Complexes or assemblages of culicid species and subspecies from the southern region of Latin America are difficult to differentiate morphologically (Harbach 2012, Laurito et al. 2017). These complexes include taxa with differences in their vectorial capacity and therefore in their epidemiological importance (Dujardin and Schofield 2004). However, morphological variation within the species and superposition of diagnostic characters based on classical structures often do not allow for a correct identification.

In Argentina, the Pipiens Assemblage includes Culex pipiens with its variant Culex molestus and Culex quinquefasciatus (Harbach 2012). Both species have been incriminated as potential vectors of several arboviruses, including the West Nile virus and the St. Louis encephalitis virus, and have differences in their eco‐physiological features and geographic distribution. Cx. quinquefasciatus , a tropical and subtropical species, is distributed from the center to the north of Argentina, whereas Cx. pipiens , which is a temperate species, is distributed from the center to the south of the country. Between latitudes 30° 36’ S (Córdoba Province) and 36° 13’ S (La Pampa Province), and between longitudes 57° 57’ W (La Plata City) and 64° 48’ W (Córdoba Province), both distributions overlap and these species co‐exist along with viable hybrids (Almirón et al. 1995, Diez et al. 2012).

Most studies have aimed to differentiate the two above‐mentioned species and the hybrids morphologically by using a morphometric index of male genitalia and/or the siphon of immature stages (Vinogradova 2003, Diez et al. 2012). Dehghan (et al. 2016) has proposed to differentiate adult females by traditional morphometry, considering the relative position of subcosta/costa intersection and R2+3 vein bifurcation. However, this method is not completely effective and the geometric shape analysis not only offers a precise and accurate description but also serves the equally important purposes of visualization and interpretation (Zelditch et al. 2004).

Geometric morphometry is a tool that has allowed identification and discrimination between species and species complexes of mosquitoes (Vidal et al. 2011, Laurito et al. 2015). The wings are most used in this tool because of their approximately flat characteristics that minimize the error when locating landmarks in the veins, facilitate the use of homologous points (Bookstein 1991), and their insights into ecological and evolutionary points of view (De Riva et al. 2001, Baylac et al. 2003). The aim of this work was to evaluate, through geometric morphometry, differences in the wing shape of female mosquitoes belonging to the Pipiens Assemblage (Cx. pipiens and Cx. quinquefasciatus ) of Argentina.

Female Cx. quinquefasciatus were obtained from several domiciliary sites of two localities, Colonia Aurora (27° 28’ S, 54° 31’ W) and Eldorado (26° 24’ S, 54° 38’ W), Misiones Province, Argentina. Cx. pipiens females were collected in cemeteries from Puerto Madryn City (42° 45’ S, 65° 02’ W) in Chubut Province, Argentina. Both species were obtained from the exclusive region of their distribution. We avoided taking samples in areas of a hybrid zone where both species and their hybrids were present (Figure 1).

Immatures of both species were collected from artificial habitats and reared in semi‐natural conditions (ambient temperature, with water from larval habitats and fed with yeast solution) until the adult stage. Dead larvae and exuviae of the 4^th instars were preserved in 97% alcohol. Taxonomic keys (Rossi et al. 2002) were used for the identification to species. To identify between Cx. quinquefasciatus and Cx. pipiens , both the geographical origin and the siphonal index (SI) were considered. The SI was calculated as the ratio between the length, measured from the siphon base to the midpoint of the siphon tip, and the siphonal width, measured at the widest point (Brogdon 1981). Species classification was made according to the ranges proposed by Brogdon (1981), S.I range: 3.1–3.7 for Cx. quinquefasciatus and S.I range: 4.3–4.7 for Cx. pipiens .

A total of 71 dead 4^th instar larvae and their exuviae were used to measure the SI, and 44 females were used for geometric morphometrics as follows: 35 larvae and 21 adults from Misiones Province (Cx. quinquefasciatus exclusive zone) and 36 larvae and 23 adults from Chubut Province (Cx. pipiens exclusive zone). Whenever possible, larval exuviae and adult traits were analyzed in the same individuals. For morphometric analysis we used those females that were in a better state of conservation after rearing. Digital photos of both the siphons and the left wings were taken with a camera coupled to a stereoscopic microscope. A total of 17 landmarks were selected on each digital image of the wings (Figure 1) and Cartesian coordinates were generated with the software tps‐DIG® 2.16. The landmark configurations were transferred, rotated, and scaled according to the Procrustes superimposition method (Bookstein 1991) by using the software MorphoJ® 1.05 (Klingenberg 2011). This allowed generating Procrustes coordinates that were used as shape variables.

