Are geometric morphometric analyses replicable? Evaluating landmark measurement error and its impact on extant and fossil Microtus classification

Geometric morphometric analyses are frequently employed to quantify biological shape and shape variation. Despite the popularity of this technique, quantification of measurement error in geometric morphometric datasets and its impact on statistical results is seldom assessed in the literature. Here, we evaluate error on 2D landmark coordinate configurations of the lower first molar of five North American Microtus (vole) species. We acquired data from the same specimens several times to quantify error from four data acquisition sources: specimen presentation, imaging devices, interobserver variation, and intraobserver variation. We then evaluated the impact of those errors on linear discriminant analysis‐based classifications of the five species using recent specimens of known species affinity and fossil specimens of unknown species affinity. Results indicate that data acquisition error can be substantial, sometimes explaining >30% of the total variation among datasets. Comparisons of datasets digitized by different individuals exhibit the greatest discrepancies in landmark precision, and comparison of datasets photographed from different presentation angles yields the greatest discrepancies in species classification results. All error sources impact statistical classification to some extent. For example, no two landmark dataset replicates exhibit the same predicted group memberships of recent or fossil specimens. Our findings emphasize the need to mitigate error as much as possible during geometric morphometric data collection. Though the impact of measurement error on statistical fidelity is likely analysis‐specific, we recommend that all geometric morphometric studies standardize specimen imaging equipment, specimen presentations (if analyses are 2D), and landmark digitizers to reduce error and subsequent analytical misinterpretations.     

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.     

Use of shape to distinguish Chamelea gallina and Chamelea striatula (Bivalvia: Veneridae): Linear and geometric morphometric methods

The commercially fished striped venus clams Chamelea gallina and C. striatula (Bivalvia: Veneridae) are difficult to distinguish by inexperienced observers and the taxonomy of these species is still an issue of discussion. The differences in shape between C. gallina and C. striatula from Portuguese coastal waters were studied through conventional linear and geometric morphometric analysis, using both contour (elliptic Fourier analysis) and landmark-based methods. The relationships shell length vs. height, width, and total weight were significantly different between species. However, because there was a considerable overlap in the data sets, the species could not be distinguished using any combination of those linear measurements. Geometric morphometric methods provided shape variables that led to 0-6% misclassification rates between species; linear morphometric measures led to 16.8% error. Contour analysis revealed differences primarily in the shell umbo and lunular area. The umbo was more "sharp" and the lunula less pronounced in C. striatula than in C. gallina. Generalized procrustes superimposition (landmark analysis) showed that the main differences between species reside in the length of the pallial sinus. Thus, an index was developed (PI: Pallial Index = pallial sinus length/shell length), which was successfully used to separate the species (with 100% correct classification), i.e., specimens with PI lower than 0.119 belonged to C. gallina, whereas greater PI values were attributed to C. striatula. The use of these geometric morphometric methods allowed the detection of differences in shape between these two species and to develop an easy-to-use identification index. We encourage the development of analogous indices that apply the methods of geometric morphometrics to distinguish between other species whose identification is complicated.     

Geometric craniometric analysis of sexual dimorphism and ontogenetic variation: A case study based on two geographically disparate species, Aethomys ineptus from southern Africa and Arvicanthis niloticus from Sudan (Rodentia: Muridae)

Non-geographic morphometric variation, particularly at the level of sexual dimorphism and ontogenetic (age-related) variation, has been documented in rodents, and useful for establishing whether to analyse sexes separately or together, and for selecting adult specimens for subsequent data recording and analysis. However, such studies have largely been based on traditional morphometric analyses of linear measurements that mainly focus on overall size, rather than shape-related morphometric variation. Unit-free, landmark/outline-based geometric morphometric analyses are considered to offer a more appropriate tool for assessing shape-related morphometric variation. In this study, we used geometric cranial morphometric analysis to assess the nature and extent of sexual dimorphism and age variation within the Tete veld rat, Aethomys ineptus (Thomas and Wroughton, 1908) from southern Africa and the African Nile rat, Arvicanthis niloticus (Desmarest, 1822) from Sudan. The results obtained were in turn compared with previously published results based on independent geometric and traditional cranial morphometric data from the same sampled populations examined in the present study. While our geometric morphometric results detected statistically significant sexual dimorphism in cranial shape within Ar. niloticus only, previously published results based on traditional morphometric data failed to detect significant sexual dimorphism within this species. However, similar to previously published traditional morphometric data, our geometric morphometric results detected statistically significant age-related variation in cranial shape and size within both Ae. ineptus and Ar. niloticus, with individuals of age classes 5 and 6 being considered to represent adult specimens. Our results highlight the importance of carefully evaluating both size- and shape-related non-geographic morphometric variation prior to the analysis of geographic variation and the delineation of species. Erroneous conclusions of non-geographic variation may have implications in the interpretation of geographic and evolutionary processes that may be responsible for morphological differences at both the inter- and intra-specific levels.     

