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Trigonid crests at the enamel-dentine surface of Atapuerca-Sima de los Huesos lower molars by means of microtomographic (mCT) techniques. Morphological expression and evolutionary inferences.

Martínez de Pinillos González, Marina

Martinón-Torres, María. Dental Anthropology Group, CENIEH, Burgos.

Keywords: permanent lower molars, Homo heidelbergensis, Middle Pleistocene, Sima de los Huesos,

microtomographic techniques, trigonid crest patterns.

Abstract

Hitherto, the analysis of lower molars dentine surface, within de genus Homo, has been basically

limited to Homo neanderthalensis and Homo sapiens. In this study, we present the analysis of the

Middle Pleistocene permanent lower molar sample (n=62) from the Sima de los Huesos (SH) site,

assigned to the Homo heidelbergensis species. The aim of our analysis is to characterize the pattern of

trigonid crests expression at the outer enamel and the enamel-dentine junction surfaces (OES and EDJ)

of the SH sample, to evaluate the concordance of expression between both surfaces and to explore

possible evolutionary meanings of the expression of this feature as it was previously done based on the

external morphology of the teeth. The sample has been scanned with medium and high resolution of

the nondestructively technique of Micro-Computed Tomography and the final 3D models were

reconstructed and analyzed using the AMIRA 5.3.3 software. Our results reveal a greater variability of

the trigonid crest pattern at the dentine compared to the enamel, and in M3s compared to M1s and M2s.

Our analysis also confirms a good correspondence in the expression of trigonid crest patterns between

the outer and inner surface of a molar that suggests the EDJ is mostly responsible for the morphology of

the OES. This aspect is particularly useful as it may imply the possibility of predicting the type of trigonid

crest pattern at the OES and thus, it will allow us to enlarge our study sample by including teeth that

were too worn to be analyzed based on the outer surface. Moreover, our study reveals that the SH

sample coincides with H. neanderthalensis in the almost constant expression of a continuous middle

trigonid crest. Other aspects of the variability of the trigonid crest expression at the dentine are

presented and discussed.

Introduction

The variation in dental form, as well as the frequency and degree of expression of many dental traits

are highly heritable, and that makes teeth more useful than other skeletal remains to explore

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resemblances between extant and extinct human populations. Thus, paleoanthropologists consider

teeth the “safe box” of the genetic code (Turner, 1963; Martinon-Torres et al., 2007), which allow to

investigate phylogenetic relationships and the scenario in which these species evolved (Martinon-Torres

et al., 2007; Martinón-Torres et al., 2012). The expression of those morphological traits have been

classically recorded in the outer enamel surface (OES), but other researchers, particularly during the last

decade, have attempted to characterize the expression of these features at the enamel dentine junction

(EDJ) (Macchiarelli et al., 2006; Skinner et al., 2008; Bailey et al., 2011). It is assumed that the

morphology of the EDJ is largely responsible of the external morphology of a tooth (e.g., Nager, 1960;

Schwartz et al., 1998). According to Korenhof (1982) the EDJ morphology is more evolutionarily

conservative than the OES morphology because “the enamel-dentine partition is much more a genetic

blueprint of the occlusal anatomy of the teeth”. However, the precise level of concordance between

both surfaces is still under study (e.g., Skinner et al., 2008, 2009; Bailey et al., 2011).

In the past, in order to analyze the EDJ surface it was necessary the application of destructive

techniques or that the teeth were broken or incomplete (e.g., Nager, 1960; Suzuki and Sakai, 1973;

Korenhof, 1982). Nowadays, with the aids of new methods like computed microtomography (mCT), it is

possible to carry out the virtual separation of the different tissues that compose a tooth, a process that

is also called segmentation. From this segmentation, it is possible to obtain the tridimensional (3D)

reconstruction of each dental surface with very high-resolutions in a non-destructive manner by means

of different software packages, like Amira (Visage Imaging, Inc.). In this sense, dental microCT studies

are providing a new source of variables, such as discrete morphological traits at the dentine surface that

could not be explored with standard classic analyses.

In this context, the expression of trigonid crests on human molars has revealed certain patterns of

variation that seem to be taxonomically and phylogenetically informative (e.g., (Turner et al., 1991;

Scott and Turner, 1997; Irish, 1998; Bailey, 2002a). From an evolutionary point of view, the primitive

mammalian cusp pattern in molars was a triangle (Figure 1). From this pattern evolution has allowed the

development of different forms. In humans, as in most primates, the mesial or anterior part of the lower

molars is called the trigonid (trigon in upper molars), and the distal or posterior part of the lower molars

is the talonid (talon in upper molars) (White and Folkens, 2005). Trigonid crest refers to the expression

of a crest that connect the first (protoconid) with the second (metaconid) main cusps in lower molars

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(Turner et al., 1991; Scott and Turner, 1997) and it can start from any lobe segment or from the marginal

ridge complex as well (Bailey et al., 2011).

Recently, and following the pioneer work of Korenhof in the 80’s, trigonid crests have been also

studied at the EDJ by means of mCT (Skinner et al., 2008; Bailey et al., 2011). These studies have

revealed that, as it happens at the OES, Neanderthals and modern humans present characteristic and

different patterns of trigonid crests expressions at the EDJ, both in terms of frequency and morphology

(Bailey et al., 2011). In particular, the expression of conspicuous and continuous middle trigonid crests in

lower molars has been interpreted as typical and distinctive of H. neanderthalensis (Bailey, 2002a;

Martinón-Torres, 2006). We now know that this species is not only characterized by the high frequencies

of expression of middle trigonid crests, but also that the structures underlining this crest in the dentine

may be different to the ones that contribute to the middle trigonid crest expression in other human

species (Bailey et al., 2011).

In this study, we aim to investigate the expression of trigonid crests at EDJ of the Atapuerca-Sima de

los Huesos lower molar sample by means of mCT. Sima de los Huesos site is a small cavity of

approximately 8m2 x 4 m2 that belongs to the Cueva Mayor-Cueva del Silo karst system (Atapuerca,

Spain). This site has provided the largest Middle Pleistocene Homo fossil record coming from the same

place and, to date, the human fossils recovered sum up more than 6.500 remains, about the 80% of the

worldwide human fossil record for the Middle Pleistocene (Bermúdez de Castro et al., 2004). This

extraordinary accumulation gives us the opportunity to study intrapopulation variability in a fossil

biological population (Arsuaga et al., 1991, 1993, 1997; Bermúdez de Castro et al., 2004; Martinón-

Torres et al., 2012). Sima de los Huesos hominins have been assigned to Homo heidelbergensis, a species

that has been interpreted as ancestral to H. neanderthalensis, although the exact relationship between

both taxa is still matter of debate (Arsuaga et al., 1997; Hublin, 2009; Martinón-Torres et al., 2012). Our

objectives are: a) to characterize the pattern of expression of trigonid crests at the EDJ of the Sima de

los Huesos lower molar sample and its correspondence with the expression at the OES and b) to make

possible evolutionary inferences about its pattern of expression particularly in relation to H.

neanderthalensis.

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Figure 1. Tribosphenic molar and its counterpart in humans; modified designs from Teeth by Simon Hillson (2005).

Materials

Although the SH dental sample consists of 213 permanent molars, of which 123 are lower ones, we

have only included in this study those that were isolated or included in a mandibular fragment small

enough to fit in the micro-CT scan. In addition, and following Bailey et al., 2011, we excluded those with

wear degree higher than category 5 (Molnar, 1971), i.e., teeth that exhibit moderate to extensive

secondary dentine but the entire tooth is still completely surrounded by enamel. Thus, the sample

analyzed here consisted of 62 permanent lower molars from the Sima de los Huesos site (Table 1).

