A mathematical landmark-based method for measuring worn molars in hominoid systematics

Materials

We selected 110 occlusal-view 2D images of lower second molars (LM2) to represent five species (eight subspecies) of extant hominoids: Gorilla beringei beringei (n = 10), G. b. graueri (n = 10), G. gorilla gorilla (n = 20), Pan troglodytes verus (n = 10), P. t. troglodytes (n = 10), P. t. schweinfurthii (n = 10), P. paniscus (n = 20) and modern Homo sapiens (n = 20, of which 8 were selected to represent hunter-gatherer groups and 12 to represent groups with other subsistence strategies). The photographs relating to Pan and Gorilla were randomly selected from the images used by Pilbrow (2003), Pilbrow (2006), Pilbrow (2007) and Pilbrow (2010), and those relating to Homo sapiens were randomly selected from the images used by Dykes (2018). The selected samples were equally balanced between males and females to ensure that variation from sexual dimorphism was represented and chosen from geographically-diverse populations to represent inter-population variation. All teeth included in this study come from previous studies and were at minimal stages of wear, with either no dentine exposed, dentine exposed as points on cusp tips, or dentine exposed as small pits on cusp tips (Pilbrow, 2003). The reason for utilizing relatively unworn teeth in this study is to allow for the methodology to be compared for accuracy with existing methodologies, which are reliant on identifying anatomical landmarks. Nevertheless, it is important to reiterate that the landmarking method presented here remains valid for both worn and unworn teeth, because the landmarks atop the surface of the enamel are calculated identically, whether or not occlusal surface features still remain visible. This holds true of most stages of advanced tooth wear, provided that the perimeter wall (the occlusal outline in each image) is still intact with cusp intersections along the perimeter edge visible. A list of images used is summarized in Table 1.

Image processing

All photographs were taken by the authors, using identical methodology, as described in Bailey, Pilbrow & Wood (2004), Pilbrow (2006), Pilbrow (2010), and Dykes (2014) and Dykes (2018). Images were then processed using GIMP^® (the freeware equivalent of Adobe Photoshop^®) as left-side teeth (right-side teeth were mirrored if necessary, in keeping with other concurrent research projects) with the mesial side of the tooth to the left of the image, the distal side to the right, the lingual side to the top of the image and the buccal side to the bottom. The mesial edge and the mesiodistal groove of the tooth in normal rotation (Goose, 1963) provides a guideline for the longitudinal axis of the tooth (Wood, 1991; Benazzi et al., 2012), and this is oriented horizontally on screen. Corrections of any interstitial wear or slight damage to the perimeter outline of the tooth are carried out digitally in Adobe Illustrator^® following the methodology of Wood & Abbott (1983). Thereafter, the rectangle tool was used to superimpose a bounding box around the perimeter of the molar, to stand proxy for the corrected mesiodistal (MD) and maximum buccolingual diameter (BL) measurements, the latter being at right angles to the MD diameter (Wood & Abbott, 1983). The bounding box can be seen in Fig. 1.

Landmarking

In total, 29 landmarks were chosen to represent the general dimensions, key points around the occlusal perimeter and the cusp arrangements of the tooth. The landmarks are depicted in Fig. 1 and described in Table 2. These landmarks also allowed for easily-interpretable wireframes to be produced for the analysis of relative warps in a Principal Components Analysis (PCA), and to characterize consensus molar shapes of the groups. Measurements taken between landmarks were also adaptable for further discriminant function and other statistical analyses. The first landmark (1) was placed at the geometric center of the tooth as calculated from the bounding box. Four further landmarks (2, 3, 4, 5) were placed around the perimeter of the box to mark the corrected mesiodistal and the maximum buccolingual diameter of the tooth. The next five landmarks (6, 7, 8, 9, 10) are Type I anatomical landmarks positioned at the points where the grooves between the cusps intersect with the perimeter of the tooth. These are the only Type I landmarks used in this study. Five additional landmarks (11, 12, 13, 14, 15) were placed at the midpoints of the lines connecting the anatomical landmarks, 6 –10. These helped to provide a general orientation of each cusp. The next five landmarks (16, 17, 18, 19, 20) were placed at the edge of the crown to mark the center point of each cusp arc around the perimeter. These were identified by drawing straight lines from the bounding box center (from landmark 1) to the edge of the crown while bisecting the lines connecting the Type I landmarks. Thus, a line from the center passed through landmark points 11–15 to reach the perimeter of the crown and provide a landmark point. The midpoints of the lines from landmark 1 to the peripheral landmarks 16 –20 themselves formed an additional five landmarks (21–25), which were used to stand proxy as the mathematical center of each cusp. Finally, the mathematically-derived centers of the mesial and distal cusps, respectively, metaconid and protoconid, and entoconid and hypoconid, were used in extending lines towards the lingual and buccal edge of the crown and provide four additional peripheral landmarks (26–29). These helped to provide an orientation of the mesial and distal cusps relative to the longitudinal axis of the crown. In all, 14 landmarks were used to provide a wireframe outline shape of the tooth: five pinpointing the cusp intersections, five marking the mathematically-derived centers of each cusp arc and four locating the orientations of the mesial and distal cusps relative to the longitudinal orientation of the crown. Features on the surface of the crown were captured in wireframes by two polygons: an outer polygon joining the five cusp intersections at the periphery, and an inner polygon formed by the five mathematical midpoints of each cusp. Landmark 1 is identified in the wireframe via the inclusion of the MD and BL diameters, which intersect at the center of the tooth in the occlusal basin of the tooth.

