Landmark-based Geometric Morphometrics Research Articles

Deciphering species-group taxonomic complexity of common, Barbodes binotatus and saddle barbs, B. banksi in Peninsular Malaysia

Correct assessment of freshwater fishes depends on the most recent taxonomic nomenclature and accurate identification. The existing of the complex between closely related species is one of the prominent issues that sometimes poorly addressed that leads to misidentification of the species and inaccurate diversity measurement. This study addressed the issue through morphometrics methods, to correctly evaluate the diversity of freshwater fish within species group of genus Barbodes , i.e., B. binotatus and B. banksi . In examining species complex of B. binotatus and B. banksi , traditional and landmark-based geometric morphometrics methods were applied. Traditional morphometric displayed characters that distinguished both species were generally located at the anterior part of the body, and specifically in the cephalic area of the fish. Landmark-based geometric morphometric revealed a highly similar body shape on both species. Variations within both species were subtle and could not be significantly distinguished by both methods. The overall outcomes of this study suggest that in order to achieve a proper assessment to freshwater fishes, one need to solve the identification problems in species complex. This effort could consequently lead to a better understanding of freshwater fish status, for a better conservation plan and management.

Symmetric and Asymmetric Components of Shape Variation in the Diatom Genus Frustulia (Bacillariophyta)

Irregularities in cell division can produce asymmetry in symmetric structures, such as outlines of diatom cells, which can reflect genetic, environmental, or random variability in developmental processes. This study examined 12 phylogenetic lineages of the diatom genus Frustulia using landmark-based geometric morphometrics to assess the variation between cell segments separated by apical and transapical axes. Although asymmetric variation within cells differed in some lineages, these irregularities most likely did not reflect the evolutionary history of the lineages. The intraclonal phenotypic plasticity of diatom frustules was induced rather by nongenetic factors, i.e., inherited valve abnormalities, constraints of siliceous cell walls, and random developmental instability during morphogenesis. The positive correlations between the symmetric and asymmetric components of shape variation indicated that the morphogenesis of diatom cell walls affected irregularities within cells and variability among the symmetrized cells to a similar extent.

Exposure to 17β estradiol causes erosion of sexual dimorphism in Bluegill (Lepomis macrochirus).

Estrogenic compounds including 17β estradiol (or E2) are known to negatively affect the reproductive system of many animals, including fish, leading to feminization, altered sex ratio, reduced fecundity, and decreased gonadosomatic index. The objective of this study was to evaluate the effects of varying concentrations of water-soluble 17β estradiol exposure on the external morphology of Bluegill. An experiment was set up where fish were individually maintained in 10-g tanks and exposed to 17β estradiol concentrations of 40 and 80 ng/L or no 17β estradiol exposure (the control). Fish were exposed for 21 days, with 17β estradiol replenished each week to account for 17β estradiol half-life. Fish were photographed laterally before and after the 21-day exposure to 17β estradiol. Landmark-based geometric morphometrics were conducted in MorphoJ and canonical variate and discriminant function analysis were used to compare the morphological changes in the fish under 17β estradiol exposure. The results showed that 17β estradiol exposure caused male dimorphic characters to change in Bluegill by becoming less prevalent. Specifically, there was a narrowing of the caudal peduncle, smaller nape protrusion, reduced opercular flap and pectoral fin, and a deeper body in the exposed groups compared with control fish under both concentrations. This research highlights the widespread effects of 17β estradiol on fish health beyond the reproductive system, which could have important conservation implications by affecting mate selection and reducing reproductive success.

Shape, condition and diet of the pike icefish Champsocephalus esox (Teleostei: Channichthyidae): evidence of phenotypic plasticity?

AbstractThe shape (derived from landmark-based geometric morphometrics), condition (Fulton index) and diet (determined through gut content analysis) were described for the pike icefish Champsocephalus esox (Channichthyidae) from Última Esperanza sound, south-west Patagonia, Chile. Based on the length-weight relationship, females were heavier at length than males. Nevertheless, the Fulton index was similar between males and females. The morphospace of C. esox showed high intraspecific variability in the dorsoventral position of the tip of the snout, anus and the ventral insertion of the pectoral fin, as well as the anteroposterior position of the premaxilla, opercle and anus. This indicates the existence of phenotypic plasticity, leading to specimens with larger jaws and heads but shorter trunks, or specimens with shorter jaws and heads but larger trunks. This phenotypic plasticity was independent of size and sex. The feeding incidence was similar between sexes (34.1% and 47.2% for males and females, respectively). Diets consisted of only fish, small notothenioids of the genus Patagonotothen (P. tessellata, P. cornucola and P. sima), showing similarities between males and females. Finally, C. esox is the second notothenioid species, and the first outside of Antarctica, to display phenotypic plasticity in its body shape.