The SI means were compared with non‐parametric Wilcoxon test for independent samples, using Infostat®statistical software. Only the wing shape (and not size) was analyzed because the shape was more evolutionarily informative and less affected by environmental factors (Dujardin 2008). However, allometry (shape‐size relation) was tested by a multivariate linear regression and a permutation test associated with the regression analysis using the null hypothesis of complete independence between the dependent and independent variables (Zelditch et al. 2004). A principal component analysis (PCA) was applied on shape variables. To evaluate differences between shapes of categorized a priori individuals, discriminant analysis (DA) and canonical variate analysis (CVA) were performed (Zelditch et al. 2004). We used a permutation test with 10,000 rounds of iterations over Procrustes and Mahalanobis distances. To visualize the relative morphological changes between populations, wing wireframe schemes were used through the MorphoJ® software (Zelditch et al. 2004, Klingenberg 2011). The multivariate analysis and permutation test were performed with the statistical package included in MorphoJ® (Klingenberg 2011). The level significance for all the statistical tests was 0.05.

The mean SI for samples from Chubut Province (mean±SD = 4.35±0.26; n = 36; min = 3.95; max = 5.16) was higher (Wilcoxon test, W = 2027; p<0.0001) than those from Misiones Province, (mean±SD 3.73±0.20; n = 35; min = 3.38; max = 3.92). The SI support that the individuals, collected from the northern and southern regions of Argentina, belonged to Cx. quinquefasciatus and Cx. pipiens respectively. Although according to Brogdon (1981), in the case of Cx. quinquefasciatus, the mean of siphonal index was at the end of the range.

The allometry test was significant (p = 0.003, r² = 0.09); thus, the residuals of the multivariate regression were used as shape variables not affected by size. The first two components of the PCA accumulated 57.3% of the total variability of shape (PC1: 35.8% and PC2: 21.5%). In the second axis (PC2), the shape of the individuals of Cx. pipiens was separated from Cx. quinquefasciatus (Figure 2). The wireframe scheme shows that the wings of Cx. pipiens along the negative values in PC2 were broader than those of Cx. quinquefasciatus along the positive values in PC2. The main relative change was observed in the center of the wing with LMs 11, 14, 15, and 10, and LM 1 in the posterior edge (Figure 2). The discrimination between the two species was successful when the individuals were categorized a priori in the CVA (Figure 3). The permutation test was significant for the discrimination, with a Mahalanobis distance of 14.08 and p‐value<0.0001 and Procrustes distance of 0.028 and p‐value<0.0001. This was consistent with the cross validation of DA, where the misclassified rate was 0% for both species (not shown).

The results indicate that the wing shape of Cx. pipiens was different from Cx. quinquefasciatus and the application of geometric morphometry allowed differentiating between them. This discrimination at the level of females provides new information that could be complemented with traditional morphological methods. Differences between these species could suggest particular ecological adaptations to the environment within its distribution. In Aedes albifasciatus , for example, the presence of morphological variations in the wing for populations that inhabit in contrasting climates seems to indicate an adaptation that favors its active dispersal (Garzón and Schweigmann 2018). Regarding the species studied in the present work, as Cx. pipiens remains in diapause during winter (Vinogradova 2003), wide wings could improve their dispersive capacity (Wootton 1992) of this species in its limited active season (summer). In butterflies, females with wider wings than males have a higher individual relative flight force that improves their flight performance and dispersive capacity (Berwaerts et al. 2002). It has been demonstrated that the edges of the wing have a lot of influence in the aerodynamic flight mechanisms (Bomphrey et al. 2017). In contrast, the thinner wings of Cx. quinquefasciatus could favor a higher beat frequency in warmer environments with higher average temperatures, as also observed in flies (Azevedo et al. 1998).

The particular wing shape of each species could act or contribute to some degree of sexual barrier in nature due to its recognized function during copulation (Gibson and Russell 2006). However, it has been shown that fertile hybrids exist between sympatric species of the Cx. pipiens complex both in Argentina (Almirón et al. 1995) and elsewhere in the world (Harbach 2012). Therefore, the shape of the wing may not contribute as a pre‐reproductive barrier directly, although it could contribute to the eco‐physiological variants between both species. For example, wider width in the wings of Cx. pipiens could favor flight performance in external environments (Berwaerts et al. 2002) since this species would be exophilic, resting outside, and eurygamous, mating in open spaces (Harbach et al. 1985).

Although molecular tools are available to differentiate these species, a simple, low‐cost alternative method may be useful in the absence of a molecular laboratory. A rapid identification using this technique between females of these species is convenient in a context of epidemiological risk or vector surveillance, especially since they differ in their vector competence. On the other hand, it would be interesting to apply this method to study the annual population dynamics in temperate areas where both species overlap and generate hybrids, since the temperature is a limiting environmental factor which affects the distribution of Cx. pipiens and Cx. quinquefasciatus .

