An Anatomical and Genetic Analysis of the Ceboid Lumbosacral Transition and Its Relevance to Upright Gait

Home , Common squirrel monkey, Spider monkey

AN ANATOMICAL AND GENETIC ANALYSIS OF THE CEBOID LUMBOSACRAL TRANSITION AND ITS RELEVANCE TO UPRIGHT GAIT

A thesis submitted to the Kent State University Honors College in partial fulfillment of the requirements for University Honors

Allison L. Machnicki

May, 2012

Thesis written by

Allison L. Machnicki

Approved by

______, Advisor

______, Chair, Department of Anthropology

Accepted by

______, Dean, Honors College

TABLE OF CONTENTS

LIST OF FIGURES v

LIST OF TABLES vi

ACKNOWLEDGEMENTS vii

CHAPTER

I. INTRODUCTION 1 A Brief Review of the Lumbosacral Spine 1

II. BACKGROUND OF ANATOMY AND DEVELOPMENT 4 Ligamentous Anatomy of the Lumbosacral Spine 4 The Development of the Lumbosacral Spine 5 Hox genes 6

III. OSTEOLOGY AND ANATOMY 10 Purpose 10 Methods 11 Osteology and Metric Data 11 Dissection 14 Results 15 Osteometrics of the Museum Sample 19 Conclusions 21

IV. GENETICS 23 Purpose 23 Methods 23 Genomic DNA 23 Polymerase Chain Reaction (PCR), Cloning, and Sequencing 24 Results 28 Discussion 32

V. SUMMARY AND FINAL CONCLUSIONS 33

WORKS CITED 34

iii

APPENDIX

1. APPENDIX A: Full Alignment 39

LIST OF FIGURES

Figure 1. Regions of Hox gene expression in the vertebral column 8

Figure 2. Measurements 13

Figure 3. Ligamentous Drawings 18

Figure 4. Relative Sacral Height 19

Figure 5. Plot of Alar Width Compared to Centrum Width 21

Figure 6. HoxD11 Enhancer Sequence 24

Figure 7. A Phylogenetic Comparison of Transcription Factor Binding Sites in Sequenced Specimens 30

LIST OF TABLES

Table 1. Osteological Measurement Definitions 12

Table 2. The Observed Conservation of Each Species 28

Table 3. Transcription Factor Binding Site Definitions 31

ACKNOWLEDGEMENTS

I would like to thank my advisor Dr. C. Owen Lovejoy for directing this study and for being the ultimate inspiration for a young anthropologist. Without his incredible knowledge and time this project would not have been possible and I would never have discovered my love for the study of anatomy. I would also like to thank him for always supporting me, encouraging me, and remembering that I played oboe.

I would like to thank Dr. Linda Spurlock for assisting with dissections, helping with research, and providing drawings. I am also thankful to her for reading drafts, for her unbelievable and unending support throughout this project, and for making this project exceedingly enjoyable to undertake.

I would like to thank Dr. Chi-hua Chiu for guiding the genetics portion of this study, for the use of her lab, and for serving on my defense committee. I would like to thank Dr. Elizabeth Howard and Dr. Douglas Kline for serving on my defense committee.

I would like to thank Daniel Sprockett for training me in the genetics lab, running procedures for me, and helping me troubleshoot; without him the genetics portion of this project would not have been possible.

I would like to thank Yohannes Haile-Selassie for selecting me as his Adopt-A-

Student intern at the Cleveland Museum of Natural History where I began this project. I would like to thank Lyman Jellema for access to the Hamann Todd Osteology collection,

vii photography, x-rays, and help finding cadavers. I would like to thank Chris Vineyard and

Mary-Ann Raghanti for dissection specimens. I would like Chris Flask, John Jesberger, and Matt Griswold for MRIs. I would like to thank Steve Ward for his support and funding in this project. I would like to thank Kelly Droney and Caitlin Sheesley for their help with this project.

I would like to thank Gerri Machnicki and Peter Koby for their unending encouragement and reading drafts. I would also like to thank Peter Koby for helping with figures.

I would like to thank the following institutions: The Kirtlandia Society, The

Cleveland Museum of Natural History, and Kent State University.