Geometric morphometric analyses of leaf variation in four oak species of the subgenus Quercus (Fagaceae)

Abstract

This paper defines a protocol for the study of leaf morphology and morphometric relationships among Quercus frainetto, Quercus robur, Quercus petraea and Quercus pubescens. Leaf shapes were investigated in an attempt to characterize and discriminate among these four species. Landmark and outline data were compared in order to ascertain their relative efficacy for describing variation in leaf morphology. MANOVA was performed to assess intra‐specific differentiation and to compare the distribution of variance in individuals, populations and species. A strong correlation was found between landmark and outline datasets of the four species: both methods proved capable of discriminating among the four species. Finally, a Two‐Block Partial Least‐Squares analysis was performed for each species in order to assess the correlation between intra‐specific shape variation and environmental factors.     

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.     

Dealing with allometry in linear and geometric morphometrics: a taxonomic case study in the Leporinus cylindriformis group (Characiformes: Anostomidae) with description of a new species from Suriname

To achieve maximum efficacy, taxonomic studies that seek to distinguish amongst species must first account for allometric shape variation within species. Two recently developed software packages (SMATR and MorphoJ) offer regression‐based allometric approaches that are notable for their statistical power and ease of use and that may prove highly useful to taxonomists working with linear or geometric morphometric data. We investigate species delimitation of the slender‐bodied fishes in the Leporinus cylindriformis group using these programs and demonstrate the utility of the allometric corrections that they provide. Without allometric correction, many pairs of species are difficult to distinguish on the basis of morphometrics, but once regressions are used to account for marked allometric variation within species, most of the recognized species in this group can be readily distinguished with linear or geometric morphometrics, particularly using variation in the depth of the body. Both approaches returned congruent patterns of separation amongst putative species, but the geometric approach in MorphoJ distinguished amongst four more pairs of species than did the linear approach in SMATR and appears to provide slightly more statistical power. Based on distinctive morphometrics, meristics, and coloration, a highly elongate species of Leporinus from the Suriname, Corantijn, and Coppename rivers of Suriname is described herein as a new species, Leporinus apollo sp. nov. The unique L. cylindriformis holotype from Porto de Moz, Brazil differs in morphology, meristics, and pigmentation from specimens commonly referred to that species from the main basin of the Amazon; the latter specimens may represent an additional undescribed species. The L. cylindriformis holotype itself may represent a rare species or a specimen collected at the edge of its native range. Measurements of the holotype and paratype of Leporinus niceforoi, which were collected in the Amazonian slope of Colombia, differ substantially from similarly pigmented and putatively conspecific specimens from Amazonian portions of Ecuador and Peru. Recently collected specimens from Colombia are needed to determine whether the observed morphometric variation encompassed by the current concept of L. niceforoi indicates a morphocline within a single species, suggests the presence of multiple cryptic species, or results from shrinkage of the types. In all these cases, linear or geometric morphometric data can reliably differentiate amongst species, but only after one accounts for allometric shape variation. The new SMATR and MorphoJ software packages both offer easy and effective approaches to such allometrically informed taxonomy, and may prove useful to any systematist working on taxa that change shape as they grow. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 162 , 103–130.     