Landmarking was carried out using ImageJ^® freeware, which has the capacity to scale images and which has a line segment tool that shows the midpoint of lines traced onto the image and a “blob” tool to mark these midpoints with a colored dot. The landmark placement tool outputs the x and y coordinates of landmarks after they have been sited, ready for export to any spreadsheet software such as Microsoft Excel^®, which may be prepopulated with formulae to calculate distances and angles between landmarks. The whole process of scaling, marking midpoints and landmarking each tooth takes on average three to four minutes to complete.

Special landmarking cases—molar crowns with four or six cusps

All molars in a landmark-based analysis require the same number of landmarks per specimen. For teeth with four cusps, for instance in certain individuals of modern H. sapiens, a modification is made to the landmarks pertaining to the absent hypoconulid (landmarks 13, 18 and 23). In this situation a hypoconulid is inferred from the small groove separating the entoconid and hypoconid (Fig. 2). Landmarks are sited on this small inter-cusp area as though it were a normal hypoconulid.

In the case of a lower molar with six cusps (C6 or tuberculum sextum), the C6 is bisected between the entoconid and hypoconulid for purposes of marking the intersection between these cusps (Wood, 1991, p. 306, Fig 8.13[j]). This allows the same number of landmarks to be maintained across specimens (Fig. 3).

Discriminant function analyses (DFA)

The same sample of 110 hominoid lower second molars was analyzed by means of stepwise discriminant function analyses (DFA) in SPSS^®, with leave-one-out cross-validation. A DFA minimizes within-group variation and maximizes between-group variation, providing good understanding of relative separation among the groups being analyzed. In a stepwise DFA, variables are included in the analysis until they no longer provide any further significant discrimination between groups, at which point redundant variables are removed from the analysis (Manly, 2005). Euclidean coordinates of landmarks are used to derive measurements for the DFA. Depending on the analysis and the sample sizes, these can include the natural log of centroid size as a proxy for overall tooth size, linear dimensions of the tooth crown, orientation of occlusal features measured in radians of angles, and shape features measured as ratios between landmarks, as shown in Fig. 4. For the present analysis, as the minimum sample size per group was 10 individuals, nine measurements that provided the highest canonical loadings were selected for the DFA. These are described in Table 3.

Table 3:

List of mathematically-derived measurements used for input into discriminant function analyses in this study.

#	Measurement	Description
1	Mesiodistal (MD) diameter	Between landmarks 2 and 4
2	Buccolingual breadth across mesial cusps	Between landmarks 26 and 29
3	Buccolingual breadth across distal cusps	Between landmarks 27 and 28
4	Breadth across buccolingual groove	Between landmarks 7 and 10
5	Length of mesial edge of buccal development groove	Between landmarks 29 and 10
6	Length of distal edge of buccal development groove	Between landmarks 10 and 28
7	Angle of mesial cusps	Angle between line connecting centers of mesial cusps (landmarks 21 and 25) and the MD diameter (line between landmarks 2 and 4)
8	Angle of distal cusps	Angle between line connecting centers of distal cusps (landmarks 22 and 24) and the MD diameter (line between landmarks 2 and 4)
9	Hypoconulid curvature ratio	The extent of the outward projection of the of the arc of the hypoconulid at the perimeter, in relation to the total length between the tooth center and the midpoint of the hypoconulid at the perimeter, to measure flatness or curvature of the hypoconulid (landmarks 13 –18, divided by landmarks 1–18)

DOI: 10.7717/peerj.6990/table-3

Results

Principal components analyses

The first two principal components in the shapespace (shape only) analysis are shown in Fig. 5. Relative warps wireframes at the ends of each axis show the average molar shape change along that axis.