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CHAPTER I

INTRODUCTION

A Brief Review of the Lumbosacral Spine:

The human trunk is characterized by three distinct curvatures: a cervical lordosis, a thoracic kyphosis, and a lumbar lordosis. In humans, the cervical portion has an erect atlas and axis, the lumbar column is long, and the sacrum is broad and short (Ishida,

2006). In contrast, the chimpanzee has only two curvatures: a cervical lordosis and a thoracic-lumbar kyphosis (Ishida, 2006). Furthermore, in chimpanzees the atlas (C1) is tilted on the axis (C2), the lumbar column is short, and the sacrum is narrow and long

(Ishida, 2006).

During human bipedalism the weight of the body is supported solely by the hindlimb and vertebral column, rather than the forelimbs (as in quadrupedalism). Of critical importance is the ability to lordose the lower lumbar spine—that is, to reverse the direction of spinal curvature in such a way as to allow the body's center of mass to be placed forward over the hip and knee joints. Only with lordosis could our ancestors have adopted a striding gait that did not involve the bent hip and bent knee joints seen in most primates that walk bipedally (McCollum et al., 2009). Such a gait pattern is exhibited by

African apes which have almost completely eliminated lower spinal mobility by shortening their spine and “submerging” their most caudal lumbar(s) between the dorsally elongated ilia of their pelvis.

Human erect walking is a unique specialization among primates and even among mammals. While many other mammals are upright during locomotion, they retain this posture because they are saltatory (hopping) or suspensory. The human gait pattern is not shared with any other primate and requires many unique features in the pelvis to have evolved in our ancestors. The hominid pelvis was undergoing changes for adaptations to bipedality 3 to 4 million years ago, when hominid brains were only slightly larger than those of chimpanzees (Lovejoy, 2009). The earliest hominid fossil with definitive anatomical adaptations for bipedality is Ardipithecus ramidus, dating to 4.4mya

(Lovejoy, 2009).

On average, Old World monkeys possess seven lumbar vertebrae while New

World monkeys possess from four to seven lumbar vertebrae with atelines having four or five. Great apes have three to four lumbars, and the lesser apes have five lumbar vertebrae (Schultz and Strauss, 1945). Modern Humans have five lumbar vertebrae with occasional variations of four or six lumbars (Schultz and Strauss, 1945). The Most

Recent Common Ancestor (circa 7-10 mya) of African apes and hominids is argued to have retained a long lower spine (most likely 6 lumbars) (McCollum et al., 2009). Such mobility would have permitted more effective bipedality than is seen in extant apes. On the other hand, a long lumbar column, with more lumbar vertebrae, might have been mechanically less capable than a short one for bridging behaviors or climbing in the manner of extant apes (Sanders, 1997).

In humans (hominids), the most caudal lumbar vertebra is free from constriction by contact with the dorsal ilia, suggesting that reduction in iliac height and an increase in

the breadth of the sacrum are early adaptations to a bipedal locomotion. Freeing of the most caudal lumbar vertebra by iliac height reduction and sacral broadening therefore could have been the earliest adaptations to upright walking in hominids (McCollum et al.,

2009).

By contrast, in Old World monkeys and apes the most caudal lumbar is partially trapped by dorsally extended ilia, restricting lordosis and preventing bipedality without a bent-hip-bent-knee (BHBK) gait (Lovejoy & McCollum, 2010). Interestingly, while no

New World monkeys are terrestrial or bipedal, one prominent group, the atelines (Ateles,

Brachyteles, Lagothrix, Alouatta; family Atelidae; Wildman et al. 2009) have flexible and highly enervated prehensile tails, with which they suspend themselves while frequently also using their forelimbs (Personal communication – K. Strier). This posture places their lower spines in lordosis, a lumbar posture similar to that exhibited in hominids. Some physical characteristics of the lumbar column in atelines and hominids, then, are convergent.

In this thesis, I explore this hypothesis of convergent evolution and subsequent impacts on locomotion in hominids by using morphometrics and genetic analysis in a range of primates with focus on members of the Atelidae.

CHAPTER II

BACKGROUND OF ANATOMY AND DEVELOPMENT

Ligamentous Anatomy of the Lumbosacral Spine:

In humans, multiple ligaments stabilize the articulation of the vertebral column with the pelvis. The lumbosacral ligament connects the lower portion of the most caudal lumbar vertebra to the lateral surface of the sacrum, blending with the sacroiliac ligament

(Gray, 1977). It is short, thick, and triangular.