Leaf morphological analyses in four European oak species (Quercus) and their hybrids: A comparison of traditional and geometric morphometric methods

Abstract

In this study, leaf morphology was assessed in a mixed oak stand (western France) using two geometric morphometric (landmark and outline) datasets and one dataset of 19 leaf measures. Adult oaks (817 oaks), comprising four white oak species (Quercus petraea, Q. robur, Q. pubescens and Q. pyrenaica), were sampled for DNA extraction and genetic analysis (nuclear microsatellites). Leaf morphology was assessed on 336 oaks, comprising pure species and hybrids as determined by genetic assignment. This comparative study of oak leaf morphology, based on the use of two free size geometric morphometric methods and a set of leaf measurements, combined with the genetic assignment of individuals to pure species or hybrids, provided information about the differences among species and the intermediate leaf morphology of their hybrids.     

A morphometric and genetic framework for the genus Gazella de Blainville, 1816 (Ruminantia: Bovidae) with special focus on Arabian and Levantine mountain gazelles

Gazella is one of the most species‐rich genera within horned ruminants. Despite overall similarity in body size and morphology, gazelles show variability in coloration and horn morphology. Unfortunately, however, species differentiation based on these characters, or on discrete skull characters, is very difficult due to high intraspecific variability. Furthermore, most species have fragmented and allopatric distributions, so that species boundaries were hard to define in the past. Mitochondrial DNA sequences have proven useful for investigating gazelle taxonomy in recent years, but especially for old museum material, i.e. type specimens, destructive sampling is often impossible. We provide a comprehensive morphometric framework for the genus Gazella based on linear skull measurements reconciled with results from molecular phylogenetic analysis based on the largest dataset available so far. In particular for males, the skull morphology shows interspecific differences concurrent with DNA data and provides a reliable tool for species identification. Based on morphometric data we synonymize G. karamii with G. marica, and confirm the identification of the G. arabica and G. a. rueppelli type skulls from analyses of mitochondrial DNA sequences. © 2013 The Linnean Society of London     

Inter- and intraspecific variation in grass phytolith shape and size: a geometric morphometrics perspective

Background and AimsThe relative contributions of inter- and intraspecific variation to phytolith shape and size have only been investigated in a limited number of studies. However, a detailed understanding of phytolith variation patterns among populations or even within a single plant specimen is of key importance for the correct taxonomic identification of grass taxa in fossil samples and for the reconstruction of vegetation and environmental conditions in the past. In this study, we used geometric morphometric analysis for the quantification of different sources of phytolith shape and size variation.MethodsWe used landmark-based geometric morphometric methods for the analysis of phytolith shapes in two extant grass species (Brachypodium pinnatum and B. sylvaticum). For each species, 1200 phytoliths were analysed from 12 leaves originating from six plants growing in three populations. Phytolith shape and size data were subjected to multivariate Procrustes analysis of variance (ANOVA), multivariate regression, principal component analysis and linear discriminant analysis.Key ResultsInterspecific variation largely outweighed intraspecific variation with respect to phytolith shape. Individual phytolith shapes were classified with 83 % accuracy into their respective species. Conversely, variation in phytolith shapes within species but among populations, possibly related to environmental heterogeneity, was comparatively low.ConclusionsOur results imply that phytolith shape relatively closely corresponds to the taxonomic identity of closely related grass species. Moreover, our methodological approach, applied here in phytolith analysis for the first time, enabled the quantification and separation of variation that is not related to species discrimination. Our findings strengthen the role of grass phytoliths in the reconstruction of past vegetation dynamics.     

Morphometric analysis of inflorescence phytoliths produced byAvena sativa L. andAvena strigos schreb

Morphometric analysis, the study of measurements of size and shape, has the potential to be an effective tool for phytolith analysis. This study reports the first attempt to apply the methodology to oats. In particular, this study was designed to determine if morphometric analysis could adequately discriminate between phytoliths produced in the inflorescence bracts of two species of oats, Avena sativa L. and Avena strigosa Schreb. Results indicate that while the taxa produce the same types of phytoliths, those phytoliths have significantly different measurements of size and shape. This suggests the technique has the potential to become a powerful research tool for investigators working in the wide variety of disciplines that utilize phytolith analysis.     

