The systematics of the Cervidae: a total evidence approach

Cranium

All cervids share several anatomical features, such as two lacrimal foramina, a preorbital vacuity, and a lacrimal fossa (Fig. 1). In lateral view, the dorsal outline is convex at the braincase, concave at the fronto-nasal transition and straight at the nasals. The anterior extension of the snout is moderate depending on the overall size of the cervid species. The basicranial outline in lateral view is flexed. The preorbital vacuity varies in size and form, the lacrimal fossa can be deep and round, covering a large proportion of the facial aspect of the skull, shallow, or barely visible (particularly in females). The position of the two lacrimal foramina on the orbita rim (more internally or externally) and the position to each other is variable. A detailed description of the craniodental morphology for each cervid species investigated is in Heckeberg (2017a).

Some Miocene cervids have a sagittal crest (e.g. Dicrocerus and Procervulus), which is absent in all other cervids (Fig. 5). The number and size of supraorbital foramina and presence and absence of the supraorbital sulcus are variable and could potentially be features to distinguish groups of cervids; however, more specimens per species need to be investigated to confirm this. The presence of an extended vomerine septum and the division between the temporal foramina is characteristic for Capreolinae (Fig. 5). Most cervids have small, oval auditory bullae, some species have large inflated bullae (e.g. Axis) (Fig. 5).

Most Miocene cervids have long pedicles, the insertion point of the pedicle is directly above the orbit and the pedicle is upright (Fig. 6). Muntiacini, Euprox and Eostyloceros have long strongly inclined pedicles. In most other cervids the pedicles originate more posteriorly to the orbit, are inclined at 45–60, and short. Mazama and Pudu have strongly inclined and short pedicles.

Antlers

Based on evidence from comparative anatomy (Heckeberg, 2017b), the cranial appendages of early Miocene cervids, including lagomerycines, were shed and followed the same principles of the antler cycle as extant cervids and are therefore to be considered as antlers. Even though antlers are species-specific, they have a high variability, intraspecifically and ontogenetically. No antler looks exactly the same, not even the left and the right antler of the same individual are identical. Also, antlers change from one year to the next; in addition pathologies, abnormal growth, and other phenomena occur.

While cervid genera and most species can be qualitatively distinguished based on antler morphology, translation of these distinctions into discrete characters for quantitative or phylogenetic analyses is difficult. Convergence, which can be distinguished by eye, but is sometimes too subtle to be scored differently in the character matrix is the reason for this. Three morphotypes can be distinguished in cervids.

Dentition

Some dental characters are highly variable and thus difficult to score unambiguously. Despite convergent modifications depending on dietary requirements, a species-specific pattern underlies these adaptations in most species (N. Heckeberg, 2017, personal observation), particularly in the lower premolars and upper molars. The difficulty is to score these species-specific patterns without scoring the convergent adaptations and the intraspecific variability.

Upper premolars and molars

The upper incisors and the P1 are absent in cervids. The upper premolar row is characterised by robust, compact, predominantly horseshoe-shaped teeth. P3 and P4 are less variable, P2 can have more rectangular or triangular outlines, particularly in early fossil cervids. All premolars have at least one prominent central fold, except for Rangifer, in which central folds are missing (Fig. 7). Sometimes there are tiny additional folds, or the main central fold is serrated. A separation of the lingual cone into an antero- and posterolingual cone is relatively common (Fig. 7). In all Miocene cervids the P2 is longer than the P4, while in extant taxa the P4 is most often longer than the P2. Several fossil cervids have a well developed lingual cingulum (Fig. 7).

The upper molars are all two-lobed and quadrangular with only little variation in morphology. The posterior lobe of the M3 is distinctively smaller than the anterior one in most species. The entostyles are variably present. In some species the entostyle(s) has/have a λ-shaped morphology, especially in later wear (Axis, Rusa, Rucervus and Elaphurus) (Fig. 7). Metaconule folds are variably present within Cervinae and Capreolinae and are mostly small. Protocone folds are usually absent in Cervinae, while they are regularly present Capreolinae, often well developed on all molars (Fig. 7). The same applies to fossil cervids, where tiny metaconule folds are much more common than protocone folds. Only in Miocene cervids protocone folds are common. However, in these species it often looks more like a bifurcation of the postprotocrista than a fold originating from the crista, particularly when the internal part of this bifurcation is longer than the external as on M2 in Dicrocerus. It is not entirely evident, whether these are two independent structures or the same structure with variable characteristics. Several species have an anterior cingulum and some fossil cervids have a lingual cingulum. The protocone and metaconule folds are variably present. In a few species, for example Rucervus eldii, the premetaconulecrista is serrated (Fig. 7) More details are in Heckeberg (2017a).

Lower premolars and molars

p1 is usually absent in cervids, it was present in individual Lagomeryx parvulus specimens. The p2 has a simpler morphology with fewer elements compared to p3 and p4. A strong reduction in p2 length could be observed in Mazama and particularly in Ozotoceros. In a few specimens the p2 is missing. Mesolingual cristids were variably present in p3 and p4 (absent in Axis, often absent in early Miocene species) (Fig. 8). p3 and p4 often show modifications of tooth elements, which make them more similar to molars to a different extent. While the p3 shows these modifications only in a few species and not to the same extent as p4, the p4 is modified in many species, especially in Rangifer and Alces (Fig. 9). The labial incision on premolars is rarely and weakly developed in p2; it is more often developed on p3, and most often occurs on p4 (Fig. 8). p4 is the most variable tooth in cervids.

Some species show a spike like extension of the posterolabial conid of the p4 towards labiad; these species are Capreolus capreolus, Capreolus pygargus, Blastocerus dichotomus, Hippocamelus spp., Hydropotes inermis, Ozotoceros bezoarticus, Croizetoceros ramosus, Procapreolus cusanus and ‘Cervus’ philisi (Fig. 8). Whether this feature can be used as a phylogenetic character and whether it is indicative of affiliation to a certain subclade has to be investigated in the future.

All lower molars have a similar morphology; m1 and m2 are two-lobed, m3 is three-lobed. The orientation of the lingual conids and cristids may be more diagonal in some species. Ectostylids are variably present on one to three molars. never high, nevertheless they become involved in wear in aged individuals (Fig. 8). In most Miocene cervids and in Cervus australis external postprotocristids are present on all molars (Fig. 8). Anterior cingulids are present in several species, usually more prominent on the anterior molar position(s). In Rucervus and Rusa the anterior cingulids are particularly prominent (Fig. 8). In Rucervus and also to a lesser extent in Rusa and Axis the anterior and posterior labial walls of the lobes of the lower molars are indented (Fig. 8). The metastylids can be bent labiad in some species, for example Alces. The third lobe on m3 is variable; most often the hypoconulid and entoconulid are connected via the postento- and posthypoconulidcristids and form a crescent-shaped structure. Sometimes the third lobe is reduced to one of these elements or has an additional fold on the posthypoconulidcristid. In a few individuals the third lobe is missing entirely. More details are in Heckeberg (2017a).

Lower incisors and canines, upper canines

All Miocene cervids have enlarged upper canines, which are curved posteriad. From the Pliocene onwards, the upper canines become reduced in size and are lost in some species. Extant muntiacines have enlarged upper canines, similar to those of Miocene cervids. Hydropotes has strongly elongated sabretooth-like upper canines, which differ in morphology from those in muntiacines and early fossil cervids. In all other extant species upper canines are reduced in size or missing entirely. Most cervines possess small upper canines. Adult capreolines rarely have upper canines, while some capreoline juveniles have deciduous upper canines.

The lower incisors, i1–i3, have a simple spatulate morphology. The crown width decreases from i1 to i3, that is i1 typically is distinctively broader than i2 and i3. Exceptions are Alces, Hippocamelus, and Pudu, where i1 is only a little broader than i2. All lower canines in Cervidae are incisiviform. More details are in Heckeberg (2017a).

Nuclear genes

Although interpretations of the systematic relationships on genus and species level was difficult in the single gene topologies due to low taxon sampling and/or lack of resolution, the combined nuclear topology was well resolved and supports the higher hierarchical clades. The BI and the ML topologies were largely congruent (Fig. 13). There was no split into Odocoileina and Blastocerina as observed in the topologies based on the mitochondrial markers. The unexpected placement of Capreolus capreolus in this topology may be caused by the possibly contaminated Sry sequence of this species.

Combined mitochondrial genes

The BI topology of the combined mitochondrial analysis showed higher support values for the majority of nodes than the Cytb only topology, but lower support values for some nodes than for the mtG analysis. The ML topology differed in generally lower support values for most nodes, but was otherwise largely congruent (Fig. 13). The placement of non-cervid ruminants differed in both topologies. The main difference concerning cervid taxa is the position of Pudu mephistophiles (based on the correct Cytb sequence (Heckeberg et al., 2016)), which was the sister taxon to Blastocerina in the BI topology and the sister taxon to Rangifer and Odocoileini in the ML topology. This combined topology includes the polyphylies for Rucervus, Hippocamelus, Odocoileus, Mazama and Pudu.

Combined molecular analyses

The BI and ML topologies of the combined nuclear and mitochondrial analyses were largely congruent, the support values were partly lower, particularly in the ML topology, in comparison to the topologies based on the mitochondrial markers (Fig. 13). Both topologies differed in the position of non-cervid ruminants, and the positions of Alces alces and Pudu mephistophiles, which remain uncertain. The split of Odocoileini into Blastocerina and Odocoileina was supported.

Bayesian inference

The BI combined topology was largely unresolved (Fig. 14). Most extant cervids formed clades; the three Axis species and two Rucervus species formed a well supported clade. There was also an supported clade including eight Miocene cervids.

Maximum likelihood

In the ML combined topology the nodes were poorly or not at all supported (Fig. 14). Some extant clades were recovered, for example Muntiacini, Odocoileina, Capreolini. Eight Miocene cervids formed a clade.

Maximum parsimony

The nodes in the MP combined topology are largely unsupported (Fig. 14). Procapreolus cusanus was unexpectedly placed as the sister taxon to Moschus. Cervinae, Cervini, Muntiacini, and Odocoileini form unsupported clades. Capreolini is a supported clade. All Miocene cervids except for Eostyloceros hezhengensis form a clade.

Figure 15 qualitatively summarises the topologies from all analyses undertaken. The topology was not generated by an analysis but was drawn to show the consensus of all topologies and which character sets support the respective nodes.