The sacroiliac ligament is made up of anterior and posterior parts (Gray, 1977).

The anterior portion is made of thin bands that connect the anterior surface of the sacrum to the ilium (Gray, 1977). The posterior portion is composed of strong interosseous fibers and is positioned in the posterior depression between the sacrum and ilium (Gray, 1977).

The human iliolumbar (IL) ligament helps stabilize the lumbosacral spine on the pelvis (Luk, 1986). It passes horizontally from the transverse process of the most caudal lumbar vertebra to the crest of the ilium in front of the articulation of the sacroiliac joint

(Gray, 1977). The iliolumbar ligament is formed by collagenation of the fibers of the quadratus lumborum muscle (Pun, 1987). Studies have emphasized the importance of the

IL in restraining extension and lateral bending of L5 and in maintaining stability of the lumbosacral junction (Pool, 2001). The IL has been shown to be present in humans and rhesus

macaques but absent in other quadrupeds (i.e. rats, rabbits, and dogs) with a horizontal spine (Pun, 1987). The IL is also present in New and Old World primates including

Ateles, Brachyteles, Alouatta, langurs, and gibbons (personal observation: this study). In the rhesus macaque, the IL connects the last, and occasionally the second-to-last, lumbar transverse process to the adjacent iliac wing (Pun, 1987). This ligament may help accommodate stresses generated around the perimeter of the lumbosacral joint when primates assume orthograde truncal positioning (Luk et al., 1986). Even though rhesus macaques frequently sit upright, they still lack a true lordosis (Pun, 1987).

The Development of the Lumbosacral Spine:

Many of the details of early embryogenesis have been gleaned from studies of chick embryos, and, although there are significant differences between humans and chick development, the deployment of structures relevant to this thesis is largely the same. The description provided here largely follows Bogduk’s general account in tetrapods (2005).

Vertebral development begins during early embryogenesis. After approximately

15 days, the embryo consists of two layers of cells: the ectoderm that will give rise to the skin and the endoderm that will form the alimentary tract. Dorsal to the notochord, the endoderm forms the neural tube that differentiates into the brain and spinal cord. On either side of the notochord, the mesoderm becomes thickened and forms the paraxial mesoderm. By 21 days, the paraxial mesoderm becomes marked by transverse clefts across the dorsal surface that separates the paraxial mesoderm into individual parcels called somites. The first somites appear in the head region, and others progressively 6

appear in the caudal direction. By 30 days of development, there are 42-44 somites in humans. Within each somite, two clusters of cells develop; the cells in the ventral and medial regions multiply rapidly to form a mass known as the sclerotome. These cells are used exclusively in the formation of vertebrae.

The development of the vertebral column occurs when the primary sclerotome forms from cells on the medial side of each somite. The streams of cells form secondary sclerotomes which are half derived from one somite and half from the next more caudal somite (Kardong, 2006). These cells grow upward, around, and above the nerve cord in order to form the neural arches and vertebral spines (Kardong, 2006). Chondrification and ossification produce the bony vertebrae (Kardong, 2006). The intervertebral discs are formed between the vertebrae within the former perichordal rings (Kardong, 2006).

Hox genes:

A homeobox is a 180 base-pair (bp) portion of DNA that encodes a homeodomain, a 60 amino acid helix-turn-helix domain that binds DNA (Gehring, 1986).

Homeodomain sequences are widely evolutionarily conserved and found in about 20 other families of homeobox containing genes; all encode DNA-binding proteins

(Gehring, 1986).

Homeotic genes regulate specific body region identity (Lewis, 1970). Hox genes, whose function was first discovered in Drosophila (Lewis, 1970), are a subset of homeobox, containing genes that occur in clusters in the genomes of metazoans (Ruddle et al., 2000; Carroll, 2001). Jawed vertebrates possess four Hox clusters (A-D; Chiu et al.,

2002). Each cluster resides on a separate chromosome (Krumlauf, 1994). There are thirty 7

nine Hox genes in humans (Krumlauf, 1994). In humans, genes in each Hox cluster are numbered 1 through 13 in the to 5 direction (Krumlauf, 1994). Recently, a group 14 paralog has been found in the HoxA and HoxD clusters of sharks and coelacanth

(Amemiya et al., 2009; Venkatesh et al., 2008) as well as a single Hox cluster in

Amphioxus (Holland, 1992).