Wing shape as a potential discriminator of morphologically similar pest taxa within the Bactrocera dorsalis species complex (Diptera: Tephritidae)

Four morphologically cryptic species of the Bactrocera dorsalis fruit fly complex (B. dorsalis s.s., B. papayae, B. carambolae and B. philippinensis) are serious agricultural pests. As they are difficult to diagnose using traditional taxonomic techniques, we examined the potential for geometric morphometric analysis of wing size and shape to discriminate between them. Fifteen wing landmarks generated size and shape data for 245 specimens for subsequent comparisons among three geographically distinct samples of each species. Intraspecific wing size was significantly different within samples of B. carambolae and B. dorsalis s.s. but not within samples of B. papayae or B. philippinensis. Although B. papayae had the smallest wings (average centroid size=6.002 mm±0.061 SE) and B. dorsalis s.s. the largest (6.349 mm±0.066 SE), interspecific wing size comparisons were generally non-informative and incapable of discriminating species. Contrary to the wing size data, canonical variate analysis based on wing shape data discriminated all species with a relatively high degree of accuracy; individuals were correctly reassigned to their respective species on average 93.27% of the time. A single sample group of B. carambolae from locality 'TN Malaysia' was the only sample to be considerably different from its conspecific groups with regards to both wing size and wing shape. This sample was subsequently deemed to have been originally misidentified and likely represents an undescribed species. We demonstrate that geometric morphometric techniques analysing wing shape represent a promising approach for discriminating between morphologically cryptic taxa of the B. dorsalis species complex.     

Morphometric analyses of Staurosira inflata comb. nov. (Bacillariophyceae) and the morphologically related Staurosira tabellaria from north-western Russia

The morphology and ontogenetic allometric trends of a rare diatom Fragilaria heidenii Østrup and the morphologically related Staurosira tabellaria (W. Smith) Leuduger-Fortmorel were compared using conventional and semilandmark-based geometric morphometric analyses. Fragilaria heidenii was studied in detail by light and electron microscopy using type material and recent samples from Lake Ladoga and Lake Ilmen (north-western Russia). The taxon is transferred into the genus Staurosira Ehrenberg as Staurosira inflata comb. nov. on the basis of its valve morphology. This taxon is characterized by the absence of rimoportulae, lack of perforated copulae, spines located on the interstriae, internal vola occlusion in the areolae and the features of the areolae and apical pore fields. Conventional morphometric analysis showed considerable overlapping of S. inflata and S. tabellaria in their frustule characteristics such as length, width, length-to-width ratio and striae density. Moreover, at later stages of the vegetative life cycle, S. inflata has a tendency to resemble S. tabellaria by its valve outline that makes it difficult to separate these two taxa. The geometric morphometric analysis revealed two shape groups corresponding to S. inflata and S. tabellaria that were separated by a clear gap. Semilandmarks representing shape of the middle part of the valve were primarily responsible for discrimination between species. Apart from differences in valve shapes, S. inflata and S. tabellaria also differed significantly in their ontogenetic allometric trajectories. Overall, our results demonstrate that the semilandmark-based geometric morphometrics is sensitive enough to distinguish species by their outlines, when traditional morphometric parameters are not able to discriminate them with confidence.     

Geographic distribution and phenetic skull variation in two close species of Graomys (Rodentia, Cricetidae, Sigmodontinae)

The interspecific differentiation of South American rodents of the genus Graomys was assayed at ecological and morphometric levels in two species. At the ecological level, niche modelling was used. At the morphometric level, the hypothesis that the size and shape of the skull vary with the geographic location was tested using geometric morphometrics by assessing the extent and spatial distribution of phenotypic skull variation within and among two species, Graomys griseoflavus and Graomys chacoensis. Our results of niche modelling indicate the spatial differentiation between the two species, with G. chacoensis inhabiting preferably the Chaco ecoregion and G. griseoflavus inhabiting mainly the Monte ecoregion. In multiple linear regressions, approximately 20% of the skull size variation is explained by latitude, altitude, and temperature seasonality. The partial least square analysis reveals strong correlation between shape and environmental variables, mainly with latitude, annual mean temperature, and annual precipitation. Discrimination between G. griseoflavus and G. chacoensis was highly reliable when using geometric morphometric tools. These results permit us to elucidate some evolutionary processes that have occurred in these species.     

