Landmark-based Geometric Morphometrics Research Articles

Geographical variation in the forewing shape of the red dwarf honeybees revealed by landmark-based geometric morphometrics

Accurate intraspecific variation assessment is the prerequisite for determining subspecies, evolutionarily significant units, or management units across a species' range. However, intraspecific variation assessment is often hindered by inaccurate morphological results from traditional methods or high-cost genetic analyses. Geometric morphometrics is a promising technique for quantifying and visualising intraspecific diversity. We used landmark-based geometric morphometrics to investigate forewing shape variation and microtaxonomy of populations of Apis florea Fabricius 1787 (Apidae: Apini), which originated from some parts of Asia and Africa based on the 19 landmarks plotted at the venation intersections of forewings. We collected samples of 162 colonies from 36 different localities. On average, 13 workers per colony were analysed (1851 bees in total). Distinct spatial structures and significant differences in wing shape between the studied populations were revealed using canonical variate analysis, and no considerable overlap was detected between the colonies belonging to the western and eastern populations. The populations at higher latitudes showed significantly larger centroid sizes than those at lower latitudes, and a clinal variation in forewing size was confirmed. Significant distance-decay relationships between the studied populations were observed so that the populations located at the maximum geographical distance showed the most significant distance from each other and vice versa. Wing geometric morphometrics was an accurate tool for the reliable estimation of the population structure of the red dwarf honeybees. It is essential to increase the reliability of these variations by combining the results with further molecular data.

Craniofacial anomalies in schizophrenia-relevant GFAP.HMOX10-12m mice.

Subtle craniofacial dysmorphology has been reported in schizophrenia patients. This dysmorphology includes midline facial elongation, frontonasal anomalies and a sexually dimorphic deviation from normal directional asymmetry of the face, with male patients showing reduced and female patients showing enhanced facial asymmetry relative to healthy control subjects. GFAP.HMOX10-12m transgenic mice (Mus musculus) that overexpress heme oxygenase-1 in astrocytes recapitulate many schizophrenia-relevant neurochemical, neuropathological and behavioral features. As morphogenesis of the brain, skull and face are highly interrelated, we hypothesized that GFAP.HMOX10-12m mice may exhibit craniofacial anomalies similar to those reported in persons with schizophrenia. We examined craniofacial anatomy in male GFAP.HMOX10-12m mice and wild-type control mice at the early adulthood age of 6-8 months. We used computer vision techniques for the extraction and analysis of mouse head shape parameters from systematically acquired 2D digital images, and confirmed our results with landmark-based geometric morphometrics. We performed skull bone morphometry using digital calipers to take linear distance measurements between known landmarks. Relative to controls, adult male GFAP.HMOX10-12m mice manifested craniofacial dysmorphology including elongation of the nasal bones, alteration of head shape anisotropy and reduction of directional asymmetry in facial shape features. These findings demonstrate that GFAP.HMOX10-12m mice exhibit craniofacial anomalies resembling those described in schizophrenia patients, implicating heme oxygenase-1 in their development. As a preclinical mouse model, GFAP.HMOX10-12m mice provide a novel opportunity for the study of the etiopathogenesis of craniofacial and other anomalies in schizophrenia and related disorders.

The effects of temperature on plasticity, shape symmetry and seasonal variation in the freshwater benthic green microalga Micrasterias thomasiana

AbstractDesmids are usually abundant in shallow peatland pools. In these localities, water temperature is closely linked to seasonal fluctuations in air temperature, so with increasing temperature extremes in temperate ecosystems, these microalgae are exposed to conditions of high-temperature stress. We investigated whether the shape, size, and growth rates of Micrasterias thomasiana, a frequently occurring species, are associated with varying temperatures in cultures and natural populations. The research was based on parallel analysis of clonal populations in temperature levels from 13 to 33 °C as well as cells from natural populations collected during the season. The effects of high temperature on morphological plasticity and fluctuating asymmetry in the shape of cellular parts were investigated by the landmark-based geometric morphometrics. The results showed that variation among individuals and fluctuating asymmetry between the lateral lobes of Micrasterias cells increased at 29 °C and in natural samples taken in July and October. In parallel, the size of semicells growing at temperatures above 25 °C decreased compared to those grown at lower temperatures. However, the temperature effects on shape and size were not directly related to the growth rates. The overall bilateral asymmetry between semicell halves did not change in relation to varying temperatures. In general, the results showed that morphological variation in natural populations of M. thomasiana reflected seasonal cycles and corresponded to plasticity associated with temperature changes in clonal cultures. It might therefore be possible to use these phenotypic markers as indicators of thermal stress in natural populations inhabiting shallow pools in peatlands.

