Were late Gomphotheres (Plio-Pleistocene) of the Siwaliks at more Stress as Compared to early Gomphotheres (middle to late Miocene)?

Gomphotheres existed in the Siwaliks from the middle Miocene (14.2Ma) to the middle Pleistocene (0.8Ma) and became extinct later on. In this paper, we tried to discuss the reasons of such extinction of gomphotheres in the lower Pleistocene time span by considering Linear Enamel Hypoplasia (LEH) among 114 isolated tooth samples to assess whether ecological changes correlate with the stress factor in gomphotheres. For this purpose, the Siwalik gomphotheres were divided into two Groups viz. early gomphotheres (middle Miocene to late Miocene) and late gomphotheres (Pliocene to middle Pleistocene). We presented the hypothesis, that as the gomphotheres are characterized by the brachydonty and relied on browsing for their feeding while inhabiting the semi forest land setting thus, expected to have higher stress in Plio-Pleistocene time span as vegetational change around ~6 Ma may have exerted stress on late gomphotheres. The results for the occurrence of frequency of LEH indicated severe ecological stress in late gomphotheres (33%). The significant differences were found (P < 0.05) among the early gomphotheres and late gomphotheres which can be correlated to the vegetational change from C3 to C4, higher aridity indices and intensified seasonality after the late Miocene vegetational shift which may have resulted in substantial faunal turnover, extinction and speciation. We assume that such palaeoecological changes forced a competition with more pronounced grazers like of family Elephantidae and Bovidae resulting in extinction of gomphotheres during the late Pleistocene in the Siwaliks of Pakistan.


INTRODUCTION
The Siwaliks of Pakistan ranges from the middle Miocene to the late Pleistocene time span and exhibit a rich vertebrate diversity including perissodactyls, artiodactyls, rodents, proboscideans, carnivores and hominids (Dennell et al., 2006;Badgley et al., 2008;Flynn et al., 2016). The Siwaliks display coarse and fine-grained sandstone, siltstone, claystone and conglomerates which are best represented in the western part of the sub-Himalaya (Roohi et al., 2015). The Potwar Plateau (Figure 1) has yielded a rich fossil record and has been a center of focus for palaeontologists from all over the globe from a century (Falconer and Cautley, 1868;Pilgrim, 1910, 1913, Sarwar, 1977Barry et al., 1982Barry et al., , 2002Barry et al., , 2013. In this study, the samples have been taken throughout the Siwalik series to assess that how palaeoecological changes forged the phylogeny of gomphotheres.

Paleogeography of the Siwalik Gomphotheres
The order proboscidea includes both extinct and extant elephants and their relatives both on land (hyraxes) and in water (manatees and dugongs). The order proboscidea comprises of 10 families, 42 genera and 175 species and subspecies known from different biogeographic regions of the world (Shoshani and Tassy 2005). The Siwalik proboscideans are known by 4 families, 11 genera and about 22 species (Sarwar, 1977).
Gomphotheres were amongst the giant proboscideans recognized by their fossil record from the entire globe except from Australia and Antarctica (Shoshani, 1998). Gomphotheres were present during the middle Miocene (14.2Ma) to the middle Pleistocene (0.8Ma) in the Siwaliks (Table 1). Usually gomphotheres have two pairs of opposing lower and upper tusks. They have appeared and predominantly found in the earliest sediments of the early Miocene of Africa (~22.0 Ma) and migrated to Asia and Europe. These proboscideans are recognized as trilophodonts because of the presence of 3-plated primary two molars (m1 and m2). Tobien (1973) explained a simplified tooth structure linked to genus Gomphotherium with several crosswise ridges, comprising of a certain cone-like rudiments. The presence of trefoil structure is believed to be a characteristic of all bunodont mastodonts (Gomphotheriids), which can only be found on the worn molars thus, supporting the idea that these mastodonts were dominant browsers or somehow mixed feeders. This study deals with the hypothesis that, how ecological changes shape up the phylogeny of the Siwalik gomphotheres after the late Miocene transition (Cerling et al., 1993;Barry et al., 2002Barry et al., , 2013. The present work is the estimation of palaeoenvironmental stress among Gomphotheres before and after the transition.