Supplemental Information on the phylogenetic analyses and topologies can be found in the Suppemental Files (S2; Data Sets S1 and S3).

Cranium

The cranial morphology of cervids is highly conservative (Lister, 1996; Merino & Rossi, 2010). Also, some morphological characters in ruminants likely are the results of convergent evolution and thus are homoplastic, which may cause difficulties in reconstructing phylogenetic relationships (Bouvrain, Geraads & Jehenne, 1989; Douzery & Randi, 1997). Despite the homoplasy, some clades were well defined and re-occurring across different data sets in the topologies here.

Differences in the size of the praeorbital vacuity are primarily species specific, but have also an ontogenetic component, since they are often smaller in aged individuals. Similarly, the lacrimal fossa varies in size and depth in different species, presumably depending on the presence, size, and usage of the lacrimal gland and sexual dimorphism. The position of the lacrimal foramina to each other and on the orbita rim can potentially be used to distinguish groups of cervids. The consistent presence of two lacrimal foramina is typical for cervids, but is also present in some bovid species. In Dremotherium feignouxi sometimes only one lacrimal foramen is present (Costeur, 2011). The contact of the lacrimal and the frontal at the orbita rim without interlocking sutures was first observed in Rössner (1995). This trait is most likely an intraspecific variability and could be an effect of ageing.

Evolutionary trends observed in Pliocene cervids include an increase of the overall body size, a decrease of the pedicle length relative to the antler length and an associated increase of the antler length (Heintz, 1970). The degree of inclination of the pedicles changes through time and is presumably a result adapting to rich vegetation. With the stronger inclination the insertion point of the pedicle on the skull moved posteriad. The pedicle in early Miocene cervids is entirely above the supraorbital process and not in contact with the braincase; the pedicles are vertical in lateral view, parallel or converging in frontal view. The shortening of the pedicles could be related to the increasing size of antlers, because a longer and heavier set of antlers would put a biomechanically unfavourable leverage on long pedicles.

Basicranial and ear region characters were not yet widely used when inferring morphological phylogenies, but were assumed to have strong potential to provide characters, which are less prone to convergent evolution caused by climatic change (Janis & Theodor, 2014). Recently, it has been shown that traits of the inner ear provide useful characters with phylogenetic signal (Mennecart et al., 2016, 2017).

Antlers

There is broad consensus that antlers originated only once (Loomis, 1928; Azanza & Morales, 1989; Azanza, 1993a, 1993b; Azanza, DeMiguel & Andrés, 2011; Heckeberg, 2017b). The antlers of most Miocene cervids have a simple bifurcating pattern, sometimes with an additional tine, or are coronate (Azanza, DeMiguel & Andrés, 2011). These antlers are relatively short, do not have a shaft and the bifurcation originates directly from a broad antler base. From the late Miocene onwards, more complex branching patterns developed, the length of antlers increased and antlers developed a shaft below the first bifurcation. Evolution of size and complexity of antlers is associated with reduction or loss of upper canines (Scott, 1937; Beninde, 1937; Geist, 1966; Brokx, 1972).

In extant cervids, short and simple antlers and long and more complex or palmated antlers are present. Many extant cervids develop exactly three tines (Heckeberg, 2017b). The three antler morphotypes have previously been associated with ecological habitats: simple antlers for the tropics, a three-tined antler plan for woodland areas typical in East Eurasia or India, and the large and complex display organs in temperate regions (Pitra et al., 2004). The simple antlers in Mazama and Pudu are considered as a secondary adaptation to dense vegetation.

There is a lot of inter- and intraspecific variation in antlers (Goss, 1983; Heckeberg, 2017b). The high variability of antlers is a problem particularly in fossil taxa, where the entire intraspecific variation cannot always be observed due to the lack of a sufficient number of specimens or the incompleteness of ontogenetic stages. The taxonomy of fossil cervids is often based on antler morphology, because antlers are easy to identify and numerous in the fossil record and antler morphology is more distinctive than other anatomical characters (Kurtén, 1968; Fry & Gustafson, 1974; Lister et al., 2010; Merino & Rossi, 2010). Thus, the validity of some fossil cervid taxa is doubtful. To base classifications just on antler morphology is problematic for the given reasons.

In contrast to Loomis (1928), Gentry, Rössner & Heizmann (1999) stated that cranial appendage morphology proved to be more suitable than tooth morphology to distinguish species of horned Pecora. It is true that different cervid species can be easily identified based on their antler morphology (branching pattern, orientation, size). Antler characters were often used to solve intra-subfamily relationships, but they are problematic because of convergent development and subsequent homoplasy in antler characters (Pitra et al., 2004).

Since Cervidae is diagnosed by the presence of antlers (Janis & Scott, 1987; Pitra et al., 2004), the reason for the absence of antlers in Hydropotes inermis species was controversially discussed; a primitive condition and secondary loss have been suggested (Bouvrain, Geraads & Jehenne, 1989; Hernández Fernández & Vrba, 2005; Hassanin et al., 2012; Schilling & Rössner, 2017). The robust placement of Hydropotes inermis as the sister taxon to Capreolus proves the secondary-loss hypothesis. However, the process of antler loss is not known, neither is the process(es), which trigger(s) the growth of the first set of antlers in antler-bearing species. Hydropotes inermis might be the key to investigate these processes.

Dentition

Variations of accessory dental elements in combination with the degree of modifications of tooth elements of premolars can be used to identify genera or species. Widely accepted evolutionary trends in cervids concerning the dentition are increasing hypsodonty, the reduction of the premolar row length and the reduction or loss of upper canines (Heintz, 1970; Dong, Pan & Liu, 2004). However, the hypsodonty index, although widely used in ruminant phylogeny, has been considered to be a misleading character due to its ambiguous definition and convergent evolution among all large herbivorous mammals (Janis & Scott, 1987; Hassanin & Douzery, 2003).

The first deer had brachyodont dentition and were considered as leaf-eaters; recent dental analyses generally support these findings, but also showed that Procervulus ginsburgi likely was a seasonal mixed feeder. Based on this a facultative leaf-grass mixed feeding strategy with preference for leaf-eating is likely the primitive dietary state in cervids and ruminants (DeMiguel et al., 2008).

Ginsburg & Heintz (1966) regarded the bifurcation of the postprotocrista into an internal and external crista as a derived cervid character based on its presence in Dicrocerus and Euprox. Amphimoschus is the only other non-cervid pecoran species that shows this trait (Janis & Scott, 1987). The bifurcated postprotocrista was regarded as an advanced cervoid character in Janis & Scott (1987), while later this character is referred to as ‘primitive presence of bifurcated protocone’. In extant cervids, this feature is present in Odocoileus, Blastocerus, Alces, Mazama, Pudu and Capreolus (Janis & Scott, 1987). These observations could be confirmed here by morphological comparisons. One specimen of Palaeoplatyceros hispanicus (MNCN 39181) shows both a bifurcating postprotocrista and a tiny protocone fold on the preprotocrista. This indicates that both structures may in fact be developmentally independent, however, as this could only be observed in one specimen, it remains speculation.

Throughout the evolutionary history of cervids the lingual cingulum, regularly present on molars and sometimes even on premolars of fossil cervids, becomes reduced and eventually lost in extant cervids. In Rucervus, Rusa, and Axis the anterior and posterior lingual walls of the molars tend to be indented; this is also observed in Axis lydekkeri, Rusa kendengensis and ‘Cervus’ sivalensis.

The lower p2 is the tooth with the fewest changes in occlusal morphology throughout cervid evolution; only a shortening is observed in most extant taxa and in a few individuals p2 was lost. The lower p3 and p4 are more variable (Fig. 9) and sometimes become more similar to molars in modern cervids.

The elongated upper canines in Hydropotes inermis are used in intraspecific fights. It is likely that the presence and/or size of upper canines is somehow genetically linked with the antlers, which leads to the question, why female deer have upper canines, too (Brokx, 1972). Even though they are often much smaller, especially in species, where males have enlarged upper canines, they are present without any obvious function. In other ungulates, where males use their canines in intraspecific fights, for example in equids, upper and lower canines are lost in almost all females. Much more research is needed to find this link and associated interactions and effects on behaviour.

Lagomeryx parvulus and ligeromeryx praestans

Qualitative morphological comparisons, especially on antler morphology, suggest that Lagomeryx parvulus and Ligeromeryx praestans and also Paradicrocerus (not included here) are closely related to each other. Only one analysis (cranial data set) here supports the sister taxon relationship of the former two taxa. Based on external comparative morphology of the cranial appendages, it was found that lagomerycines had antlers, which were shed (Heckeberg, 2017b). Therefore, they are included in Cervidae and a subfamily Lagomerycinae would be justified based on morphological qualitative comparisons, but is not strongly supported in the topologies. Data completeness or presence of specific characters that are absent in the other taxon could be the reasons for this. Also, whether lagomerycines(-ids) form a family as the sister taxon to Cervidae could not be entirely ruled out, but the tendency of Ligeromeryx, Lagomeryx, and Palaeoplatyceros to form a clade within a clade consisting of Miocene taxa (Figs. 14 and 15) indicates that lagomerycines form a subfamily of Cervidae in a stem position.

The systematic position of lagomerycines, has always been controversial. They have been considered as a family between Giraffidae and Cervidae (De Chardin, 1939), as part of the superfamily Cervoidea (Romer, 1966; Viret, 1961; Young, 1964), as a separate subfamily within Cervidae (Vislobokova, Changkang & Sun, 1989), as a family of aberrant giraffoids, as a junior synonym of Palaeomerycidae (Pilgrim, 1941; Simpson, 1945; Young, 1964), as junior synonym of Muntiacini/-ae (Chow & Shih, 1978), as more closely related to Antilocapridae (Ginsburg, 1985; Solounias, 1988), or as representing an entirely independent clade (Bubenik & Bubenik, 1986; Azanza, 1993b; Azanza & Ginsburg, 1997).

The discussions on the taxon in the literature and the new insights resulting from the analyses here clearly show that the systematic position of Lagomerycinae represents one of the most controversial of ruminant families, so far without unambiguous consensus. However, cranial and postcranial morphology and particularly the presence of antlers support the affiliation as stem Cervidae (Chow & Shih, 1978; Leinders & Heintz, 1980; Vislobokova, Changkang & Sun, 1989; Azanza & Ginsburg, 1997; Heckeberg, 2017b; Mennecart et al., 2017).