A Geometric Morphometrics Approach to the Study of Natural Variations and Hybrid Detection in Populations of Alnus incana (L.) Moench and Alnus rohlenae Vít, Douda and Mandák.

Landmark-based geometric morphometrics (GM) was used to examine, for the first time, spontaneous hybridization between Alnus incana (L.) Moench and Alnus rohlenae Vít, Douda and Mandák, and to assess inter- and intrapopulation variability in leaf shape, leaf size and venation in natural populations in Serbia (Western Balkans). Two geographically distant (30 km) and two close (1.2 km) populations were selected to examine hybridization. The variability in leaf shapes was assessed by canonical variate analysis and linear discriminant analysis performed on the symmetric component of variation. Covariation between the symmetric component of shape variation and the number of pairs of secondary leaf veins was investigated with partial least squares analysis. Static allometry was examined for the first time in the genus Alnus Mill. A higher proportion of A. incana leaves was classified as A. rohlenae in geographically close populations, which is in accordance with the hypothesis about spontaneous hybridization. No single leaf of A. rohlenae was classified as A. incana, indicating that putative hybrids can only be found in grey alder populations. This study demonstrates that GM is a powerful tool for species delimitation and hybrid detection in the genus Alnus and it can be used for preliminary screening in hybrid zones.

Wing geometric morphometrics and DNA barcoding to distinguish three closely related species of Armigeres mosquitoes (Diptera: Culicidae) in Thailand

Armigeres subalbatus, a mosquito species widely found in Thailand and other Asian countries, serves as a vector for filarial parasites, affecting both humans and animals. However, the surveillance of this vector is complicated because of its morphological similarity to two other species, Armigeres dohami and Armigeres kesseli. To differentiate these morphologically similar species, our study employed both wing geometric morphometrics (GM) and DNA barcoding, offering a comprehensive approach to accurately identify these closely related Armigeres species in Thailand. Our GM analyses based on shape demonstrated significant accuracy in differentiating Armigeres species. Specifically, the outline-based GM method focusing on the 3rd posterior cell exhibited an accuracy rate of 82.61%, closely followed by the landmark-based GM method with 81.54%. Both these GM techniques effectively distinguished Ar. subalbatus from Ar. dohami and Ar. kesseli. Regarding DNA barcoding, our investigation of pairwise intra- and interspecific divergences revealed a "barcoding gap". Furthermore, the results of species confirmation using both species delimitation methods including the automatic barcode gap discovery method (ABGD) and the Multi-rate Poisson tree process (mPTP) were consistent with those of morphological identification, sequence comparisons with the GenBank and Barcode of Life Data System (BOLD) databases, and the neighbor-joining tree construction. These consistent results emphasize the efficacy of DNA barcoding in the precise identification of Armigeres species.

Clovis point allometry, modularity, and integration: Exploring shape variation due to tool use with landmark-based geometric morphometrics.

Landmark-based geometric morphometrics (LGM) is most often used in archaeology to characterize and differentiate groups of artifacts, but it can be used for much more. We demonstrate LGM's power to uncover new insights by exploring stone-tool allometry, modularity, and integration using a sample of 100 western North American Clovis points. Here, allometry concerns how stone tools change in shape as their size changes through their use-lives, and modularity and integration concern how the constituent parts of a tool work together. We show that Clovis points are surprisingly complex tools. When their blades and hafts are defined technologically, rather than arbitrarily, they unambiguously exhibit allometry, and their hafts and blades are modular and highly integrated. We use these analyses to further explore questions about Clovis points, including the differences between cache and non-cache points. Finally, we use heuristic haft-size categories to examine functional constraints on the shape and size of hafts and blades. This work illustrates the importance of using accurate measurements of point components rather than estimates or proxies, which can lead to unfounded inferences. These analytical approaches and accompanying R code are easily transferable to other research questions of stone-tool use.

Function, Style, and Standardization: Is the Proximal or Distal End of a Middle Stone Age Point More Variable?

