated reclassification data Declaration of Competing Interest provided indirect support to that hypothesis. None. 4.4.4. Incipient speciation in the oriental region? To be complete, a less likely hypothesis of local speciation should be Acknowledgment discussed. In the Vishnui subgroup, species are morphologically very close, subject to difficultiesin performing morphological determination We appreciate the assistance of Toshihiko Sunahara to collect mos­ (Weeraratne et al., 2018). Recently, DNA barcoding could not separate quito samples in Nagasaki, Japan, and the collaboration of Takeo

8 R. Morales Vargas et al. Infection, Genetics and Evolution 90 (2021) 104764

Yamauchi in collecting mosquitoes from Toyama, Japan. We also thank Klingenberg, C.P., Leamy, L.J., 2001. Quantitative genetics of geometric shape in the – Nguyen Thu Thuy for helping to collect samples in Hanoi and Thai Binh mouse mandible. Evolution 55, 2342 2352. Klingenberg, C.P., Leamy, L.J., Cheverud, J.M., 2004. Integration and modularity of in Vietnam. The authors gratefully acknowledge the NOAA Air Re­ quantitative trait locus effects on geometric shape in the mouse mandible. Genetics sources Laboratory (ARL) for the provision of the HYSPLIT transport and 166, 1909–1921. dispersion model and/or READY website (https://www.ready.noaa. Kovarovic, K., Aiello, L., Cardini, A., Lockwood, C., 2011. Discriminant function analysis in archaeology: are classification rates too good to be true? J. Archaeol. Sci. 38, gov) used in this publication. This research was supported by the 3006–3018. Japan Agency for Medical Research and Development (AMED) under Mitteroecker, P., Bookstein, F., 2011. Linear discrimination, ordination, and the Grant Number JP20wm0125006. visualization of selection gradients in modern morphometrics. Evol. Biol. 38, 100–114. https://doi.org/10.1007/s11692-011-9109-8. Morales Vargas, E.R., Ya-umphan, P., Phumala-Morales, N., Komalamisra, N., References Dujardin, J.P., 2010. Climate associated size and shape changes in Aedes aegypti (Diptera: Culicidae) populations from Thailand. Infect. Genet. Evol. 10 (4), 580–585. Airi, M., Kaur, S., 2015. Confirmation of Culex (Culex) tritaeniorhynchus summorosus Morales Vargas, R.E., Phumala-Morales, N., Tsunoda, T., Apiwathnasorn, C., Jean- – (Diptera: Culicidae) as a separate species. J Vector Borne Dis 52, 219–223. Pierre, D., 2013. The phenetic structure of Aedes albopictus. IGE 13, 242 251. Asahina, S., 1970. Transoceanic flight of mosquitoes on the Northwest Pacific. Japanese Nabeshima, T., Loan, H., Inoue, S., Sumiyoshi, M., Haruta, Y., Nga, P.T., Huong, V.T., J. Med. Sci. Biol. 23, 35–38. Parquet, M., Hasebe, F., Morita, K., 2009. Evidence of frequent introductions of Atkinson, D., 1994. Temperature and organism size - a biological law for ectotherms? Japanese encephalitis virus from south-east asia and continental east asia to Japan. – Adv. Ecol. Res. 25, 1–58. J. Gen. Virol. 90, 827 832. Barraud, P., Christophers, S., 1931. On a collection of Anopheline and Culicine Perrard, A., Baylac, M., Carpenter, J.M., Villemant, C., 2014. Evolution of wing shape in mosquitoes from Siam. Records of the Malaria Survey of India 2 (2), 269–285. hornets: why is the wing venation efficient for species identification? J. Evol. Biol. – Becker, N., Petri´c, D., Zgomba, M., Boase, C., Madon, M., Dahl, C., Kaiser, A., 2010. 27, 2665 2675. Mosquitoes and their Control, Second ed. Springer-Verlag, Berlin Heidelberg. Phanitchat, T., Apiwathnasorn, C., Sungvornyothin, S., Samung, Y., Dujardin, S., Bookstein, F.L., 1991. Morphometric Tools for Landmark Data. Geometry and Biology. Dujardin, J., Sumruayphol, S., 2019. Geometric morphometric analysis of the effect Cambridge University Press, NY. of temperature on wing size and shape in Aedes albopictus. Med. Vet. Entomol. Couette, S., Escarguel, G., Montuire, S., 2005. Constructing, bootstrapping, and https://doi.org/10.1111/mve.12385. comparing morphometric and phylogenetic trees: a case study on new world Rajavel, A., Pradeep Kumar, N., Natarajan, R., Vanamail, P., Rathinakumar, A., monkeys (Platyrrhini, primates). J. Mammal. 85 (4), 773–781. Jambulingam, P., 2015. Morphological and molecular characterization of the Dujardin, J.P., 2011. Modern morphometrics of medically important insects. In: ecological, biological and behavioural variants of the JE vector Culex Tibayrenc, M. (Ed.), Chapter 16, 473–501. In: Genetics and Evolution of Infectious tritaeniorhynchus: an assessment of its taxonomic status. J Vector Borne Dis 52, – diseases. Elsevier, p. 749. ISBN: 978–0–12-384890-1. 