Procervulus dichotomus and procervulus praelucidus

In most analyses here, Procervulus was placed in a stem position and Procervulus and Dicrocerus were more closely related to each other than to other cervids. A sister taxon relationship of Procervulus and Heteroprox was not observed. In the combined morphological and TE analyses, a close relationship of Procervulus dichotomus and Procervulus praelucidus to Dicrocerus elegans was confirmed.

Procervulus was assumed to be the Miocene descendant of Amphitragulus and Dremotherium (Gentry, 1994; Rössner, 1995). Presumably, transitional forms existed, which were not documented in the fossil record (Rössner, 1995). Procervulus has often been hypothesised to be the sister taxon to all other cervids (Janis & Scott, 1987; Groves, 2007). In previous studies, Procervulus was placed as the sister taxon to Heteroprox Azanza Asensio (2000), Mennecart et al. (2016) and both were the sister taxon to the clade containing Dicrocerus elegans. In Mennecart et al. (2017) Procervulus dichotomus was the sister taxon to Heteroprox larteti and Procervulus praelucidus the sister taxon to both of them; this clade was placed between Lagomeryx parvulus and all other cervids, which is similar to the results here.

Heteroprox larteti

In the analyses here, Heteroprox larteti was most often placed in an unresolved position, between the outgroup and cervids, as the sister taxon to Euprox furcatus or Dicrocerus elegans, or in a clade with other Miocene taxa (morphology, TE). Some topologies indicated a potential closer relationship to Muntiacini based on apomorphic characters, similar to Euprox furcatus.

Heteroprox was assumed to be the descendant of Procervulus (Rössner, 1995). In Azanza Asensio (2000) Heteroprox was most often placed as the sister taxon to Procervulus or as an (unresolved) stem lineage. Similarly, in Mennecart et al. (2017) Heteroprox larteti was the sister taxon to Procervulus dichotomus.

Dicrocerus elegans

In the analyses here, Dicrocerus elegans was most often placed closely related to Procervulus, sometimes as the sister taxon to Heteroprox larteti, or between the outgroup and cervids. Based on the results here and discussions in the literature, Dicrocerus is most certainly a stem cervid with affinities primarily to Procervulus and secondarily to other Miocene cervids. In a few analyses a potentially closer relationship to Muntiacini was observed.

Azanza, DeMiguel & Andrés (2011) suggested that Dicrocerus is a transitional form between the Procervulinae and crown Cervidae, which had also been hypothesised by Vislobokova (1990). In Azanza Asensio (2000) Dicrocerus elegans was placed as the sister taxon to Acteocemas and Stehlinoceros (=Paradicrocerus) and this clade was the sister taxon to all burr-bearing antlered cervids. In Mennecart et al. (2017) Dicrocerus elegans was the sister taxon to Eostyloceros hezhengensis in a sister taxon position to the crown cervids.

Euprox furcatus

In the TE analyses here, Euprox furcatus was most often placed in an unresolved position or as the sister taxon to Heteroprox larteti; in the TE analyses it was placed in a clade with other Miocene cervids. The results indicate that Euprox furcatus shares characters with other Miocene cervids. However, antler and pedicle morphology is apomorphic and resembles that of extant Muntiacini.

It was suggested that modern Muntiacus and fossil muntiacines such as Eostyloceros, Metacervulus and Paracervulus diverged from Euprox (Vislobokova, 1990; Croitor, 2014). Euprox was the first cervid with burr-bearing antlers and a pedicle inclination similar to that of muntjacs. Therefore, it has been suggested in several studies that Euprox may be the earliest representative of crown cervids (Azanza, 1993b; Gentry, Rössner & Heizmann, 1999; Dong, 2007; Azanza, Rössner & Ortiz-Jaureguizar, 2013; Mennecart et al., 2016, 2017). It was often considered as a member of Muntiacini, which would imply that Muntiacini is the sister taxon to all other cervids. In Azanza Asensio (2000), Euprox is variably placed closely related to Amphiprox, to extant Muntiacus and Elaphodus, to Eostyloceros, or to Metacervulus, or as the sister taxon to a clade containing all five of the above species or a subset thereof. In Mennecart et al. (2016), Euprox furcatus was placed as the sister taxon to Cervus elaphus. They further stated that Dicrocerus elegans, Euprox furcatus and Cervus elaphus differ from the other Miocene cervids, that is Procervulinae, in certain inner ear characters; Euprox furcatus had the most derived characters among them. In Mennecart et al. (2017) Euprox furcatus was placed as the sister taxon to all crown cervids.

There is a large temporal gap in the early putative fossil muntjac-like cervid lineage between the first representatives, Euprox, and the presumed direct ancestors of muntiacines, for example Eostyloceros (Azanza Asensio & Menendez, 1989; Azanza, 1993b), and additionally an even larger gap between those early fossils and the first members of extant Muntiacus, which appear in the Pleistocene. For more certainty of the systematic relationships it would be crucial to find more fossil material that would link the early presumed muntiacines with the crown muntiacines.

Palaeoplatyceros hispanicus

In most analyses here Palaeoplatyceros hispanicus was placed between the outgroup and cervids, as the sister taxon to Lagomeryx parvulus or as the sister taxon to most other Miocene taxa. Palaeoplatyceros is highly incomplete and has a combination of plesiomorphic traits and apomorphic traits, such as ‘presence of a burr’.

Palaeoplatyceros hispanicus can be distinguished from all other contemporaneous cervid species based on the palmation of antlers; however, its systematic position is problematic (Azanza Asensio, 2000). In Azanza Asensio (2000), Palaeoplatyceros was mostly placed as the sister taxon to all other cervids with burr-bearing antlers. Unless more material becomes available, its systematic position will remain controversial. Based on the analyses here, Palaeoplatyceros is likely a stem cervid with burr-bearing antlers.

Pliocervus matheronis

Pliocervus matheronis is known from the Messinian (upper Turolian, MN13). In the analyses here, Pliocervus matheronis was most often placed in an unresolved position, mostly between the outgroup and cervids and sometimes related to other Miocene taxa.

Although Simpson (1945) included Pliocervinae, comprising Cervocerus, Cervavitus, Procervus, and Pliocervus, which were regarded as the immediate crown Cervini precursors (Gentry, 1994; Groves, 2007), in Cervinae, others could not find any phylogenetic relationship of Pliocervus with Cervini/Cervinae (Petronio et al., 2007). Gentry, Rössner & Heizmann (1999) placed Cervavitus and Pliocervus among Cervoidea, whereas Azanza & Montoya (1995) and Azanza Asensio (2000) classified Pliocervus as Cervinae. It was suggested to be closely related to the holometacarpal Cervavitus within Pliocervini, which was included in Cervinae (Czyżewska, 1968; Vislobokova, 1990; Azanza Asensio, 2000).

The high morphological similarity of Pliocervus matheronis to the late Miocene Pavlodaria orlovi implies that these two genera could be closely related or possibly even synonymous. It was suggested that the subfamily Pliocervinae Symeonidis (1974), containing Pliocervus and Pavlodaria is a synonym of Capreolinae. In Azanza Asensio (2000) Pliocervus matheronis was variably placed and seems to have the highest proportion of apomorphic characters compared to other Miocene cervids. In most recent studies Pliocervus was regarded as incertae sedis (Croitor, 2014, 2018).

A definite morphological characterisation of Pliocervus is still missing and its systematic position remains controversial (Godina et al., 1962; Czyżewska, 1968; Korotkevich, 1970; Azanza Asensio, 2000; Petronio et al., 2007; Croitor, 2014). More and new morphological and biometric data are needed to solve the systematic relationships of ‘pliocervines’ (Di Stefano & Petronio, 2002).

Eostyloceros hezhengensis

Eostyloceros hezhengensis from the late Miocene of China was used for scoring characters (Deng et al., 2014). In the analyses here, Eostyloceros hezhengensis was most often placed in an unresolved position or within Muntiacini, suggesting that it is probably more closely related to muntjacs than to other cervids, which would support results from comparative morphology. Thus, Eostyloceros hezhengensis can be considered as a direct ancestor of muntjacs.

Euprox is considered as the direct ancestor of Eostyloceros, Metacervulus, and Paracervulus; after a change from subtropical to more temperate climate and Euprox-like cervids were replaced by representatives of Eostyloceros (Azanza Asensio & Menendez, 1989; Azanza, 1993b; Pitra et al., 2004). This lineage is assumed to lead to extant Muntiacus (Vislobokova, 1990; Croitor, 2014). In Azanza Asensio (2000), Eostyloceros was always closely related to Muntiacus and Metacervulus, while in Mennecart et al. (2017) Eostyloceros hezhengensis was not placed within Muntiacini but was the sister taxon to Dicrocerus elegans.

Pliocene and Plio-Pleistocene Cervids

There is no generally accepted classification of Plio- and Plio-Pleistocene cervids Pfeiffer (1999); however, for Villafranchian cervids (MN16) the following classifications were suggested: Croizetoceros ramosus, Metacervocerus pardinensis, ‘Cervus’ philisi, ‘Cervus’ perolensis, Eucladoceros ctenoides were considered as Cervini, Arvernoceros ardei as Megacerini, and Libralces gallicus (not included here) and Procapreolus cusanus were considered as Capreolinae.

In most morphological topologies here, Plio- and Pleistocene cervids were placed within crown cervids, sometimes forming subclades. Some Plio- and Pleistocene cervids were placed more closely related to extant Cervini. Most of them were nested in a clade together with Pleistocene cervids. In a few topologies the majority of Pliocene cervids were in an unresolved sister taxon position to all other Cervinae.

Cervus australis

In the phylogenetic analyses here, Cervus australis was most often placed in an unresolved position, sometimes closer to Muntiacini than to other cervids; it was also placed between the outgroup and cervids, as the sister taxon to Eostyloceros hezhengensis and Praeelaphus etueriarum, to Hippocamelus bisulcus, or Muntiacus muntjak. Based on qualitative morphological comparisons it is most likely a stem cervid, potentially closer to Muntiacini.