ABSTRACT Middle Stone Age (MSA) point styles may reflect the development of regional identities of human social groups. MSA people likely used these points for various functions including as weapon tips. Recent work in cultural evolution suggests that functional aspects of technology tend to be more standardized and subject to negative selective pressures, while stylistic aspects are highly visible and tend to be more variable. This study examines the patterns of segmented (proximal vs. distal) standardization for unretouched MSA points from six archaeological assemblages using landmark-based geometric morphometrics to quantify shape variation. Contrary to expectations, our results show that the proximal end has greater shape variation than the distal end. Segregating within point variation provides clues to past function and style patterns in the MSA. This approach also opens new avenues for exploring ethnographic and recent archaeological projectile points.

Molecular and morphometric differentiation of secondary filariasis vector Coquillettidia mosquitoes (Diptera: Culicidae) in Thailand.

Coquillettidia mosquitoes are important nuisance-biting pests and a vector of brugian filariasis in Thailand. However, comprehensive information about these mosquitoes remains unavailable such as molecular and morphometric differences among species. The lack of vector knowledge on Coquillettidia species could affect future disease control. This study aims to investigate differences in molecular variations based on mitochondrial cytochrome oxidase subunit I (COI) gene and wing geometric traits of three Coquillettidia species, namely Cq. crassipes, Cq. nigrosignata, and Cq. ochracea in Thailand. The results of molecular analyses revealed the differences among three Coquillettidia species. The genetic difference measure based on the Kimura two-parameter model among three Coquillettidia species showed low intraspecific distances (0%-3.05%) and large interspecific distances (10.10%-12.41%). The values of intra- and inter-genetic differences of three Coquillettidia species did not overlap which showed the existence of a barcoding gap indicating the efficiency of the identification based on the COI gene. As with molecular analysis, the landmark-based geometric morphometrics approach based on wing shape analysis indicated three distinct species groups which were supported by the high total performance score of cross-validated classification (97.16%). These results provide the first evidence of taxonomic signal based on molecular and wing geometric differences to support species identification and biological variations of Coquillettidia mosquitoes in Thailand for understanding these rare vector mosquitoes in depth and leading to effective further mosquito control.

Wing geometric morphometrics to distinguish and identify Haematobosca flies (Diptera: Muscidae) from Thailand

The hematophagous flies of the genus Haematobosca Bezzi, 1907 (Diptera: Muscidae) are important ectoparasites in domestic animals and wildlife. Two species of this genus have been recorded in Thailand, viz., Haematobosca sanguinolenta (Austen, 1909) and Haematobosca aberrans (Pont, Duvallet & Changbunjong, 2020). They have a similar morphology and coexist in the same habitat. The correct species identification of these flies is extremely important for understanding disease epidemiology and developing effective control measures. Geometric morphometrics (GM) has been confirmed to be a useful tool for differentiating and identifying morphologically similar insect species. Therefore, GM was used to distinguish and identify H. sanguinolenta and H. aberrans in Thailand. Adult flies of both sexes were collected using Nzi traps, morphologically identified, and analyzed by landmark-based GM of the wing. Results showed that GM was highly effective in distinguishing the two Haematobosca species based on their wing shape, with an overall accuracy score of 99.3%. We also revealed that our study material could be used as reference data to identify new field specimens collected from other geographic locations. We propose that wing GM can be used as a supplement to conventional morphology identification, particularly for Haematobosca specimen that has been damaged or has lost its diagnostic characteristics due to specimen collection and processing in the field.

Parallel Evolution of Skull Form in Three Rodent Genera Inhabiting Steep Elevational Gradients of Ethiopian Highlands

Several rodent genera radiated in Ethiopian highlands during Pleistocene, giving rise to species specialized to different elevation zones. The zonation is accompanied by vegetation change from savannahs or forests in lower elevations, through the mid-elevation belt of ericaceous shrublands to Afroalpine moorlands on the mountain tops. Such evolution on elevation gradient is expected to promote phenotypic adaptations and several genera are known to include highly distinctive high-elevation specialists. In this study, we systematically examined shape and size differentiation of skull and mandible in three genera, Lophuromys, Arvicanthis and Stenocephalemys, each including from six to eight species inhabiting different elevation zones on both sides of the Great Rift Valley. Employing computed tomography, three-dimensional virtual models, landmark-based geometric morphometrics and a powerful toolkit of partial least squares analysis, we demonstrated the elevation-related differentiation is a prominent feature of skull and mandible shape variation in all three genera. We identified the parts of skull and mandible which change most between the successive zones. These include, notably, orbits and insertion areas of masticatory muscles, which suggests adaptive shifts in visual perception and diet. We also showed that while the same parts are modified in all three genera, they are modified in different ways and to a different degree. The evidence for size differentiation was mixed despite the universal trend of larger size in higher elevations. Our study confirms the high-elevation specialists represent the most distinctive forms, which is especially true for the species from Bale and Arsi Mountains, East of the Great Rift Valley.

