Comparative distal limb anatomy reveals a primitive 2 trait in 2 breeds of Equus caballus . 3

The 55-million-year history of equine phylogeny has been well documented from the 25 skeletal record, however not the soft tissue structures that are now vestigial in modern horse. A 26 recent study reported 2 ligamentous structures resembling functional 3rd and 4th interosseous 27 muscles were evident in Dutch Konik horses. The current study investigates this finding and 28 compares it to members of the genus Equus to identify either a breed anomaly or functional 29 primitive trait. Distal limbs (n=571) were dissected from 4 species of Equus; E. caballus, E. asinus, E. 30 przewalskii and E. burchelli beohmi. Breed representatives of E.caballus (n=18) included the primitive 31 Dutch Konik. The 2nd and or 4th interosseous muscle was evident in all 4 species, but only 2 breeds 32 of E.caballus expressed this trait the Dutch Konik and Bosnian Mountain Horse. These 2 breeds 33 were the only close descendants of the extinct Tarpan (Equus ferus ferus) represented in this study. 34 In conclusion, the 2nd and 4th interosseous muscles originated from the distal nodule of respective 35 splint bones and inserted into the corresponding branches of the 3rd interosseous muscle proximal 36 to the sesamoids. Suggesting a functional role in medial and lateral joint stability and a primitive 37 trait in modern equids. 38


Introduction
Aristotle (384-322BC) is regarded by many as the founder of comparative anatomy because of his methodical and systematic approach to the study of animals [1].His rigorous and comprehensive methods provided the basis for numerous original theories, including Charles Darwin's 1859 publication 'On the Origin of Species' [1,2].Aristotle believed that 'form and function' were integral parts of the same science and his in-depth knowledge of bodily systems were likely derived from direct observations and dissections [1].As medical practitioners began to embrace post mortem instruction, medieval practices were abandoned and the modernisation of medicine as we know it today was founded [3].
By the 1800's, this new anatomic enlightenment inspired many scholars and scientists, whilst simultaneously invoking strong academic debate and controversy [4,5].Nonetheless, the science of comparative anatomy prevailed and shaped the fundamental principles of taxonomy in extinct and extant species [6].Even when geographically isolated, the similarity between species was undeniable and furthermore, it became evident that structural conformity was in direct response to the environment [2,6].These distinctions were developed through the study of comparative anatomy and formed the basis of sound phylogenetic interpretation and evolutionary taxonomy [6].
It was this methodical approach to comparative observation that palaeontologist O. C. Marsh utilised when he correlated fossil evidence and established the 55-million-year history of equine phylogeny [7].His research deciphered the skeletal transformation from polydactyl to monodactyl, including connective soft tissue structures operating the distal digits [7,8].These skeletal archives presented a convoluted, but connected history, originating with the polydactyl Eohippus, until the present-day monodactyl E. caballus [7][8][9].The adaptations of the distal digits through the millennia corresponded to a period in history when the climate became progressively drier and open plains expanded at the expense of forests [8].
For the polydactyl horse, living in an open environment exposed it to greater stressors, namely foraging competition and predative pressures; hence speed and endurance became a vital trait in order to survive.However, as its distal limb was designed for stability and not speed, the mass of the extra digits became an energetic cost that hindered locomotive efficiency [8,10,11].Consequently, the distal limb required new adaptions that favoured efficient fore and aft linear movement with less flexibility [8,11].So, in direct response to its environment, the polydactyls distal limb evolved by favouring the reduction of the medial and lateral digits, whilst elongating the middle; thus, becoming the tight jointed, rigid hoofed monodactyl that we encounter today [7][8][9][10][11].
In modern horse, polydactyl atavism or primitive characteristics have been reported in the appendicular skeleton and although considered rare, so have reports of atavistic or vestigial soft tissue structures in the distal limb.[12][13][14][15].Evidently, these soft tissue structures were once strong and functional in the polydactyl, but now the morphology is regarded as rudimentary and ineffectual (Figure 1) [12,15].However, two atavistic soft tissue structures reported in the forelegs; the medial interosseous muscle (IM2) and lateral interosseous muscle (IM4) are of interest to this study.Both are vestigial remnants of medial and lateral limb reduction, but in the modern horse, there is only one interosseous muscle considered functional -the middle or 3 rd interosseous muscle (IM3) [12][13][14][15][16].This muscle (IM3) evolved even further and now contains a large number of strong ligamentous fibres with elastic properties that functionally support the fetlock joint when the limb is either standing, or during locomotion [15,16].When present in the modern horse, the IM2 and IM4, have been described as thin, pale and fleshy ligamentous structures originating from the distal nodule of the 2 nd and or 4 th metacarpal that ends inconspicuously near the fetlock joint (3 rd metacarpal, 1 st phalanx and sesamoids) [12,15].In contrast, a recent study revealed strong chord-like bands in primitive Dutch Konik horses originating from the distal nodules of the 2 nd and 4 th metacarpals, and 2 nd and 4 th metatarsals [17].
Therefore, the aim of this study is to investigate the strong chord-like bands reported in the primitive Dutch Konik horse and compare them to domestic horse breeds along with other available species in the genus Equus.We describe the morphology, anatomic origin and insertion, and postulate the function of the bands, with the objective to provide a better understanding of whether they are a rare finding, a breed anomaly or a primitive trait.We conclude the presence of an IM2 and or IM4 in its current morphological form as noted in this study, relates to a primitive trait and furthermore, one that is possibly, a functional remnant of limb reduction in response to an undulating environment.