Vertebral identity is specified by Hox gene expression in individual somites; however, the correspondence between specific somite number and identity of different vertebrae can vary among organisms (Burke et al., 1995; McCollum et al., 2009;

Wolpert, 2002). For example, the thoracic vertebrae begin at somite 20 in chick versus somite 12 in mouse (Burke et al., 1995; Wolpert, 2002). In a given taxon, the transition from one vertebral region to another is consistent with a shift in the Hox gene expression

(Wolpert, 2002). For example, in chick, HoxC5 and HoxC6 are expressed on either side of the cervical and thoracic vertebral boundary, and HoxD9 and HoxD10 are expressed at the boundary of the lumbar and sacral vertebrae (Burke et al., 1995). Regardless of differences between species in numbers of vertebrae with a specific identity (e.g. cervical versus thoracic) in the adult, the correlation between Hox gene expression and vertebral identity is conserved throughout different species (Schoenwolf, 2009) although Hox gene expression dynamics can vary. For example, HoxC6 and HoxC8 are involved in thoracic vertebra development and in specifying forelimb position and shoulder development in tetrapods, whereas in the limbless python, HoxC6 and HoxC8 are expressed throughout the entire trunk (Cohn, 1999). 8

Even though each somite has a specific combination of Hox genes, only the most

5 Hox genes expressed within a somite play a role in specifying its identity (McCollum et al., 2009; see Figure 1).

Figure 1: Regions of Hox gene expression in the vertebral column. From McCollum et al., 2009.

Given this pattern of "posterior prevalence," transformations of vertebral identity reflect changes in the anterior boundaries of particular Hox gene expression domains along the anteroposterior (AP) body axis (McCollum et al., 2009). Hox gene products seem to

anipulate cell attri utes in a way that ensures that cells e pressing ore 5 genes enter into the e ryonic para ial esoder later than the cells e pressing genes, thus setting up the spatial colinearity along the AP body axis of Hox gene expression domains

(McCollum et al., 2009; Pilbeam, 2004). This pattern of expression means that genes that are ore are expressed with older somites (Pilbeam, 2004). 9

Over-expression and knockout experiments have demonstrated the important role played by Hox genes in axial patterning (Pilbeam, 2004). Functional studies show that minor changes in Hox gene expression dynamics can transform one to three caudal lumbar vertebrae into sacral vertebrae. Furthermore, null mutations of either Hox8 or

Hox10 lead to cranialization of the somatic segments of the trunk (Schoenwolf, 2009).

For example, the loss of the Hox10 paralogs leads to the loss of vertebrae with lumbar characteristics and results instead in the development of thoracic vertebrae with ribs

(Schoenwolf, 2009). In addition, gain of Hox gene expression results in caudalization of the vertebrae (Schoenwolf, 2009). For example, if HoxA10 is misexpressed in the presomatic mesoderm, the thoracic vertebrae are respecified to form vertebrae with characteristics of lumbar vertebrae that lack ribs (Schoenwolf, 2009).

The HoxD11 gene is critical to the position of the lumbosacral transition (Gerard et al., 1997) along the AP axis. In mice, a bipartite enhancer (DNA cis-regulatory sequence) that controls the precise spatiotemporal expression of HoxD11 at the lumbosacral transition has been functionally defined (Gerard et al., 1997). Gerard et al. show that changes in the enhancer sequence of this area of HoxD11 in mice result in the changes in length of the lumbar vertebral column. Thus, changes in the expression pattern of this gene could possibly play a role in freeing trapped lumbar vertebrae (Gerard et al.,

1997). However, atelines have fewer lumbar vertebrae than do other New World monkeys, which suggests that changes in the proportion and growth of the ilium are more likely to underlie this important anatomical shift.