PERMANENT RIGTH MOLARS (N) PERMANENT LEFT MOLARS (N)

Tooth N Individual Sigla Sex Age Tooth N Individual Sigla Sex Age

M1 right 10 II AT-2 M 12.5-14.5 M1 left 12 III AT-22 F 15-17

III AT-101 F 15-17 IV AT-14 F 26-32

X AT-141 F 15-17 VI AT-1759 F 16-18

XI AT-272 F 13-15 VII AT-21 M 24-30

XIV AT-2276 - 12.5-14.5 X AT-556 F 15-17

XVIII AT-943 M 9.5-11.5 XI AT-286 F 13-15

XX AT-3175 M 12.5-14.5 XIV AT-1459 - 12.5-14.5

XXIV AT-2438 - 12.5-14.5 XVIII AT-829 M 9.5-11.5

XXV AT-3933 F 11-13 XIX AT-576 F 16-18

XXVI AT-561 F 16-18 XX AT-4318 M 12.5-14.5

M2 right 7 II AT-142 - 12,5-14,5 XXIV AT-1458 - 12.5-14.5

III AT-271 F 15-17 XXV AT-3934 F 11-13

XI AT-1761 F 13-15 M2 left 11 II AT-3179 - 12,5-14,5

XIV AT-284 - 12,5-14,5 III AT-273 F 15-17

XVIII AT-1752 M 9,5-11,5 X AT-169 F 15-17

XX AT-3890 M 12,5-14,5 XI AT-557 F 13-15

XXVI AT-1756 M 16-18 XIV AT-2272 - 12,5-14,5

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M3 right 12 IV AT-811 F 26-32 XVIII AT-941 M 9,5-11,5

XVIII AT-2277 M 9,5-11,5 XX AT-946 M 12,5-14,5

XXIV AT-2438 - 12,5-14,5 XXIV AT-2396 - 12,5-14,5

XXV AT-3943 F 11-13 XXV AT-6579 F 11-13

XXVI AT-30 F 16-18 XXVI AT-2270 F 16-18

- AT-143 - - XXVII AT-3176 M 20-26

- AT-599 - - M3 left 10 IV AT-100 F 26-32

- AT-942 - - VII AT-13 M 24-30

- AT-1468 - - XVIII AT-2271 M 9,5-11,5

- AT-1959 - - XXIV AT-2385 - 12,5-14,5

- AT-2777 - - XXV AT-6580 F 11-13

- AT-3182 - - - AT-598 - -

- AT-1473 - -

- AT-1945 - -

- AT-2273 - -

- AT-2760 - -

Table 1. List of molars analyzed in the present study with the number of the individual they are assigned to, sex and age at death estimated by death, estimated by Bermúdez de Castro et al. (2004)

Methods

Every SH molar was been scanned with high or medium resolution of a Scanco Medical AG Micro-

Computed Tomography 80, housed at the CENIEH. The Scanco Medical Micro-CT80 system is

characterized by a maximum scan size diameter of 75.8mm and a maximum scan length of 140mm, with

image matrix from 512 x 512 to 4096 x 4096 pixels. Scans of the Homo heidelbergensis teeth from Sima

de los Huesos were performed by X-ray source with peak energy of 70 kVp and intensity of scanning of

114µA. The sampletime of one projection is 800.000µs, and the Micro-CT takes nearly 500 parallel

projections for each 180. The slices thicknesses are 18µm or 36µm depending on whether they were

scanned at high or medium resolution, respectively. Final sections were reconstructed and analyzed by

means of AMIRA 5.3.3 (Visage Imaging, Inc.), the segmentation of the volume was done semi-

automatically with manual corrections, and no filters were applied.

Terminological considerations

In order to ensure the reproducibility of our work, we believe it is crucial to summarize the different

terminologies employed by researches when they analyze the expression of trigonid crests and their

correspondence with the terms chosen for this study (Korenhof, 1982; Carlsen, 1987; Turner et al., 1991;

Wu and Turner, 1993; Scott and Turner, 1997; Bailey et al., 2011; Martinón-Torres et al., 2012).

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Following Carlsen’s terminology, the crown of a tooth is divided in different structures such as lobes,

lobe segments, marginal ridges complexes, cingulum derivatives and supernumerary coronal structures

(Carlsen, 1987). According to him, in posterior teeth, what usually researchers refer as a cusp (Cope,

1888; Osborn, 1888) it is indeed a lobe. A lobe is made up of different lobe segments: two accessory

lobes segments (referred by Scott and Turner as accessory ridges (Scott and Turner, 1997)) and one

essential lobe segment (referred by Scott and Turner as an essential ridge (Scott and Turner, 1997)) with

an occlusal cusp with a free apex, known as an essential cusp (Carlsen, 1987). For him, a ridge is the

elevation of enamel that does not extend across the central sulcus, and a crest is the bridge of enamel

resulting for the join of two ridges from opposing lobes (Carlsen, 1987). All the crown traits that usually

take the form of accessory occlusal tubercles are called supernumerary coronal structures (Carlsen,

1987).

In 1997, Scott and Turner claimed that a tooth crown is made up of four secondary macroscopic

units: lobes, marginal ridges, cingulum derivatives and supernumerary coronal structures (Scott and

Turner, 1997). According to them, and following Carlsen´s terminology (Carlsen, 1987), one lobe is

divided in two sections -buccal and lingual- and three segments –mesial accessory lobe, essential lobe

and distal accessory lobe -, and a cusp is the part of a lobe segment with a free apex at the occlusal edge

(Scott and Turner, 1997). The marginal ridge complex occurs on the mesial and distal surface of the

crown but it may not be always clear. When a free apex occurs on the occlusal surface of a marginal

ridge complex, it is called marginal tubercle (Scott and Turner, 1997). Finally, a cingulum derivative is a

lingually or buccally projection at the cervical section of a tooth crown (Scott and Turner, 1997).

In this study, we will basically follow the terminology employed in Martinón-Torres et al., 2012, that

combines terms and concepts from both Carlsen and Scott and Turner (Carlsen, 1987; Scott and Turner,

1997) (Figure 2). Thus, we consider that the main structures of a molar crown are cusps -with their

corresponding lobes or ridges-, grooves and crests, and that these may be expressed or not at the

dentine surface with the same score as at the enamel one (Skinner et al., 2008). In addition, we can also

find secondary structures such as marginal ridges, cingula or tuberculum projections, and

supernumerary structures, for which we will use the same terminology as Scott and Turner (Scott and

Turner, 1997). Thereby, in accordance with the traditional literature (Cope, 1888; Osborn, 1888) and in

order to avoid possible confusions, we will refer as a cusp what Carlsen (Carlsen, 1987) and Scott and

Turner (Scott and Turner, 1997) referred as an entire lobe, and thus, we consider it like the occlusal

projection of the crown (White and Folkens, 2005). A cusp can be divided in three different segments or

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lobes or ridges (hereof we will call it lobe) which can be mesial, middle –or essential- and distal (Scott

and Turner, 1997) depending on their anatomical position. The free apex on the top of the essential lobe

will be called cusp tip (note that Carlsen, 1987, called it essential cusp, but Scott and Turner, 1997,

simply call it cusp). The shallow and longitudinal depressions in the surface of enamel will be called

grooves (Carbó Ayala, 2005).

CARLSEN (1987) SCOTT & TURNER (1997) MARTINÓN ET AL. (2012)

Lobe Lobe Cusp

Accessory Lobe Segment (ALS) Mesial Accessory Lobe (MAL) Mesial Lobe (ML)

Essential Lobe Segment (ELS) Essential Accessory Lobe (EAL) Essential Lobe (EL)

Accessory Lobe Segment (ALS) Distal Accessory Lobe (DAL) Distal Lobe (DL)

Essential Cusp (EC) Cusp (C) Cusp Tip (CT)

Figure 2. Definitions and schemes (below) of some of the molar crown structures.

Arising from these concepts about the basic components of a molar we can now discuss the different

terminologies published so far to refer to the trigonid crests.

In 1982, Korenhof defined a mesial (sic) trigonid crest -although Wu and Turner (Wu and Turner,

1993) and Bailey et al. (Bailey et al., 2011) assumed that Korenhof also called it middle trigonid crest-

and a distal trigonid crest. These crests were joining the mesial and the distal parts, respectively, of the

two main mesial cusps of a lower molar. In 1993, Wu and Turner defined a middle and a distal trigonid

crest, but they did not found individuals expressing both crests at the same time. According to them,

when a complete crest connects the middle portions of the mesial cusps and it lies mesialward, but not

on the marginal border, it is a middle trigonid crest (Wu and Turner, 1993); otherwise it is a distal

trigonid crest. In 1997, Scott and Turner only described a distal trigonid crest, and they defined it as a

crest or ridge that courses buccolingually along the distal aspect of the primitive trigonid, now

represented by the protoconid and metaconid (Scott and Turner, 1997).

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Finally, in recent studies, Skinner et al., 2008 and Bailey et al., 2011, scored crests at the EDJ based on

their origin and thus, stated that a mesial trigonid crest could occur between the mesial marginal ridge

and the middle trigonid crest. However, these authors scored trigonid crests mostly based on the

relative position of one to another, so a mesial trigonid crest might occur between the cusp tips

(although according to Carlsen’s terminology (Carlsen, 1987) that would be a middle-middle crest) as far

as its position is substantially mesialward within the tooth contour. Although we acknowledge the

atypical mesial position of some middle crests (Figure 9b from Bailey et al., 2011), in order to

standardize terms to discuss anatomy in such a detail, we have preferred to subscribe to the notion that

individual cusps are divided into three elements (mesial, middle and distal lobes) and that the middle or

essential one is the one bearing the cusp tip. In this frame, the three types of crests should be scored

independently of the presence/absence of the other, in a similar way as they are scored at the OES (e.g.,

(Guatelli-Steinberg and Irish, 2005; Martinón-Torres et al., 2012). Anyhow, although we subscribe to the

notion that there are mesial, middle and distal trigonid crests we believe that, at the OES, it is not

possible to reliably differentiate a mesial (the one where at least one of the extremes goes clearly to the

marginal ridge) from a middle trigonid. Thus, at the OES we will simply score a middle trigonid crest and

a distal trigonid crest, following traditional literature and classic scoring systems (Korenhof, 1982; Scott

and Turner, 1997).

