Palaeoecological differences underlie rare co-occurrence of Miocene European primates

RESEARCH ARTICLE Open Access Palaeoecological differences underlie rare co-occurrence of Miocene European primates Daniel DeMiguel 1,2* , Laura Domingo 3,4 , Israel M. Sánchez 2 , Isaac Casanovas-Vilar 2 , Josep M. Robles 2 and David M. Alba 2 Abstract Background: The two main primate groups recorded throughout the European Miocene, hominoids and pliopithecoids, seldom co-occur. Due to both their rarity and insufficiently understood palaeoecology, it is currently unclear whether the infrequent co-occurrence of these groups is due to sampling bias or reflects different ecological preferences. Here we rely on the densely sampled primate-bearing sequence of Abocador de Can Mata (ACM) in Spain to test whether turnovers in primate assemblages are correlated with palaeoenvironmental changes. We reconstruct dietary evolution through time (ca. 12.6–11.4 Ma), and hence climate and habitat, using tooth-wear patterns and carbon and oxygen isotope compositions of enamel of the ubiquitous musk-deer Micromeryx. Results: Our results reveal that primate species composition is strongly correlated with distinct environmental phases. Large-bodied hominoids (dryopithecines) are recorded in humid, densely-forested environments on the lowermost portion of the ACM sequence. In contrast, pliopithecoids inhabited less humid, patchy ecosystems, being replaced by dryopithecines and the small-bodied Pliobates toward the top of the series in gallery forests embedded in mosaic environments. Conclusions: These results support the view that pliopithecoid primates preferred less humid habitats than hominoids, and reveal that differences in behavioural ecology were the main factor underpinning their rare cooccurrence during the European Miocene. Our findings further support that ACM hominoids, like Miocene apes as a whole, inhabited more seasonal environments than extant apes. Finally, this study highlights the importance of high-resolution, local investigations to complement larger-scale analyses and illustrates that continuous and densely sampled fossiliferous sequences are essential for deciphering the complex interplay between biotic and abiotic factors that shaped past diversity. Keywords: Hominoids, Pliopithecoids, Primate evolution/adaptation, Palaeodiet, Stable isotopes, Tooth wear, Feeding behaviour, Palaeobiology © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. * Correspondence: [email protected] 1 ARAID foundation / Universidad de Zaragoza, Departamento de Ciencias de la Tierra, and Instituto Universitario de Investigación en Ciencias Ambientales de Aragón (IUCA), Pedro Cerbuna 12, 50009 Zaragoza, Spain 2 Institut Català de Paleontologia Miquel Crusafont, Universitat Autònoma de Barcelona, Edifici ICTA-ICP, C/ Columnes s/n, Campus de la UAB, 08193 Cerdanyola del Vallès, Barcelona, Spain Full list of author information is available at the end of the article DeMiguel et al. BMC Biology (2021) 19:6 https://doi.org/10.1186/s12915-020-00939-5 Background Fossil primates from the Miocene of Europe are generally rare and absent from most sites, and when recorded, different primate species only seldom co-occur within a single locality (stratigraphic horizon). As a result, there is an ongoing debate about the factors underpinning the geographic and chronostratigraphic distribution of Miocene primates in this continent [1–6]. Before the dispersal of cercopithecoids (Old World monkeys) into Europe by the early Turolian (ca. 8.5 Ma, late Miocene), two main groups are recorded there: pliopithecoids, generally considered a Eurasian clade of stem catarrhines (i.e. preceding the cercopithecoid-hominoid split [7]), and hominoids (crown catarrhines more closely related to extant apes and humans than to cercopithecoids [8–10]). Both groups presumably dispersed from Africa to Eurasia following the closure of the Tethys Seaway during the late middle Miocene and subsequently diversified across the continent giving rise to multiple genera and species. There are approximately one hundred known localities recording either or both of these groups—almost 20% corresponding to Abocador de Can Mata (ACM) in Spain—although they only co-occur in less than 10% of them, with pliopithecoid-bearing localities