Discrimination of Eubazus (Hymenoptera, Braconidae) sibling species using geometric morphometrics analysis of wing venation

Abstract.  Complexes of sibling and cryptic species are encountered frequently in parasitic Hymenoptera. Geometric morphometrics is a useful tool to detect minimal morphological variations, which often are undetectable by traditional morphological studies and even by classical morphometric approaches. We applied geometric morphometrics to wing venation to assess a complex case of sibling species in the genus Eubazus (Hymenoptera, Braconidae), parasitoids of conifer bark weevils of the genus Pissodes (Coleoptera, Curculionidae). The results and methods were compared with previous taxonomic studies on the same species, involving classical multivariate morphometrics, isoenzyme analyses, cross-mating experiments and biological observations. Geometric morphometrics confirmed the previous division into four distinct species. However, this approach enabled the four species to be separated simultaneously, with a reliability of 98.6% for well-classified females and 93.1% for males. A similar result in previous studies was obtained only by combining isoenzyme analyses and several canonical variate analyses, including many morphometric characters. Furthermore, measurements of wing venation were less time-consuming, more reliable and required less prior knowledge of braconid taxonomy than the measurements needed for the classical morphometrics methods. Geometric morphometrics was used also to test the effect of host species on wing shape. Several female populations of Eubazus semirugosus originating from three different Pissodes spp. were compared. Significant differences were found in wing shape between conspecific Eubazus from different host species. The results are discussed in relation to reproductive isolation and genetic flow between the four species.     

Getting into Shape: An Empirical Comparison of Traditional Truss-Based Morphometric Methods with a Newer Geometric Method Applied to New World Cichlids

Body shape is a difficult, but important, trait to quantify. Researchers have traditionally used multivariate analysis of several linear measures ('trusses') across the body form to quantify shape. Newer geometric morphometric methods claim to better estimate shape because they analyze the geometry among the locations of all landmarks simultaneously rather than the linear distances between pairs of landmarks. We tested this claim by comparing the results of several traditional morphometric analyses against a newer geometric analysis involving thin-plate splines (TPS), all applied to a common data set of morphologically variable new world cichlids Amphilophus citrinellus and A. zaliosus. The TPS method yielded slightly stronger evidence of morphological differences among forms, although traditional methods also distinguished the two species. Perhaps our most important result was the idiosyncratic interpretation of shape variation among the traditional truss-based methods, whereas the generation of deformation grids using the TPS approach yielded clear and visually interpretable figures. Our results indicate that geometric morphometrics can be a more effective way to analyze and interpret body form, but also that traditional methods can be relied upon to provide statistical evidence of shape differences, although not necessarily accurate information about the nature of variation in shape.     

Phylogenetic Signal in the Wing Shape in the Subfamily Dolichopodinae (Diptera,Dolichopodidae)

Interspecific and sex-related variations in the wing shape of 22 species of the fly subfamily Dolichopodinae, family Dolichopodidae were analyzed using geometric morphometrics. Mapping morphometric traits onto phylogeny revealed a clear phylogenetic signal in the interspecific variation and sexual dimorphism of wing shape. In some cases, not too closely related species occupied the same portion of the morphometric space, indicating some degree of homoplasy. Interspecific variation was associated with an increased wing area due to both elongation and widening or only elongation of the wing. An increase in wing area was accompanied by extension of the posterior crossvein to the apical part of the wing. The variation in wing shape related to sexual dimorphism involved the same structures (the posterior crossvein and the apical part of CuA₁), but variation associated with sexual dimorphism was distributed in fewer dimensions than interspecific variation. The allometric component of sexual dimorphism varied between species, and in most cases it was not the leading factor in wing shape variability.     

Fused traditional and geometric morphometrics demonstrate pinniped whisker diversity

Vibrissae (whiskers) are important components of the mammalian tactile sensory system, and primarily function as detectors of vibrotactile information from the environment. Pinnipeds possess the largest vibrissae among mammals and their vibrissal hair shafts demonstrate a diversity of shapes. The vibrissae of most phocid seals exhibit a beaded morphology with repeating sequences of crests and troughs along their length. However, there are few detailed analyses of pinniped vibrissal morphology, and these are limited to a few species. Therefore, we comparatively characterized differences in vibrissal hair shaft morphologies among phocid species with a beaded profile, phocid species with a smooth profile, and otariids with a smooth profile using traditional and geometric morphometric methods. Traditional morphometric measurements (peak-to-peak distance, crest width, trough width and total length) were collected using digital photographs. Elliptic Fourier analysis (geometric morphometrics) was used to quantify the outlines of whole vibrissae. The traditional and geometric morphometric datasets were subsequently combined by mathematically scaling each to true rank, followed by a single eigendecomposition. Quadratic discriminant function analysis demonstrated that 79.3, 97.8 and 100% of individuals could be correctly classified to their species based on vibrissal shape variables in the traditional, geometric and combined morphometric analyses, respectively. Phocids with beaded vibrissae, phocids with smooth vibrissae, and otariids each occupied distinct morphospace in the geometric morphometric and combined data analyses. Otariids split into two groups in the geometric morphometric analysis and gray seals appeared intermediate between beaded- and smooth-whiskered species in the traditional and combined analyses. Vibrissal hair shafts modulate the transduction of environmental stimuli to the mechanoreceptors in the follicle-sinus complex (F-SC), which results in vibrotactile reception, but it is currently unclear how the diversity of shapes affects environmental signal modulation.     