This species was originally described by De Serres (1832) and all known specimens are from Montpellier, France (Gervais, 1852; Czyżewska, 1959). Little further information is available in the literature concerning this species. Many entries point to muntiacines, for example Paracervulus australis (Gentry, 2005); however, there are no obvious similarities to muntiacines in the investigated specimens. Croitor (2018) also confirms an incertae sedis status for this cervid based on comparative morphology. Thus, the systematic position of Cervus australis remains uncertain.

Arvernoceros ardei

In the analyses here, Arvernoceros ardei was placed in an unresolved position, often close to or within Cervini. In some topologies it was placed as the sister taxon to Metacervocerus pardinensis, Praeelaphus perrieri, and Metacervocerus rhenanus. It was placed as the sister taxon to Dama dama in several topologies.

Arvernoceros was part of the first radiation of Cervinae/-i together with Metacervocerus, Praeelaphus, Axis and Rucervus (Croitor, 2014). The systematic position of Arvernoceros ardei has been subject to speculation for decades, its definition is still incomplete and affinities to other cervids unclear. Depéret (1884) found similarity to Axis, but no affiliation to Dama; it was suggested that it is most similar to Megacerini (Heintz, 1970; Vislobokova, 1990, 2012). Arvernoceros ardei was considered to be closely related to modern Elaphurus (De Chardin & Piveteau, 1930), declared as incertae sedis genus by Lister (1987), closely related to Axis Di Stefano & Petronio (2002), closely related to Rucervus (Croitor, 2009; Croitor, 2018). Despite some uncertainties in the morphological analyses, a closer relationship to Dama dama than to other cervids was suggested here.

Croizetoceros ramosus

In most of the analyses here, Croizetoceros ramosus was placed in an unresolved position; it was sometimes the sister taxon to Procapreolus cusanus, Alces alces, Ozotoceros bezoarticus, or Odocoileus. The results suggest a placement within Capreolinae and most likely within Odocoileini.

The antler morphology of Croizetoceros ramosus does not share similarities with any extant cervid species or with other cervid species from the Villafranchian (Heintz, 1970). Unfortunately, there is not much known about its skull morphology (Croitor, 2014). In Mennecart et al. (2017) Croizetoceros was placed as the sister taxon to Capreolinae.

‘Cervus’ perolensis

In the analyses here, ‘Cervus’ perolensis was placed in an unresolved position and as the sister taxon to several cervine taxa. Repeated placements within Cervini suggest that ‘Cervus’ perolensis almost certainly belongs to Cervini and is likely closely related to and/or an ancestor of Cervus.

‘Cervus’ perolensis, Metacervocerus rhenanus, and ‘Cervus’ philisi were found to be similar to each other and ‘Cervus’ perolensis and Metacervocerus pardinensis were classified as Pseudodama Azzaroli (1953); Azzaroli & Mazza (1992a). Later, ‘Cervus’ perolensis was considered as a descendant of ‘Cervus’ philisi by Stefaniak & Stefaniak (1995). Spaan (1992), however, concluded that ‘Cervus’ philisi and ‘Cervus’ perolensis are junior synonyms of Metacervocerus rhenanus and should be renamed as such, which was supported by Pfeiffer (1999). If this were true, ‘Cervus’ philisi and ‘Cervus’ perolensis should come out in a similar systematic position as Metacervocerus rhenanus.

Procapreolus cusanus

In the analyses here, Procapreolus cusanus was placed between the outgroup and cervids, within Capreolinae, sometimes within Odocoileini, and as the sister taxon to both Capreolus. Thus, Procapreolus cusanus most likely belongs to Capreolinae and the previously suggested close relationship to Capreolus was confirmed in some analyses.

Despite the widely accepted assumption that Procapreolus cusanus is closely related to or even a direct ancestor of Capreolus, the origin of Capreolus within Procapreolus is still under debate (Lechner-Doll et al., 2001). Some authors hypothesise that it may be assigned to Capreolus rather than Procapreolus (Valli, 2010). Others place it in an intermediate position between lower Pliocene and Pleistocene Procapreolus species and extant Capreolus (Czyżewska, 1968; Heintz, 1970; Lechner-Doll et al., 2001).

Metacervocerus pardinensis

In the analyses here, Metacervocerus pardinensis was most often closely related to or within Cervini, which suggests that Metacervocerus pardinensis is a member of Cervini and probably a close relative and/or ancestor of Cervus.

The temporal distribution of Metacervocerus pardinensis suggests that it could be an ancestor of ‘Cervus’ philisi. Metacervocerus pardinensis and Metacervocerus rhenanus have enough morphological differences to justify two different species (Spaan, 1992). Dietrich (1938) proposed that Metacervocerus pardinensis is synonymous with etueriarum, perrieri, issiodorensis and rhenanus. Based on similarities to Rusa deer, the genus Metacervoceros was erected to represent European rusine deer (Croitor, 2006a). However, their systematic position remained controversial. Metacervocerus pardinensis was classified as Pseudodama by Azzaroli & Mazza (1992a), while De Vos & Reumer (1995) assigned Metacervocerus pardinensis and Metacervocerus rhenanus to Cervus, Pfeiffer (1999) to Dama, and Di Stefano & Petronio (2002) to Rusa. Differences in the skull morphology suggest that Metacervocerus does not belong to the Cervus–Rusa evolutionary lineage, which needs stronger evidence from the fossil record. Croitor (2014) suggested it is more likely that Metacervocerus pardinensis represents an ancestor of Dama.

Praeelaphus perrieri

In the analyses here, Praeelaphus perrieri was placed close to or within Cervini, which suggests that Praeelaphus perrieri is a member of Cervini and probably closely related to and/or the ancestor of Cervus.

The teeth and postcranial material from Praeelaphus perrieri and Eucladoceros are indistinguishable; however, Praeelaphus perrieri and Eucladoceros ctenoides do not coexist in any of the known localities, although they occupy the same niches. The systematic relationships remained uncertain (Croitor, 2014). Already Portis (1920) proposed a new subgenus Praeelaphus for ‘Cervus’ perrieri, as well as for C. avernensis, C. etueriarum from the early Villafranchian (Croitor, 2014). Praeelaphus perrieri was considered as the earliest representative of Cervus in Europe by Di Stefano & Petronio (2002), however, even though it is an early cervine, there is no clear evidence that it is directly related to Cervus and it more likely represents an extinct lineage within the early cervine evolution (Croitor, 2014).

Praeelaphus etueriarum

In the analyses here, Praeelaphus etueriarum was placed between Eostyloceros hezhengensis and Eucladoceros ctenoides, as the sister taxon to Metacervocerus rhenanus, Eostyloceros hezhengensis, or Eucladoceros ctenoides. Placements as the sister taxon to the Cervus-clade and within Muntiacini suggest that Praeelaphus etueriarum belongs to Cervinae and most likely to Cervini.

There is consensus that Praeelaphus is a member of the early radiation of Cervini and perrieri, warthae, and lyra may be synonyms as they represent similar and contemporaneous cervids (see above) (Croitor, 2014). Heintz (1970) suggested that Praeelaphus etueriarum was established based on a juvenile Praeelaphus perrieri, which is yet to be proven.

Eucladoceros ctenoides

Here, Eucladoceros ctenoides was most often placed within Cervinae and/or Cervini. which also indicate a potentially close relationship to Cervus.

Most of the previously defined Eucladoceros species were synonymised with Eucladoceros ctenoides (Azzaroli & Mazza, 1992a; De Vos & Reumer, 1995; Pfeiffer, 1999; Croitor & Bonifay, 2001; Valli & Palombo, 2005). ‘E. senezensis’ has been suggested to be an ancestor of Megaceroides or Megaloceros giganteus in particular (Azzaroli & Mazza, 1992a, 1992b; Kuehn et al., 2005). Pfeiffer (2002) proposed that Eucladoceros, Megaloceros and Cervus form a group. Flerov (1952) suggested that Eucladoceros is an ancestor of Alces, which is not supported by others (Heintz, 1970; Croitor, 2014). The comb-shaped antler morphology is unique and more similar to Cervus elaphus or Cervus albirostris than to any other living cervid (N. Heckeberg, 2017, personal observation). Because upper canines in Eucladoceros ctenoides are absent it was interpreted that the genus most likely does not belong to the Cervus-Rusa-lineage (Croitor, 2014); instead, Eucladoceros ctenoides was hypothesised as a descendant of an early three-tined ancestor of Axis or Metacervocerus (Croitor, 2014). In Mennecart et al. (2017) Eucladoceros ctenoides was placed as the sister taxon to the Cervus-Rusa-clade, which confirms the results from the analyses here.

Metacervocerus rhenanus

In the analyses here, Metacervocerus rhenanus was mostly placed as the sister taxon to Cervini and/or within Cervinae, which suggests that Metacervocerus rhenanus is a member of Cervini and potentially is either a close relative and/or ancestor of Cervus or Axis.

The genus Metacervocerus was established by Dubois (1904) as Cervus (Axis) rhenanus for the small sized deer from Tegelen. Spaan (1992) synonymised ‘Cervus’ philisi from Senèze with ‘C’. rhenanus based on dentition and antler morphology. Croitor & Bonifay (2001) assigned it to the genus Metacervocerus. Several three-tined cervids were described from the early Pleistocene of Europe (De Vos & Reumer, 1995); Metacervocerus rhenanus was considered to include ‘C’. philisi, ‘C’. perolensis, C. ischnoceros and Pseudodama lyra and ‘Cervus’ philisi was suggested to be a junior synonym of Metacervocerus rhenanus (Azzaroli et al., 1988; Spaan, 1992). Metacervocerus rhenanus was hypothesised to be an ancestor of Dama dama (Pfeiffer, 1999; Di Stefano & Petronio, 2002); however, this hypothesis was ruled out by the coexistence of both genera in the early Pleistocene (Croitor, 2014).

From the analyses based on the present data sets, the synonymy of ‘Cervus’ philisi and ‘Cervus’ perolensis with Metacervocerus rhenanus could not be confirmed. All analyses placed the three taxa differently and not closely related to each other. This may be caused by the differing availability of characters for each taxon and should be tested based on exclusively overlapping characters.

‘Cervus’ philisi

In the analyses here, ‘Cervus’ philisi was most often placed within Cervinae or Cervini sometimes within the extant Cervus-clade, which suggests that ‘Cervus’ philisi belongs to Cervini with a potentially closer relationship to Cervus. The results further support previous findings that ‘Cervus’ philisi cannot be assigned to any extant cervid (except maybe Cervus nippon). ‘Cervus’ philisi together with Praeelaphus perrieri potentially represents an extinct clade leading to Cervus. The suggested synonymy of Metacervocerus rhenanus, ‘Cervus’ philisi, and ‘Cervus’ perolensis could not be supported in the analyses.