Landmark-based geometric morphometrics as a tool for the characterization of biogeographically isolated populations of the pollinator scarab beetle Erioscelis emarginata (Coleoptera: Melolonthidae)

Erioscelis emarginata is a species of pollinator scarab beetle whose adults are predominantly associated with night-blooming aroid inflorescences (Araceae) of the Neotropical genus Thaumatophyllum, towards which they are attracted by volatile organic compounds (VOCs). It is assumed that olfactory preferences towards species-specific floral VOCs emitted by different plant hosts, as well as geographical segregation of natural populations of E. emarginata, might reinforce their reproductive isolation and differentiation, but external morphological dissimilarities between them are not evident. In the present work, body shapes of geographically isolated populations of E. emarginata associated with different species of Thaumatophyllum were compared using landmark-based geometric morphometrics. A total of 86 adult specimens of E. emarginata were analyzed, collected in inflorescences of Thaumatophyllum spp. at four locations in Brazil distant 333–912 km from one another. Eighteen landmarks and semi-landmarks were digitized to capture the shapes of the elytra and pronotum separately, and landmarks configurations were compared through Canonical Variates and pairwise Discriminant Function Analyses. There were significant shape differences between specimens from the four different populations of E. emarginata (p < 0.05) in all paired comparisons, for both the shape of the elytra and pronotum and correct classification levels were high for most pairwise comparisons. Further investigations on the morphological variation across these populations and assessments of the specificity of their olfactory-oriented relationship with host plants are important next steps in understanding the full scenario of the segregation and diversification of E. emarginata. In addition, a lectotype male of Apogonia emarginata (ZIN) was designated to fix the identity of E. emarginata.

Molecular Identification and Geometric Morphometric Analysis of Haematobosca aberrans (Diptera: Muscidae).

The genus Haematobosca Bezzi, 1907 (Diptera: Muscidae) contains haematophagous flies of veterinary importance. A new fly species of this genus was recognised from northern Thailand based on morphological characters and described as Haematobosca aberrans Pont, Duvallet & Changbunjong, 2020. In the present study, the mitochondrial cytochrome c oxidase I (COI) gene was used to confirm the morphological identification of H. aberrans. In addition, landmark-based geometric morphometrics was used to determine sexual dimorphism. The molecular analysis was conducted with 10 COI sequences. The results showed that all sequences were 100% identical. The sequence was not highly similar to reference sequences from GenBank and did not match any identified species from Barcode of Life Data Systems (BOLD). Phylogenetic analysis clearly differentiated this species from other species within the subfamily Stomoxyinae. For geometric morphometric analysis, a total of 16 wing pictures were analysed using the landmark-based approach. The results showed significant differences in wing shape between males and females, with a cross-validated classification score of 100%. The allometric analysis showed that wing shape has no correlation with size. Therefore, the COI gene is effective in species identification of H. aberrans, and geometric morphometrics is also effective in determining sexual dimorphism.

Landmark-based geometric morphometrics analysis of body shape variation among populations of the blue jack mackerel, Trachurus picturatus, from the North-East Atlantic

Phenotypic variation in fish can indicate exposure to different environmental conditions, affecting species growth and maturation rates and/or be a consequence of genetic factors that allow fish adaptation to different environments. Understanding population structure and dynamics is extremely important for the establishment of sustainable fisheries. The blue jack mackerel, Trachurus picturatus, is an important fishery resource of the North-East (NE) Atlantic. Fish were captured in six sampling locations in the NE Atlantic - Azores, Madeira, Canaries and Portugal mainland - Matosinhos, Peniche and Portimão. The phenotypic diversity of the species was studied using body landmark-based geometric morphometrics. A truss box method was followed and 30 distance variables were extracted from digital images. Additionally, the temporal stability of the morphological characters was investigated by comparing samples from two different years: 2016 and 2019. The transformed distances were analysed by univariate and multivariate statistics. The Macaronesia region – Azores, Madeira and Canaries – and Portugal mainland displayed a clear phenotypic pattern. Distances related to the body width and caudal peduncle were the most significant variables explaining the observed phenotypic heterogeneity. Furthermore, this study also showed that T. picturatus can exhibit fast phenotypic adaptations to the surrounding environment, observed by the discrimination of Peniche in 2019. Nevertheless, the high overall allocation of individuals to the original location observed for both years (78% in 2016 and 83% in 2019) suggests that the population of T. picturatus in the NE Atlantic is phenotypic heterogeneous. These findings corroborate previous studies and have implications for fishery management.