CHAPTER III

OSTEOLOGY AND ANATOMY

PURPOSE

The purpose of the anatomical portion of this study is to examine variables that affect and determine the relationship between the dorsal ilia and sacrum as they relate to mobility of the most caudal lumbar vertebra(e) and its effect on potential lordosis, especially in atelines and hominids. As noted previously, it has been argued that the origin of bipedality involved reduction and eventual elimination of contact between the superior portion of the ilia and the lowest lumbar (usually the fifth in humans, but most likely the sixth in Australopithecus), such that the most caudal lumbar could participate to an increasing degree in lordosis. However, there are few fossil ancestral columns on which to base judgment of actual number of lumbars (McCollum et al., 2009; Lovejoy,

2010). The goal of this research is to record variation in the position of the sacrum and its bearing on the potentially restrictive role of the dorsal ilia with respect to the most caudal lumbar in atelines, hominids, and other primates, including the African apes. As described earlier, I anticipated similar degrees of laxity in the lumbosacral regions of hominids and atelines because both exhibit lumbosacral lordotic behaviors, even though they differ in lumbar number.

METHODS

Osteology and Metric Data:

Metric and nonmetric data were collected from the pelves of 140 adult, skeletonized specimens housed in the Hamann-Todd Osteological collection and CMNH collection of the Cleveland Museum of Natural History. This sample included twenty-five New World monkeys (ceboids), twenty-seven Old World monkeys (cercopithecoids), twenty prosimians, thirty great apes (Pan, Pongo, Gorilla), ten lesser apes (Hylobates), one

Australopithecus afarensis (A.L.288-1 "Lucy"), and twenty modern humans of both sexes. Measurements using small and large digital calipers were taken from the individual bones and assembled pelves and the last lumbar vertebra of each specimen

(see figures below). Lumbar vertebrae were defined by the “Schultz method” (Table 1).

In addition to the measurements in Table 1, particular attention was paid to the position of sacral promontory in relation to the iliac crest and the ischio-pubic ramus.

Their relationship was found using several metrics (see Figure 2 below). These metrics included measurements of the vertical distance from the sacral promontory to the most superior portion on the iliac crest and the vertical distance from the sacral promontory to the most distal portion of the ishiopubic ramus. The most superior and distal portions of the pelvis were defined by determining the total length of the os coxa from the most superior point of the iliac crest to the most distal point of the ischiopubic ramus along an imaginary vertical body axis.

TABLE 1: Osteological Measurement Definitions

Measurements of the sacrum: o The maximum mediolateral breadth of the first sacral centrum.

o The maximum diameter between the lateral most points of the sacral alae.

o The total vertical height of sacrum from the most superior point of either ala (whichever is more dorsal) to the most distal point on the sacrum, the latter being the most distal point at midline of the last sacral centrum, as defined by the “Schultz method” of discriminating sacral from caudal centra (Schultz and Strauss, 1945). Specifically, a centrum was considered sacral if it was joined to the centrum above it by lateral processes that were completely fused bilaterally (Schultz and Strauss, 1945). If either lateral process or both contain any gaps in fusion, the centrum was considered caudal.

Measurements of the ilium: o The horizontal breadth of the ilium from its lateral edge to the most superior point of the auricular surface.

o The horizontal breadth of the ilium from its posteriomedial edge to the same point on the auricular surface as used in previous metrics and a horizontal line from the lateral most point on the ilium to the vertical midpoint along midline of the first sacral segment is inscribed.

o The maximum distance across the acetabulum was taken from two points on the acetabular rim.

Measurements of the terminal lumbar vertebra: o The maximum mediolateral breadth of the centrum, the maximum vertical height of the centrum.

o The maximum span (breadth) between the lateral tips of the two transverse processes.

o The total vertical height from anterior longitudinal ligament scar to the

most superior point on the spinous process.

o The horizontal distance from the side of the centrum with the superior articular processes to the transverse process, the horizontal distance from the side of the centrum with the inferior articular process to the transverse process, and the total length of centrum from anterior to posterior side. The angle the transverse process makes with a horizontal passing through the centroid of the centrum of the last lumbar vertebra was taken from photos uploaded to Image J.