来自拉丁美洲的南部地区络合物或culicid物种的组合和亚种都难以区分形态（哈巴克2012，Laurito等人2017年）。这些复合物包括分类单位，其矢量能力不同，因此其流行病学重要性也不同（Dujardin和Schofield 2004）。但是，基于经典结构的物种内的形态变异和诊断特征的叠加通常无法正确识别。

在阿根廷，“ Pipiens组合”包括带有各种变种Culex molestus和Culex quinquefasciatus的Culex pipiens（Harbach 2012）。这两种物种均被认为是几种虫媒病毒的潜在载体，包括西尼罗河病毒和圣路易斯脑炎病毒，并且在生态生理特征和地理分布上也存在差异。Cx。quinquefasciatus是一种热带和亚热带物种，分布在阿根廷中部和北部，而Cx。pipiens属于温带种，从该国的中部到南部分布。在南纬30°36'（科尔多瓦省）和36°13'S（拉潘帕省）之间，以及经度57°57'W（拉普拉塔市）和64°48'W（科尔多瓦省）之间这些物种与可行的杂种共存（Almirón等，1995； Diez等，2012）。

大多数研究旨在通过使用雄性生殖器和/或未成熟阶段的虹吸管的形态计量指数在形态上区分上述两个物种和杂种（Vinogradova 2003，Diez等人2012）。Dehghan（等人2016）已经提出来区分成年女性由传统形态，考虑subcosta /科斯塔交点和R2 + 3静脉分叉的相对位置。但是，这种方法并不完全有效，几何形状分析不仅提供了准确而准确的描述，而且还具有可视化和解释同样重要的目的（Zelditch等，2004）。

几何形态计量学是一种可以识别和区分蚊子物种和物种复合体的工具（Vidal等，2011； Laurito等，2015）。机翼最常用于此工具，因为机翼具有近似平坦的特性，可最大程度地减少在静脉中定位地标时的误差，便于使用同源点（Bookstein 1991）以及对生态和进化观点的见识（De Riva等）。等人，2001； Baylac等人，2003）。这项工作的目的是通过几何形态学评估属于Pipiens组合（Cx。pipiens和Cpi.pipiens的雌性蚊子的翅膀形状的差异）。Cx。quinquefasciatus）。

女Cx。quinquefasciatus是从阿根廷米西奥内斯省的两个地区的几个定居点获得的，这两个地区分别是Colonia Aurora（南纬27°28'，54°31'）和Eldorado（南纬26°24'，54°38'）。Cx。蚊雌性在从马德林市（42°45' S，65°02' W）在丘布特省阿根廷墓地收集。两种物种均从其分布的专有区域获得。我们避免在存在物种及其杂种的杂种区（图1）中取样。

从人工栖息地收集两种物种的未成熟体，并在半自然条件下（环境温度，用幼虫栖息地的水和酵母溶液喂养）饲养直至成年阶段。死亡幼虫和4的蜕皮^日龄在97％的醇被保存。分类键（Rossi等。2002）用于识别物种。在Cx之间进行识别。quinquefasciatus和Cx。pipiens，考虑了地理起源和虹吸指数（SI）。SI的计算公式为：从虹吸管底部到虹吸管尖端中点的长度与最宽点处的虹吸管宽度之间的比值（Brogdon 1981）。根据Brogdon（1981）提出的范围对物种进行分类，SI范围：Cx为3.1–3.7 。quinquefasciatus和SI范围：Cx为4.3–4.7 。pipiens。

总共71死4的^第龄幼虫及其蜕皮分别测量的SI，并用于几何形态测量女性44如下：35幼虫和从米西奥内斯省21名成年人（CX倦。专属区）和36幼虫和23 Chubut Province（Cx。pipiens）的成年人专属区域）。只要有可能，就对同一个体中的幼虫虫体和成年性状进行分析。对于形态分析，我们使用了饲养后处于更好保存状态的雌性。虹吸管和左翼的数码照片都是用与立体显微镜耦合的相机拍摄的。在机翼的每个数字图像上共选择了17个地标（图1），并且使用软件tps-DIG®2.16生成了笛卡尔坐标。通过使用软件1.05（Klingenberg 2011）根据Procrustes叠加方法（Bookstein 1991），对地标配置进行传输，旋转和缩放。这样就可以生成用作形状变量的Procrustes坐标。