Combining geometric morphometrics with pattern recognition for the investigation of species complexes

The parasitoid Hymenoptera contain a large number of species complexes that are as yet unresolved by traditional taxonomic studies. Evolutionary studies as well as biological control programmes often require further investigations which cannot rely on simple qualitative morphological characters; in many cases, particularly with dried specimens housed in museums, molecular approaches cannot be used. Recent developments in geometric morphometrics and statistical exploratory approaches open new perspectives for the objective evaluation of morphological characters in this taxonomic context. In this study, geometric morphometrics and pattern recognition approaches were applied to the wing shape and venation of two closely related braconid species considered to differ by subtle qualitative morphological head characters. Exploratory analyses such as kernel density estimates and Gaussian mixture analyses were used to explore the structure of the data in the multivariate morphometric space. Discrimination techniques (linear discriminant functions and neural networks combined with cross‐validations) were used to estimate the taxonomic value of qualitative characters. Gaussian mixtures highlighted the existence of two non‐overlapping groups. A good congruence was found between one of the two groups and the a priori defined Bassus tumidulus. The misclassification rate was higher for B. tegularis specimens, which also appeared morphometrically heterogeneous. Discrimination between the two a priori defined species was incomplete with misclassification rates higher than, or equal to, 6%. In most cases, the lack of congruence between species and morphometrically defined subgroups could be related to specimens that exhibited ambiguous qualitative character states. In summary, if two entities are present, they still need to be defined morphologically, while B. tegularis heterogeneity calls for further investigation of specimens of known origin and hosts. © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 89–98.     

Learning to see the wood for the trees: machine learning,decision trees,and the classification of isolated theropod teeth

Taxonomic identification of fossils based on morphometric data traditionally relies on the use of standard linear models to classify such data. Machine learning and decision trees offer powerful alternative approaches to this problem but are not widely used in palaeontology. Here, we apply these techniques to published morphometric data of isolated theropod teeth in order to explore their utility in tackling taxonomic problems. We chose two published datasets consisting of 886 teeth from 14 taxa and 3020 teeth from 17 taxa, respectively, each with five morphometric variables per tooth. We also explored the effects that missing data have on the final classification accuracy. Our results suggest that machine learning and decision trees yield superior classification results over a wide range of data permutations, with decision trees achieving accuracies of 96% in classifying test data in some cases. Missing data or attempts to generate synthetic data to overcome missing data seriously degrade all classifiers predictive accuracy. The results of our analyses also indicate that using ensemble classifiers combining different classification techniques and the examination of posterior probabilities is a useful aid in checking final class assignments. The application of such techniques to isolated theropod teeth demonstrate that simple morphometric data can be used to yield statistically robust taxonomic classifications and that lower classification accuracy is more likely to reflect preservational limitations of the data or poor application of the methods.     

Ontogeny, phylogeny, and morphology in anuran larvae: morphometric analysis of cranial development and evolution in Rana tadpoles (Anura: Ranidae)

Comparative studies of chondrocranial morphology in larval anurans are typically qualitative in nature, focusing primarily on discrete variation or gross differences in the size or shape of individual structures. Detailed data on chondrocranial allometry are currently limited to only two species, Rana sylvatica and Bufo americanus. This study uses geometric morphometric and multivariate statistical analyses to examine interspecific variation in both larval chondrocranial shape and patterns of ontogenetic allometry among six species of Rana. Variation is interpreted within the context of hypothesized phylogenetic relationships among these species. Canonical variates analyses of geometric morphometric datasets indicate that species can be clearly discriminated based on chondrocranial shape, even when whole ontogenies are included in the analysis. Ordinations and cluster analyses based on chondrocranial shape data indicate the presence of three primary groupings (R. sylvatica; R. catesbeiana + R. clamitans; and R. palustris + R. pipiens + R. sphenocephala), and patterns of similarity closely reflect phylogenetic relationships. Analysis of chondrocranial allometry reveals that some patterns are conserved across all species (e.g., most measurements scale with negative allometry, those associated with the posterior palatoquadrate tend to scale with isometry or positive allometry). Ontogenetic scaling along similar allometric trajectories, lateral transpositions of individual trajectories, and variable allometric relationships all contribute to shape differences among species. Overall patterns of similarity among ontogenetic trajectories also strongly reflect phylogenetic relationships. Thus, this study demonstrates a tight link between ontogeny, phylogeny, and morphology, and highlights the importance of including both ontogenetic and phylogenetic data in studies of chondrocranial evolution in larval anurans.