Table 1: Descriptions of osteological measurements

Figure 2: Measurements:

Figure 2A: (left; assembled pelvis). A: Height of iliac crest above most lateral point on sacral ala; B: Distance from sacral promontory to lateral-most point on sacrum; C: Sacral promontory to inferior pubis along midline; D: Sacral promontory to most inferior point on ischiopubic ramus along vertical; E: Maximum coxal length. Figure 2B: (right, lumbar vertebra in superior and lateral views). A: Maximum distance between transverse processes; B: Breadth of centrum; C: Anteroposterior length of vertebra; D: Anteroposterior length of centrum; E: Angle of transverse process; F: Distance from the face of the centrum to the base of the superior articular process; G: Distance from the face of the centrum to the base of the inferior articular process; H: Anteroposterior height of centrum in lateral view

Dissection:

The lower backs and pelves of a howler monkey, three spider monkeys, a woolly spider monkey, a langur, and a gibbon were dissected in order to assess the musculature and ligaments of pelves with free and trapped last lumbar vertebrae1. Howler monkeys

(Alouatta), spider monkeys (Ateles), and woolly spider monkeys (Brachyteles) are all

Atelines and are arboreal primates with fully prehensile tails (Ankel-Simmons, 2000).

Langurs (Presbytis) are arboreal Old World monkeys with non-prehensile tails (Ankel-

Simmons, 2000). Gibbons (Hylobates) are lesser apes that specialize in a form of swinging locomotion called brachiation (Ankel-Simmons, 2000). They are primarily arboreal, and they do not have a tail (Ankel-Simmons, 2000). The spider monkeys and howler monkey (both atelines) exhibit a free most caudal lumbar vertebra while the langur’s is trapped (i.e., restricted by immediate physical contact) between the dorsal ilia.1

The howler monkey and langur were frozen cadavera procured from the

Cleveland Museum of Natural History; they were originally captive specimens from the

Cleveland Metroparks Zoo. The spider monkeys were obtained from Kent State

University. Two of the spider monkeys were of unknown origin, and the third was a

1 During dissections, the quality of the preservation of some of the specimens was not ideal. Though the spider monkeys all yielded successful results, two were very desiccated and their results had to be confirmed with another fresher and better preserved specimen. The woolly spider monkey was previously partially dissected and was also very desiccated. I am confident in the identification of the ligamentous tissue that I found, but, due to the preservation of the specimen, there could have been additional ligaments that were missing or destroyed. Another specimen would be needed in order to determine if there were more ligaments.

captive specimen from the Cleveland Metroparks Zoo. The woolly spider monkey and gibbon were acquired from the Northeast Ohio Medical University and had been frozen.

The howler monkey and langur were injected with standard embalming fluid at multiple sites before and during dissection; the spider monkeys, woolly spider monkey, and gibbon were dissected without this treatment. In order to document musculature prior to maceration, MRIs were taken at Case Western Reserve Medical Center of the howler monkey and langur. Following MRI and/or photographic documentation, the appendages of the specimens were dismembered, and the backs of the specimens were skinned. The vertebropelvic musculature of each specimen was then subjected to detailed dissection.

The muscles of the erector spinae (spinalis, iliocostalis, and longissimus) were removed, along with multifidus and quadratus lumborum, in order to reveal the ligamentous tissue.

Following this first layer dissection, specimens were soaked in embalming fluid for several days in order to assure complete fixation of ligamentous tissue. Ligament dissections were then conducted using scissors technique with occasional cautious use of a scalpel. The process of dissection was documented in photographs. Following dissections, pelves were cleaned of all remaining muscular tissue and dried. X-rays were taken of the dried pelves.

RESULTS

The dissections revealed distinct differences between the ligamentous tissue associated with free and trapped most caudal lumbars. One of the spider monkeys (Figure

3A) had three lumbars and the other two had four lumbars (Figure 3B). Their relevant ligamentous tissue consisted of a thin intertransverse ligament running vertically between

the transverse processes of their L3 or L4, respectively (as in the howler monkey), and ilium. The fibers of these ligaments ran vertically between the transverse processes of the vertebrae. An iliolumbar ligament also spanned L2 and L3, respectively, and the ilium had fibers running at an oblique angle from the vertebra to the iliac crest. This tissue was broader and covered more surface area on the iliac crest than in the howler monkey (see later), and again, likely restricted excessive motion, while nevertheless permitting a substantial range of motion. The spider monkeys also possessed a narrow ligament that ran from the body of the terminal lumbar vertebra (L3 or L4) to the ilium of the pelvis.

This ligament was more substantial in cross sectional area than the other ligamentous tissue. Finally, the spider monkeys had ligamentous tissue with horizontal fibers that spanned the sacroiliac joint. These ligaments were lax enough to allow motion and lordosis, but were substantial enough in number to prevent excessive motion.