being slightly more abundant (ca. 55 vs. 45%) than hominoid-bearing ones [4,5]. European hominoids are generally larger than pliopithecoids and considered great apes (hominids), except for the small-bodied Pliobates, interpreted as a stem hominoid [11]. There are very few sites in the European Miocene where hominoids and pliopithecoids co-occur [1,4,5], and in most cases, fossils of each group come from different localities within the same site (e.g. different karstic fissure fillings from La Grive) or it is uncertain whether their remains came from the same stratigraphic horizon (e.g. Castell de Barberà [6]). Strong taphonomic evidence supporting sympatry is only available from Rudabánya in Hungary [12] and ACM (locality ACM/C5-C3 [5]). These localities therefore offer a unique opportunity to evaluate the palaeoenvironmental conditions that enabled the coexistence of pliopithecoids and hominoids. Given the rarity of primate remains among mammalian assemblages from the European Miocene, the infrequent co-occurrence of two different primate species at a single locality might be, at least in part, a sampling artefact [1]. However, the lack of co-occurrence in many wellsampled primate-bearing localities would rather support the view that their infrequent coexistence is a real phenomenon that requires an explanation. The competitive exclusion principle [13] predicts that species occupying the same ecological niche cannot coexist on the long-term, ultimately leading to the prevalence of one over the other, or to the progressive divergence of their respective niches. This explanation is unlikely to hold for different clades such as pliopithecoids and hominoids, characterised among others by different locomotor adaptations—leading to the proposal that these two groups probably had different habitat preferences, which only enabled their coexistence under particular ecological conditions [1]. Early ecomorphological analyses based on ungulate hypsodonty (a proxy for vegetation structure also used to infer palaeoprecipitation) have concluded that both hominoid and pliopithecoid-bearing localities from the European Miocene were more humid than those lacking primates [2]. More recent work based on hypsodonty further showed that pliopithecoids generally inhabited more humid environments (i.e. with higher moisture and/or rainfall) than hominoids, although probably less humid than those in which both groups co-occur [4]. However, given the small number of fossil localities recording both taxa, such comparisons lack statistical power and may fail to consider palaeoenvironmental differences across geography and time throughout the Miocene, especially at the regional and local scales. Focusing on faunal elements that accompany primates within a single area and over a restricted time span would allow us to test whether turnovers in the primate assemblage are correlated to local changes in palaeoenvironmental conditions. The composite stratigraphic sequence of ACM, located in the area of els Hostalets de Pierola within the Vallès-Penedès Basin (NE Iberian Peninsula [14]) (Fig. 1a–c), and spanning more than 1 Myr (12.6–11.4 Ma [5,15]), offers an unparalleled opportunity to test this hypothesis for several reasons. First, the ACM sequence has delivered one of the most diverse primate assemblages from the European Miocene, including both hominoids and pliopithecoids [5]. Second, the co-occurrence of hominoids and pliopithecoids has only been recorded in one out of the 19 ACM primate-bearing localities, and the distribution of each group throughout the series does not appear random [5]. Finally, thanks to continuous palaeontological surveillance during the construction of a landfill, most of the fossil finds are accurately dated based on detailed litho-, bioand magnetostratigraphic correlations [5,16, 17]. This offers the opportunity to test alternative explanations for the variable temporal distribution of both primate groups during a restricted time span and within a uniform depositional setting. With this aim in mind, here we present a reconstruction of the local climate and palaeoenvironments through the ACM sequence based on tooth wear and dental enamel stable carbon and oxygen isotope values (represented by the notation δ 13 Candδ 18 O) of the ruminant Micromeryx—(Fig. 