Scoring procedures

For this study, the crest that courses along the distal aspect of the protoconid and metaconid will be

scored as a distal trigonid crest (DTC). In addition and on the enamel, the one that courses along the

mesial aspect of the protoconid and metaconid will be registered as a middle trigonid crest (MdTC)1

(Figure 3). However, on the dentine we sometimes find another type of crest that we recognize as a

mesial trigonid crest (MeTC), which arises from the middle or mesial lobe to the mesial marginal ridge

(Figure 4). In order to score these crests, we draw an imaginary straight line from the protoconid to the

metaconid tip; any crest lying distal to this line would be a distal trigonid crest, and over or mesial to this

line it can be a middle or mesial.

1 What Korenhof calls mesial trigonid crest, in our study is considered as middle trigonid crest.

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Figure 3. Enamel from the third lower molar of the Sima de los Huesos site where we can see a Middle Trigonid Crest (MdTC) and a Distal Trigonid Crest (DTC) in AT-2271. Green line is the imaginary line from protoconid tip to metaconid tip employed to assess the position of the crests. Virtual reconstruction of the enamel computer model based on microCT.

Figure 4. Dentine from the third lower molars of the Sima de los Huesos site where we can see a Middle Trigonid Crest (MdTC) and a Distal Trigonid Crest (DTC) in AT-2271, and Mesial Trigonid Crest (MeTC) in AT-143. Green line is the imaginary line from protoconid tip to metaconid tip employed to assess the position of the crests. Virtual reconstruction of the dentine computer models based on microCT.

Summarizing, and taking into account all the anatomical considerations above, at the OES we

recognize four basic types of trigonid crest expression (Figure 5 and Figure 6):

Type A: a continuous MdTC and an absent or discontinuous DTC

Type B: continuous DTC and absent or discontinuous MdTC

Type C: continuous MdTC and DTC

Type D: absent or discontinuous MdTC and DTC

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Figure 5. Scheme (based on Korenhof, 1982) of the four basic types of trigonid crests on enamel identified in our study. Explanation in the text. Scheme represents the occlusal surface at the OES including the tips of the cusps (represented by the circles) of the protoconid and metaconid.

Figure 6. Examples of different types of Trigonid Crests on enamel: type A (AT-946); type B (AT-2385); type C (AT-2271); type D (AT-943). Virtual reconstruction of the enamel computer models based on microCT.

Although both antimeres were analyzed, we employed the unilateral count method (Turner, 1987)

and in case of asymmetry we have chosen the tooth with the highest degree of expression for the trait

because we consider that is the one that has better expressed the genetic signal.

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The next table (Table 2) displays the list of teeth, as well as the antimere, that was chosen to

calculate the frequencies of expression analysis. We will provide the frequencies of the different types

of trigonid crest expression in total and by tooth class. In addition, we will also present the inventory of

the trigonid crest patterns of variation and combination found at the EDJ in the SH molar sample, the

frequencies and percentages per molar class and how the different types found correspond with the

expression of the trigonid crest at the OES.

Table 2. Molars selected in the present study for the frequency analysis and their corresponding antimere.

TOOTH ANTIMERE M1 AT-2 (Right)

AT-14 (Left) AT-21 (L) AT-101 (R) AT-141 (R) AT-272 (R) AT-561 (R) AT-576 (L) AT-829 (L) AT-1759 (L) AT-2276 (R) AT-2438 (R) AT-3175 (R) AT-3933 (R)

- - - AT-22 (L) AT-556 (L) AT-286 (L) - - AT-943 (R) - AT-1459 (L) AT-1458 (L) AT-4318 (L) AT-3934 (L)

M2 AT-142 (R) AT-169 (L) AT-271 (R) AT-284 (R) AT-557 (L) AT-1752 (R) AT-1756 (R) AT-2396 (L) AT-3176 (L) AT-3890 (R) AT-6579 (L)

AT-3179 (L) - AT-273 (L) AT-2272 (L) AT-1761 (R) AT-941 (L) AT-2270 (L) - - AT-946 (L) -

M3 AT-13 (L) AT-30 (R) AT-143 (R) AT-598 (L) AT-599 (R) AT-811 (R) AT-942 (R) AT-1468 (R) AT-1473 (L) AT-1945 (L) AT-1959 (R) AT-2271 (L) AT-2273 (L) AT-2438 (R) AT-2760 (L) AT-2777 (R) AT-3182 (R) AT-6580 (L)

- - - - - AT-100 (L) - - - - - AT-2277 (R) - AT-2385 (L) - - - AT-3943 (R)

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Results

Trigonid crest types at the OES

Table 3 displays the results for the trigonid crest types found at the OES for all the Sima de los Huesos

molar specimens (n=62) by tooth class.

ENAMEL (OES) Specimen Tooth Side Wear Type A Type B Type C Type D

AT-2 M1 R 3 X

AT-14 M1 L 5 X

AT-21 M1 L 5 X

AT-22 M1 L 3 X

AT-101 M1 R 3 X

AT-141 M1 R 3 X

AT-272 M1 R 3 X

AT-286 M1 L 3 X

AT-556 M1 L 3 X

AT-561 M1 R 3 X

AT-576 M1 L 4 X

AT-829 M1 L 2 X

AT-943 M1 R 2 X

AT-1458 M1 L 3 X

AT-1459 M1 L 3 X

AT-1759 M1 L 4 X

AT-2276 M1 R 3 X

AT-2438 M1 R 3 X

AT-3175 M1 R 3 X

AT-3933 M1 R 3 X

AT-3934 M1 L 3 X

AT-4318 M1 L 3 X

AT-142 M2 R 3 X

AT-169 M2 L 2 X

AT-271 M2 R 2 X

AT-273 M2 L 2 X

AT-284 M2 R 2 X

AT-557 M2 L 2 X

AT-941 M2 L 1 X

AT-946 M2 L 2 X

AT-1752 M2 R 1 X

AT-1756 M2 R 2 X

AT-1761 M2 R 2 X

AT-2270 M2 L 2 X

AT-2272 M2 L 2 X

AT-2396 M2 L 2 X

AT-3176 M2 L 3 X

AT-3179 M2 L 2 X

AT-3890 M2 R 2 X

AT-6579 M2 L 2 X

AT-13 M3 L 3 X

AT-30 M3 R 2 X

AT-100 M3 L 3 X

AT-143 M3 R 1 X

AT-598 M3 L 2 X

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AT-599 M3 R 1 X

AT-811 M3 R 2 X

AT-942 M3 R 1 X

AT-1468 M3 R 1 X

AT-1473 M3 L 2 X

AT-1945 M3 L 1 X

AT-1959 M3 R 1 X

AT-2271 M3 L 1 X

AT-2273 M3 L 1 X

AT-2277 M3 R 1 X

AT-2385 M3 L 1 X

AT-2438 M3 R 1 X

AT-2760 M3 L 1 X

AT-2777 M3 R 1 X

AT-3182 M3 R 1 X

AT-3943 M3 R 1 X

AT-6580 M3 L 1 X

Table 3. Different types of trigonid crest developed on enamel of all the molar sample from the Sima de los Huesos. Bold

type samples refer to the molars selected for the frequency analysis.

As we can see in Figure 8, the most frequent type in the SH molar sample is the trigonid crest type A,

which represents the 77% of the total data that means a frequency of 48 out of 62 lower molars (21 out

of 22 for the M1; the totality of the M2; 9 out of 22 for the M3). The next percentage corresponds to TC

type C whose frequency falls to a 15% with 9 specimens (9 out of 22 for the M3). Regarding TC type B,

the percentage is a 5% of the overall sample, with only 3 molars with that type of crest (3 out of 22 for

the M3). Finally, TC type D is the one with the lowest percentage, only 2 molars of the entire sample

which corresponds to the 3% of the total (1 out of 22 for the M1; 1 out of 22 for the M3). Graphs below

(Figure 7) show the rates of the different types of trigonid crests on enamel for the overall of the molars

from the Sima de los Huesos site.

Figure 7. Graphic with the TC types at OES by tooth class for the overall molar sample from the SH site.

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ENAMEL (OES) N / %

Type A 48 (77%)

Type B 3 (5%)

Type C 9 (15%)

Type D 2 (3%)

Table 4.Table with the frequencies and percentages of all molar sample from the SH for the different types of TC on enamel.

Figure 8. Graphic with the frequencies of all the molar sample from the SH for the different types of TC on enamel.