Ethical statement
No horses were euthanized for the purpose of this study and all observations were obtained post mortem.

Animal Details
Dissections hindlegs with each DL displaying 2 splint bones; the 2nd and 4th metacarpals in the forelegs (MC2 and MC4); the 2nd and 4th metatarsals in the hindlegs (MT2 and MT4).In total, 1142 splint bones were examined and associated soft tissues (interosseous muscles).

Dissections
The forelegs and hindlegs were skinned from the proximal carpus and tarsus to the coronet band.Disarticulation of the distal limbs occurred between the 1st and 2nd row of carpals in the foreleg, and the tibial talus joint in the hindleg.This exposed the superficial fascia of the DL, and the extensor and flexor tendons.Resection of the flexor tendons, the inferior check ligament, nerves and various vessels from the palmer aspect of the 3rd metacarpal (MC3) and 1st phalanx was next; exposing the greater part of MC2 and MC4 including the IM3 (Figure 2).continued distally until insertion onto the respective medial and lateral sesamoids.However, when the IM2 and IM4 were present the resection required slow and precise strokes so not to compromise the origin or insertion of the IM2 and IM4.Any connective fascia was then carefully removed revealing the IM2 and IM4 in its entirety.The same process of resection was repeated for the metatarsals in the hindleg from the plantar surface.

Results
All Equus expressed the IM3, however the IM2 and IM4 were noted only in Dutch Konik horses, Przewalskis, donkeys and Bosnian Mountain horse.The Grant's zebra expressed the IM4 only in the forelegs (Table1).
Anatomically, the IM2 and IM4 originated from the nodules of the MC2 and MC4, and the MT2 and MT4, respectively.The point of insertion for the IM2 was the medial branch of the IM3; the point of insertion for the IM4 was the lateral branch of the IM3.All points of insertion were dorsal to the sesamoids.The thickness of the IM2 and IM4 appeared to remain constant from origin to insertion (Figure 3).At the point of insertion, the fibres of the IM2 and IM4 appeared to interconnect with those of the IM3 (Figure 4).These fibres were longitudinal in arrangement from origin to insertion and appeared consistent with a collagenous protein with elastic properties, such as that found in tendons or special ligamentous structures such as the IM3.