Enamel Hypoplasia as Stress Indicator
Enamel is the outermost whitish shell of tooth crown which is extremely mineralized and toughest tissue of the mammalian body (Shawashy and Yaeger, 1986;Kierdorf et al., 2012). Hypoplasia is the low or underdevelopment of a tissue or organ. Enamel Hypoplasia (EH) is a deficit of enamel thickness because of physiological faults that negotiate the deposition of ameloblasts through the secretory period of amelogenesis (Sarnat and Schour, 1941;Yaeger, 1980;Shafer et al., 1983;Guita, 1984). Simply, EH is known as the failure of tooth enamel to reach its normal thickness during its development on the tooth crown (Goodman et al., 1987;Goodman and Rose, 1991). It can appear as vertical or horizontal grooves or as discrete pin prick cavities, more or less encircling the tooth crown in an ordinary enamel (Figure 2,3,4), supposed to resemble with less severe stress (Ainamo, 1982). Typically, this kind of EH is attributed to an inadequate secretions of ameloblasts. It can be easily linked to myriad but is typically associated with malnutrition, febrile disease or infections (Suckling, 1989), which embraces an everlasting mark on the tooth. These everlasting marks can be evaluated both in bones and teeth and also in similar type of tissues in mammals.
The dispersal and morphology of EH on different types of mammalian dentition may identify the type of environmental stress, trauma and disease. These tooth developmental defects in mammals are believed to be noticeable components for the scale of the environmental stress from mild to severe morbidity and mortality (Skinner, 1996). These perturbations of EH can occur via three causal conditions: (1) systematic or physiological stress (nutritional stress, illness, weaning, parturition, and stress during cow calf separation) (2) hereditary anomalies or (3) localized trauma in emerging teeth at a specific ontogenetic age (Weinmann et al., 1945;Shawashy and Yaeger, 1986;Suckling, 1989) and are typically identified as linear enamel hypoplasia (LEH) (Neiburger, 1990;Mead, 1990;Goodman and Rose, 1990;Dobney and Ervynck, 2000;Lukacs, 2001;Franz-Odendaal et al., 2004). LEH is usually produced by the physiological or environmental stresses at a specific phase in an animal's life, at which the development, growth and eruption of tooth was taking place. On the basis of ease of examination, chronological array of its development, sensitivity, and failure to remodel, tooth enamel is endorsed to be a perfect tissue for recording any change in an animal physiology during its development (Kreshover, 1940;Massler et al., 1941;Sarnat and Schour, 1941). Hence, the width of depression mark, depth, and frequency of LEH reveal the duration, strength of severity, and the episodes of ecological stress (Shklar and McCarthy, 1976;Guita, 1984;Rose et al., 1985) respectively, which have been faced by an animal during its tooth enamel development. Thus, in this study we have utilized LEH to evaluate our hypothesis.

MATERIALS AND METHODS
The present study was conducted on a total of 114 tooth specimens from the fossilized family gomphotheriidae for the evaluation and correlation of dental enamel paleopathology (Linear Enamel Hypoplasia) to the Siwalik palaeoenvironment. The studied fossil material utilized for the analysis of LEH was taken from the Dr. Abu Bakr Fossil Display and Research Centre, and Fossil Research Centre Jhelum Campus, University of the Punjab, Lahore, Pakistan. The gomphotheres fossilized material was identified following the dental terminology of Tassy (1996). The tooth specimens consisting of worn enamel were omitted. The selected tooth specimens were examined by two raters having expertise in LEH analysis for the precision and to avoid any ambiguity. A 10-X hand lens for magnification was used for each tooth examination.
A 50-watt variable incandescent light was used in laboratory examination of LEH, as samples were analyzed both in artificial and natural sunlight. The location of LEH on enamel of the tooth crown was measured starting from root crown junction (RCJ) towards the tip by using a White Worth digital vernier caliper. The distance was recorded in millimeters (mm). After quantitative examination of LEH, samples were shifted for pictorial presentation of paleopathology marks. A digital handheld camera "canon ECOS-350D" was being used for snapshots. The tooth specimens with unknown and erased catalogue were newly catalogued by giving the first alphabet of the family after Punjab University Palaeontological Collection (PUPC), and the numeric denominators for each unknown specimen as PUPC-G 1 , PUPC-G 2 and so on.
The old abbreviation (UZ, University Zoology) was also considered. Cohen's Kappa (1960) statistics was performed as a statistical tool to observe the differences in opinion among both raters. The details of LEH results on gomphotheres teeth are provided in Table  1.