If we calculate the frequency of expression of the trigonid crest types at the OES counting only one

antimere per individual (n=43), we observe that the totality of the M1 and M2 specimens analyzed

present a MdTC, and for the M3 the percentage reaches to 83.33%, which means a frequency of 15 out

of 18 lower molars (Table 5). All the MdTC present in the sample show continuous crests at the OES.

ENAMEL (OES)

Middle Trigonid Crest Total molar sample M1 sample M2 sample M3 sample

Present N 40 14 11 15

% 93.02% 100% 100% 83.33%

Absent N 3 0 0 3

% 6.97% 0% 0% 16.66%

Total 43 14 11 18

Table 5. Frequencies and percentages of the middle trigonid crest (MdTC) at the OES for the SH lower permanent molars analyzed (only one antimere included).

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Table 6 shows us that the overall M3 molars sample analyzed exhibit distal trigonid crest (DTC), half of

which present continuous crests and the other half discontinuous ones. For the M2 the percentage

reaches 72.72% that means a frequency of 8 out of 11 lower molars, all of them with a discontinuous

crest; and for the M1 just only one molar (7.14%) present this type of trigonid crest that is also

discontinuous.

ENAMEL (OES)

Distal Trigonid Crest Total molar sample M1 sample M2 sample M3 sample

Present N 27 1 8 18

% 62.79% 7.14% 72.72% 100%

Absent N 16 13 3 0

% 37.20% 92.85% 27.27% 0%

Total 43 14 11 18

Table 6. Frequencies and percentages of the distal trigonid crest (DTC) at the OES for the SH lower permanent molars analyzed (only one antimere included).

Trigonid crest types at the EDJ

In the dentine of the SH molar sample, the variety of different trigonid crest types and combinations

is clearly higher and more complex that the ones established on enamel. This has led us to develop an

inventory of all the types of trigonid crests combinations that may be useful for other researchers in

forthcoming studies about these traits. Contrary to what occurred with the enamel, at the EDJ we were

able of being more precise in discerning the origin of the crests and in differentiating a mesial trigonid

crest (MeTC) from a middle trigonid crest (MdTC). As made clear on page 8, a MeTC is present when at

least one of the extremes of the crest ends at the mesial marginal ridge, and a MdTC is the one that runs

in the mesial aspect of the trigonid and originates from the middle or mesial lobe. In addition, and as we

explained above, we could also found a DTC that arise from the essential or middle lobes (from the cusp

tip or very close to it), or DTC that clearly arise from the distal lobes. These crests can be continuous or

discontinuous and, in the SH sample (Figure 9 and Figure 10), were combined in 13 different types such

as:

Type 1: continuous MdTC with an absent or discontinuous DTC.

Type 2: continuous MeTC with an absent or discontinuous DTC.

16

Type 3: continuous DTC when at least one of the origins is the distal lobe, with an absent or

discontinuous MeTC or MdTC.

Type 4: discontinuous DTC starting from the distal lobes, with an absent or discontinuous MeTC or

MdTC.

Type 5: continuous MdTC and a continuous DTC arising from the middle lobes.

Type 6: continuous MdTC and a continuous DTC when at least one of the origins is the distal lobe.

Type 7: continuous MdTC with a discontinuous DTC when at least one of the origins is the distal

lobe.

Type 8: continuous MdTC with a discontinuous DTC arising from the distal lobes.

Type 9: continuous MeTC and continuous DTC when at least one of the origins is the distal lobe.

Type 10: continuous MdTC, discontinuous DTC when at least one of the origins is the distal lobe,

and a pronounced distal ridge on one side.

Type 11: continuous MdTC, a discontinuous DTC when at least one of the origins is the distal lobe,

and a discontinuous DTC originated from the distal lobes.

Type 12: continuous MdTC, a discontinuous DTC arising from the middle lobes and discontinuous

DTC arising from the distal lobes.

Type 13: continuous MeTC, a discontinuous DTC arising from the middle lobes and discontinuous

DTC arising from the distal lobes.

Figure 9. Scheme of all the different types of trigonid crests on dentine identified in our study from the SH molar sample. Explanation in the text. View is towards the occlusal surface of the EDJ including the tips of the dentine horn (represented by circles) of the protoconid and metaconid and the mesial marginal ridge (represented by the continuous black line).

17

Figure 10. Examples of different types of Trigonid Crests on dentine: type 1 (AT-169); type 2 (AT-1761); type 3 (AT-1945); type 4 (AT-1473); type

5 (AT-1959); type 6 (AT-942); type 7 (AT-141); type 8 (AT-829); type 9 (AT-2438); type 10 (AT-3934); type 11 (AT-2276); type 12 (AT-2270); type

13 (AT-557). Virtual reconstruction of the dentine computer models based on microCT.

18

Table 7 displays the results for the trigonid crest types found at the dentine (EDJ) for the entire Sima

de los Huesos molar sample by tooth class. We also present the side and degree of wear of each one.

DENTINE (EDJ)

Specimen Tooth Side Wear Type

1 Type

2 Type

3 Type

4 Type

5 Type

6 Type

7 Type

8 Type

9 Type

10 Type

11 Type

12 Type

13

AT-2 M1 R 3 X

AT-14 M1 L 5

X

AT-21 M1 L 5

X

AT-22 M1 L 3 X

AT-101 M1 R 3

X

AT-141 M1 R 3

X

AT-272 M1 R 3

X

AT-286 M1 L 3

X

AT-556 M1 L 3

X

AT-561 M1 R 3

X

AT-576 M1 L 4

X

AT-829 M1 L 2

X

AT-943 M1 R 2

X

AT-1458 M1 L 3

X

AT-1459 M1 L 3

X

AT-1759 M1 L 4

X

AT-2276 M1 R 3

X

AT-2438 M1 R 3

X

AT-3175 M1 R 3

X

AT-3933 M1 R 3

X

AT-3934 M1 L 3

X

AT-4318 M1 L 3

X

AT-142 M2 R 3 X

AT-169 M2 L 2 X

AT-271 M2 R 2

X

AT-273 M2 L 2

X

AT-284 M2 R 2

X

AT-557 M2 L 2

X

AT-941 M2 L 1

X

AT-946 M2 L 2

X

AT-1752 M2 R 1

X

AT-1756 M2 R 2

X

AT-1761 M2 R 2

X

AT-2270 M2 L 2

X

AT-2272 M2 L 2

X

AT-2396 M2 L 2

X

AT-3176 M2 L 3

X

AT-3179 M2 L 2

X

AT-3890 M2 R 2

X

AT-6579 M2 L 2

X

AT-13 M3 L 3

X

AT-30 M3 R 2

X

AT-100 M3 L 3

X

AT-143 M3 R 1

X

AT-598 M3 L 2

X

AT-599 M3 R 1

X

AT-811 M3 R 2

X

AT-942 M3 R 1

X

AT-1468 M3 R 1

X

AT-1473 M3 L 2

X

19

AT-1945 M3 L 1

X

AT-1959 M3 R 1

X

AT-2271 M3 L 1

X

AT-2273 M3 L 1

X

AT-2277 M3 R 1

X

AT-2385 M3 L 1

X

AT-2438 M3 R 1

X

AT-2760 M3 L 1

X

AT-2777 M3 R 1

X

AT-3182 M3 R 1

X

AT-3943 M3 R 1

X

AT-6580 M3 L 1

X

Table 7. Different types of trigonid crest developed in dentine of the entire molar sample from the Sima de los Huesos.

Bold type samples refer to the molars selected for the frequency analysis.

As we can see in Figure 12, the most frequent pattern in the SH molar sample at the EDJ is TC type

11, which represents the 18% of the total sample with 11 out of 62 lower molars (4 out of 22 for the M1;

5 out of 18 for the M2; 2 out of 22 for the M3). The next percentage corresponds to the TC type 10 with

the 13% or a frequency of 8 molars out of 62 (5 out of 22 for the M1; 1 out of 18 for the M2; 2 out of 22

for the M3). In decreasing order, the TC type 12 (3 out of 22 for the M1; 4 out of 18 for the M2) and TC

type 13 (5 out of 18 for the M2; 2 out of 22 for the M3) present an equal value of 11% which corresponds

to 7 molars out of the total sample. Regarding the TC type 8, the percentage falls down to a 10% with a

frequency of 6 molars (5 out of 22 for the M1; 1 out of 22 for the M3). The TC type 1 (2 out of 22 for the

M1; 2 out of 18 for the M2), TC type 3 (4 out of 22 for the M3) and TC type 6 (4 out of 22 for the M3) are

represented by 6% each one, followed by the and the TC type 5 with a 5% (3 out of 22 for the M3).

Finally, for the TC type 2 (1 out of 18 for the M2; 1 out of 22 for the M3), TC type 4 (1 out of 22 for the

M1; 1 out of 22 for the M3), TC type 7 (2 out of 22 for the M1) and TC type 9 (2 out of 22 for the M3) the

percentage reduce to 3% of the overall sample, this means just only a frequency of 2 molars with those

types of crests (Figure 11).