A study of wing morphology and fluctuating asymmetry in interspecific hybrids between Drosophila buzzatii and D. koepferae

In this work we investigate the effect of interspecific hybridization on wing morphology using geometric morphometrics in the cactophilic sibling species D. buzzatii and D. koepferae. Wing morphology in F₁ hybrids exhibited an important degree of phenotypic plasticity and differs significantly from both parental species. However, the pattern of morphological variation between hybrids and the parental strains varied between wing size and wing shape, across rearing media, sexes, and crosses, suggesting a complex genetic architecture underlying divergence in wing morphology. Even though there was significant fluctuating asymmetry for both, wing size and shape in F₁ hybrids and both parental species, there was no evidence of an increased degree of fluctuating asymmetry in hybrids as compared to parental species. These results are interpreted in terms of developmental stability as a function of a balance between levels of heterozygosity and the disruption of coadaptation as an indirect consequence of genomic divergence.     

The use of geometric morphometrics in understanding shape variability of sclerotized haptoral structures of monogeneans (Platyhelminthes) with insights into biogeographic variability

The sclerotized attachment organ of monogeneans has been widely used to address fundamental questions in ecology and evolution. However, traditional morphometric techniques appear to be partially inadequate and non-optimal. Traditional linear measurements mainly provide information on the size of sclerites but provide very little information, if any, on their shape. The shape of sclerites is indeed virtually unexplored and its implication for ecological and evolutionary processes remains to be analyzed. This study aims to both introduce and illustrate the use of geometric morphometrics in order to study sclerites of monogeneans in a biogeographic context. To do this, we investigated morphological variation patterns among four populations from the Pacific Ocean and six monogenean species through traditional and geometric morphometric techniques. Unlike the traditional method, the geometric morphometric method yielded a high percentage of individuals correctly classified to the four populations, providing strong evidence for phenotypic variability, divergence and local adaptation among islands without evolutionary constraint. Moreover, the traditional method also resulted in inconsistent interpretations of shape variations. This study highlighted the limitations that may arise when using traditional morphometric techniques and emphasizes that considerable information about the shape of sclerotized haptoral parts is added by using geometric morphometrics. Given the prominent taxonomic, ecological and evolutionary role of the haptor for characterizing monogeneans, we ultimately discuss the potential broad use of geometric morphometrics in a wide variety of ecological and evolutionary contexts. This powerful approach might allow a more robust estimation of the extent to which traditional evolutionary theories based on size of sclerites are congruent with their shape.     

Wing size and shape variation of Phlebotomus papatasi (Diptera: Psychodidae) populations from the south and north slopes of the Atlas Mountains in Morocco

The wing shape and size morphology of populations of the medically important phlebotomine sand fly, Phlebotomus papatasi, were examined in two endemic (south of the Atlas Mountains) and nonendemic (north of the Atlas Mountains) foci of cutaneous leishmaniasis by using geometric morphometrics in Morocco. Although it is present in all of Morocco, P. papatasi is the main vector of Leishmania major in only southern part of the Atlas Mountains. There are four major mountain ranges that serve as geographical barriers for species distribution in the study area and at least four gaps were recognized among these barriers. We found statistically significant differences in wing shape morphology between southern and northern populations. Analysis clearly recognized two main groups of populations on both sides of the mountains. The graphical depiction of Principal Component Analysis (PCA) and Canonical Variates Analysis (CVA) confirmed our morphometric study suggesting that the difference in wing morphology between the populations indicates that the population of P. papatasi shows phenotypic plasticity in the study area. According to centroid size analyses, which were used as measures of wing size differences among different sites, the north population of P. papatasi had relatively larger wings than the south population.     

Evolution of wing shape in hornets: why is the wing venation efficient for species identification?