Reversible morphological changes in a juvenile marine fish after exposure to predatory alarm cues.

Chemical cues from predators induce a range of predator-induced morphological defences (PIMDs) observed across fish taxa. However, the mechanisms, consistency, direction and adaptive value of PIMDs are still poorly studied. Here, we have tested if predatory cues can induce changes in the body shape of the juvenile marine fish Sparus aurata reared under controlled conditions without the presence of predators by exposing individuals to the olfactory stimulus of a fish predator. We tested our hypothesis using a nested replicated before-after-control-impact experiment, including recovery (potential reversibility) after the cessation of the predator stimulus. Differences in the size-independent body shape were explored using landmark-based geometric morphometrics and revealed that, on average, individuals exposed to a predatory cue presented deeper bodies and longer caudal regions, according to our adaptive theoretical predictions. These average plastic responses were reversible after withdrawal of the stimulus and individuals returned to average body shapes. We, therefore, provide evidence supporting innate reversible PIMDs in marine naive fish reared under controlled conditions. The effects at the individual level, including fitness and the associated applied implications, deserve further research.

Wing morphometric variability of the malaria vector Anopheles (Cellia) epiroticus Linton et Harbach (Diptera: Culicidae) for the duration of the rainy season in coastal areas of Samut Songkhram, Thailand.

In Thailand, Anopheles (Cellia) epiroticus Linton et Harbach (Diptera: Culicidae) is the secondary vector of human malaria along coastal regions. While there are some studies of phenotypic variability and population structure of A. epiroticus, more information on morphological variation would enhance epidemiological understanding of medically important mosquito vectors. This research examined morphological variation at three different distances from coastlines of Samut Songkhram Province, Thailand, using landmark-based geometric morphometrics. Wing shape of A. epiroticus was significantly different in the area 0.2 km away from the sea compared to areas 2 and 4 km away from the sea (p < 0.05). Phenotypic variability in wing shape is associated with distance from the sea. Morphological variations in the area closest to the sea were most pronounced, showing a relationship between A. epiroticus and the ecosystem that affects wing geometry. These results provide important information to understand morphological variation of A. epiroticus in coastal areas.

Comparison of Wing, Ovipositor, and Cornus Morphologies between Sirex noctilio and Sirex nitobei Using Geometric Morphometrics.

Sirex noctilio F. (Hymenoptera: Siricidae) is an invasive woodwasp from Europe and North Africa. Globalization has led to an expanding global presence in pine forests. S. noctilio has been previously introduced outside of its native range and now co-occurs in trees with native S. nitobei Matsumura (first discovered in 2016). Damage to Pinus sylvestris var. mongolica Litv in northeast China can be attributed to two types of woodwasp. To distinguish the two species by the traditional taxonomic morphology, we mainly differentiate the color of the male’s abdomen and the female’s leg. There remains intraspecific variation like leg color in the delimitation of related genera or sibling species of Sirex woodwasps. In this study, we used landmark-based geometric morphometrics including principal component analysis, canonical variate analysis, thin-plate splines, and cluster analysis to analyze and compare the wings, ovipositors, and cornus of two woodwasps to ascertain whether this approach is reliable for taxonomic studies of this group. The results showed significant differences in forewing venation and the shapes of pits in the middle of ovipositors among the two species, whereas little difference in hindwings and cornus was observed. This study assists in clarifying the taxonomic uncertainties of Siricidae and lays a foundation for further studies of the interspecific relationships of the genus Sirex.