Prevalence of LEH by Taxon
The detailed information of LEH incidences in the family gomphotheriidae by taxon is grouped and summarized in table 1. The height, location, and magnitude of the LEH on each tooth specimen were greatly variable in the examined material (

Prevalence of LEH by Tooth
Our results related to the frequency of LEH by tooth revealed considerable variations as summarized in Table 3. A total of 114 gomphotheriid isolated teeth were analyzed consisted of 24 affected individual teeth comprising of 20.05% of LEH. All the five species, C. corrugatus, G. browni, P. chinjiensis, A. sivalensis, and A. osborni belonging to the Siwalik family gomphotheridae were showed 16.36%, 28.57%, 15.79%, 37.50%, and 25.0 %, of the LEH, respectively. The findings showed that the lowest number of teeth defected with LEH were found 15.79% in P. chinjiensis and 16.36% in C. corrugatus whereas, the species A. sivalensis having 37.50% of LEH was found to have the highest number of defected teeth. The results for these observed defects of LEH in dentition of gomphotheres showed that the members of A. sivalensis were affected more severely by prevailing environmental conditions compared to other species analyzed in this Siwalik family. The frequency for the occurrence of LEH determined that there are maximum of three LEH's were found on a single tooth (PUPC-15/239) which showed that three different types of ecological stress incidents may have addressed that animal during his life history.

Interpretation of kappa: an Inter-rater reliability
The software version "IBM SPSS statistics-20" was used to calculate the significant/ non-significant differences in the analyzed data. The Cohen's Kappa (1960) statistics was used for the description of agreement or disagreement between the two raters, observing the presence of LEH, a major descriptive of ecological stress in mammals. The calculation of "K" value indicated that there was a perfect agreement in both analyses where K=0.898 was present in the tooth material analyzed in natural sunlight whereas the value K=0.874 for the samples observed in artificial 50-watt incandescent light. The value p > 0.05 indicated that the difference in opinion between the two observers was nonsignificant during the analysis of LEH in gomphotheres.