The woolly spider monkey had five lumbars (Figure 3C). The relevant ligamentous tissue consisted of thin intertransverse ligaments running vertically between the transverse processes of the lumbar vertebrae. The woolly spider monkey also had iliolumbar ligaments running from L3, L4, and L5 to the tip of the ilium of the pelvis.

These fibers ran at an oblique angle to the ilium. The ligament from L5 to the ilium ran at an oblique angle and attached along the sharpest curve of the iliac crest. Furthermore, the fibers of these iliolumbar ligaments were thinner than those of the spider monkeys. The woolly spider monkey also had ligamentous tissue with horizontal fibers that spanned the sacroiliac joint. While it was narrower in cross sectional area, it was also denser in girth than the sacroiliac ligamentous tissue in the other atelines. Like the spider monkeys, the

fibers of this ligament were lax enough to allow motion and lordosis, but substantial enough in number to prevent excessive motion.

The howler monkey had five lumbars (Figure 3D). Union of its free L5 and ilium was restricted to a thin ligamentous raphe, which was sufficiently lax to allow significant motion. This tissue was similar to that seen in the spider and woolly spider monkeys. A ligamentous connection of the iliac crest and transverse process of the L4 appeared to be essentially a restriction of excessive potential motion. The howler monkey also had ligamentous tissue with horizontal fibers that spanned the sacroiliac joint.

The gibbon had five lumbars (Figure 3E). The ligamentous tissue connecting the

L5 to the ilium was thick and consisted of horizontal and oblique iliolumbar ligament fibers. The gibbon also had horizontal fibers running between the transverse processes of its lumbar vertebrae and iliolumbar ligaments that ran from L3 and L4 to the crest of the ilium at an oblique angle. The overall thickness and orientation of the ligamentous fibers were more reminiscent of the spider monkey and howler monkey than the langur (see later). The ligamentous tissue would not have been as restrictive in the gibbon as it was in the langur.

The langur had seven lumbars (Figure 3F). The ligamentous union of the langur’s

L7 and ilium was via thick ligamentous tissue that joined both structures to the transverse processes of L6. Because the L7 was positioned deeply between the iliac blades, its motion was highly restricted. The ligamentous tissue of the langur was substantially denser than that of all the other specimens dissected.

Figure 3: Drawing showing observations made during dissections of ligamentous union among the ilium, sacrum and most caudal lumbar vertebrae of several primate species. (A) spider monkey; (one of two similar specimens), (B) third spider monkey with a slight variation in anatomy and four lumbar vertebrae; (C) woolly spider monkey; (D) howler monkey; (E) gibbon; (F) langur. The more superficial ligamentous unions between vertebral column and pelvis are shown for each animal's right, while those on the specimen’s left show patterns of the deepest tissues. Ligamentous union was considerably greater in the langur than in the four atelines and gibbon.

Osteometrics of the museum sample:

The most relevant metric comparison in the large sample of museum skeletons described earlier was the vertical distance from the sacral promontory to the most superior point of the iliac crest compared to the distance from the same point on the sacrum to the most inferior extent of the pelvis (invariably along the ischio-pubic ramus).

The more caudal the sacrum promontory’s location, the higher the probability that at least one caudal lumbar would be restricted from lordosing by partial ligamentous union with the adjacent iliac blades (as seen in the dissections--see above). This ratio is shown in Figure 4 (relative sacral height). Note that the Ateline group differs significantly in the predicted direction from Old World monkeys and especially gibbons and great apes. Data for humans are not shown in this figure because their pelves have been further radically shortened for additional adaptations to upright walking.

Figure 4: Relative sacral height

Soft tissue correlates in the lumbosacral complex are reflective of lordotic capacity, and also have osteological correlates. For example, sacral breadth was likely a morphological aspect of "freeing" the deepest (most caudal) lumbar from contact with the iliac blades. This result can be seen in Figure 5, which shows the breadth of the sacral alae normalized by the breadth of the sacral centrum (as an indicator of general body size). Again, it appears likely that increased alar breadth has been part of the morphological "strategy" that freed the most caudal lumbar in both atelines and hominids for active lordosis at the sacral promontory. Note in this figure that hominoids have narrower sacra than all other species, but humans have similar sacral breadth compared to both New and Old World monkeys due to the presence of a tail in these lower primates.