Identification of Acrossocheilus fasciatus (Teleostei: Cyprinidae) from Three Different Phylogeographic Sources using Morphology and COI DNA Barcoding

Background: Accurate species delimitation is essential for phylogenetic, phylogeographic, ecological, conservation and biogeographic investigations. Acrossocheilus species classification has long been contentious, owing to their uncertain evolutionary relationships, high levels of morphological homoplasy and a lack of knowledge of the taxonomic validity of conventionally utilized morphological characters. Methods: A final data set consisting of 102 mitochondrial cytochrome oxidase I (COI) gene sequences was analysed for K2P intraspecific and interspecific divergence in the current study, using reconstruction methods (maximum likelihood) and various models (ABGD, PTP, GMYC) to divide the sequences according to the pattern of genetic variation. We used landmark-based geometric morphometrics to identify specimens. Result: Two taxa were identified according to the phylogenetic tree (maximum likelihood method) and three different models (ABGD, PTP, GMYC). The molecular taxonomy of Acrossocheilus agreed with the morphological identification of specimens from the same collection locality. Therefore, the COI gene and geometric morphometrics were effective in species identification of A. fasciatus.

Alone in a cave: Examination of a 5200 BCE skeleton from the Judean Desert, Israel

The remains of a >50-years-old male, thus far representing the only complete skeleton dated to the Early Chalcolithic (Wadi Rabah) period in Israel, were recovered in a cave in the Judaean desert (Nahal Mishmar, F1-003). The old male suffered abscesses in the maxilla following tooth caries, and a well-healed trauma in the left tibial midshaft. Skull and mandibular morphology were described using plain measurements, indices and angles, and compared with similarly taken Chalcolithic data. In addition, mandibular morphology was captured using a landmark-based geometric morphometrics method and compared to Natufian hunter-gatherers, Pre-Pottery Neolithic early farmers, and Late Chalcolithic populations. The results, although cautionary, reveal similarity to the succeeding Ghassulian Chalcolithic period populations and suggest population continuity from the Early to the Late (Ghassulian) Chalcolithic period. Future ancient DNA study may clarify this hypothesis and further reveal population affinity in this period in Israel.

Semicircular canal shape diversity among modern lepidosaurs: life habit, size, allometry

BackgroundThe shape of the semicircular canals of the inner ear of living squamate reptiles has been used to infer phylogenetic relationships, body size, and life habits. Often these inferences are made without controlling for the effects of the other ones. Here we examine the semicircular canals of 94 species of extant limbed lepidosaurs using three-dimensional landmark-based geometric morphometrics, and analyze them in phylogenetic context to evaluate the relative contributions of life habit, size, and phylogeny on canal shape.ResultsLife habit is not a strong predictor of semicircular canal shape across this broad sample. Instead, phylogeny plays a major role in predicting shape, with strong phylogenetic signal in shape as well as size. Allometry has a limited role in canal shape, but inner ear size and body mass are strongly correlated.ConclusionsOur wide sampling across limbed squamates suggests that semicircular canal shape and size are predominantly a factor of phylogenetic relatedness. Given the small proportion of variance in semicircular canal shape explained by life habit, it is unlikely that unknown life habit could be deduced from semicircular canal shape alone. Overall, semicircular canal size is a good estimator of body length and even better for body mass in limbed squamates. Semiaquatic taxa tend to be larger and heavier than non-aquatic taxa, but once body size and phylogeny are accounted for, they are hard to distinguish from their non-aquatic relatives based on bony labyrinth shape and morphology.