Figure 11.Graphic with the frequencies of TC types at EDJ by tooth class for the overall molar sample from the SH site.

20

Table 8. Table with frequencies and percentages of all the molar

sample from SH for the different types of TC on dentine.

Figure 12. Graphic with the frequencies of the entire molar sample from SH for the different types of TC on dentine.

The next tables present the frequencies of expression of the MeTC (Table 9), MdTC (Table 10) and

DTC (Table 11) continuous and discontinuous for each dental class if we choose only one antimere per

individual. The largest number of MeTCs is displayed by the M3s (5 molars out of 18), followed by M2s (3

molars out of 11) and ending with no MeTCs for the M1s. The totality of the molars with MeTCs (19%)

presents a continuous trigonid crest type.

DENTINE (EDJ) N / % Type 1 4 (7%)

Type 2 2 (3%)

Type 3 5 (8%)

Type 4 2 (3%)

Type 5 3 (5%)

Type 6 4 (6%)

Type 7 2 (3%)

Type 8 6 (10%)

Type 9 1 (2%)

Type 10 8 (13%)

Type 11 11 (18%)

Type 12 7 (11%)

Type 13 7 (11%)

21

DENTINE (EDJ)

Mesial Trigonid Crest Total molar sample M1 sample M2 sample M3 sample

Present N 8 0 3 5

% 19% 0% 27.27% 27.77%

Absent N 35 14 8 13

% 81% 100% 72.72% 72.22%

Total 43 14 11 18

Table 9.Frequencies and percentages of the mesial trigonid crest (MeTC) at the EDJ for the SH lower permanent molars analyzed.

In Table 10, we can observe that on dentine the middle trigonid crest (MdTC) is present in 74% of the

overall sample analyzed and represented by a continuous trigonid type. The highest percentage

correspond to the M1s with the totality of the sample, followed by a 72.72% for the M2s with a

frequency of 8 molars out of 11, and 55.55% for the M3s with 10 molars out of 18.

DENTINE (EDJ)

Middle Trigonid Crest Total molar sample M1 sample M2 sample M3 sample

Present N 32 14 8 10

% 74% 100% 72.72% 55.55%

Absent N 11 0 3 8

% 26% 0% 27.27% 44.44%

Total 43 14 11 18

Table 10. Frequencies and percentages of the middle trigonid crest (MdTC) at the EDJ for the SH lower permanent molars analyzed.

In relation to the distal trigonid crest (DTC) on dentine, the Table 11 shows us how this pattern

presents the highest value of the overall sample analyzed with a percentage of 91% that means a

frequency of 39 out of 43 lower molars. The values are a 94.44% for the M3s, 92.85% for the M1s and

90.90% for the M2s. All molars present discontinuous crests except 10 M3s that exhibit continuous ones.

DENTINE (EDJ)

Distal Trigonid Crest Total molar sample M1 sample M2 sample M3 sample

Present N 39 13 10 17

% 91% 92.85% 90.90% 94.44%

Absent N 4 1 1 1

% 9% 7.14% 9.09% 5.55%

Total 43 14 11 18

Table 11. Frequencies and percentages of the distal trigonid crest (DTC) at the EDJ for the SH lower permanent molars analyzed.

22

Variability of the trigonid crest types at the EDJ for the SH sample

As we mentioned above, the four basic types of trigonid expression at the OES correspond to a higher

variability, with a wider spectrum of crests types and combinations at the EDJ. As we can see in the

following table (Table 12), the TC Type A at the OES is the one showing higher variability at the EDJ,

followed by the TC Type C, TC type B and TC type D. All the teeth present a good correlation between

the enamel and dentine surface, with the exception of four M3 lower molars sample whose morphology

differs from the inner and outer level. This correspondence is measured a general match between the

expression of continuous/discontinuous middle and distal crests at both surfaces.

ENAMEL (OES) DENTINE (EDJ)

Type A 1 - 2 - 6 - 7 - 8 - 9 - 10 - 11 - 12 - 13

Type B 3

Type C 3 - 5 - 6 - 9- 10

Type D 4

Table 12. Variability of the TC types found at the EDJ from SH samples.

TC type A at the OES (continuous MdTC with absent or discontinuous DTC) could be represented at

the dentine surface by a continuous MeTC or MdTC in combination with a discontinuous DTC. However,

we found that for AT-2273 there is a continuous MeTC with a continuous DTC -which correspond at the

EDJ with a TC type 9- and for AT-2760 there is a continuous MdTC with a continuous DTC -which

correspond at the EDJ with a TC type 6. This means that just only two molars of the overall sample

(3.22%) present a different combination that does not match with the morphology found at the OES.

In our sample, a TC Type B (continuous DTC with absent or discontinuous MdTC) at the OES

corresponds with an absent or discontinuous MeTC or MdTC and a continuous DTC.

TC type C at the OES (continuous MdTC and DTC) exhibits the co-expression of both continuous

crests also at the EDJ, except in AT-100 where the MeTC or MdTC is absent -TC type 3- and in AT-811

where the MdTC is continuous -TC type 10. These two molars represent the 3.22% of the total sample

with a different morphology regarding to the OES.

TC type D (absent or discontinuous MdTC and DTC) is also related on dentine with an absent or

discontinuous MeTC or MdTC and a discontinuous DTC.

23

Frequencies of expression for the TC types at the OES and EDJ by tooth class

The frequencies of expression for trigonid crests are given by tooth class (M1, M2 and M3). Table 13,

Table 14, Table 15 and Table 16 provide the frequencies and percentages of each different type of

trigonid crest on both enamel and dentine surfaces, but using the individual count method. We have

detailed the frequency of the different types of trigonid crest expression from the Sima de los Huesos

sample in the 43 lower permanent molars analyzed (Table 13), in the M1 permanent ones (Table 14), in

the M2 (Table 15) and in the M3 (Table 16).

In Table 13, we present the frequencies and percentages for the different trigonid crest types, both

on enamel and dentine, of the total molar sample. At the enamel, the TC type A represents the 77% of

the total data, which means a frequency of 33 molars with this type of trigonid crest, the highest value.

Regarding to the TC type C the percentage decrease to 16% with a total frequency of 7 molars samples.

For TC type B, the percentage reduces to the number of 5% for the overall sample, this means just only

a frequency of 2 molars with that type of crest. The lowest value corresponds to the TC type D with a 2%

that represent only 1 molar of the entire analyzed sample. Talking about the dentine, the highest value

stands for the TC type 11 (16%) with a frequency of 7 molars, following by the TC type 10 (14%) with a

frequency of 6 molars, and the TC type 8 and 13, both with 12% or 5 lower molars of frequency. Next

value is represented by the TC type 1, 5, 6 and 12, with a frequency of 3 molars that means the 7% of

the overall sample for each type. For the TC type 3, 7 and 9 (5%) there are just only 2 molars; one more

than the frequency represented by the TC type 2 and 4 whose percentage fall down to 2% of the total

analyzed sample.

ENAMEL TYPES N / % DENTINE TYPES N / % A 33 (76.74%) 1 3 (6.97%) B 2 (4.65%) 2 1 (2.32%) C 7 (16.27%) 3 2 (4.65%) D 1 (2.32%) 4 1 (2.32%)

TOTAL 43 5 3 (6.97%) 6 3 (6.97%) 7 2 (4.65%) 8 5 (11.62%)

9 2 (4.65% 10 6 (13.95%) 11 7 (16.27%) 12 3 (6.97%) 13 5 (11.62%) TOTAL 43

Table 13. Frequencies of expression and percentages for the different types of trigonid crests on both enamel and dentine from the Sima de los Huesos lower permanent molars sample analyzed.

24

For the M1 (Table 14) M2 (Table 15) and M3 (Table 16) permanent molars, we can observe different

frequencies and percentages for each trigonid crest type depending on the enamel or dentine surface.

The next table (Table 14) shows us that for the M1s there is a complete correlation between the enamel

trigonid crest types and the dentine ones. Treating the data separately, we can see that for the enamel

the TC type A represents the overall percentage (100%), meanwhile at the dentine the values are

distributed between the TC type 8 with the highest percentage of the sample (29%), following by the TC

type 10 and TC type 11 both with a frequency of 3 molars (21%), the TC type 7 (14%) with 2 lower

molars, and lastly the TC type 1 and TC type 12 with only one sample (7%).

ENAMEL TYPES N / % DENTINE TYPES N / % A 14 (100%) 1 1 (7.14%) B 0 2 0 C 0 3 0 D 0 4 0

TOTAL 14 5 0 6 0 7 2 (14.28%) 8 4 (28.57%) 9 0 10 3 (21.42%) 11 3 (21.42%) 12 1 (7.14%) 13 0 TOTAL 14

Table 14. Frequency of expression of trigonid crests and their percentages on both enamel and dentine for the M1 permanent molars analyzed.