Wing venation has long been used for insect identification. Lately, the characterization of venation shape using geometric morphometrics has further improved the potential of using the wing for insect identification. However, external factors inducing variation in wing shape could obscure specific differences, preventing accurate discrimination of species in heterogeneous samples. Here, we show that interspecific difference is the main source of wing shape variation within social wasps. We found that a naive clustering of wing shape data from taxonomically and geographically heterogeneous samples of workers returned groups congruent with species. We also confirmed that individuals can be reliably attributed to their genus, species and populations on the basis of their wing shape. Our results suggested that the shape variation reflects the evolutionary history with a potential influence of other factors such as body shape, climate and mimicry selective pressures. However, the high dimensionality of wing shape variation may have prevented absolute convergences between the different species. Wing venation shape is thus a taxonomically relevant marker combining the accuracy of quantitative characters with the specificity required for identification criteria. This marker may also highlight adaptive processes that could help understand the wing's influence on insect flight.     

Can members of the south‐western Gila robusta species complex be distinguished by morphological features?

The goal for this project was to re‐examine key morphological characters hypothesized to differentiate Gila intermedia, Gila robusta and Gila nigra and outline methods better suited for making species designations based on morphology. Using a combination of meristic counts, morphological measurements and geometric morphometrics, morphological dissimilarities were quantified among these three putative species. Traditional meristic counts and morphological measurements (i.e. distances between landmarks) were not useful for species identification. Geometric morphometrics, however, identified differences among species, while also suggesting an effect of geographic location on morphological variation. Using canonical variate analysis for the 441 fish sampled in this study, geometric morphometrics accurately predicted true group membership 100% of the time for G. nigra, 97% of the time for G. intermedia and 91% of the time for G. robusta. These results suggest that geometric morphometric analysis is necessary to identify morphological differences among the three species. Geometric morphometric analysis used in this study can be adopted by management officials as a tool to classify unidentified individuals.     

Geometric morphometrics in entomology: Basics and applications

The recent expansion of a variety of morphometric tools has brought about a revolution in the comparison of morphology in the context of the size and shape in various fields including entomology. First, an overview of the theoretical issues of geometric morphometrics is presented with a caution about the usage of traditional morphometric measurements. Second, focus is then placed on two broad approaches as tools for geometric morphometrics; that is, the landmark‐based and the outline‐based approaches. A brief outline of the two methodologies is provided with some important cautions. The increasing trend of entomological studies in using the procedures of geometric morphometrics is then summarized. Finally, information is provided on useful toolkits such as computer software as well as codes and packages of the R statistical software that could be used in geometric morphometrics.     

Habitat variation and wing coloration affect wing shape evolution in dragonflies

Habitats are spatially and temporally variable, and organisms must be able to track these changes. One potential mechanism for this is dispersal by flight. Therefore, we would expect flying animals to show adaptations in wing shape related to habitat variation. In this work, we explored variation in wing shape in relation to preferred water body (flowing water or standing water with tolerance for temporary conditions) and landscape (forested to open) using 32 species of dragonflies of the genus Trithemis (80% of the known species). We included a potential source of variation linked to sexual selection: the extent of wing coloration on hindwings. We used geometric morphometric methods for studying wing shape. We also explored the phenotypic correlation of wing shape between the sexes. We found that wing shape showed a phylogenetic structure and therefore also ran phylogenetic independent contrasts. After correcting for the phylogenetic effects, we found (i) no significant effect of water body on wing shape; (ii) male forewings and female hindwings differed with regard to landscape, being progressively broader from forested to open habitats; (iii) hindwings showed a wider base in wings with more coloration, especially in males; and (iv) evidence for phenotypic correlation of wing shape between the sexes across species. Hence, our results suggest that natural and sexual selection are acting partially independently on fore‐ and hindwings and with differences between the sexes, despite evidence for phenotypic correlation of wing shape between males and females.     

Differentiation at a microgeographical scale within two species of ground beetle, Carabus auronitens and C. nemoralis (Coleoptera, Carabidae): a geometrical morphometric approach

New morphometric methods, the geometrical morphometrics, offer promising perspectives to appraise morphological variation among organisms and open up, to a large extent, the field of morphometrics for the study of systematics and evolution. Until now, however, few studies have explored the potential of these methods at a microgeographical scale. In the present work, we applied them to quantify morphological (size and shape) differentiation among populations of two forest species of ground beetles: Carabus auronitens and C. nemoralis . We found a significant shape variation among sites, as well as among sexes, for both species. Additionally, for C. auronitens , we found significant positive correlations in both sexes between morphological (shape) and geographical distances between populations. In contrast, significant size differences were found between sexes, but not between sites. We conclude that geometrical morphometric methods provide valuable tools for the study of morphological variation among populations and therefore offer, on the whole, interesting perspectives for the study of biodiversity patterns.     