Phenotypic Plasticity in Juvenile Frogs That Experienced Predation Pressure as Tadpoles Does Not Alter Their Locomotory Performance

Anuran species can respond to environmental changes via phenotypic plasticity, which can also result in ecological impacts across the life history of such species. We investigated the effects of predation pressure (i.e., the non-consumption effect) from the dragonfly larva (Anax parthenope) on the phenotypical change of tadpoles into juvenile frogs (specifically the black-spotted pond frog, Pelophylax nigromaculatus), and also analyzed the impact of morphological changes on locomotory performance after metamorphosis. The experiments on predator impact were conducted in the laboratory. Body length, weight, development timing, and metamorphosis timing in the presence of dragonfly nymphs were measured in both tadpoles and juvenile frogs. The body and tail shapes of the tadpoles, as well as the skeletal shape of the juvenile frogs, were analyzed using landmark-based geometric morphometrics. Furthermore, the locomotory performance of the juvenile frogs was tested by measuring their jumping and swimming speeds. Tadpoles that had grown with predators possessed smaller bodies, deeper tail fins, and slower development rates, and they waited longer periods of time before commencing metamorphosis. Having said this, however, the effect of predator cues on the body length and weight of juvenile frogs was not found to be significant. These juvenile frogs possessed longer limbs and narrower skulls, with subtle morphological changes in the pelvis and ilium, but there was no subsequent difference in their swimming and jumping speeds. Our results showed that the changes in anatomical traits that can affect locomotor performance are so subtle that they do not affect the jumping or swimming speeds. Therefore, we support the view that these morphological changes are thus by-products of an altered tadpole period, rather than an adaptive response to predator-escape ability or to post-metamorphosis life history. On the other hand, delayed metamorphosis, without an increase in body size, may still be disadvantageous to the reproduction, growth, and survival of frogs in their life history following metamorphosis.

Diversification of the shell shape and size in Baikal Candonidae ostracods inferred from molecular phylogeny

Ostracod shells are used extensively in paleontology, but we know little about their evolution, especially in ancient lakes. Lake Baikal (LB) is the world’s most important stronghold of Candonidae diversity. These crustaceans radiated here rapidly (12–5 Ma) and with an unprecedented morphological diversity. We reconstruct their molecular phylogeny with 46 species and two markers (18S and 16S rRNA), and use it to estimate the evolution of the shell shape and size with landmark-based geometric morphometrics (LBGM). High posterior probabilities support four major clades, which differ in node depth and morphospace clustering. After removing a significant allometry, the first three principal components (PCs) describe about 88% of total variability, suggesting a strong integration. Reconstructed ancestral shapes are similar for all four clades, indicating that diversification happened after colonization. Major evolutionary changes occurred from trapezoidal to elongated shapes. Sister species are separated in morphospace, by centroid size, or both, as well as by vertical and horizontal distributions in LB. Ostracod shell is a strongly integrated structure that exhibits high evolvability, with some extreme shapes, although mostly along the first PC. This is the first study that combines molecular phylogeny and LBGM for ostracods and for any LB group.

Species Discrimination of Stomoxys Flies S. bengalensis, S. calcitrans, and S. sitiens (Diptera: Muscidae) Using Wing Geometric Morphometrics.

The flies of the genus Stomoxys Geoffroy, 1762 (Diptera: Muscidae), are regarded as pests of veterinary and medical importance. In Thailand, Stomoxys calcitrans (Linnaeus, 1758) is the most abundant species and is widely distributed throughout the country. This Stomoxys species can coexist with two other morphologically similar species: Stomoxys bengalensis Picard, 1908, and Stomoxys sitiens Rondani, 1873. Hence, discriminating using morphological characteristics is difficult, especially if the specimen is damaged or loses its diagnostic characteristics. In this study, we evaluated the effectiveness of the landmark-based geometric morphometric (GM) approach to discriminate among the three Stomoxys spp.: S. bengalensis, S. calcitrans, and S. sitiens. Left-wing images of S. bengalensis (n = 120), S. calcitrans (n = 150), and S. sitiens (n = 155) were used for the GM analyses. The results of the wing shape analyses revealed that the GM approach was highly effective for discriminating three Stomoxys, with high accuracy scores ranging from 93.75% to 100%. This study adds to the evidence that landmark-based GM is an excellent alternative approach for discriminating Stomoxys species.

A landmarking protocol for geometric morphometric analysis of squamate endocasts.