1d and Figure S1)—a representative of the family Moschidae (musk-deer) [18,19]. Because the diet of any plant-eating mammal is a direct DeMiguel et al. BMC Biology (2021) 19:6 Page 2 of 15 Fig. 1 (See legend on next page.) DeMiguel et al. BMC Biology (2021) 19:6 Page 3 of 15 link with the habitats in which it lives, we used the diet (i.e. ecology) of this ruminant to inform about ACM primate ecological preferences and habitats. The selection of Micromeryx is based on the following reasons: (1) the record of this taxon throughout the ACM stratigraphic sequence, characterised by abundant isolated teeth and dentognathic fragments, allows us to construct a continuous tooth-wear and isotopic record; (2) by focusing on a single genus, we can characterise more consistently changes in the vegetation cover, food abrasiveness, etc., across the selected time interval, avoiding thus biases due to different physiologies; and (3) Micromeryx was ubiquitous in the Miocene of Iberia, inhabiting a varied range of biomes from more or less open savannas to (sub) tropical forests [18,20], and exhibiting an extraordinary versatility in terms of exploitation of nutrients and resources. The combination of all these factors justifies the suitability of employing Micromeryx as a case study to investigate the environmental and climatic shifts that took place during the latest middle Miocene in the area of els Hostalets de Pierola. Results Tooth wear The fossil material studied consists of dentognathic remains and isolated teeth of Micromeryx. Although initially a single species of Micromeryx was reported from ACM [15], the currently available dental material indicates the presence of three different morphotypes that likely represent different species (Additional file 1: Supplementary information, Note 1). For mesowear, we measured individuals and provide the results for the three morphotypes separately (Additional file 1: Supplementary information, Note 2). All Micromeryx morphotypes (Table 1) show occlusal surfaces with predominance of high relief (pH = 95–100%) and sharpened cusps (pS = 69–87%), although there is a considerable proportion of rounded apices (pR = 13– 31%). Morphotypes do not have any incidence of blunt cusps or, except for Micromeryx morphotype 3 (pL = 5%), low occlusal relief (which relates to a low height difference between tooth cusps and valleys). Average mesowear score (MS) for morphotypes ranges from 0.19 to 0.31 (Table 1). We do find significant differences with the chi-square test (χ 2 ) but marginally non-significant with the Fisher exact test. For the chi-square test, the results show that Micromeryx morphotype 1 is different from morphotype 2 (p= 0.0283), whereas non-significant differences are between morphotypes 2 and 3 (p= 0.2255) and between morphotypes 1 and 3 (p= 0.2019). On average, mesowear results indicate a browsing on soft vegetation and low levels of abrasives (endogenous phytolith-rich grasses and dicotyledonous, and exogenous dust and grit), although Micromeryx morphotype 1—with more rounded cusps and higher MS (Table 1)— shows a shift toward the exploitation of tougher and more abrasive foods than the others. Stable isotope data The difference between carbonate (δ 18 O CO3 ) and phosphate (δ 18 O PO4 ) oxygen isotopic composition can be used to monitor possible bioapatite diagenetic alteration. Micromeryx tooth enamel did not undergo extensive post-burial alteration since the difference calculated between δ 18 O CO3 and δ 18 O PO4 (Δ 18 O CO3 - PO4 =δ 18 O CO3 − δ 18 O PO4 ) values for the whole dataset (8.6 ± 0.8‰)is within the range obtained when considering modern mammals (~ 8.6–9.1‰[22,23]) (Additional file 1: Supplementary information, Note 3). Micromeryx morphotypes (Table 1and Additional file 6: Table S1) yielded tooth enamel δ 13 C values indicative of woodland to woodland-mesic C 3 grassland conditions (see Additional file 1: Supplementary information, Note 4 for a detailed explanation of the calculated δ 13 C cutoff values among different habitats). Significant differences in δ 13 C values have been only found between morphotypes 1 and 2 of Micromeryx (t= 4.250, p< 0.001) (Additional file 7: Table S2). Tooth enamel δ 18 O CO3 and δ 18 O PO4 values do not show significant differences among the three morphotypes (δ 18 O CO3 :F= 0.845, df = 2, p= 0.439, and δ 18 O PO4 :F= 0. 