Once more, the TC type A represents the whole percentage (100%) at enamel (Table 15), whereas at

dentine the values are distributed between the TC type 11 and 13, both with a frequency of 3 molars

(27%), following by the TC type 1 and 12 with 18% that means 2 lower molars for each one, and the TC

type 10 with the lowest percentage of the M2 sample represented by only one molar (9%).

ENAMEL TYPES N / % DENTINE TYPES N / % A 11 (100%) 1 2 (18.18%) B 0 2 0 C 0 3 0 D 0 4 0

TOTAL 11 5 0 6 0 7 0 8 0 9 0 10 1 (9.09%) 11 3 (27.27%) 12 2 (18.18%) 13 3 (27.27%) TOTAL 11

Table 15. Frequency of expression of trigonid crests and their percentages on both enamel and dentine for the M2 permanent molars analyzed.

25

With regard to the M3 molar samples analyzed there is a greater variability between the trigonid

crest types on enamel and dentine than the previous molars samples (Table 16). In this way, the overall

percentage at enamel are divided into TC type A (44%), TC type C (39%), TC type B (11%) and TC type D

(6%). The values at dentine correspond with the highest percentage to TC type 5 and 6 (17%) following

by the TC type 3, 9, 10 and 13 (11%), and finally with the TC type 2, 4, 8 and 11 (6%).

ENAMEL TYPES N / % DENTINE TYPES N / %

A 8 (44.44%) 1 0 B 2 (11.11%) 2 1 (5.55%) C 7 (38.88%) 3 2 (11.11%) D 1 (5.55%) 4 1 (5.55%)

TOTAL 18 5 3 (16.66%) 6 3 (16.66%) 7 0 8 1 (5.55%) 9 2 (11.11%) 10 2 (11.11%) 11 1 (5.55%) 12 0 13 2 (11.11%) TOTAL 18

Table 16. Frequency of expression of trigonid crests and their percentages on both enamel and dentine for the M3 permanent molars analyzed.

Discussion

Due to the many discrepancies found in the traditional classification systems, one of the most

important inconveniences we have found when analyzing the lower molar sample from the Sima de los

Huesos site was to establish single and uniform criteria to classify them (see Terminological

considerations on page 5). This problem prevents the direct comparison of our results with those

obtained by Bailey et al., 2011 for the Neanderthal and the H. sapiens groups.

Among all the molar samples from SH, the M3s ones are those with more variability and complexity.

They are rather atypical (in some of them cusps are rotated within the tooth contour, some other have

reduced main cusps in combination with accessory cusps…), preventing an easy identification of the

morphological features. As an example, in AT-100 the marginal ridge is atrophied and ends at the mesial

part of the molar, making difficult to distinguish whether there is a continuous MeTC or not (Figure 13).

Most of the cases showing a “mismatch” between surfaces correspond to atypical teeth. Despite of this

inconvenience, there is a huge correlation between the OES and EDJ, but sometimes happens that what

26

seems to be a continuous crest on the enamel surface actually is a discontinuous one at dentine level. As

stated above, it happens rarely, and when this occurs it is usually in quite atypical teeth and with C7s.

Figure 13.Dentine surface of a M3 (AT-100) with an atrophied MMR.

Variability and frequencies of expression at the EDJ

Our results confirm a greater variability in the dentine than in the enamel surface, but with a good

general correspondence between them. Thus, at the OES a MdTC could be a MeTC or MdTC at the EDJ.

Similarly, a DTC at the EDJ could have different origins such as middle-middle, distal-middle, distal-distal,

or even exhibit double distal ridges. But in all cases there is generally great concordance, in whatever

form, between the presence of a continuous crest at the OES and the presence of a continuous crest at

the EDJ.

Regarding the distal trigonid crest at the EDJ, and unlike what other researchers have stated -Wu and

Turner did not found individuals with MdTC and DTC at the same time (Wu and Turner, 1993); and

Bailey and colleagues affirmed that they have not seen molars connecting distal segments of the C1 and

C2 (Bailey et al., 2011)- we can affirm that in the 43 lower molar sample analyzed there are large

number of molars with distal origins (36 specimens that means a 83.72%) and, that we have also found

cases with “true” distal trigonid crest although they are not very common (3 molars or 6.97%) and only

M3s display it (Figure 14) which connects the distal portions of the protoconid and metaconid (Bailey et

al., 2011). Concerning the crests that connect middle and distal lobe segments, other researchers have

found a small number of molars but only in Homo sapiens (Bailey et al., 2011), while in our sample there

are 4 molars, which mean a 9.30% of the sample, that present this type of trigonid crest pattern.

27

Figure 14. Illustration of a “true” distal trigonid crest at EDJ and OES in an M3 molar specimen (AT-2385). Virtual reconstruction of dentine and enamel computer models based on microCT.

Talking about the “true” middle trigonid crest (Figure 15) which connects the essential lobes of the

protoconid and metaconid (Bailey et al., 2011), there are 13 out of 32 molars sample that present this

type of trigonid crest (40.62%), which means that the remaining 19 samples present a middle trigonid

crest connecting the mesial and essential lobes (59.37%). According to Bailey et al. (2011) the mesial

trigonid crest originates most often from the middle lobe segment; however, our results reveals that of

the 8 lower molars which present a mesial trigonid crest, 5 of them have their origin at the mesial lobe

(62.5%) and the other 3 specimens have an origin at the middle segment origin (37.5%).

Figure 15. Illustration of a “true” middle trigonid crest at EDJ and OES in an M2 molar specimen (AT-946). Virtual reconstruction of dentine and enamel computer models based on microCT.

If we pay attention to the trigonid crests types by tooth class, we can observe two aspects that could

be relevant for the correct classification of isolated molars. One of them relates to the TC types at the

OES where the TC type B and C are only found in M3s samples (see pag. 13) The other relevant feature is

related to the TC types at the EDJ, since there are not M1s with MeTC in the overall SH sample (see pag.

28

20). If these notions are confirmed in this and other fossil samples, it may become an advantage for the

correct classification of some isolated remains in a particular sample.

Taxonomical implications

At this point we are obliged to limit our debate to the Sima de los Huesos Pleistocene samples, for

which we have analyzed the trigonid crests patterns on both the enamel (OES) and dentine (EDJ).

Although the results of our analyses corroborate a high correspondence in the percentages and

frequency of this trait between the enamel and dentine surface, (Bailey et al., 2011) the expression and

variability of some trigonid crests at the EDJ can be masked by the enamel deposition at the OES (Bailey

et al., 2011; this study). We have used some traditional criteria -like Wu and Turner used in 19932 (Wu

and Turner, 1993)- to score the trigonid crest at the OES, but we also recorded the full variability

expressed at the EDJ to have a more precise classification of our fossil dental sample. According to

Bailey (Bailey, 2002a, 2002b) and Martinón-Torres (Martinón-Torres et al., 2012) the expression at the

OES of a continuous MdTC is a typical Neanderthal feature, but this feature is also present in the Middle

Pleistocene hominins fossils from Europe like Sima de los Huesos and Arago –but not in Mauer despite

being the holotype of the species Homo heidelbergensis that group them- and in variable percentages in

earlier species like H. erectus, H. georgicus and H. antecessor (Martinón-Torres et al., 2007, 2008, 2012).

Previous studies about the outer surface on Homo heidelbergensis teeth from Sima de los Huesos site

have shown that those fossils present the classic Neanderthal traits (Bermúdez de Castro, 1986;

Bermúdez de Castro et al., 1999; Martinón-Torres et al., 2007, 2012). Now, with the new techniques of

microCT and virtual segmentations, we are allowed to compare these characteristics present on the

enamel (OES) with those offered at dentine (EDJ) in order to be more accurate when considering the

resemblances found between them, and thus attempt to answer the currently assumptions whether the

SH hominids were the starting point at the origin of the classic Neanderthals, or if on the contrary were

a different lineage (Tattersall and Schwartz, 2009; Dennell et al., 2010; Martinón-Torres et al., 2012).

In absolute terms, we can affirm that at the EDJ there is a greater number of SH molars with middle

or mesial trigonid crest than without it, that the middle crest is more frequent than the mesial one and

that there is a high proportion of lower molars with a continuous mesial (MeTC) or middle trigonid crest

2 “If only one trigonid crest occurs, and if the crest lies slightly mesialward, but not on the marginal border, it is said

to be a middle trigonid crest.”