Relationship between body size and flying-related structures in Neotropical social wasps (Polistinae,Vespidae, Hymenoptera)

Body size influences wing shape and associated muscles in flying animals which is a conspicuous phenomenon in insects, given their wide range in body size. Despite the significance of this, to date, no detailed study has been conducted across a group of species with similar biology allowing a look at specific relationship between body size and flying structures. Neotropical social vespids are a model group to study this problem as they are strong predators that rely heavily on flight while exhibiting a wide range in body size. In this paper we describe the variation in both wing shape, as wing planform, and mesosoma muscle size along the body size gradient of the Neotropical social wasps and discuss the potential factors affecting these changes. Analyses of 56 species were conducted using geometric morphometrics for the wings and lineal morphometrics for the body; independent contrast method regressions were used to correct for the phylogenetic effect. Smaller vespid species exhibit rounded wings, veins that are more concentrated in the proximal region, larger stigmata and the mesosoma is proportionally larger than in larger species. Meanwhile, larger species have more elongated wings, more distally extended venation, smaller stigmata and a proportionally smaller mesosoma. The differences in wing shape and other traits could be related to differences in flight demands caused by smaller and larger body sizes. Species around the extremes of body size distribution may invest more in flight muscle mass than species of intermediate sizes.     

Geographic phenetic variation of two eastern-Mediterranean non-commensal mouse species, Mus macedonicus and M. cypriacus (Rodentia: Muridae) based on traditional and geometric approaches to morphometrics

We tested the hypothesis that skull shape within the genus Mus may vary with geographic location by assessing the extent and spatial distribution of phenotypic skull variation within and among two wild mouse species, M. macedonicus and M. cypriacus, using traditional and geometric morphometrics including a rather novel application of sliding semilandmarks. Shape was shown to be significantly correlated both with longitude and latitude in M. macedonicus, yet the correlation between morphometric and geographic distances was not significant, and morphometric differences between Asian and European populations were not higher than those within the particular continents. The phylogenetic signal was found to be stronger in dental characters than in cranial ones, however, overall concordance between the pattern of morphometric variation and the presumed history of M. macedonicus was rather weak. In both species, the dorsal and ventral sides of the skull were shown to covary in many aspects though there were also some differences between them, making the functional interpretation of these differences difficult. Discrimination between M. cypriacus and M. macedonicus as well as discrimination between two M. macedonicus subspecies was highly reliable using both traditional and geometric morphometric tools to analyze skull measurements.     

Wing geometry of Phlebotomus stantoni and Sergentomyia hodgsoni from different geographical locations in Thailand

Geographic populations of the two main sandflies genera present in Thailand were studied for species and population identification. Size and shape of Phlebotomus stantoni and Sergentomyia hodgsoni from different island and mainland locations were examined by landmark-based geometric morphometrics. Intraspecific and interspecific wing comparison was carried out based on 12 anatomical landmarks. The wing centroid size of P. stantoni was generally larger than that of S. hodgsoni. Within both species, wings from the continent were significantly larger than those from island populations. Size variation could be significant between geographic locations, but could also overlap between genera. The wing venation geometry showed non-overlapping differences between two species. The within-species variation of geometric shape between different geographical locations was highly significant, but it could not interfere with the interspecies difference. The lack of species overlapping in shape, and the high discrimination between geographic populations, make geometric shape a promising character for future taxonomic and epidemiological studies.     

Delimitation difficulties in species splits: a morphometric case study on the Euxoa tritici complex (Lepidoptera, Noctuidae)

Abstract. Genital characteristics tend to vary greatly between Lepidoptera species, providing helpful features for species delimitation. The differences between species are usually remarkable and suspicions about species identity never arise. However, fairly often, and possibly increasingly, taxa are elevated to species rank on the basis of very slight morphological differences, often without quantitative support. Euxoa tritici (Linnaeus) is a typical example of a variable species split into several morphologically similar species. The present study tested whether the diagnostic genital characters of the current classification, based on nonquantitative methodology, provide safe identification of species. Both traditional distance morphometrics as well as modern geometric morphometrics, which also enables quantitative shape exploration, were used. Moreover, whether the study specimens can be unambiguously categorized into several species with visual comparisons was tested independently using four specialist entomologists. Genital types of several named species as well as considerable variation in genitalia were found, but no support was found for the presence of several morphologically distinguishable species with quantitative morphometric analyses. Neither were study specimens categorized unambiguously by specialists. The results suggest that pure visual comparisons may lead to unsound taxonomic conclusions and that a quantitative approach in critical cases should be used more frequently.