562, df = 2, p= 0.577) (Additional file 7: Table S2). Relationship between mesowear and δ 13 C values A scatter plot showing the correlation between mean MS and mean δ 13 C(‰VPDB) among Micromeryx morphotypes by localities was constructed (Fig. 2). Niche domains are visually presented for each variable. This approach (described in Additional file 1: Supplementary information, Notes 2 and 4) allowed us to contextualise (See figure on previous page.) Fig. 1 Abocador de Can Mata (ACM) and the moschid Micromeryx.aGeographical situation and general geological context of the Vallès-Penedès Basin. bDetailed geological map of the basin and the sequence of Abocador de Can Mata (ACM) (black dot). cCorrelation of the composite local magnetostratigraphy of ACM series with the Geomagnetic Polarity Time Scale (modified from Alba et al. [5]). European Land Mammal Ages, Mammal Neogene (MN) units and local biozones of the Vallès-Penedès Basin are shown on the left. The shadowed region indicates an unsampled interval of the Vallès-Penedès record. The stratigraphic positions of the ACM localities studied in this work are shown to the right on the composite lithostratigraphic column. Note that the bottom boundary of the lowermost local biozone is unknown. dLife reconstruction of a Micromeryx azanzae male. Art by I.M.S DeMiguel et al. BMC Biology (2021) 19:6 Page 4 of 15 Table 1 Summary of mesowear and isotopic values of Micromeryx from the ACM sequence according to morphotypes and environmental phases Morphotypes #MpS pR pH MS #Cδ 13 CSD δ 13 C δ 18 O CO3 SD δ 18 O CO3 #Pδ 18 O PO4 SD δ 18 O PO4 Δδ 18 O CO3 - δ 18 O PO4 Morphotype 1 8 69.2 30.8 100 0.31 8 −10.5 0.7 27.9 1.0 6 19.6 1.4 8.4 Morphotype 2 24 80.6 19.4 100 0.19 21 −11.8 0.8 27.7 1.5 19 19.1 1.9 8.7 Morphotype 3 11 86.7 13.3 95 0.21 6 −11.0 1.2 27.0 0.6 5 18.5 1.1 8.3 Environmental phases #MpS pR pHMS#Cδ 13 CSD δ 13 C δ 18 O CO3 SD δ 18 O CO3 #Pδ 18 O PO4 SD δ 18 O PO4 Δδ 18 O CO3 - δ 18 O PO4 δ 13 C diet, mequ MAP a (mm/ year) MAP b (mm/ year) MAT (°C) Phase III (11.70–11.60 Ma) 23 71.9 28.1 97.3 0.32 19 −11.1 1.2 27.2 1.2 19 18.5 1.6 8.7 −27.2 801 608 17.1 Phase II (11.90–11.79 Ma) 15 83.3 16.7 100 0.16 13 −11.4 1.2 27.7 1.2 6 19.8 1.7 8.2 −27.5 992 765 20.3 Phase I (12.33–11.95 Ma) 7 90.9 9.1 100 0.09 5 −12.0 0.3 28.9 0.8 5 20.4 0.9 8.5 −28.1 1190 928 21.8 #M (number of samples for mesowear); percentage of specimens with sharp (pS) and rounded (pR) cusps; percentage of specimens with high (pH) occlusal relief; mesowear score (MS); #C (number of samples for stable isotope analyses on the carbonate fraction); mean δ 13 C(‰VPDB); standard deviation (SD) δ 13 C(‰VPDB); mean δ 18 O CO3 (‰VSMOW); standard deviation (SD) δ 18 O CO3 (‰VSMOW); #P (number of samples for stable isotope analyses on the phosphate fraction); mean δ 18 O PO4 (‰VSMOW); standard deviation (SD) δ 18 O PO4 (‰VSMOW); Δδ 18 O CO3 −δ 18 O PO4 , mean δ 13 C diet, mequ (‰VPDB); inferred mean MAP (mm/year) (from Kohn [21]) without (estimated MAP a ) and with (estimated MAP b ) altitude and latitude correction; and inferred mean MAT (°C) DeMiguel et al. BMC Biology (2021) 19:6 Page 5 of 15 the niche occupation per morphotype and locality given the variables investigated. MS and δ 13 C values point to a frequent ingestion of C 3 plants in woodland to mesic C 3 grasslands. Mean MS of some individuals of morphotypes 1 and 2 from a few localities (those with MS = 0.33 to 0.5) is also compatible with regular consumption of C 4 vegetation. C 4 plants have never been documented as an important component of plant communities in the Iberian Neogene (despite being recorded there since the Oligocene) [24,25]. However, they may well have been present to some extent in some areas and/or time intervals (e.g. ACM/C4-C1 and ACM/C5-C2 at ~ 11.8 Ma). Discussion Micromeryx diet at ACM Our results indicate that the bulk of Micromeryx diet at ACM consisted of foliage with a particular emphasis on forbs, dicots and woody leaves. The various morphotypes generally maintain sharpened, high-relief cusp apices and low MS—a signal that informs that foods were of relatively low abrasion, as seen in extant forestdweller browsers [26]. We rule out a regular consumption of fruits and/or seeds in ACM, as teeth