使用Infostat®统计软件，将SI均值与独立样品的非参数Wilcoxon检验进行比较。仅分析机翼的形状（而不是尺寸），因为该形状更具进化性，并且受环境因素的影响较小（Dujardin 2008）。但是，异方差（形状与大小的关系）通过多元线性回归和与回归分析相关联的置换检验，使用因变量和自变量之间完全独立的零假设进行检验（Zelditch等人，2004年）。主成分分析（PCA）应用于形状变量。评估先验分类形状之间的差异对个体进行判别分析（DA）和规范变量分析（CVA）（Zelditch等，2004）。我们对Procrustes和Mahalanobis距离进行了10,000次迭代的置换测试。为了可视化种群之间的相对形态变化，通过MorphoJ®软件使用了机翼线框方案（Zelditch等，2004； Klingenberg，2011）。使用MorphoJ®（Klingenberg 2011）中包含的统计软件包进行多元分析和置换检验。所有统计检验的水平显着性为0.05。

来自丘布特省的样本的平均SI（平均值±SD = 4.35±0.26; n = 36;最小值= 3.95;最大值= 5.16）（Wilcoxon检验，W = 2027; p <0.0001）比密西昂斯省的样本高（平均值±SD 3.73±0.20； n ＝ 35；最小值＝ 3.38；最大值＝ 3.92）。SI支持从阿根廷北部和南部地区收集的个人属于Cx。quinquefasciatus和Cx。pipiens。尽管根据Brogdon（1981）的说法，对于Cx。quinquefasciatus，虹吸指数的平均值在范围的末端。

异体测试显着（p = 0.003，r ² = 0.09）；因此，将多元回归的残差用作不受大小影响的形状变量。PCA的前两个组件累积了形状总变化的57.3％（PC1：35.8％和PC2：21.5％）。在第二轴（PC2）中，Cx的形状。pipiens与Cx分离。quinquefasciatus（图2）。线框方案显示了Cx的翅膀。在PC2中，沿着负值的pipiens比Cx宽。西洋参沿PC2中的正值。在机翼中心观察到主要的相对变化，LM 11、14、15和10，LM 1在后缘（图2）。当对个体进行CVA的先验分类时，两个物种之间的区分成功了（图3）。排列检验对于区分具有重要意义，马氏距离为14.08，p值<0.0001，Procrustes距离为0.028，p值<0.0001。这与DA的交叉验证一致，DA的交叉分类率均为2％（未显示）。

结果表明机翼形状为Cx。pipiens与Cx不同。quinquefasciatus和几何形态计量学的应用可以区分它们。对女性的这种歧视提供了可以用传统形态学方法补充的新信息。这些物种之间的差异可能表明其分布范围内对环境有特殊的生态适应。例如，在白纹伊蚊中，居住在不同气候条件下的种群的机翼中存在形态变异，似乎表明这种适应有利于其主动扩散（Garzón和Schweigmann 2018）。关于目前工作中研究的物种，Cx。琵鹭在冬季仍处于滞育状态（Vinogradova 2003），在有限的活动季节（夏季），宽翅可以提高其在该物种中的分散能力（Wootton 1992）。在蝴蝶中，具有比雄性更大的翅膀的雌性具有更高的个体相对飞行力，从而改善了它们的飞行性能和分散能力（Berwaerts等，2002）。已经证明了翼的边缘有空气动力学飞行机制很大的影响力（Bomphrey等人2017年）。相比之下，Cx的机翼更薄。在苍蝇的平均温度较高的温暖环境中，金枪鱼可能会喜欢更高的拍频（Azevedo等人，2006）。1998）。

由于其在交配过程中的公认功能，每个物种的特定翼形可能在自然界中发挥某种作用或起到一定作用（Gibson and Russell 2006）。然而，已经表明Cx的同胞种之间存在可育的杂种。pipiens complex在阿根廷（Almirón等，1995）和世界其他地方（Harbach，2012）。因此，机翼的形状虽然可能会导致两个物种之间的生态生理变异，但可能不会直接作为繁殖前屏障。例如，Cx机翼的宽度更宽。pipiens可能会有利于外部环境中的飞行性能（Berwaerts等，2002）），因为这种将exophilic，静息外，和eurygamous，在开放的空间配合（哈巴克等人1985）。

尽管可以使用分子工具来区分这些物种，但是在没有分子实验室的情况下，一种简单，低成本的替代方法可能会有用。在流行病学风险或媒介监测的情况下，使用这种技术在这些物种的雌性之间进行快速鉴定非常方便，特别是因为它们的媒介能力不同。另一方面，将这种方法用于研究两个物种重叠并产生杂种的温带地区的年度种群动态将是有趣的，因为温度是影响Cx分布的限制性环境因素。pipiens和Cx。quinquefasciatus。