Ontogenetic and interspecific variation in skull morphology of two closely related species of toad, Bufo bufo and B. spinosus (Anura: Bufonidae)

Using micro-CT and 3D landmark-based geometric morphometrics, I investigated postmetamorphic shape variation in the skull of Bufo bufo and Bufo spinosus, two widespread European toad species with small phenotypic differences. Two ontogenetic series were compared, for a total of 58 individuals. They exhibited similar allometric growth patterns, characterised by cranial widening with relative shortening and dorsoventral compression. However, some interspecific shape divergence was observed, particularly among adults: a relatively shorter skull and a more dorsally extended snout distinguished B. spinosus from B. bufo. This disparity, which gives further support to species separation, can probably be ascribed to changes in the allometric trajectories, and seen in light of the evolutionary history of the two lineages.

Shell and appendages variability in two allopatric ostracod species seen through the light of molecular data

Ostracod crustaceans are among the most abundant microfossil animals. Understanding intra- and interspecific variability of their shell is of pivotal importance for the interpretation of paleontological data. In comparison to appendages, ostracod shell displays more intraspecific variability (in shape, size, and ornamentation), often as a response to environmental conditions. Shell variability has been studied with sophisticated methods, such as geometric morphometrics (GM), but the conspecificity of examined specimens and populations was never tested. In addition, there are no GM studies of appendages. We build on previously published high cytochrome c oxidase subunit I (COI) divergence rates among populations of a brackish water species, Ishizakiella miurensis (Hanai, 1957). With landmark-based GM analyses of its shell and appendages, and additional genetic markers (ITS, 28S, 18S), we test if the genetic variability is structured in morphospace. This approach is the core of integrative taxonomy paradigm which has been proposed to bring the gap between traditional taxonomy and other disciplines such as evolutionary biology. The results show that it is the shell shape, and not the shape of appendages, that mirrors the molecular phylogeny, and we describe a new species. Our results suggest that the landmark-based GM studies may be useful in paleontological datasets for closely related species delineation. We implement molecular clock and population statistics to discuss speciation processes and phylogeography of the two congeners in Korea and Japan.

Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) development by landmark-based geometric morphometrics of cephalopharyngeal skeleton: a preliminary assessment for forensic entomology application

BackgroundConsidering the practicality of geometric morphometrics which could discriminate insect species, this application was extended to the analysis of blow fly larval growth based on cephalopharyngeal skeleton. In forensic entomology, cephalopharyngeal skeleton plays a crucial role in species identification but the morphometric information of this part is scarce. In this study, Chrysomya megacephala (Fabricius, 1794) was reared in two study replicates in natural conditions and samplings were conducted at fixed daily intervals. Cephalopharyngeal skeletons were removed from larvae and mounted on glass slides. Images were obtained from the specimens; digitized and geometric morphometric analysis on C. megacephala cephalopharyngeal skeletons was performed with MorphoJ software based on the ordination of five landmarks. The assessments of this analysis were based on centroid size measurements, visualization on the landmarks displacements, classification of the relative landmarks by using canonical variate analysis, and ontogenetic allometry determination.FindingsCentroid size was strongly correlated with developmental time (p < 0.05) and significantly different between daily intervals (p < 0.05). Ontogenetic allometric effect based on multivariate regression on Procrustes coordinates and centroid size was significant (p < 0.0001), indicating that shape was influenced by growth (60.3%). Disposition occurred on all landmarks during development and was further discriminated based on age groups.ConclusionsOther than discriminating between species, geometric morphometrics was found to be practical to visualize larval growth based on cephalopharyngeal skeletons which can be useful in forensic entomology.

Morphological assessment raises the possibility of cryptic species within the Luristan newt, Neurergus kaiseri (Amphibia: Salamandridae)

One of the main challenges in the conservation of biodiversity is to overcome inadequate knowledge about species and their intra-specific diversity. In the present study, we attempted to assess morphological distinction of the two previously identified genetic clades within the Luristan newt (Neurergus kaiseri, Schmidt 1952) endemic to Iran, which is essential for its conservation planning. Signals of the morphological variation in N. kaiseri were evaluated using landmark-based geometric morphometrics of body shape and characters of osteological structures. Morphological approaches revealed consistent groupings within the species, confirming the presence of two distinct lineages (previously named as the northern and southern clades). The morphological and genetic data provide evidence for the possible co-existence of two species in N. kaiseri and we recommend assigning the newly recognised forms to the species level.