In the past, ‘Cervus’ philisi was considered to be related to Axis (Depéret & Mayet, 1911), to Rusa (Stehlin, 1923; Viret, 1954), and to Cervus nippon (Schaub, 1941). Heintz (1970) suggested an evolutionary Metacervocerus pardinensis-‘Cervus’ philisi-‘Cervus’ perolensis-lineage. However, the temporal occurrence of these species in the fossil record contradicts this hypothesis. It was suggested that ‘Cervus’ perolensis is the descendant of ‘Cervus’ philisi (Stefaniak & Stefaniak, 1995; Croitor, 2006a, 2014) and that Metacervocerus rhenanus from Tegelen and ‘Cervus’ philisi from Senèze are synonymous and that ‘Cervus’ philisi and ‘Cervus’ perolensis are junior synonyms of Metacervocerus rhenanus (Spaan, 1992). Later, ‘Cervus’ philisi was included in the genus Metacervocerus (Croitor & Bonifay, 2001; Croitor, 2006a). In Mennecart et al. (2017) ‘Cervus’ philisi was placed closely related to Axis and Rucervus duvaucelii.

‘Cervus’ sivalensis

The remains of ‘Cervus’ sivalensis resemble Rucervus duvaucelii in morphology and size and Rucervus eldii in antler morphology (Azzaroli, 1954). Here, ‘Cervus’ sivalensis was placed as the sister taxon to Megaloceros giganteus to a clade consisting of Axis lydekkeri, Rusa kendengensis and Metacervocerus pardinensis to Metacervocerus pardinensis, to the Elaphurus, or in a polytomy with Metacervocerus pardinensis and Cervus canadensis within the Cervus-clade. The placements within Cervini and close to the Cervus-clade show that ‘Cervus’ sivalensis belongs to Cervini and is most likely closely related to Cervus, Rusa, and/or Rucervus. Together with Axis lydekkeri it could belong to the ancestral group of cervids that leads to Axis, Cervus, Rusa and Rucervus. Although the tooth morphology of ‘Cervus’ sivalensis resembles that of Rucervus (N. Heckeberg, 2017, personal observation), a placement closely related to Rucervus could not be found. There is still a lot of confusion concerning the taxonomy and systematics of this taxon and a revision is needed (Lydekker, 1884; Azzaroli, 1954; Arif, Shah & Vos, 1991; Samiullah & Akhtar, 2007).

Axis lydekkeri

Even though Axis lydekkeri is a fairly complete fossil and despite the morphological similarities to Axis, Axis lydekkeri was not placed as closely related to extant Axis in the analyses here, instead it was mostly placed as the sister taxon to or within Cervini, or within the Cervus-clade, which shows that Axis lydekkeri belongs to Cervini.

Axis lydekkeri was suggested to be more closely related to the smaller Axis species of today (‘Hyelaphus’) than to Axis axis (Zaim et al., 2003; Meijaard & Groves, 2004; Gruwier, De Vos & Kovarovic, 2015), but a clear systematic relationship to any of them could not yet be confirmed.

Rusa kendengensis

In the analyses here, Rusa kendengensis was most often placed within Cervini and sometimes as the sister taxon to the Cervus-clade, which shows that Rusa kendengensis belongs to Cervini. Even though based on comparative anatomy it is more similar to Rusa, the analyses placed it more closely to Cervus. Rusa kendengensis potentially belongs to an extinct group of ancestors including also Axis lydekkeri and ‘Cervus’ sivalensis, which gave rise to modern Axis, Cervus, and Rusa.

There is little information about Rusa kendengensis in the literature; it was suggested that it belongs to Rusa and not to Cervus as previously assumed for most Pleistocene cervids from Java (Dubois, 1908; Zaim et al., 2003). Recently, this was confirmed by morphometric analyses (Gruwier, De Vos & Kovarovic, 2015). More material of this species is needed to further investigate its systematic relationships.

Candiacervus ropalophorus

In the analyses here, Candiacervus ropalophorus was often placed close to several fossil cervine taxa and/or within Cervinae; in the SFA it was placed within Odocoileini. The investigated Candiacervus ropalophorus specimens were fairly complete; therefore, it was unexpected that this taxon was difficult to place. Frequent placements as the sister taxon to Cervini or within Cervini indicated that Candiacervus ropalophorus belongs to Cervini. The often hypothesised close relationship to megacerine/damine deer could only be found in one topology.

For Candiacervus ropalophorus, up to six different size groups representing six taxonomic units, sometimes even eight morphotypes have been suggested, but with differing views on the actual taxonomic affiliations Simonelli (1907, 1908), Kuss (1975), Kotsakis & Palombo (1979), De Vos (1979, 1984, 2000) and Van der Geer et al. (2006). Candiacervus ropalophorus is the smallest species of the eight morphotypes. Since no cranial material can be unambiguously assigned to Candiacervus cretensis or Candiacervus rethymnensis, only Candiacervus ropalophorus can be considered as clearly recognisable species based on cranial and postcranial elements (De Vos, 1984).

The systematic position of Candiacervus is controversial; a close relationship to Megaceros, Praemegaceros, Eucladoceros, Cervus, or Croizetoceros, as has been suggested before (Kuss, 1975; De Vos, 1984). It remains difficult to determine the ancestor of the Greek island deer, and data are still insufficient to establish robust phylogenetic relationships of Cretan deer (Van der Geer et al., 2006).

Megaloceros giganteus

In the morphological analyses here, Megaloceros giganteus was placed in varying positions, within Cervinae, as the sister taxon to Dama dama, and often closely related to Rangifer tarandus (presumably due to similarities in antler morphology) . A close relationship to Dama, as strongly suggested by molecular analyses (Lister et al., 2005), is also supported in the TE BI and ML topologies. Together with the evidence from comparative morphology a close relationship of Megaloceros giganteus to Dama is almost certain.

There is a broad consensus today that Megaloceros consists of only one species, Megaloceros giganteus (Vislobokova, 1990, 2012, 2013; Azzaroli & Mazza, 1993; Croitor, Bonifay & Bonifay, 2006; Croitor & Bonifay, 2001; Croitor, 2014). All recent phylogenetic analyses consistently placed Megaloceros giganteus within Cervinae (Lister et al., 2005; Hughes et al., 2006; Vislobokova, 2009). In some studies Megaloceros giganteus was placed closely related to Cervus elaphus based on molecular data (Kuehn et al., 2005) and morphological data (Geist, 1998; Pfeiffer, 1999, 2002; Vislobokova, 2009). Lönnberg (1906) put it close to Rangifer because of a completely ossified vomer and palmated brow tines; however, it was found that the division of the nasal cavity is only ossified in the anterodorsal part of the vomerine septum, which is different from the condition in Capreolinae and presumably is a side effect of the cranial pachyostosis (Lister, 1994; Croitor, 2006b, 2014). Already Lydekker (1898) suggested an affiliation of Megaloceros giganteus to the damine group, which was supported in several subsequent studies using morphological, molecular or both types of data (Gould, 1974; Kitchener, 1987; Lister, 1994; Lister et al., 2005; Vislobokova, 2009). In the topology of Marcot (2007) Megaloceros giganteus was the sister taxon to all cervine taxa, and in Pfeiffer (2002) it was the sister taxon to two extant Cervus. In Mennecart et al. (2017) Megaloceros giganteus was the sister taxon to Dama.

Odocoileus

In the analyses here, both fossil Odocoileus specimens were most often placed as the sister taxon to odocoileine taxa, within Blastocerina, and sometimes to the other fossil Odocoileus.

The results for both fossil Odocoileus suggest that they are included within Capreolinae and within Odocoileini. However, only a few analyses placed them as sister taxa or closely related to their presumed living descendants Odocoileus virginianus and Odocoileus hemionus. Particularly the BSPG specimen was more often placed closely related to Mazama species. In Mennecart et al. (2017) the fossil Odocoileus BSPG specimen was placed in a trichotomy with the extant Odocoileus species.

Muntiacus

The fossil Muntiacus muntjak was often placed within Muntiacini, mostly as the sister taxon to Muntiacus atherodes. The results show that the fossil Muntiacus is certainly a member of Muntiacini.

Axis

The study of Meijaard & Groves (2004) was so far the only one to include the three species, Axis porcinus and Axis kuhli, for which molecular data were available. In the supertree analysis of Hernández Fernández & Vrba (2005) all four Axis species were included. Axis was not monophyletic in some studies (Pitra et al., 2004; Marcot, 2007; Agnarsson & May-Collado, 2008). This is most likely caused by re-analysing the same misidentified sequences (see discussion in Gilbert, Ropiquet & Hassanin (2006)).

In the analyses here Axis formed a well supported clade. Axis axis was always the sister taxon to the other two Axis species. Based on craniometrics and morphological similarities Axis calamianensis, Axis kuhli, Axis porcinus were considered to be closely related to each other and distinct from Axis axis (Meijaard & Groves, 2004). This was confirmed by the molecular and combined topologies here. In most of the topologies here Axis was closely related to Rucervus, which differs from the results in Pitra et al. (2004) and the supertree analysis in Hernández Fernández & Vrba (2005).

Cervus

The morphological analyses here, resulted in varying positions for the four Cervus species. All of them have a very similar cranial and dental morphology (N. Heckeberg, 2017, personal observation). In the nuclear analyses, Cervus elaphus, Cervus canadensis and Cervus nippon were more closely related to each other than to Cervus albirostris. In the mtG analyses Cervus albirostris and Cervus nippon formed a clade and Cervus elaphus was the sister taxon to them; if Cervus canadensis was included it was the sister taxon to Cervus nippon (and Cervus albirostris, if it was a trichotomy) and Cervus elaphus was the sister taxon to all of them. The same was found in the combined molecular and TE analyses. This is also confirmed in recent studies using mtDNA (Marcot, 2007; Hassanin et al., 2012; Heckeberg et al., 2016; Zurano et al., 2019).