Discussion
In this study, the presence of a strong cord-like band resembling a tendon or ligament in the distal limb was identified and described in four species of the genus Equus.The bands originated from the distal nodules of the splint bones, matching the previously reported description of the atavistic IM2 and IM4 [12,15].However, in contrast to a thin and feeble ligamentous structure, morphological variations in size and insertion were noted, with conspicuous cord-like bands inserting into corresponding branches of the IM3 (Figures 3 and 4).The described bands were observed in 1 domestic breed (BMH) from 17; 1 primitive breed (DK); donkeys; Przewalskis and 1 Grant's zebra.
Of the 18 breeds representing E. caballus, only the close descendants of the extinct Tarpan (E. ferus ferus), the DK and BMH expressed the IM2 and IM4 [19,20].This coincides with a previous study, where the DK and BMH were the only 2 from 20 breeds of E.caballus that exhibited a full nuchal ligament lamellae [21].Thus, implying the ligamentous structures noted in the current and previous study, may well be attributed to the Tarpan as a heritable characteristic [21].
The strong cord-like bands described in this study were not vestigial, but displayed clear demarcations associated with origins and insertions as would be expected of a functional structure.
Somewhat like the inferior check ligament that originates from the palmar surface of the distal row of carpals and inserts into the deep digital flexor tendon providing stability and support (Figure 3) [12,15].In fact, both structures have proximal skeletal origins with distal soft tissue insertions and identical orientation, implying similar function (Figures 3 and 4).Furthermore, the morphology of the strong bands described in this study, present surprising similarities within members of the genus Equus that are geographically isolated, suggesting heritably from a common ancestor.
As previously described, vestigial or atavistic soft tissue structures in the distal limb are reported as rudimentary and non-functional.Yet, identifying new soft tissue structures that are functional in an equid's distal limb, is not unprecedented [12,15,22,23].A recent study in a miniature donkey (E.asinus), introduced a new ligament on the palmar surface of the 3 rd metacarpal with functional capabilities of stabilising the superficial digital flexor tendon [22].An earlier study reported fascial bundles in 5 horses descending distally from the nodule of the splint bone and attaching to the 3 rd metacarpal, securing the smaller metacarpal to the larger one [23].Although reports of soft tissue structures like those described in this study have not been found for extant members of Equus, this does not apply to families closely related to Equidae or polydactyls.In fact, all 3 interosseous muscles have been reported in extant and extinct polydactyls [24][25][26][27][28][29].
Studies have identified the presence of the IM2, IM3 and IM4 in the hippopotamus, tapir and canine, including marsupials such as the thylacine; of which, the origins and insertions displayed similar morphology in the hippopotamus and tapir to those found in this study [24,25,27].The IM3 and IM4 were noted in pigs, sheep and llamas, but not cattle or camels, where only the IM3 was mentioned per digit [24,[28][29][30][31][32].There were no definite anatomic origins or insertions noted for pigs or sheep, however, the llama's presentation corresponded anatomically to the current study [24,28,29].
Many of the species previously mentioned belong to the unguligrade mammals within the orders perissodactyl and cetartiodactyl [33].The latter order provides evidence of skeletal entheses patterns in the distal limb, where the IM3 and IM4 attached in an extinct species of palaeomerycid, a deer like creature from the Miocene epoch [34,35].During the same epoch, the perissodactyl forebears of Equidae, the tridactyl Mesohippus and Merychippus had anatomic structures comparable to the tapir, including interosseous muscles [36].The 3 digits in the tridactyl were referred to as II, III and IV (medial, middle and lateral respectively), with III being the largest, widest and dominant during weightbearing [36,37].Each interosseous muscle corresponded to its metacarpal or metatarsal, hence the interosseous muscles in the Mesohippus and Merychippus would be labelled IM2, IM3 and IM4 from which they originated [12,15].In current literature this anatomical arrangement remains constant between species, whether extinct or extant.
Furthermore, it has been suggested that functionally the 2 nd and 4 th digits in prehistoric tridactyls assisted in the prevention of lateral dislocation of the fetlock joint while increasing agility and maneuverability; whereas in Merychippus, these digits also helped increase traction over soft ground and savannas [36].This concurs with the adaptive responses that feature in the camel's specialized distal limb to its sandy environment [30]; and applies to donkeys, where specific adaptations were in direct response to a specific environment, which has already been reported in ungulate morphology [2,6,38].Unlike domestic horse, the distal limb in the donkey ends in a small boxy upright hoof with

Figure 1 .
Figure 1.The white arrow denotes the atavistic soft tissue structure located along the medial palmar border of the 2nd metacarpal in a 17-year-old Thoroughbred horse.The black arrow denotes the resected 3rd interosseous muscle.

Figure 2 .
Figure 2. (a) Lateral view of the distal forelimb revealing the flexor and extensor structures of the MC3 and phalanges.(b) Caudal view of the distal forelimb revealing the IM3 on the palmar surface of MC3.Adapted from Stashak [18].

Figure 4 .
Figure 4. Physical appearance at the point of insertion of a dissected 2 nd interosseous muscles (IM2) into the medial branch of the 3 rd interosseous muscle (IM3) in the left hindleg of a Dutch Konik horse.Arrows indicate: IM2 (red); and IM3 ligament (black).

Table 1 .
The documented occurrence of 2 nd and 4 th interosseous muscles (IM2 and IM4) in the distal limbs (n = 571) of four Equus species, dissected post mortem.