DISCUSSION
Linear enamel hypoplasia is a systemic physiological perturbation which has been associated to the palaeoenvironmental stability of a taxon in a particular time span. In the Siwaliks of Pakistan, the stratigraphic range of Gomphotheriidae has been considered from the middle Miocene (14.2Ma) to the middle Pleistocene (0.8 Ma) as given in Table 5. Total four genera of the Siwalik Gomphotheres were used for the analysis of LEH. Among these four lineages, Choerolophodon, Gomphotherium, and Protanancus are reported from the middle Miocene deposits while the fourth lineage, Anancus is reported from the Pliocene deposits of the Siwaliks. The two taxa, Gomphotherium, and Protanancus disappeared during the late Miocene whereas; Choerolophodon and Anancus extinct during the Pliocene and Middle Pleistocene of the Siwaliks, respectively, as indicated in Figure 5. The overall results of linear enamel hypoplasia in both early and late gomphotheres are sufficient to understand the stressful palaeoclimatic and paleoecological conditions which were responsible for the either migration or extinction of gomphotheres from the Siwalik sub-Group of Pakistan. The comparisons of LEH showed that each of the taxa had different levels of adaptations to the changing ecological conditions, depending on their dietary habits. The tooth examination of Protanancus and Gomphotherium indicated that these are the primitive taxa with the presence of lower incisors and less advanced lophodont teeth. The loss of lower incisors in proboscideans is one of the characteristics supposed towards novelty. The early gomphotheres, gomphotherium and protanancus clear have lower incisors, which can be considered as primitive character (Göhlich, 1999). The pronounced occurrence of LEH (20.05%) in gomphotheres similar to the family giraffidae (Ahmad et al., 2018) with 34% indicated that the less advanced teeth in these animals (brachydont) were responsible to impede their feeding habits on hard and coarse vegetation during the late Miocene. Herbert et al., (2016) reported that the late Miocene had warm and much drier environmental conditions with enhanced seasonality (Nelson, 2005). In the Siwaliks sub-Group, C 3 forests were gradually replaced by C 4 savannahs during the late Miocene (8.5 to 6.0 Ma) (Morgan et al., 2009;Waseem et al. 2021). During this vegetational turnover, few mammalian lineages evolved and strived for their survival but several others went extinct (Barry et al., 2002;Badgley et al., 2008). The simplest tooth structure of protanancus and gomphotherium out of all these Siwalik gomphotheres indicated that they browsed on leaves or shoots. Similarly, the long term warm and dry ecological conditions of the late Miocene also reduced the amount of available water resources both from environment and diet, forcing them towards extinction or migration (Böhme et al., 2008;Scheiter et al., 2012).
The lineage, Choerolophodon from the late gomphotheres was found with 16.36% of LEH close to Protanancus (15.97%). The absence of lower incisors can be considered as the more advanced character compared to Protanancus and Gomphotherium as it enabled Choerolophodon maintained its survival throughout the middle Miocene and disappeared by the Pliocene strata (15.2 -5.3 Ma) of the Siwaliks (Abbas et al., 2018). The existence of this genus until the Pliocene indicates that this group of gomphotheres has strived because of having more advanced tooth structure with higher hypsodonty indices. However, these land mammals somehow shifted their dietary niche from browsing towards mixed feeding (browsing/grazing). The extinction of Choerolophodon by the early Pliocene of the Siwaliks can be attributed to the large scale C 3 /C 4 transition and climatic variations. The Miocene -Pliocene boundary ~5.3 Ma demonstrated the stages of many C 4 ecological events (Hynek et al., 2012). The fourth studied Siwalik lineage, Anancus appeared during the late Pliocene (~3.6 Ma) and became extinct during the Pleistocene (~0.8 Ma) (Khan et al., 2011). The highest occurrence of LEH (33.33%) in the genus Anancus shows that this lineage was remained under highly intense ecological conditions which can also be observed in the late Miocene. The episodes of climatic variation profoundly affected the distribution and evolution of both plant and animal communities during the mid-Pleistocene times (Head et al., 2008). On average, the long-term ice volume of the globe was increased between 1.25 Ma to 700 ka which was linked with major cooling episodes (Clark et al., 2006). During the Middle Pleistocene (1.6 Ma), the forest succession was subsequently interrupted by a brief period of open vegetation of colder and drier continental climate, followed by a new vegetational cycle (Ravazzi and Strick, 1995). The unstable ecological conditions of the middle Pleistocene with substantial glaciation, prevailing ice sheaths and cooling regimes were responsible for the vanishing of Anancus. The simple and smooth tooth structure (Khan et al., 2011) indicate that the evolution was not as fast as vegetational change, hence confronted complications in shifting themselves on C 4 food choices and became extinct from the Siwaliks during the Pleistocene. The stable isotope values of carbon ( Figure 6) indicate that the late Miocene vegetational transition (Cerling et al., 1993;Nelson, 2007;Badgley et al., 2008;Sanyal et al., 2010) forced the gomphotheres to adapt towards the C 4 grazing behavior while their low hypsodonty indices and less specialized teeth were not much in line with this transition. We assume, that due to such disadvantages, gomphotheres could not compete the coexisting specialized grazers (e.g. elephantids and bovids) which had specialized dentition and thus, became extinct towards the end of the Pleistocene.

CONCLUSION
Current study concludes that the proboscidean family Gomphotheriidae was severely affected because of changing paleoecological condition, during the late Miocene to the middle Pleistocene of the Siwalik sub-Group of Pakistan. The maximum occurrence of LEH found in the genus Gomphotherium and Anancus can been associated with their migration or extinction during the late Miocene and middle Pleistocene, respectively. Very high prevalence of LEH in the late Gomphotheres signals towards the stressful paleoecological conditions with limited food and water resources, limiting the survival of gomphotheres in the Siwaliks. Hence, it can be assumed that the late Miocene (7.2 -5.3 Ma) and middle Pleistocene (2.58-1.6 Ma) time spans were the time of substantial modification in regional climate and ecology that may have reduced suitable habitats for gomphotheres pushing them gradually towards migration and extinction from the Siwalik sub-Group of Pakistan.

Authors Contribution
MA and AMK generated the idea, MA and RMA conducted the analysis as two raters, MA and MTW drafted the manuscript, MI and MA formatted the file, AI and AR organized the taxonomic identifications, and MA and MTW made the tables and figures.