In previous studies, Cervus elaphus was the sister taxon to Cervus nippon (Lister, 1984; Randi et al., 1998), or Cervus nippon was the sister taxon to Cervus canadensis, with Cervus elaphus and Rusa as the sister taxa to them (Randi et al., 2001; Pitra et al., 2004; Hughes et al., 2006). Cervus canadensis was the sister taxon to Cervus nippon with Cervus albirostris and Cervus elaphus as the sister taxon to all of them in Kuwayama & Ozawa (2000), Groves (2006) and Zachos et al. (2014). This contradicts results from traditional morphology, where Cervus elaphus and Cervus canadensis were usually sister taxa (Kuwayama & Ozawa, 2000). However, Polziehn & Strobeck (2002) stated that the divergence of mtDNA noted for Cervus nippon, Cervus canadensis, and Cervus elaphus is congruent with geographical, morphological, and behavioural distinctions.

In some studies, Cervus albirostris was the sister taxon to the other Cervus species (Hernández Fernández & Vrba, 2005; Hu et al., 2019); it was the sister taxon to Cervus nippon, with Cervus canadensis as the sister taxon to both and Cervus elaphus the sister taxon to all of them . In Agnarsson & May-Collado (2008) Cervus albirostris was the sister taxon to Cervus elaphus, and Cervus nippon to both of them. In contrast to this, Flerov (1952) suggested that Cervus albirostris diverged from Rusa in the late Pliocene and Koizumi et al. (1993) considered it more closely related to Rucervus. However, all recent molecular studies placed it closer to the Cervus species (Leslie, 2010). Cervus albirostris almost certainly evolved in temperate northern Eurasia; Epirusa hilzheimeri or Eucladoceros may have been its Pleistocene ancestors (Di Stefano & Petronio, 2002; Flerov, 1952; Zdansky, 1925; Geist, 1998; Grubb, 1990; Leslie, 2010).

The difference between mitochondrial and nuclear genes may indicate an ancient hybridisation event. It is known that hybridisation between Cervus nippon and Cervus elaphus (mainly Cervus elaphus females and Cervus nippon males) occurs and that hybrids are fertile. Hybridisation may lead to extensive introgression (Zachos & Hartl, 2011). Studies on population genetics and subspecies of red deer exclusively used mtDNA, which may suggest relationships that are not reproducible when using paternal genes. Hu et al. (2019) provide another insight into Cervus phylogeny using single nucleotide polymorphisms (SNP). Hybridisation could have occurred frequently in Cervus. The topologies here suggested varying sister taxon relationships across the four Cervus species.

Dama

In the analyses here, Dama dama and Dama mesopotamica were always sister taxa to each other and in most cases placed as the sister taxon to a clade consisting of Cervus, Rusa, Elaphurus davidianus, and Rucervus eldii. In previous studies, both Dama species were also sister taxa to each other (Randi et al., 2001; Lister et al., 2005; Hughes et al., 2006; Hassanin et al., 2012; Heckeberg et al., 2016; Zurano et al., 2019).

Elaphurus

In the nuclear analyses here, Elaphurus davidianus was mostly placed close to Cervus, while it was consistently placed as the sister taxon to Rucervus eldii in all mitochondrial, molecular combined, and TE analyses. In the morphological analyses it was placed closer to Cervus based on cranial characters and closer to Rucervus and Rusa, particularly Rucervus schomburgki, based on the dentition and the morphological combined data set.

The oldest known fossils of the Elaphurus davidianus lineage are known from the late Pliocene or slightly earlier (Taru & Hasegawa, 2002) and the first certain Elaphurus davidianus fossils date from the mid Pleistocene (Ji, 1985). The speciation of Elaphurus has been discussed as an ancient (late Pliocene or earlier) hybridisation event (Meijaard & Groves, 2004). Cervus canadensis or a closely related ancestor supposedly was the male parent and Rucervus eldii or a very close ancestral relative the female parent (Taru & Hasegawa, 2002; Meijaard & Groves, 2004; Pitra et al., 2004; Groves, 2006). The unique antler morphology and the overall phenotype of Elaphurus davidianus is distinct from all other cervids (Lydekker, 1898; Emerson & Tate, 1993; Meijaard & Groves, 2004; Pitra et al., 2004). Although some similarities to Rucervus eldii were stated (Meijaard & Groves, 2004), morphological scrutiny does not necessarily support that. The morphology of Elaphurus contains apomorphic character states and is not intermediate between its two parent taxa (Groves, 2014; N. Heckeberg, 2017, personal observation). This phenomenon is called transgressive segregation and the new phenotypes may be favoured in the new hybridogenetic population (Rieseberg, Archer & Wayne, 1999; Groves, 2014).

Because of this hybridisation molecular phylogenetic analyses result in conflicting systematic positions as clearly shown here, but also in earlier studies. Analyses of mitochondrial data placed Elaphurus davidianus as the sister taxon to Rucervus eldii (Randi et al., 2001; Pitra et al., 2004), while Electrophoretic patterns of 22 proteins and κ-casein DNA, and the karyotype placed Elaphurus closer to Cervus (Emerson & Tate, 1993; Cronin et al., 1996; Meijaard & Groves, 2004).

Rucervus

Rucervus species have a unique antler morphology and their teeth are uniquely folded indicating a specialisation for graminivory (Grubb, 1990; Meijaard & Groves, 2004); both provide useful morphological characters. The hypothesis that Rucervus is more closely related to Rusa than to Cervus was partly supported in the nuclear analyses and the morphological analyses here, while in the mitochondrial, molecular combined, and TE analyses Rucervus was polyphyletic with Rucervus eldii more closely related to Elaphurus davidianus and the other two species more closely related to Axis. Based on this it was suggested that Rucervus eldii may represent a different evolutionary lineage than the other two Rucervus species (Meijaard & Groves, 2004) and was sometimes put into a separate genus Panolia (Pocock, 1943; Groves, 2006). It is now widely regarded as Rucervus eldii (Wilson & Reeder, 2005; Timmins et al., 2008; Angom & Hussain, 2013). This is also supported by the topologies here, particularly the morphological topologies show the close relationship to the other two Rucervus species. The placement of Rucervus eldii separate from its two congeners in molecular topologies (especially mtDNA) is most likely artificially caused by the hybridisation of Rucervus eldii and Cervus canadensis in the past.

Rucervus duvaucelii and Rucervus schomburgki were sister taxa to each other in the analyses here and were mostly the sister taxon to Axis. The last specimen of Rucervus schomburgki became extinct in 1938. The first accounts on the species were by Blyth (1863), who noted the distinctive antler pattern. According to Gühler (1936), the geographical distribution of Rucervus schomburgki was restricted to Siam. It was assumed to be closely related to Rucervus duvaucelii and potentially interbreeding with Rucervus eldii in its natural habitat. The earliest fossils of Rucervus date back to 2.9 mya (Azzaroli et al., 1988; Meijaard & Groves, 2004).

Rusa

In the morphological analyses here, Rusa was more closely related to Rucervus (rarely to Axis). In the nuclear analyses, it was close to Rucervus or within Cervini, while it was more closely related to Cervus in the mitochondrial, combined molecular, and TE analyses. When all four Rusa were included, Rusa timorensis and Rusa unicolor were sister taxa to each other and to Cervus, and Rusa marianna and Rusa alfredi were sister taxa to each other and to all of the above. This was also found in recent studies (Hassanin et al., 2012; Heckeberg et al., 2016; Zurano et al., 2019) and confirms the controversial monophyly of Rusa (Meijaard & Groves, 2004; Hernández Fernández & Vrba, 2005; Randi et al., 2001; Leslie, 2011).

Thus, despite some new insights into the systematic relationships of Rusa, uncertainties remain. The Philippine Rusa alfredi and Rusa marianna share morphological similarities, and are distinct from the other two Rusa because of the overall smaller size. Rusa unicolor and Rusa timorensis from the mainland and Indonesia were considered to be more derived (Groves & Grubb, 2011), which is in contrast to the assumption that based on the high similarity of Rusa unicolor to pliocervines, an extinct lineage of Pliocene cervids, it is the most ancestral of the four extant rusine deer (Petronio et al., 2007; Leslie, 2011).

The first appearance of R. unicolor was recorded from the middle Pleistocene (Zong, 1987; Dong, 1993; Meijaard & Groves, 2004). The oldest R. timorensis is reported from the late Pleistocene (Van Mourik & Stelmasiak, 1986; Dong, 1993) and suggested to have then dispersed south-eastwards to Taiwan and Java (Meijaard & Groves, 2004).

Elaphodus

Elaphodus cephalophus was always the sister taxon to the other muntiacine species in all molecular and TE analyses presented here, which is also widely supported in the literature (Wang & Lan, 2000; Hernández Fernández & Vrba, 2005; Agnarsson & May-Collado, 2008; Hassanin et al., 2012). In contrast, in Marcot (2007) Elaphodus cephalophus is the sister taxon to all cervids.

Elaphodus cephalophus has the smallest known antlers, which are completely covered by tufts (Leslie, Lee & Dolman, 2013). Groves & Grubb (1990) considered Elaphodus cephalophus as the most primitive representative of living muntiacines. However, this is in contrast to the absence of fossils with such diminutive antlers. The first Elaphodus fossils are known from the Pleistocene of China, which were larger than Elaphodus cephalophus; therefore, the decrease in size can be considered as evolutionary trend in this species (Leslie, Lee & Dolman, 2013).

Muntiacus

All muntjacs have long pedicles, facial crests, and bifurcating antlers (N. Heckeberg, 2017, personal observation; e.g. Ma, Wang & Xu, 1986). In the morphological analyses here, muntiacine taxa were placed as the sister taxa to most other cervids or in an unresolved position. In most of the combined morphological analyses Muntiacini was monophyletic except for the BI analyses. In the MP analyses, Muntiacini were placed more closely related to other small cervids, such as Mazama and Pudu.

The earliest fossil of the Muntiacus lineage is Muntiacus leilaoensis from Yunnan, China and was dated to the late Miocene 9–7 mya (Dong, Pan & Liu, 2004). All Muntiacus species consistently formed a clade as the sister taxon to Cervini in the mitochondrial, molecular combined, and TE analyses here. A clade consisting of Muntiacus crinifrons, Muntiacus feae, and Muntiacus muntjak and a clade consisting of Muntiacus putaoensis, Muntiacus truongsonensis, Muntiacus rooseveltorum, Muntiacus vuquangensis, and Muntiacus reevesi were recovered in the mitochondrial and combined molecular analyses. Muntiacus atherodes was placed in a polytomy with these clades. In the TE analyses Muntiacus reevesi was placed between Elaphodus cephalophus and the other muntjacs and Muntiacus atherodes was the sister taxon to Muntiacus feae.