Genetic analysis of the mandible morphology in DDD.Cg-$$A^{y}$$/Sgn and C57BL/6J inbred mice

Quantitative trait loci (QTL) mapping analysis was performed for the mandible morphology in DDD.Cg-Ay/Sgn and C57BL/6J inbred mice. The size and shape of the mandible was analysed by landmark-based geometric morphometrics as the centroid size and principal components (PCs), respectively. The Ay allele at the agouti locus significantly reduced the mandible size in DDD/Sgn background, and substantially altered the mandible shape in both strain backgrounds. Single-QTL scans, by including the agouti locus genotype (Ay or non-Ay) as an additive covariate, identified three significant QTL for the centroid size on chromosomes 5, 6 and 17, along with four suggestive QTL on chromosomes 2, 12, 18 and 19. These QTLs explained 46.85% of the centroid size variation in F2 mice. When the F2Ay and F2 non-Ay mice were analysed separately, additional significant QTL were identified on chromosomes 12 and 15 in F2 non-Ay mice. Single-QTL scans also identified 15 significant QTL for the PC1, PC2 and PC3. When the agouti locus genotype was included as an interactive covariate, nine significant QTLs were identified. Unexpectedly, these agouti-interacting QTLs were identified for relatively minor PCs, for which no significant single-QTL were identified. Therefore, it was suggested that the alteration of the mandible shape in Ay mice was the consequence of interactions between the Ay allele and genes that themselves have relatively small phenotypic effect. Although further in vivo studies are required, we postulated Pkd1 as a possible candidate gene underlying QTL for the centroid size on chromosome 17.

Geometric Morphometrics Dismiss the Polymorphic Hydrocotyle quinqueloba (Araliaceae) from the Neotropics

Abstract—Hydrocotyle taxonomy is poorly resolved due the traditional assessment of leaf morphology that has imposed difficulties to species delimitation and prompted the recognition of several infraspecific taxa. Hydrocotyle quinqueloba comprises ten infraspecific taxa that differ mainly by the shape of their leaf blade. The species complex occurs in montane forests of the Andes and eastern Brazil, along the Atlantic Forest and Cerrado domains. However, the geographic distribution of the taxa under H. quinqueloba is poorly known, and their supposedly continuous morphological variation remains unverified. Here we analyze the morphological variation and documented correlated differences in geographic distribution of H. quinqueloba to assess whether they support the delimitation of distinct species or the recognition of infraspecific taxa. For this task, we applied landmark-based geometric morphometrics (GM) to leaves and traditional morphometrics to other structures of specimens of H. quinqueloba. Procrustes analysis of variance (Procrustes ANOVA) indicated that variation of leaf blade shape was weakly associated with geography and was mainly explained by taxa themselves. In contrast, variation of leaf blade size was significantly (p &lt; 0.001) associated with geography, but variation at the specimen level could account for it. Principal component analysis (PCA) of leaf blade shape indicated that taxa differed mainly by the angle and relative size between median and lateral primary veins, by the space between the petiole insertion and middle lobe’s sinuses, and by the width of the middle lobe’s base. Canonical variate analysis (CVA) indicated significant (p &lt; 0.01) differences among seven infraspecific taxa that formed groups defined by leaf blade shape. Linear discriminant analysis (LDA) of 11 morphological characters separated five groups of taxa, which displayed significant (p &lt; 0.001) differences among each other. Based on that, we propose an updated taxonomic treatment that restricts the circumscription of H. quinqueloba to plants from the Andes and accepts four species from eastern Brazil.

A Typology of Florida Fluted Points Using Landmark-based Geometric Morphometrics

ABSTRACTThis typology of Florida fluted points was created from 164 images of fluted-point bases using landmark-based geometric morphometrics (LGM). Three highly distinctive types were discriminated using the shapes of the point bases. LGM is a powerful method for discriminating shapes, so paying close attention to analytical details is crucial for meaningful analyses. In archaeology, LGM is in its relative infancy compared to its use in biology, and archaeologists have not settled on standard procedures for its use in artifact shape analysis. Several general issues in the use of LGM for artifact-shape discrimination are discussed and illustrated, especially the effects of sample size and the Pinocchio Effect, in which highly variable aspects of an artifact shape, such as the fluted point blade, can affect analyses in unintended ways. The choices made in creating this typology are discussed in detail and alternatives tested to show how choices can significantly change results and archaeological interpretations.