show no signs of strong rounding or blunt apices—and frugivorous taxa have significant percentages of rounded and blunt cusps because of tip-crushing [27]. These results contrast with some previous data for middle and late Miocene Micromeryx from elsewhere in Europe, which appear strictly frugivorous [28–30]. However, these results are in agreement with the leaf browsing inferred for other Micromeryx [29,31]. Therefore, it seems that Micromeryx, since its oldest occurrences in the middle Miocene of Eurasia, was capable of feeding alternatively on fruits, seeds and soft leaves, depending on habitatspecific circumstances (e.g. ecologic niche partitioning or food availability). In the case of ACM, the unusual secondary crests of the upper molars of Micromeryx are compatible with an adaptation for heavier reliance on leaves and stems, as seen in other mammalian groups [32]—an anatomical trait that is consistent with the Fig. 2 Scatter plot of mesowear and δ 13 C values (‰VPDB) of Micromeryx. Stippled areas show the transition between C 3 -dominated diets, mixed C 3 -C 4 diets and C 4 -dominated diets. Colour informs about Micromeryx morphotype (green for Micromeryx morphotype 1, blue for Micromeryx morphotype 2 and yellow for Micromeryx morphotype 3), and symbol refers to the temporal range within the sedimentary sequence (diamond for 12.38–11.95 Ma, circle for 11.90–11.79 Ma and square for 11.70–11.60 Ma). See Additional file 1: Supplementary information, Notes 2 and 3 for further details DeMiguel et al. BMC Biology (2021) 19:6 Page 6 of 15 folivorous signal retrieved from mesowear analyses. The Micromeryx from ACM are therefore the only ones in which these features are recognised, probably showing a regional adaptation associated with the particularity of these environments. Moreover, Micromeryx had a wider dietary plasticity than modern Moschus, whose diet comprises mainly arboreal lichens (a resource rarely exploited by other ruminants), forbs and woody leaves [33]. Within such a generalised soft, leafy browsing, there are differences among morphotypes in tooth wear and isotopic values through time (Additional file 6: Table S1). In other words, the same morphotype behaves differently when the temporal gradient is considered. The less sharp and more rounded cusps of Micromeryx morphotypes 1 and 2 recorded from 11.81 Ma onwards (and a signal of browse-dominated mixed feeding for some individuals) reflect a more pronounced abrasion than in older specimens and indicate that more abrasive browse and/or some dust/grit-infested foliage was eaten—as extant browsers that feed on leafy, soft foods generally maintain sharpened/high relief cusps [27,31] (Additional file 6: Table S1). That is, abrasive browse and encroachments by exogenous dust/grit content was only slight or even absent in the diet of older Micromeryx, whereas it significantly increased in later forms along the ACM sequence. δ 13 C data support dietary inferences based on mesowear, as all Micromeryx morphotypes depict values that are within woodland to woodland-mesic C 3 grassland conditions, thereby indicating a consumption of both soft leaves and more abrasive grasses. Temporal patterns in Micromeryx tooth wear and stable isotopes along the ACM sequence The primate assemblage recorded at ACM [5] includes three great ape (dryopithecine) species from different genera (Pierolapithecus catalaunicus,Anoiapithecus brevirostris and Dryopithecus fontani)[5,10,16,34], the pliopithecoid Pliopithecus canmatensis [35], probably a second pliopithecoid unassigned to species [36]anda putative stem hominoid (Pliobates cataloniae)[11], alternatively interpreted as a pliopithecoid [37]. The distribution of these taxa is not homogeneous along the ACM sequence, with Pliopithecus postdating most great ape finds but preceding Pliobates [5] (Fig. 3a). We show mesowear and isotopic data of Micromeryx teeth in chronological order of the localities for a time span ranging from 12.38 to 11.63 Ma (Fig. 3), to compare their chronostratigraphic distribution with palaeoenvironmental changes through time. While remaining within a browsing dietary category based on C 3 plants, differences observed in both mesowear and isotopic values among Micromeryx specimens provide insight on the environmental changes along the ACM