Several new muntiacine species have been discovered in the 1990s; subsequently, five to possibly six new muntjac species were established, Muntiacus gongshanensis, Muntiacus crinifrons, Muntiacus feae, Muntiacus reevesi, Muntiacus muntjak (Lan, Wang & Shi, 1995). Ma, Wang & Xu (1986) stated that Muntiacus crinifrons and Muntiacus rooseveltorum derived from Muntiacus reevesi, whereas Muntiacus feae and Muntiacus muntjak derived from a different lineage. The species status of Muntiacus rooseveltorum has been controversial for decades (Amato et al., 1999b); for example Groves & Grubb (1990) suggested that Muntiacus rooseveltorum is the synonym of Muntiacus feae and that Muntiacus feae is the sister taxon to Muntiacus muntjak and Muntiacus crinifrons. This is supported by most molecular studies and the topologies of this work. Sometimes, Muntiacus crinifrons and Muntiacus gongshanensis are considered as a single species (Amato et al., 1999b). It was proposed that Muntiacus atherodes should be included in Muntiacus muntjak based on morphological evidence, because the holotype of Muntiacus atherodes is a subadult male with single-tined antlers (Ma, Wang & Xu, 1986). The two specimens investigated here were indeed subadult individuals with not yet fully developed antlers (N. Heckeberg, 2017, personal observation). However, molecular topologies here and in the literature indicate a separate species status for Muntiacus atherodes (Heckeberg et al., 2016). The genus status of Megamuntiacus is not justified demonstrated by the sequence divergence estimated for the mitochondrial variation and by morphological comparisons; therefore, it is referred to as Muntiacus (Schaller, 1996; Giao et al., 1998; Amato, Egan & Rabinowitz, 1999a, Rabinowitz et al., 1999; Wang & Lan, 2000). Apart from the larger size, there are no morphological features that would justify a separate genus (N. Heckeberg, 2017, personal observation).

Alces

Alces has a highly derived skull morphology with an elongated viscerocranial proportion and antlers that protrude horizontally. The dentition, particularly the lower premolars, shows similar modifications as in Rangifer. In the morphological analyses here, Alces alces was in an unresolved position or placed as the sister taxon to Odocoileus hemionus, Mazama chunyi, Ozotoceros bezoarticus or Cervus canadensis. In the mitochondrial, combined molecular and TE analyses Alces alces was consistently placed as the sister taxon to Capreolini, except for the BI combined molecular topology, where it was placed between Capreolini and Odocoileini plus Rangifer.

In most recent studies, Alces was placed as the sister to Capreolini (Randi et al., 1998; Pitra et al., 2004; Hughes et al., 2006; Agnarsson & May-Collado, 2008; Hassanin et al., 2012) or as the sister taxon to Capreolus (Hernández Fernández & Vrba, 2005). In Marcot (2007) Alces was the sister taxon to Capreolini and Odocoileini and Rangiferini, while it was in a polytomy with Odocoileini plus Rangifer and Capreolini or the sister taxon to Odocoileini plus Rangifer in Gilbert, Ropiquet & Hassanin (2006). More controversial positions included Alces as the sister taxon to Cervini or Dama dama in Kuehn et al. (2005) and the sister taxon position to Rangifer in Pfeiffer (2002). Alces was in a polytomy with Odocoileini and Rangiferini in Lister (1984) and took up variable positions in previous studies as summarised in Lister (1998). Thus, the systematic position of Alces remains unresolved.

The first Alces alces is known from the Riss glaciation 200–100 kya; those late Pleistocene moose were larger than their extant representatives (Franzmann, 1981).

Capreolini

Most analyses based on the combined morphological data set supported monophyletic Capreolini. However, the systematic position of Capreolini varied and could not be determined with certainty using morphological data only. In the molecular analyses here, Capreolini was always monophyletic and mostly placed closely related to or in most cases as the sister taxon to Odocoileini plus Rangifer.

Miyamoto, Kraus & Ryder (1990) suggested that Capreolini probably originated in the late Miocene in the Old World. The assumption of a late Miocene Old World origin of Capreolinae is in congruence with the findings here considering the placement of Procapreolus. Cronin (1991) hypothesised that Alces and Rangifer split earlier than the Capreolus lineage, but after the separation of Cervinae and Capreolinae.

Capreolus

In the morphological, molecular, and TE topologies Capreolus capreolus and Capreolus pygargus both species were consistently placed as sister taxa. In the mitochondrial, molecular combined and TE topologies, Capreolus was always the sister taxon to Hydropotes with strong support. Molecular studies of the past decades support the consistent placement of Hydropotes as the sister taxon to Capreolus forming monophyletic Capreolini (Douzery & Randi, 1997; Randi et al., 1998; Hassanin & Douzery, 2003; Pitra et al., 2004; Hughes et al., 2006; Gilbert, Ropiquet & Hassanin, 2006; Marcot, 2007; Agnarsson & May-Collado, 2008; Hassanin et al., 2012; Heckeberg et al., 2016; Zurano et al., 2019).

Hydropotes

Here, Hydropotes and Capreolus were sister taxa in the morphological combined, nuclear, mtDNA, molecular combined and TE analyses. In the past, Hydropotes was considered as a separate subfamily Hydropotinae as the sister taxon of all other cervids (Groves & Grubb, 1987; Janis & Scott, 1987; Hernández Fernández & Vrba 2005; Kuznetsova, Kholodova & Danilkin, 2005). Schilling & Rössner (2017) extensively reviewed the taxon. Already Bouvrain, Geraads & Jehenne (1989) favoured the hypothesis that Hydropotes and Capreolus are sister taxa. The first molecular studies indicated that Hydropotes is included in monophyletic Cervidae (Kraus & Miyamoto, 1991). From this follows that Hydropotes lost the antlers secondarily and developed enlarged upper canines as compensation (Douzery & Randi, 1997; Randi et al., 1998; Hassanin & Douzery, 2003).

Randi et al. (1998) demonstrated that the two Capreolus species and Hydropotes share a G at position 525 of Cytb, which occurs only rarely in other mammal species, and stated that ‘this replacement represents a nearly exclusive synapomorphy for the Hydropotes-Capreolus-clade’. Further, the telemetacarpal condition and a large medial opening of the temporal canal are morphological features that Hydropotes shares with other Capreolinae (Bouvrain, Geraads & Jehenne, 1989; Douzery & Randi, 1997; Randi et al., 1998). Behavioural characters also suggested that Hydropotes inermis is closely related to Capreolus (Cap, Aulagnier & Deleporte, 2002). Thus, increasing evidence from mitochondrial and nuclear DNA, morphology, and behaviour confirm a sister taxon relationship of Hydropotes and Capreolus.

Rangifer

The systematic position of Rangifer was variable in the morphological analyses here. Rangifer has some apomorphic characters, not shared by other cervids, which is likely the cause of the difficulties to place the taxon based on morphology only. In the molecular and TE topologies Rangifer tarandus was consistently placed as the sister taxon to Odocoileini. This is supported by the most recent literature (Randi et al., 1998; Hassanin & Douzery, 2003; Pitra et al., 2004; Hernández Fernández & Vrba, 2005; Gilbert, Ropiquet & Hassanin, 2006; Hughes et al., 2006; Agnarsson & May-Collado, 2008; Duarte, González & Maldonado, 2008; Hassanin et al., 2012; Gutiérrez et al., 2017).

Rangifer appeared in the fossil record in the Pleistocene; based on its arctic specialisations it is hypothesised that it dispersed to America during the Pleistocene contemporaneously with Alces (Gilbert, Ropiquet & Hassanin, 2006).

Blastocerus

In the analyses here, Blastocerus dichotomus was positioned in an unresolved position based on morphological data and consistently placed within Blastocerina in the molecular and TE analyses. Most often it was positioned between Pudu puda (sometimes also Mazama nemorivaga) and the other Blastocerina. In previous studies Blastocerus took up variable positions, most likely depending on the taxon sampling. for example as the sister taxon to Hippocamelus bisulcus plus Mazama gouazoubira (Duarte, González & Maldonado, 2008), as the sister taxon to Mazama gouazoubira (Agnarsson & May-Collado, 2008), in a polytomy with Mazama gouazoubira, Pudu puda, Hippocamelus antisensis (Gilbert, Ropiquet & Hassanin, 2006), as the sister taxon to Pudu puda (Hughes et al., 2006), and as sister taxon to Mazama nemorivaga (Hassanin et al., 2012). Studies with a more extensive taxon sampling (Heckeberg et al., 2016; Gutiérrez et al., 2017) and the analyses of this work indicated a systematic position of Blastocerus as the sister taxon to most blastocerine species, with Mazama nemorivaga as the sister taxon to them and Pudu puda as the sister taxon to all other Blastocerina. A few analyses placed Blastocerus as the sister taxon to all other Blastocerina. These differing placements of Blastocerus most likely resulted from a differing taxon sampling.

The first Blastocerus fossils are known from the Pleistocene of Brazil and Paraguay. The populations in central Brazil most likely expanded between 28 and 25 kya and it was assumed that there were no geographical barriers until about 300 years ago (Merino & Rossi, 2010).

Hippocamelus

In several of the morphological topologies, both Hippocamelus species were monophyletic, sometimes with Ozotoceros as the sister taxon. Two of the four sequences for Hippocamelus antisensis formed a clade with Hippocamelus bisulcus, while the other two formed a clade with Ozotoceros bezoarticus (Heckeberg et al., 2016). This makes it almost certain that two of the four sequences are misidentified or mislabelled; a less likely possibility is that this polyphyly represents a valid split within the genus. Without knowing the exact provenance of the samples it cannot be determined which sequences are truly Hippocamelus antisensis. In the molecular combined and TE analyses here, those Hippocamelus antisensis mt-sequence(s) were included, with which the genus is monophyletic (Heckeberg et al., 2016). Hippocamelus was the sister taxon to Mazama gouazoubira (plus Mazama chunyi, if included).