Sexual size and shape dimorphism in two ground beetle taxa, Carabus (Procrustes) coriaceus cerisyi and C. (Morphocarabus) kollari praecellens (Coleoptera: Carabidae) - A geometric morphometric approach

We investigated morphometric variation in size and shape of the head, pronotum, and abdomen between the taxa and sexes of two ground beetles, Carabus coriaceus cerisyi Dejean, 1826 and C. kollari praecellens Palliardi, 1825. These two taxa differ in overall size, and both of them are characterized by significant sexual size dimorphism. In many taxa, allometry, the relationship between changes in shape and changes in size, can be a major component of intra- and interspecific variation in body shape. In the present study, we applied landmark-based geometric morphometrics to explore allometric and non-allometric components of shape variation between the taxa and more importantly between sexes within the taxa. We were able to show that the differences of shape between the taxa cannot be explained by allometric changes, as allometry explains only a small amount of total shape variation between the taxa, which was expected due to the fact that the taxa belong to separate subgenera. Between the sexes, on the other hand, allometry contributes largely to the variation, particularly in abdomen shape. However, the differences of head and pronotum shape between the sexes cannot be entirely explained in terms of allometric scaling. Our results indicate that allometry contributes largely to differences of body shape between the sexes, while differences between the taxa are influenced by other factors and processes.

3d point cloud model of human bio form created by the application of geometric morphometrics and method of anatomical features: human tibia example

Morphometrics refers to the quantitative analysis of a biological form and it can be used to describe its shape. Common types of geometric morphometrics are Landmark-based Geometric Morphometrics which describe shape by using anatomical landmarks (e.g. points), and Outline-based geometric morphometrics which uses envelope curves to describe shape of the biological form (e.g. bone), and they are not absolutely exclusive. Geometric morphometrics can be used for the creation of statistical models which represent shape variation of specific bio form. In this paper, novel application of geometric morphometrics for the creation of personalized models of unique bio-forms, i.e. models which are created for the specific patient is presented. Personalized model is defined as 3D point cloud model of biological form (in this case human tibia). Positions of points in 3D space are determined by using set of parametric functions defined by applying geometrical morphometrics, morphology properties and statistical analysis on the input set of human tibia samples. By using this technique, anatomically correct and geometrically accurate personalized models of bio forms can be created and used in pre, intra, and post-operative procedures in clinical practice.

The cultural trajectories of Aurignacian osseous projectile points in Southern Europe: Insights from geometric morphometrics

Studies on European population dynamics during the Early Upper Palaeolithic heavily rely on evidence gathered from the analysis of material culture. Spatial occurrence of an artefact type allows to delimit the territories occupied by their bearers, and, when a chronological perspective is added to the study, to follow shifts in land occupation through time. In this paper, we review first-hand data gathered from 129 Aurignacian osseous projectile points yielded by 11 sites that are distributed along the southern dispersal route assumed to have facilitated the initial dispersal of Homo sapiens into Western Europe, i.e., the Mediterranean coastline. In this region, clear differences are observed with regard to the relative distribution of split- and massive-based points. The Adriatic Sea is identified as the geographical limit dividing the western and eastern regions of the Mediterranean coastline. In both regions, massive-based points occur contemporaneously with split-based points at some sites. In the western region, split-based points appear at the end of the Proto-Aurignacian, and overall outnumber massive-based points. In the eastern region, split-based points are associated with the end of Early Aurignacian occupations, and it is massive-based points that are found in greater number. The geographic distribution of the data obtained from a landmark-based geometric morphometrics analysis of the forms for both projectile point types suggests the high mobility of Aurignacian populations. However, the rules of production seem more flexible for the manufacture of massive-based points compared to those reproduced during the manufacture of split-based points. A parallel is drawn between this pattern and the complexity of the reduction sequence of both types. These results cast doubts on the hypothesis according to which Aurignacian osseous projectile points, and more specifically split-based points, would represent a proxy for the initial dispersal of Homo sapiens into Europe. On the contrary, the present review indicates the adoption of osseous projectile technologies at a continental scale signals the development of novel socio-economic strategies by groups already present on the continent and linked to one another.