29

(MdTC) and a discontinuous distal trigonid crest (DTC), which means a clear predominance of the TC

type A. We have also identified a wide spectrum of trigonid crests types at the EDJ, but future studies in

larger samples are necessary in order to know whether all the types are present and/or typical of other

hominin groups or some of them are particular to the SH group. In this sense, the comparison with other

hominins samples is necessary to ensure if the manifestation of these types of trigonid crest pattern in

the Sima de los Huesos sample are derived or primitive in Homo, particularly with regard to H. sapiens

and H. neanderthalensis. However, and although a direct comparison between both studies is not

possible because of the different scoring systems employed (this study and Bailey et al., 2011),it was

possible to make some general assessments between both analysis. It seems that the SH sample

coincides with H. neanderthalensis in the almost constant expression of a continuous mesial or middle

trigonid crest and a discontinuous DTC, and therefore, in the high percentages3 of this pattern (although

Bailey et al., 2011 use a different terminology).

ENAMEL (OES) SH sample H. neanderthalensis H. sapiens N % N % N %

MdTC continuous (Grade 2-3, Bailey et al.) 40 93.02 61 100 8 11.76 MdTC discontinuous (Grade 1, Bailey et al.) 3 6.97 0 0 13 19.11

DENTINE (EDJ) SH sample H. neanderthalensis H. sapiens N % N % N %

MdTC continuous (Grade 2-3, Bailey et al.) 40 93.02 72 98.63 24 35,29 MdTC discontinuous (Grade 1, Bailey et al.) 3 6.97 1 1.36 8 11.76

Figure 16. Illustrations of differences among specimens for grades of presence of middle trigonid crest (MdTC) at the OES and EDJ. Note that H. neanderthalensis and H. sapiens dates have been taken from Bailey and colleagues (Bailey et al., 2011).

3 Bailey et al. (Bailey et al., 2011) found a 100% of a trigonid crest at the OES and 98.6% at the EDJ for the H.

neanderthalensis and we found a 100% at both OES and EDJ for the SH sample.

30

Conclusions

In accordance with Bailey and colleagues (Bailey et al., 2011) and because the dentine exhibits

greater variability and more different trigonid crest types than the enamel, it was necessary to use

different scoring system than the traditional ones (Korenhof, 1982; Wu and Turner, 1993) in order to

characterize in a more precise manner the morphology of this sample. Also in accordance with that

study, our analysis has revealed a higher variability of trigonid crest expressions at the EDJ compared to

the OES.

However, and in general terms, our results broadly ratify the strong harmony of trait expression

between the enamel (OES) and the dentine (EDJ) (Skinner et al., 2008; Bailey et al., 2011). Because of

that, we may also point out that both the expression and the grade of development of the trigonid crest

patterns at the enamel is mostly determined by the manifestation at the dentine. This fact is particularly

relevant as it implies the potential possibility of predicting the type of trigonid crest pattern at the OES

in the case of excessively worn teeth. This also maximizes the number of teeth that can be analyzed,

enlarging the sample size particularly in the case of the scarce fossil human remains.

Based on the results of the 43 lower molars sample analyzed from the Sima de los Huesos site, the

complete lack of trigonid crest type B, type C and type D in SH M1s and M2s , could be an interesting

feature to help classify isolated dental specimens, but we must contrast it with other fossils hominins.

Additionally, we observe a greater variability of trigonid crest patterns in M3s than in M1s and M2s for

the overall molars sample. Future studies may clarify if these are general patterns of certain species or

lineages or is a particularity of the SH group.

We also think is desirable to analyze other Pleistocene hominins in order to have greater fossil record

that allows us to get more taxonomic and phylogenetic information about these traits expression as well

as to make possible evolutionary inferences about the expression of this trait in relation with other

ancestral hominins.

31

Bibliography

Arsuaga, J.L., Carretero, J.M., Lorenzo, C., Gracia, A., Martínez, I., Bermúdez de Castro, J.M., Carbonell, E., 1997. Size variation in middle Pleistocene humans. Science 277, 1086–1088.

Arsuaga, J.L., Carretero, J.M., Martínez, I., Gracia, A., 1991. Cranial remains and long bones from Atapuerca/Ibeas (Spain). Journal of Human Evolution 20, 191–230.

Arsuaga, J.-L., Martínez, I., Gracia, A., Carretero, J.-M., Carbonell, E., 1993. Three new human skulls from the Sima de los Huesos Middle Pleistocene site in Sierra de Atapuerca, Spain. Nature 362, 534–537.

Bailey, S.E., 2002a. Neandertal dental morphology: implications for modern human origins. Arizona State University.

Bailey, S.E., 2002b. A closer look at Neanderthal postcanine dental morphology: I. The mandibular dentition. New Anatomist 269, 148–156.

Bailey, S.E., Skinner, M.M., Hublin, J.-J., 2011. What lies beneath? An evaluation of lower molar trigonid crest patterns based on both dentine and enamel expression. American Journal of Physical Anthropology 145, 505–518.

Bermúdez de Castro, J.M., 1986. Dental remains from Atapuerca (Spain) I. Metrics. Journal of Human Evolution 15, 265–287.

Bermúdez de Castro, J.M., Martinón-Torres, M., Carbonell, E., Sarmiento, S., Rosas, A., Van der Made, J., Lozano, M., 2004. The Atapuerca sites and their contribution to the knowledge of human evolution in Europe. Evolutionary Anthropology: Issues, News, and Reviews 13, 25–41.

Bermúdez de Castro, J.M., Martinón-Torres, M., Lozano, M., Sarmiento, S., Muela, A., 2004. Paleodemography of the Atapuerca-Sima de los Huesos hominin sample: a revision and new approaches to the paleodemography of the European Middle Pleistocene population. Journal of Anthropological Research 60, 5–26.

Bermudez de Castro, J.M., Rosas, A., Carbonell, E., Nicolas, M.E., Rodríguez, J., Arsuaga, J.L., 1999. A modern human pattern of dental development in Lower Pleistocene hominids from Atapuerca-TD6 (Spain). Proceedings of the National Academy of Sciences 96, 4210.

Carbó Ayala, J., 2005. Anatomía dental y de la oclusión. Ciudad de La Habana: Editorial de Ciencias Médicas.

Carlsen, O., 1987. Dental morphology. Munksgaard. Cope, E.D., 1888. The mechanical causes of the origin of the dentition of the Rodentia. The American

Naturalist 22, 3–13. Dennell, R.W., Martinón-Torres, M., Bermúdez de Castro, J.M., 2010. Out of Asia: The initial colonization

of Europe in the Early and Middle Pleistocene. Quaternary international 223. Guatelli-Steinberg, D., Irish, J.D., 2005. Brief Communication: Early Hominin Variability in First Molar

Dental Trait Frequencies. American Journal of Physical Anthropology 128, 477–484. Hublin, J.J., 2009. The origin of Neandertals. Proceedings of the National Academy of Sciences 106,

16022 –16027. Irish, J.D., 1998. Diachronic and synchronic dental trait affinities of Late and Post-Pleistocene peoples

from North Africa. Homo 49, 138–155. Korenhof, C.A.W., 1982. Evolutionary trends of the inner enamel anatomy of deciduous molars from

Sangiran (Java, Indonesia). Teeth: form, function and evolution. New York: Columbia University Press. p 350–365.

Macchiarelli, R., Bondioli, L., Debénath, A., Mazurier, A., Tournepiche, J.-F., Birch, W., Dean, M.C., 2006. How Neanderthal molar teeth grew. Nature 444, 748–51.

Martinón-Torres, M., 2006. Evolución del aparato dental en homínidos: estudio de los dientes humanos

32

del Pleistoceno de la Sierra de Atapuerca (Burgos). Universidad de Santiago de Compostela. Martinon-Torres, M., Bermudez de Castro, J.M., Gomez-Robles, A., Arsuaga, J.L., Carbonell, E.,

Lordkipanidze, D., Manzi, G., Margvelashvili, A., 2007. Dental evidence on the hominin dispersals during the Pleistocene. Proc Natl Acad Sci U S A 104, 13279–13282.

Martinón-Torres, M., Bermúdez de Castro, J.M., Gómez-Robles, A., Arsuaga, J.L., Carbonell, E., Lordkipanidze, D., Manzi, G., Margvelashvili, A., 2007. Dental evidence on the hominin dispersals during the Pleistocene. Proceedings of the National Academy of Sciences 104, 13279 –13282.

Martinón-Torres, M., Bermúdez de Castro, J.M., Gómez-Robles, A., Margvelashvili, A., Prado, L., Lordkipanidze, D., Vekua, A., 2008. Dental remains from Dmanisi (Republic of Georgia): morphological analysis and comparative study. Journal of human evolution 55, 249–273.

Martinón-Torres, M., Bermúdez de Castro, J.M., Gómez-Robles, A., Prado-Simón, L., Arsuaga, J.L., 2012. Morphological description and comparison of the dental remains from Atapuerca-Sima de los Huesos site (Spain). Journal of Human Evolution 62, 7–58.

Molnar, S., 1971. Human tooth wear, tooth function and cultural variability. American Journal of Physical Anthropology 34, 175–190.