temporal sequence during the latest middle Miocene. This is particularly relevant given that the lack of other palaeoenvironmental proxies from this area (such as pollen or macroplant remains) hinders a more precise reconstruction of the vegetation structure and other characteristics of the various habitats occupied by primates. Overall, the data reported here indicate that ACM habitats became progressively less humid and more Fig. 3 Correlation of mesowear and isotopic values of Micromeryx arranged temporally along the ACM sequence. aEnvironmental phases, separated by dashed lines, and stratigraphic ranges of the primates recovered at ACM based on occurrence in localities. bAverage mesowear scores (MS) by locality for Micromeryx morphotypes. cMicromeryx tooth enamel raw and mean δ 13 C(‰VPDB). Calculated average modern equivalent of diet composition (δ 13 C diet, meq ,‰VPDB) and estimated MAP (mm/year) values (without [MAP a ] and with [MAP b ] altitude and latitude correction) are given in parentheses. dMicromeryx tooth enamel raw and mean δ 18 O CO3 and δ 18 O PO4 (‰VSMOW) values. Calculated MAT (°C) values are given in parentheses. Locally weighted polynomial lines are fitted to isotopic data. Calculations further include the 95% confidence region. Colour symbols are for raw data and grey symbols are for mean values. See Additional file 1: Supplementary information, Note 4 for further details DeMiguel et al. BMC Biology (2021) 19:6 Page 7 of 15 heterogeneous (or, at least, are characterised by a gradient toward less dense canopy structure and more open patches), as reflected by (i) a trend toward higher values of mesowear—from the sharpest cusps and low MS of 0 at 12.38 Ma (ACM/C1-Ee) to more intermediate (more rounded) cusp morphologies and higher MS around 0.5 at 11.60 Ma (ACM/C5-D1) (Fig. 3(b)); (ii) an increase in mean Micromeryx δ 13 C values (from −12.4‰in ACM/ C1-Ee to −11.1‰in ACM/C5-D1; Fig. 3(c)); and (iii)a high variability in the type of vegetation consumed—revealed by a wider range of δ 13 C values in the youngest localities and fuelled by the coexistence of the three morphotypes (see a change in standard deviation values in Table 1). The increase in mean δ 13 C values may have been driven by two phenomena: a shift toward drier habitats, including non-forest patches, or, alternatively, a greater reliance on fruits through time. A change toward a more frugivorous diet would have led to a slight increase in Micromeryx tooth enamel δ 13 C, as a significant consumption of fruits ultimately results in higher bioapatite δ 13 C values [38,39]. There is, however, little reason to support strong frugivory for ACM Micromeryx,as this is contradicted by their attrition-dominated mesowear patterns. This does not mean that fruits were unavailable at ACM. In fact, all of the primate species recorded relied on frugivory to a large extent, even if with a different emphasis on hard-object feeding depending on the species [11,40,41]. However, a drop in the estimated mean annual precipitation (MAP) values throughout the sequence (from ~ 1395 to ~ 762 mm/ year or from ~ 1097 to ~ 575 mm/year with altitude and latitude correction; Table 1, Fig. 3(c), Additional file 6: Table S1) supports the fact that fruits were preferentially exploited by arboreal, or at least semiterrestrial, species instead of terrestrial taxa such as Micromeryx. Although the oxygen isotope composition does not vary significantly through time, there is a slight decrease in both δ 18 O CO3 and δ 18 O PO4 values (from −28.8‰and 20.3‰in ACM/C1-Ee to 27.4‰and 18.7‰in ACM/ C5-D1; Fig. 3(c, d)). Oxygen isotope composition of carbonate (δ 18 O CO3 ) and phosphate (δ 18 O PO4 ) fractions of tooth enamel reflects δ 18 O of body water (δ 18 O bw )[42, 43]. Changes associated with δ 18 O bw value mirror variations in the isotopic composition of ingested water, either through drinking or plant water (in the case of herbivores). Micromeryx δ 18 O values are less likely to vary according to physiological factors like fractionated water loss through the lungs or skin, since this is a relatively mesic environment overall, and the species are closely related over a narrow time window. When considering extinct mammals such as Micromeryx, it is difficult to assess the type of water economy they may