Pan, H. sapiens and Gorilla separate well in morphospace, but species and subspecies of Pan overlap with each other, as do species and subspecies of Gorilla. PC1 accounts for 66.1% of variance and relative warps wireframes indicate that broad teeth with distally-oriented hypoconulids and buccodistally-oriented hypoconids group towards the negative end of the x-axis (Pan and H. sapiens), while relatively narrow teeth with buccodistally-oriented hypoconulids and buccally-oriented hypoconids (Gorilla) group towards the positive end. PC2 (y-axis) accounts for 11.6% of variance, with broad molars, having reduced or absent hypoconulids, grouping towards the negative end of the axis (certain H. sapiens molars), and narrow molars with larger, well-defined hypoconulids grouping towards the positive end of the axis. Most Pan molars plot above the x-axis, with the exception of a few P. t. verus molars that are slightly broader across the crown and plot below the x-axis. Separation in morphospace is therefore good at the level of genus, but lacking at the species and subspecies level. There is also no separation in shapespace between molars belonging to male and female gorillas, indicating that in the absence of a size component, there is little to determine sexual dimorphism in shape alone.

In the size-versus-shape (formspace) analysis (Fig. 6), with size added back as a variable, small molars group towards the negative end of the x-axis and large molars group towards the positive end (PC1). This first component accounts for 93.1% of variance and PC2 (y-axis) accounts for 2.3% of variance. In formspace, P. troglodytes, P. paniscus and H. sapiens now separate well and can be differentiated spatially. Species and subspecies of Gorilla still overlap with each other, but not as much as in the shape-only PCA. The molars of G. b. graueri group as generally larger than molars of G. b. beringei, which in turn group as generally larger than those of G. gorilla. Males with larger molars tend to group at more positive values along the axis, although the separation between males and females is most discernable in the sexually dimorphic gorillas. Along PC2 (the y-axis), Gorilla species mostly plot above the axis (narrower teeth) with a few molars grouping just below the x-axis (slightly broader across the crown). Examining the plot and the wireframes, small, relatively narrow molars with pronounced hypoconulids group in the top left quadrant of the graph, with Pan paniscus well separated to the top left-hand side of the plot, being the smallest molars in the sample. Pan troglodytes molars generally group in this quadrant as well, particularly those belonging to P. t. troglodytes and P. t. schweinfurthii, which show considerable overlap. In the bottom left quadrant, relating to molars that are progressively broader across the crown, H. sapiens is generally separated from the few P. troglodytes molars notably those belonging to P. t. verus, which are generally relatively broader across the crown than are specimens from the other two Pan subspecies. In addition to the relative broadness of the crowns, the hypoconulid becomes less pronounced in the individuals grouping towards negative values of PC2.

Discussion

In the context of fossil hominin taxonomic studies, molars play an important role as these are the most abundantly preserved element in fossil assemblages and are well represented in museum and academic collections. However, many of these teeth are heavily worn or damaged on the crown, and researchers are usually forced to reduce limited samples of fossil specimens even further by discarding specimens without easily-identifiable anatomical features on the crown enamel. Some researchers have suggested approximating cusp peaks from the shapes of wear facets (Martinón-Torres et al., 2006), but this results in an analysis with a mixture of approaches to cusp-peak location.