Duarte, González & Maldonado (2008) stated that it is surprising that members of morphologically cohesive genera such as Hippocamelus, Mazama, or Pudu were not monophyletic based on molecular data. Hippocamelus antisensis and Hippocamelus bisulcus were found to be osteologically nearly indistinguishable (Flueck & Smith-Flueck, 2011; N. Heckeberg, 2017, personal observation). Based on this, a monophyly for Hippocamelus is more likely than a polyphyly as suggested by some of the molecular data. Thus, the potential polyphyly within Hippocamelus cannot be confirmed or ruled out yet; new sequences and more investigations are needed to clarify this phenomenon (see also discussion in Gutiérrez et al. (2017)).

The first Hippocamelus bisulcus is known from the late Pleistocene of Chile, Argentina, and Bolivia (Canto, Yáñez & Rovira, 2010; Merino & Rossi, 2010). Odocoileus lucasi is considered to be the ancestor of Hippocamelus bisulcus.

Mazama

Only little is known about the rarer Mazama species (and neotropical cervids in general), which represent the least studied organisms and many aspects of their life history are poorly understood (Duarte et al., 2012a, 2012b, 2012d, 2012e, 2012f; Lizcano et al., 2010; Gutiérrez et al., 2015, 2017). The first fossil Mazama are known from the Pleistocene of Argentina, Ecuador, Peru and Brasil (Merino & Rossi, 2010).

While the monophyly of Mazama has never been questioned based on morphological characters, previous molecular studies and the topologies here repeatedly showed polyphyletic relationships (Gilbert, Ropiquet & Hassanin, 2006; Duarte, González & Maldonado, 2008; Gutiérrez et al., 2015; Escobedo-Morales et al., 2016; Heckeberg et al., 2016; Gutiérrez et al., 2017).

Recent molecular studies showed that Mazama americana is a polyphyletic species, splitting into several lineages (Heckeberg et al., 2016; Gutiérrez et al., 2017). The genetic distance between the two Mazama americana-clades was higher than the genetic difference of Mazama bororo and Mazama nana; therefore, at least two species were assumed to be within the Mazama americana-complex, with a separate evolution of the two clades starting 1 mya and 2 mya, respectively (Duarte, González & Maldonado, 2008; Abril et al., 2010). The topology Gutiérrez et al. (2017) also shows the geographic distribution of the different Mazama americana-linegaes. Mazama bororo and Mazama nana were sister taxa to each other and are closely related to (this study) or nested within Mazama americana (Heckeberg et al., 2016; Gutiérrez et al., 2017). Mazama temama is the sister taxon to the Mazama americana group 1 (sensu Gutiérrez et al. (2017)), which was confirmed here. Mazama pandora was the sister taxon to Odocoileus here, which has also been found previously (Escobedo-Morales et al., 2016); a closer relationship to the columbianus-group of Odocoileus hemionus was found in (Gutiérrez et al., 2017), who suggested to assign Mazama pandora to the genus Odocoileus. Mazama chunyi, Mazama gouazoubira, and Mazama nemorivaga were consistently placed within Blastocerina, which confirms previous findings (Escobedo-Morales et al., 2016; Heckeberg et al., 2016; Gutiérrez et al., 2017). Already Duarte, González & Maldonado (2008) suggested that Mazama gouazoubira and Mazama nemorivaga should be assigned to a different genus. Mazama chunyi was independently found to be the sister taxon to Mazama gouazoubira based on two different Mazama chunyi sequences (Heckeberg et al., 2016; Gutiérrez et al., 2017), which was confirmed here. Therefore, Gutiérrez et al. (2017) suggested that Mazama chunyi and Mazama gouazoubira should be assigned to a genus different from Mazama. Mazama nemorivaga was the sister taxon to all other blastocerine taxa except for Pudu or nested unresolved within Blastocerina. Similar findings in Gutiérrez et al. (2017) led them to suggest that Mazama nemorivaga should be assigned to a different genus.

The low morphological diversity among Mazama is not correlated with the genotypic diversification, which leads to the problematic taxonomy; thus, a varying number of species were established based on different types of data (Groves & Grubb, 1987, 1990; Duarte & Merino, 1997; Duarte, González & Maldonado, 2008).

In the morphological analyses here most Mazama species were placed as closely related to each other most likely because of their small size and because they are morphologically almost indistinguishable (González et al., 2009; N. Heckeberg, 2017, personal observation). Gutiérrez et al. (2015) tested whether the degree of concavity of the dorsal outline in lateral view and the shape of the lacrimal fossa can distinguish Mazama bricenii and Mazama rufina, but found that these characters are too variable to discriminate species. In the molecular and TE analyses here, Mazama bricenii was placed as the sister taxon to Mazama rufina, while in Gutiérrez et al. (2015) and Gutiérrez et al. (2017) Mazama bricenii was nested within Mazama rufina and the opposite was the case in Heckeberg et al. (2016). Gutiérrez et al. (2015, 2017) stated based on their results that Mazama bricenii is not a valid taxon, but a junior synonym of Mazama rufina. Further, Gutiérrez et al. (2017) suggest to assign Mazama rufina and the Mazama americana group 2 to two different genera.

As discussed in Gutiérrez et al. (2017), the complex taxonomy of Mazama clearly needs a thorough revision, taking into account not only molecular data, but also (palaeo)biogeography, karyotypic and morphological data.

Odocoileus

In the morphological analyses based on the combined data set here Odocoileus hemionus was the sister taxon to , and in several topologies Odocoileus virginianus was the sister taxon to them. In all other morphological topologies, odocoileine taxa were placed in unresolved or varying positions. In the analyses including mitochondrial markers and a broad taxon sampling, both species were polyphyletic. In the analyses based on the nuclear markers, Odocoileus was not monophyletic in the topology based on the Prnp and Prkci marker and the combined nuclear analyses (Fig. 13; Supplemental Information 3).

Despite all the research undertaken on the genus, the taxonomy remains difficult. There are numerous subspecies (8–10 for O. hemionus, 37–38 for O. virginianus; Wilson & Reeder, 2005; Mattioli (2011)), which possibly, at least partly, represent separate species (Groves & Grubb, 2011; Gutiérrez et al., 2017).

Latch et al. (2009) demonstrated that there are two different morphotypes of O. hemionus, the mule deer and black-tailed deer, which is supported by a strong genetic discontinuity across the spatial distribution. Early investigations of mtDNA data demonstrated that O. hemionus is polyphyletic because the sequences of the mule deer (O. hemionus) and O. virginianus are more similar than the DNA of the black-tailed deer (O. hemionus columbianus) is to both of them (5–7% different) (Carr et al., 1986; Cronin, Vyse & Cameron, 1988; Cronin et al., 1996; Latch et al., 2009; Gutiérrez et al., 2017).

Similarly, the genetic divergence within O. virginianus is remarkably high, even higher than the genetic distance between other subspecies and between O. virginianus and mule deer. This led to the classification of white tailed deer into two distinct groups, the cariacou-division and the virginianus-division (Wilson, Carlson & White, 1977; Smith et al., 1986; Groves & Grubb, 1987; Grubb, 1990). Odocoileus virginianus is a highly plastic species occupying a great variety of geographically and ecologically extensive habitats between Canada and Peru, however, extreme habitat differences do not necessarily lead to large morphological divergence (Smith et al., 1986; Moscarella, Aguilera & Escalante, 2003; Merino & Rossi, 2010; Duarte et al., 2012c). Introgression seems to be the likely explanation because natural hybridisation and interbreeding between both species of Odocoileus have been documented (Groves & Grubb, 2011; Hassanin et al., 2012).

The first Odocoileus is from the early Pliocene (3.5 mya) of North America, where they were the most common cervids until the Pleistocene. Odocoileus virginianus appeared 2 mya presumably as the descendant of O. brachyodontus, which originated in Central America and dispersed to higher latitudes only recently (Hershkovitz, 1972; Smith, 1991; Merino & Rossi, 2010). It has been assumed that Odocoileus virginianus evolved in North America; it was further suggested that all South American cervid fossils belong to Odocoileus and that Mazama later diverged as a consequence of isolation within South America (Smith et al., 1986; Moscarella, Aguilera & Escalante, 2003). This is in contrast with the most recent molecular topologies (Escobedo-Morales et al., 2016; Heckeberg et al., 2016; Gutiérrez et al., 2017) and this work (Figs. 13 and 14), from which it appears that Odocoileus originated from the odocoileine Mazama-clade.

Ozotoceros

Similar to Blastocerus, the systematic position of Ozotoceros varied with the taxon sampling. With an extensive taxon sampling Ozotoceros bezoarticus was relatively consistently placed as the sister taxon to Hippocamelus, Mazama gouazoubira and Mazama chunyi (if included) in the analyses here. Other studies also demonstrated a variable position of Ozotoceros bezoarticus (Agnarsson & May-Collado, 2008; Duarte, González & Maldonado, 2008; Hassanin et al., 2012, Escobedo-Morales et al., 2016; Heckeberg et al., 2016; Gutiérrez et al., 2017; Zurano et al., 2019), which presumably is caused by varying taxon sampling, data analysed, and parameter settings.

The origin of Ozotoceros bezoarticus possibly dates back to 2.5 mya coinciding with a substantial cooling event; fossils are known from the late Pleistocene and Holocene of Brazil, the late Pleistocene of Uruguay, and the Holocene of Argentina (Gonzalez et al., 1998; Merino & Rossi, 2010).

Pudu

Both Pudu species are almost indistinguishable based on morphology, but do not evidently form a monophyletic group based on molecular data (Heckeberg et al., 2016; Gutiérrez et al., 2017; Zurano et al., 2019). Pudu puda was placed as the sister taxon to all Blastocerina in almost all of the analyses here and in previous studies with a sufficient taxon sampling. The systematic position of its congener, unfortunately, is much less certain. Here, Pudu mephistophiles was most often placed as the sister taxon to all Odocoileini plus Rangifer or to Odocoileini. Only in one topology Pudu mephistophiles was included within Blastocerina. In Zurano et al. (2019) it was the sister taxon to all other blastocerine taxa, while in Gutiérrez et al. (2017) it was placed in a polytomy with Mazama nemorivaga and all other blastocerine taxa.

The spatial and chronological origin of Pudu is unknown. Pudu most likely diverged from an odocoileine lineage, which existed in America since the Miocene-Pliocene-boundary (Merino & Rossi, 2010; Gonzalez et al., 2014). Pudu was probably restricted to South America since the Pliocene (Escamilo et al., 2010).