have had, due to the lack of modern analogues. The extant sister group of Micromeryx within the Moschidae is the genus Moschus (musk deer) [44], which inhabits forest and mountainous parts of Asia [33,45] and has a browsing diet, although it also has the ability to cope with poorer, less nutritious foods when high-quality forage is in short supply, such as in winter [33,46]. Independently from its dietary behaviour, Moschus has been observed to drink water on a daily basis (Prikhod’ko, pers. comm.); therefore, its tooth enamel δ 18 O signal will largely be dependent on drinking water δ 18 O values. On the assumption that the water reliance of Moschus is applicable to Micromeryx, mean annual temperature (MAT) values have been estimated based on Micromeryx tooth enamel δ 18 O PO4 values. They show a decreasing trend along the ACM sequence from 21.4 °C in ACM/ C1-Ee to 17.4 °C in ACM/C5-D1 (Table 1, Fig. 3(d), Additional file 6: Table S1). This trend toward lower temperatures may be framed within the gradual cooling that started by 14 Ma after the Mid-Miocene Climatic Optimum [47]. In the Iberian Peninsula, this long interval, which coincided with the expansion of mesothermic deciduous vegetation and the extinction or significant decrease in abundance of thermophilous evergreen plants [48,49], witnessed an increase in the diversity of moschids [18]. Primate assemblage composition in relation to palaeoenvironmental changes Our analyses further show a fluctuation in diet composition for Micromeryx individuals, revealing the existence of three distinct environmental phases in ACM (Table 1, Fig. 3(a–d)), with temporal patterns in precipitation, temperature and aridity that relate to changes in primate assemblage composition. Phase I A first phase ranges from the beginning of the sequence (12.38 Ma, ACM/C1-Ee) to ~ 11.95 Ma (ACM/C4-Cp), where only Micromeryx morphotype 2 is recorded. Overall, Micromeryx maintained sharp apices, high-relief cusp and low average MS of 0.1, and tooth enamel δ 13 C values of −12.0 ± 0.3‰(VPDB) (Table 1) that point to the consumption of plant resources from relatively dense wooded areas (see Additional file 1: Supplementary information, Note 4 for explanation of the calculated δ 13 C cut-off values among different habitats). Estimated MAP ranges from 928 to 1190 mm/year (depending on whether a correction for altitude and latitude is applied or not) (Table 1). In phase I, Micromeryx tooth enamel δ 18 O CO3 and δ 18 O PO4 values are the highest among the three environmental phases, with calculated MAT values reaching 21.8 °C (Table 1). According to Whittaker’s biome classification [50], estimated MAP and MAT for phase I would correspond to those of a tropical seasonal forest/savanna (Additional file 5: Figure S4). This agrees DeMiguel et al. BMC Biology (2021) 19:6 Page 8 of 15 with the soft-leafy browsing diet inferred from mesowear and indicates a humid climate with rainfall seasonality (although not marked) and the development of longstanding forests with bushy and woody vegetation [23, 31,51]. This type of environment at the beginning of the ACM series, characterised by humid and warm forests with a dense upper canopy, is somewhat more seasonal than previous inferences for ACM as a whole [52] and would be suitable for the multiple large-bodied hominoids—A. brevirostris,D. fontani and P. catalaunicus— recorded during phase I. The abundance of trees may have allowed hominoids to eat a diverse array of vegetation, ranging from leaves and soft fruits (Anoiapithecus and Dryopithecus) to harder and brittle fruits (Pierolapithecus)[11,41]. This also fits with the postcranial morphology of Pierolapithecus, which indicates an orthograde bodyplan with adaptations for arboreal vertical climbing [10,21,34,53]. Only a single Micromeryx specimen from ACM/C2-A3 (IPS29396) displays more rounded cusps and slightly higher δ 13 C and δ 18 O values. This specimen might reflect the exploitation of more abrasive elements probably located along less humid— but still forested—patches in specific localities. It is noteworthy that only one pliopithecoid is recorded in this first phase (Fig. 3(a)) (Pliopithecoidea indet. from ACM/C3-B2 at 12.06 Ma). This immediately precedes a first short pulse of decreased humidity as documented by ACM/C2-A3, which