40 51. Dujardin, S., Dujardin, J., 2019. Geometric morphometrics in the cloud. Infect. Genet. Rattanarithikul, R., Harbach, R., Harrison, B., Panthusiri, P., Jones, J., Coleman, R., Evol. 70, 189–196. https://doi.org/10.1016/j.meegid.2019.02.018. 2005. Illustrated keys to the mosquitoes of Thailand. II. Genera Culex and Lutzia. The – Dujardin, J.-P., Slice, D.E., 2006. Contributions of morphometrics to medical Southeast Asian J. Trop. Med. Public Health 36, 1 97. entomology. In: Tibayrenc, M. (Ed.), Encyclopedia of Infectious Diseases. John Wiley Reynolds, D.R., Smith, A.D., Mukhopadhyay, S., Chowdhury, A., De, B., Nath, P., & Sons, Inc, pp. 435–447. ISBN 9780470114209. Mondal, S., Das, B., Mukhopadhyay, S., 1996. Atmospheric transport of mosquitoes – Dujardin, J.P., Beard, C.B., Ryckman, R., mar, 2007. The relevance of wing geometry in in Northeast India. Med. Vet. Entomol. 10, 185 186. entomological surveillance of Triatominae, vectors of Chagas disease. Infect. Genet. Rohlf, F.J., 1990. Rotational fit (Procrustes) methods. In: Rohlf, F., Bookstein, F. (Eds.), Evol. 7 (2), 161–167. Proceedings of the Michigan Morphometrics Workshop. Special Publiation Number Dujardin, J.P., Kaba, D., Henry, A.B., 2010. The exchangeability of shape. BMC Res. 2. The University of Michigan Museum of Zoology. University of Michigan Museums, – Notes 3, 266. https://doi.org/10.1186/1756–0500–3–266. Ann Arbor, MI, pp. 227 236, 380. – Foster, W., Walker, E., 2009. Mosquitoes (Culicidae). In: Mullen, G.R., Durden, L.A. Rohlf, F.J., Marcus, L.F., 1993. A revolution in morphometrics. TREE 8 (4), 129 132. (Eds.), Medical and Veterinary Entomology, 2nd, eds. Elsevier, Amsterdam, Rolph, G., Stein, A., Stunder, B., 2017. Real-time environmental applications and display – pp. 201–253. system: READY. Environ. Model. Softw. 95, 210 228. https://doi.org/10.1016/j. Garcia, C., 2012. A simple procedure for the comparison of covariance matrices. BMC envsoft.2017.06.025. Evol. Biol. 12, 222. Rosen, L., 1986. The natural history of Japanese encephalitis virus. Annu. Rev. – Hamon, J., 1953. Etude biologique et syst´ematique des Culicidae de l’île de la Reunion.´ Microbiol. 40, 395 414. ’ M´emoires de L’institut Scientifique de Madagascar - Serie´ E. - Tome IV. I. 521–541. Royston, P., 1982. An extension of Shapiro and Wilk s W test for normality to large – Hayashi, K., H., S., S., A, 1978. Note on the transoceanic insects captured on East samples. Appl. Stat. 31, 115 124. Sea in 1977. Tropical Med. 20, 131–142. Sellers, R., 1980. Weather, host and vector - their interplay in the spread of insect-borne – Ji-Guang, M., Hua, J., Riley, J., Reynolds, D., Smith, A., Ren-Lai, W., Ji-Yi, C., Xia virus diseases. J. Hyg. 85, 65 102. Nian, C., 1993. Autumn southward ‘return’ migration of the mosquito Culex Sirivanakarn, S., 1976. Medical entomology studies - III. A revision of the subgenus Culex tritaeniorhynchus in China. Med. Vet. Entomol. 7, 323–327. in the oriental region (Diptera: Culicidae). Contrib Am Entomol Inst 1976 (12), – Kanojia, P., Paingankar, M., Patil, A., Gokhale, M., Deobagkar, D., 2010. Morphometric 1 272. ’ and allozyme variation in Culex tritaeniorhynchus mosquito populations from India. Stein, A., Draxler, R., Rolph, G., Stunder, B., Cohen, M., Ngan, F., 2015. NOAA s HYSPLIT J. Insect Sci. 10, 138. https://doi.org/10.1673/031.010.13801. atmospheric transport and dispersion modeling system. Bull. Amer. Meteor. Soc. 96, – Karthika, P., Vadivalagan, C., Thirumurugan, D., Kumar, R., Murugan, K., Canale, A., 2059 2077. https://doi.org/10.1175/BAMS-D-14-00110.1. Benelli, G., 2018. DNA barcoding of five japanese encephalitis mosquito vectors Weeraratne, T., Surendran, S., Parakrama Karunaratne, S., 2018. DNA barcoding of (Culex fuscocephala, Culex gelidus, Culex tritaeniorhynchus, Culex pseudovishnui and morphologically characterized mosquitoes belonging to the subfamily Culex vishnui). Acta Trop. 183, 84–91. https://doi.org/10.1016/j. from . Parasit. Vectors 11 article number: 266. actatropica.2018.04.006. Zelditch, M.L., Swiderski, D.L., Sheets, H.D., Fink, W.L., 2004. Geometric Morphometrics for Biologists: A Primer. Elsevier Academic Press, New-York.

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