Nager, G., 1960. [Comparison between the spatial relations of the dentin crown relief and the enamel relief of dental crowns.]. Acta Anatomica 42, 226.

Osborn, H.F., 1888. The evolution of mammalian molars to and from the tritubercular type. The American Naturalist 22, 1067–1079.

Schwartz, G.T., Thackeray, J.F., Reid, C., van Reenan, J.F., 1998. Enamel thickness and the topography of the enamel–dentine junction in South African Plio-Pleistocene hominids with special reference to the Carabelli trait. Journal of Human Evolution 35, 523–542.

Scott, G.R. and Turner, C.G., 1997. The anthropology of modern human teeth: dental morphology and its variation in recent human populations. Cambridge University Press, Cambridge.

Skinner, M.M., Gunz, P., Wood, B.A., Boesch, C., Hublin, J.J., 2009. Discrimination of extant Pan species and subspecies using the enamel-dentine junction morphology of lower molars. American Journal of Physical Anthropology 140, 234–243.

Skinner, M.M., Gunz, P., Wood, B.A., Hublin, J.-J., 2008. Enamel-dentine junction (EDJ) morphology distinguishes the lower molars of Australopithecus africanus and Paranthropus robustus. Journal of Human Evolution 55, 979–988.

Skinner, M.M., Wood, B.A., Boesch, C., Olejniczak, A.J., Rosas, A., Smith, T.M., Hublin, J.J., 2008. Dental trait expression at the enamel-dentine junction of lower molars in extant and fossil hominoids. Journal of human evolution 54, 173–186.

Suzuki, M. and Sakai, T., 1973. Occlusal surface pattern of the lower molars and second deciduous molar among the living polynesians. American Journal of Physical Anthropology 39, 305–316.

Tattersall, I., Schwartz, J.H., 2009. Evolution of the Genus Homo. Annual Review of Earth and Planetary Sciences 37, 67–92.

Turner, C.G., 1987. Late Pleistocene and Holocene population history of East Asia based on dental variation. American Journal of Physical Anthropology 73, 305–321.

Turner, C.G., Nichol, C.R., Scott, G.R., 1991. Scoring procedures for key morphological traits of the permanent dentition: the Arizona State University Dental Anthropology System., in: Kelley, M., Larsen, C. (Eds.), Advances in Dental Anthropology. Wiley Liss, New York, pp. 13–31.

Turner, E.P., 1963. Crown development in human deciduous molar teeth. Archives of Oral Biology 8, 523–IN8.

White, T.D. and Folkens, P.A., 2005. The human bone manual. Academic Press. Wu, L. and Turner, C.G., 1993. Brief communication: Variation in the frequency and form of the lower

permanent molar middle trigonid crest. American Journal of Physical Anthropology 91, 245–248.

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Illustration list

Tables Table 1. List of molars analyzed in the present study with the number of the individual they are assigned

to, sex and age at death estimated by death, estimated by Bermúdez de Castro et al. (2004) .................. 5

Table 2. Molars selected in the present study for the frequency analysis and their corresponding

antimere. ..................................................................................................................................................... 11

Table 3. Different types of trigonid crest developed on enamel of all the molar sample from the Sima de

los Huesos. Bold type samples refer to the molars selected for the frequency analysis. .......................... 13

Table 4.Table with the frequencies and percentages of all molar sample from the SH for the different

types of TC on enamel. ............................................................................................................................... 14

Table 5. Frequencies and percentages of the middle trigonid crest (MdTC) at the OES for the SH lower

permanent molars analyzed (only one antimere included). ...................................................................... 14

Table 6. Frequencies and percentages of the distal trigonid crest (DTC) at the OES for the SH lower

permanent molars analyzed (only one antimere included). ...................................................................... 15

Table 7. Different types of trigonid crest developed in dentine of the entire molar sample from the Sima

de los Huesos. ............................................................................................................................................. 19

Table 8. Table with frequencies and percentages of all the molar sample from SH for the different types

of TC on dentine. ......................................................................................................................................... 20

Table 9.Frequencies and percentages of the mesial trigonid crest (MeTC) at the EDJ for the SH lower

permanent molars analyzed. ...................................................................................................................... 21

Table 10. Frequencies and percentages of the middle trigonid crest (MdTC) at the EDJ for the SH lower

permanent molars analyzed. ...................................................................................................................... 21

Table 11. Frequencies and percentages of the distal trigonid crest (DTC) at the EDJ for the SH lower

permanent molars analyzed. ...................................................................................................................... 21

Table 12. Variability of the TC types found at the EDJ from SH samples. ................................................... 22

Table 13. Frequencies of expression and percentages for the different types of trigonid crests on both

enamel and dentine from the Sima de los Huesos lower permanent molars sample analyzed. ............... 23

Table 14. Frequency of expression of trigonid crests and their percentages on both enamel and dentine

for the M1 permanent molars analyzed. ..................................................................................................... 24

Table 15. Frequency of expression of trigonid crests and their percentages on both enamel and dentine

for the M2 permanent molars analyzed. ..................................................................................................... 24

Table 16. Frequency of expression of trigonid crests and their percentages on both enamel and dentine

for the M3 permanent molars analyzed. ..................................................................................................... 25

Figures Figure 1. Tribosphenic molar and its counterpart in humans; modified designs from Teeth by Simon

Hillson (2005). ............................................................................................................................................... 4

Figure 2. Definitions and schemes (below) of some of the molar crown structures. .................................. 7

34

Figure 3. Enamel from the third lower molar of the Sima de los Huesos site where we can see a Middle

Trigonid Crest (MdTC) and a Distal Trigonid Crest (DTC) in AT-2271. Green line is the imaginary line from

protoconid tip to metaconid tip employed to assess the position of the crests. Virtual reconstruction of

the enamel computer model based on microCT. ......................................................................................... 9

Figure 4. Dentine from the third lower molars of the Sima de los Huesos site where we can see a Middle

Trigonid Crest (MdTC) and a Distal Trigonid Crest (DTC) in AT-2271, and Mesial Trigonid Crest (MeTC) in

AT-143. Green line is the imaginary line from protoconid tip to metaconid tip employed to assess the

position of the crests. Virtual reconstruction of the dentine computer models based on microCT. ........... 9

Figure 5. Scheme (based on Korenhof, 1982) of the four basic types of trigonid crests on enamel

identified in our study. Explanation in the text. Scheme represents the occlusal surface at the OES

including the tips of the cusps (represented by the circles) of the protoconid and metaconid. ............... 10

Figure 6. Examples of different types of Trigonid Crests on enamel: type A (AT-946); type B (AT-2385);

type C (AT-2271); type D (AT-943). Virtual reconstruction of the enamel computer models based on

microCT. ...................................................................................................................................................... 10

Figure 7. Graphic with the TC types at EDJ by tooth class for the overall molar sample from the SH site. 13

Figure 8. Graphic with the frequencies of all the molar sample from the SH for the different types of TC

on enamel. .................................................................................................................................................. 14

Figure 9. Scheme of all the different types of trigonid crests on dentine identified in our study from the

SH molar sample. Explanation in the text. View is towards the occlusal surface of the EDJ including the

tips of the dentine horn (represented by circles) of the protoconid and metaconid and the mesial

marginal ridge (represented by the continuous black line). ....................................................................... 16

Figure 10. Examples of different types of Trigonid Crests on dentine: type 1 (AT-169); type 2 (AT-1761);

type 3 (AT-1945); type 4 (AT-1473); type 5 (AT-1959); type 6 (AT-942); type 7 (AT-141); type 8 (AT-829);

type 9 (AT-2438); type 10 (AT-3934); type 11 (AT-2276); type 12 (AT-2270); type 13 (AT-557). Virtual

reconstruction of the dentine computer models based on microCT. ........................................................ 17

Figure 11.Graphic with the frequencies of TC types at EDJ by tooth class for the overall molar sample

from the SH site. ......................................................................................................................................... 19

Figure 12. Graphic with the frequencies of the entire molar sample from SH for the different types of TC

on dentine. .................................................................................................................................................. 20

Figure 13.Dentine surface of a M3 (AT-100) with an atrophied MMR. ....................................................... 26

Figure 14. Illustration of a “true” distal trigonid crest at EDJ and OES in an M3 molar sample (AT-2385).

Virtual reconstruction of dentine and enamel computer models based on microCT. ............................... 27

Figure 15. Illustration of a “true” middle trigonid crest at EDJ and OES in an M2 molar sample (AT-946).

Virtual reconstruction of dentine and enamel computer models based on microCT. ............................... 27

Figure 16. Illustrations of differences among specimens for grades of presence of middle trigonid crest

(MdTC) at the OES and EDJ. Note that H. neanderthalensis and H. sapiens dates have been taken from

Bailey and colleagues (Bailey et al., 2011). ................................................................................................. 29