Benazzi et al. (2011); Benazzi et al. (2012) have successfully avoided having to locate cusp tips on worn teeth, and have shown good taxonomic discrimination between worn Neanderthal and modern human teeth, using cervical crown outlines and occlusal crown outlines, which are still present in worn teeth. The present study differs from these studies, firstly in that 3D images are not required for the determination of cervical crown outlines: the surface enamel crown outline visible from 2D images is sufficient for the calculation of all landmarks used in this study, provided that the points where the grooves between cusps meet the outline shape are visible (or readily inferable) in the images used. This would include worn teeth up to and including wear stage 7 as defined by Smith (1984), wherein dentin is exposed on the entire molar crown surface, but the enamel rim remains largely intact; but not late wear stage 7 and wear stage 8, where the enamel rim is severely broken down. A second difference is that by using midlines between cusp grooves and midpoints to stand proxy for the mathematical centers of each cusp, rather than semi-sliding landmarks around the outline (Bookstein, 1996/7), it becomes possible to calculate the relative orientations of cusps through these cusp centers, both in relation to each other and to the longitudinal axis of the tooth. This study capitalizes on the fact that linear measurements and cusp angles, which have proven to be diagnostic measurements for taxonomic studies (e.g., Pilbrow, 2006, Pilbrow, 2007 and Pilbrow, 2010), are readily calculated from the landmarks located both around the perimeter and on the surface itself. Over and above analyses based on Euclidean coordinates of landmarks (GPA, PCA and EDMA –Euclidean Distance Matrix Analysis), this methodology allows for other types of analyses to be carried out that make use of raw measurements, including DFA, CV (coefficients of variation) analyses and other types of odontometric studies. The results of analyses based on these mathematically-derived measurements would then be useful for comparison with existing studies.

The aim of the study was to establish whether mathematically-sited landmarks and the raw measurements derived from these, based on the occlusal crown outline of lower second molars, which is intact in both unworn and worn teeth, would lead to good separation in morphospace and high classification accuracy levels in a DFA. Further to this, the results should be at least equivalent in accuracy to the results achieved by other researchers. DFA classification accuracy outputs were compared to those achieved in two existing studies (modified from Pilbrow, 2006; Skinner et al., 2009).

PCA separation in morphospace and DFA classification accuracy

The results of the geometric morphometric and discriminant function analyses on 110 lower second molars of five species of extant hominoid (n ≥ 10 per subspecies) show good success in group separation, with 96.4% classification accuracy at the species level and 88.2% classification accuracy at the subspecies level. Specimens visualized on shape-only (shapespace) and size-and-shape (formspace) PCA plots grouped according to morphological differences that are diagnostic for each species and genus. Shape differences were observed between Gorilla, Pan and Homo sapiens at the genus level in the shape-only analysis, but as expected in a shapespace plot, there was no sexual dimorphism evident between molars belonging to male and female gorillas, and no interspecific general shape differences between P. paniscus and P. troglodytes (Singleton et al., 2011), with the exception of specimens of P. t. verus, which are on average relatively wider across the crown than other subspecies (Uchida, 1996; Pilbrow, 2006; Dykes, 2018). There was some overlap between P. t. troglodytes and P. t. schweinfurthii molars in shape space, which is to be expected as these two subspecies interbred until relatively recently (Hey, 2010; Gonder et al., 2011). Shape variation in H. sapiens lower second molars exceeded that of other species, undoubtedly a factor of differential evolution in molar cusp simplification (reduction in size, or absence of a hypoconulid), due to regional differences in basic subsistence strategies of hunter-gatherers and agriculturalists over many millennia (Bailit & Friedlaender, 1966; Brace & Mahler, 1971; Sofaer, 1973; Brace, Rosenberg & Hunt, 1987; Corruccini, Potter & Dahlberg, 1983; Corruccini, 1984; Larsen, 1995; Dempsey & Townsend, 2001; Grine, 2002; Grine, 2005; Brown & Maeda, 2004; Pinhasi, Eshed & Shaw, 2008; Emes, Aybar & Yalcin, 2011; Hodder, 2017; Ungar, 2017; Dykes, 2018). This unique shape variation in modern H. sapiens will be examined in detail in a further study, based on larger sample sizes.

When size is added to the analysis, there is excellent spatial separation at the species level on a PCA formspace plot. Gorilla molars were reasonably separated between the two species represented, with sexual dimorphism being observed between molars belonging to males and females at the species and subspecies level. Eastern lowland gorillas, G. b. graueri, have the largest body size of all gorillas (Jungers & Susman, 1984), and this is reflected in the grouping of their molars, towards the positive extreme of the x-axis in the plot. Western lowland gorillas, G. g. gorilla, grouped at lower values along the x-axis, with only two male specimens overlapping with males of the Eastern mountain gorillas, G. b. beringei. Gorilla gorilla as a species showed more shape variability along PC2 in their lower second molars than G. beringei, with some molars that are relatively narrow buccolingually belonging both to males and females grouping at higher values along the y-axis. On the other extreme of the x-axis, P. paniscus grouped cohesively in the top left-hand quadrant, having the smallest molars that are generally relatively narrow. Most P. troglodytes molars grouped above the x-axis, in a similar range along PC2 as that observed for Gorilla species with relatively narrow molars; however, the broader molars of P. t. verus fell below the x-axis, in the quadrant occupied by H. sapiens molars. Overall, groupings in morphospace followed expected patterns of molar shape and size differences between genera, species, subspecies, sexes and subsistence strategies.