Landmark-based geometric morphometrics is widely used to study the morphology of the endocast, or internal mold of the braincase, and the diversity associated with this structure across vertebrates. Landmarks, as the basic unit of such methods, are intended to be points of correspondence, selected depending on the question at hand, whose proper definition is essential to guarantee robustness and reproducibility of results. In this study, 20 landmarks are defined to provide a framework to analyze the morphological variability in squamate endocasts. Ten species representing a cross-section of the diversity of Squamata from both phylogenetic and ecological (i.e., habitat) perspectives were considered, to select landmarks replicable throughout the entire clade, regardless of the degree of neuroanatomical resolution of the endocast. To assess the precision, accuracy, and repeatability of these newly defined landmarks, both intraobserver and interobserver error were investigated. Estimates of measurement error show that most of the landmarks established here are highly replicable, and preliminary results suggest that they capture aspects of endocast shape related to both phylogenetic and ecologic signals. This study provides a basis for further examinations of squamate endocast disparity using landmark-based geometric morphometrics.

Sexual dimorphism in shell shape: is Pomacea canaliculata an exception or an example among Neotropical apple snails?

Sexual dimorphism has often been recorded in apple snails (Caenogastropoda Ampullariidae), but reports are concentrated in a few species, either invasive or with biocontrol potential, which sugests some taxonomic bias. To find out evolutionary and ecological correlates of sexual dimorphism it is necessary to detect and quantify it but also is important to detect its absence. Our aims were to confirm or not the existence of sexual dimorphism in shell shape of Felipponea neritiniformis and Asolene platae, using Pomacea canaliculata as reference and applying the same methodology (landmark-based geometric morphometrics) and statistical power. Significant intersexual differences were only found in P. canaliculata and, in a lesser degree, in F. neritiniformis: males have larger apertures relative to body whorl and more rounded apertural outer edges than females. Female shells are larger in F. neritiniformis and P. canaliculata, but not in A. platae. Using comparable methodologies and statistical power, sexual dimorphism in shell shape is detectable in some apple snails but not in others. Interspecific variation in sexual dimorphism in the Ampullariidae is not only due to taxonomic bias and deserves more research to establish the main patterns and possible causes.

Population Structure and Morphological Pattern of the Black-Spotted Pond Frog (Pelophylax nigromaculatus) Inhabiting Watershed Areas of the Geum River in South Korea

Black-spotted pond frogs (Pelophylax nigromaculatus), widely distributed in East Asia, can be suitably used for the study of population genetic patterns and ecosystem monitoring. To systematically manage, conserve, and study this species, it is necessary to understand its habitat range. We analyzed the genetic and morphological range of black-spotted pond frog populations within a watershed of the Geum River, one of the main rivers in South Korea. We genotyped the frogs based on seven microsatellite loci and defined the skull shape based on landmark-based geometric morphometrics. One watershed area was divided into 14 sub-watershed areas, the smallest unit of the Geum River basin. The genetic structure of frogs among the 14 sub-watershed areas did not differ significantly, nor was correlated with geographic distance. Therefore, frogs within these watershed areas constitute a single population. Morphologically, they differed between some sub-watershed areas, but morphological distance did not correlate with genetic distance but rather with geographic distance. This morphological change differs depending on the environmental gradient rather than the genetic structure. As a single population, frogs in this watershed area need to be managed in an integrated way. We suggest that the identification of response and adaptation by population genetics must be compared across and beyond the watershed range.

Geometric morphometric and molecular techniques for discriminating among three cryptic species of the Anopheles barbirostris complex (Diptera: Culicidae) in Thailand

Anopheles members of the Barbirostris complex are important vectors of malaria in Thailand. However, they are morphologically indistinguishable because they are closely related species. In this study, wing geometric morphometrics (GM) and DNA barcoding based on the cytochrome c oxidase subunit 1 (COI) gene were applied to differentiate cryptic species of the Barbirostris complex in Thailand. Three cryptic species of the Barbirostris complex, Anopheles dissidens (19.44%), Anopheles saeungae (24.54%), and Anopheles wejchoochotei (56.02%) were initially identified using the multiplex polymerase chain reaction assay. DNA barcoding analyses showed low intraspecific distances (range, 0.27%–0.63%) and high interspecific distances (range, 1.92%–3.68%), consistent with the phylogenetic analyses that showed clear species groups. While wing size and shape analyses based on landmark-based GM indicated differences between three species (p < 0.05). The cross-validated reclassification revealed that the overall efficacy of wing size analysis for species identification of the Barbirostris complex was less than the wing shape analysis (56.43% vs. 74.29% total performance). Therefore, this study's results are guidelines for applying modern techniques to identify members within the Barbirostris complex, which are still difficult to distinguish by morphology-based identification and contribute to further appropriate malaria control.