might explain the lack of great ape record between their first appearance in the sequence at 12.4–12.3 and their more abundant record at 12.0–11.9 [5]. This shifting climatic pattern at 11.98 Ma toward less humid conditions might have also influenced (preferred) food availability (as seen in Micromeryx) and impelled hominoids to exploit alternative sources, especially as fallback foods [41], not consumed before. Phase II ACM localities experienced a different environmental phase from ~ 11.90 to 11.79 Ma. There was a rapid increase in Micromeryx phenotypic diversity and population abundance after ACM/C3-Ak (11.88 Ma), with the first co-occurrence of all morphotypes (at least three) being recorded at ACM/C4-C1 (Fig. 3(b–d)). Compared to phase I, from 11.88 Ma onwards the less sharp and more rounded cusp shapes, higher average MS of 0.16 of Micromeryx and the increase in the mean δ 13 C(−11.4 ± 1.2‰, VPDB) (Table 1, Fig. 3(b, c)) are consistent with less humid and more open areas during this part of the ACM sequence. The broader range of δ 13 C observed in phase II (Fig. 3(c)) is congruent with a phase of increased habitat heterogeneity. Estimated MAP ranges from 765 to 992 mm/year, whereas estimated MAT is 20.3 °C (Table 1). The biomes of phase II would be in the domains of tropical seasonal forest/savanna and subtropical desert [50] (Additional file 5: Figure S4). In the light of the fauna recorded at ACM [15], we consider the latter inference as unrealistic and most likely attributable to a preservational bias toward drier ecosystems [54]. Alternatively, higher CO 2 levels during the Miocene might produce a similar bias in biome reconstructions, given their documented relationship not only with higher temperatures but also enhanced water-use efficiency and leaf-level productivity [55]. This “forest fertilization effect”, resulting from higher CO 2 levels in the Miocene, might have resulted in more forested environments than indicated by estimated MAP and TAP based on current standards. Discerning whether such potential biases apply uniformly to the whole ACM sequence would require a taxonomically broader isotopic sampling in selected ACM localities—as averaging values from multiple taxa from the same locality would arguably provide more robust MAP estimates [54]. Nevertheless, we consider that the palaeoenviromental changes recorded by Micromeryx isotopic values through time are at least valid in relative terms, even if their exact interpretation in terms of extant biomes should be subject to further scrutiny. The development of mosaic environments (with the earlier forested habitats containing for the first time partial clearing as new open patches) in ACM might have allowed the local evolution of new Micromeryx morphotypes (i.e. species) adapted to more open landscapes and with different dietary preferences (e.g. more abrasive forbs, shrubs and other ligneous vegetation rich in phytoliths, and even some grass). Such an interpretation is reinforced by the record at ACM/ C3-Ak of the bovid Tethytragus, a common faunal element in the more open and arid palaeoenvironments from inner Iberia that is otherwise not documented from the Vallès-Penedès Basin [56]. Our results support greater habitat heterogeneity, rather than a complete change in the palaeoenvironment compared to the previous phase. On the one hand, Micromeryx morphotype 2 (with affinity for humid conditions) persists in phase II with little variation in MS and δ 13 C—with higher values at the end of phase II (at ACM/C4-C1, 11.81 Ma) likely indicating that more abrasive foods and/or some grit loaded foliage was eaten at this time. On the other hand, the new morphotypes 1 and 3 appear for the first time with higher mean MS and δ 13 C values (Fig. 3(b, c)). These changes toward habitat (canopy) fragmentation, leading to a mosaic of forest patches interrupted by more open woodlands and maybe even shrublands, would have represented a challenge for the frugivorous and presumably arboreal great apes from ACM—especially in dietary terms (given the impossibility of maintaining a year-round supply of ripe fruits), and perhaps also from a locomotor viewpoint (at least for the highly arboreal Pierolapithecus, given the need to travel across DeMiguel et al. BMC Biology (2021) 19:6 Page 9 of 15