Comparing the PCA and DFA results, it can be seen that in both the shape-only (shapespace) and the shape-and-size (formspace) PCA analyses, there are similarities between the factors affecting the first two principal components of the PCAs on the one hand, and the factors accounting for the main canonical loadings along the first two functions of the DFAs on the other. The relative warps of the wireframes traced from the negative to the positive end of the axes of the PCA plots show a shape change between relative breadth of tooth (both axes), the extent of the buccal development groove (x-axis), the amount of curvature of the hypoconulid (y-axis), and along both axes, a change in the relative dimensions (raw measurements and orientations) between the distal cusps and other breadth and length variables. While size is shown to be an important discriminating factor between groups, relative dimensions and angles between variables therefore also play significant diagnostic roles. The methodology of landmarking worn teeth discussed in this paper makes provision for landmarks to be placed not only at selected inflexion points around the outline of the tooth but also on the enamel surface itself, irrespective of how worn that might be, thus providing for the inclusion of key cusp-related data (dimensions and angles) into the analysis that would otherwise be lost, if only perimeter-shape landmarks are to be used.

Comparisons of accuracy between mathematically-derived measurements and anatomically-derived measurements

Mathematically-derived measurements compare well with anatomically-derived measurements in both comparative analyses, based on classification accuracy outputs from stepwise DFAs. In the first analysis of 110 specimens from five species and eight subspecies, using only nine variables, the mathematically-derived measurements produced a classification accuracy of 96.4% at the species level and 88.2% at the subspecies level, versus 93.6% at the species level and 83.6% at the subspecies level for the anatomically-derived measurements taken from the same 110 teeth. In the second analysis of 25 specimens from P. t. troglodytes (n = 5), P. t. verus (n = 10) and P. paniscus (n = 9), the 100% classification accuracy of lower second molars based on anatomically-based landmarks on the EDJ was matched with 100% classification accuracy of lower second molars based on mathematically-derived measurements on and around the OES. The improvement in classification accuracy compared to the previous comparison of 110 specimens is an artefact of sampling: smaller sample sizes and a smaller subset of taxa were used to match those used in Skinner et al. (2009). In particular, the exclusion of P. t. schweinfurthii, which overlaps in molar size and shape with P. t. troglodytes improves classification accuracy. Given this identical result in classification, the mathematical landmarking method might therefore provide a means to assess how well the EDJ taxonomic signal relates to that of the OES, as discussed by Skinner et al. (2009), providing a solution to both of the frustrations experienced by this group of researchers—the scarcity of unworn teeth in museum collections and the problem of the need for increased subjectivity in the placement of landmarks on a worn OES, as compared to the well-defined ridges of the EDJ. Since landmarking only takes a few minutes to complete, this might enable quick verifications to be made, confirming the results of the EDJ analyses.

The results from the comparative analyses provide a reasonable level of comfort that the mathematically-based methodology of landmarking the crown surface might readily replace more traditional methods based on anatomical landmarks, to enable worn teeth to be included on an equal basis with unworn teeth in morphometric analyses. Worn teeth might include individual specimens of importance (particularly fossil hominin holotypes or paratypes) and important groups (e.g., modern human hunter-gatherer groups, whose teeth tend to be more heavily worn than groups with soft diets). Inclusion of worn teeth allows for the augmentation of poorly-represented fossil hominin species and for the balance between groups to be improved (e.g., between males and females, or between different extant populations). Provided that the outline shape and the five cusp intersections are identifiable on the image, landmarking is possible, irrespective of the lack of sharpness of features on the surface. Although the present study necessarily made use of relatively unworn teeth, to enable comparison of classification accuracy with previous studies that used unworn teeth, further papers are being prepared where the methodology is applied in classifying worn fossil hominin and modern human molars. The methodology is designed to be adaptable for all extant hominoid and extinct hominin molars, for all types of analysis involving landmarks or measurement variables, and for all levels of group comparison at the level of genus, species, subspecies, population and sex.