Humans have unusually broad sacra, even though they do not have a tail.

Thus, reduction in iliac height appears, therefore, to be the primary factor that permits lordosis, a significant adaptation in atelines that rely on a prehensile tail and in hominids that rely on bipedality, even though the former of these taxa have a reduced number of lumbar vertebrae compared to humans.

The sacral breadth data in Figure 5, reveal an interesting question. Hominids and monkeys both have relatively broad alae compared to those of apes. Alar breadth is substantial in both New World monkeys and Old World monkeys because they retain tails. Proconsul was tailless by the mid-Miocene (Ward et al., 1991) but was still an above branch quadruped (Ward, 1993; 2007). Alar breadth in our last common ancestor with African apes may have been retained and may, therefore, be primitive in hominids.

Alternatively, it may have been reduced in mid-Miocene hominoids but later rebroadened

in hominids for bipedality while narrowed in apes as an adaptation to suspension and/or vertical climbing.

Figure 5: Plot of alar width compared to centrum width

CONCLUSIONS

Overall, atelines and hominids share similar adaptations of reduced iliac height, which appears to be the primary adaptation permitting lordosis in both taxa. The lumbar columns of atelines and humans display free lumbar vertebrae at the lumbosacral junction as well as ligamentous tissue sufficiently lax to permit lordosis at the joint. In contrast to these features, the Old World monkeys and apes display both osteological and ligamentous features that prevent similar motion at this joint, including thick ligamentous

tissue that restricts motion and prevents lordosis. Inasmuch as humans retain five lumbar vertebrae and australopithecines are likely to have exhibited six, while atelines have as few as three, it would appear that modifications of the dorsal ilia have been the primary site of adaptation to lordosis in both taxa.

CHAPTER IV

GENETICS

PURPOSE

Two characteristic that bear on the locomotor mode of primates are 1) the lengths of the lumbar and sacrocaudal columns, and 2) the degree to which motion of the most caudal lumbar is restricted by the ilium. These characters vary extensively within hominoids and platyrrhines (New World monkeys), most specifically the atelines. Using a HoxD11 enhancer functionally defined by Gerard et al., 1997 (Figure 5), I investigated whether HoxD11 enhancer variation and length of the lumbar column are correlated in primates.

METHODS

Genomic DNA:

Genomic DNAs of New World monkeys including, black spider monkey (Ateles paniscus) and common squirrel monkey (Saimiri sciureus), were obtained from the

Coriell Institute for Medical Research in Camden, New Jersey. Genomic DNAs of apes, including white-handed gibbon (Hylobates lar), of Old World monkeys, including rhesus macaque (Macaca mulatta), and of New World monkeys, including common marmoset

(Callithrix jacchus), capuchin monkey (Cebus apella), and white-faced saki monkey

(Pithecia pithecia), are from Chi-hua Chiu's personal collection at Kent State University.

Genomic DNA of the ring-tailed lemur (Lemur catta) was obtained from the Duke

Lemur Facility in Durham, North Carolina.In addition to previously extracted DNA, I extracted genomic DNA from a hair sample of the langur (Presbytis francoisi) specimen that I previously used for dissection using the QIAGEN Genomic-tip kit (catalog #) and associated protocol for isolation of genomic DNA from blood, cultured cell, tissue, yeast, or bacteria. The sample was first prepared according to the QIAGEN tissue extraction protocol using the protocol for a 20/G tip and <20mg of tissue from the pelt.

Polymerase Chain Reaction (PCR), Cloning, and Sequencing:

Using the HoxD11 enhancer region defined in mouse and chick (Gerard et al.,

1997), I made an alignment of the enhancer region with humans, great apes, and mammals from the NCBI database.

Figure 6: Mouse HoxD11 Enhancer Sequence (Gerard et al. 1997)

This alignment was subsequently used to design primers to amplify this region in additional primate taxa using the Polymerase Chain Reaction (PCR).

Primers:

HsaHoxD11F-2: 5’ – TGT TTA CAA AAC CTT GAA CTG TC – 3’

HsaHoxD11R-2: 5’ – GGA AAT TAA GGT TAT TGA TCC TG – 3’

PCR is a technique that can be used to amplify a single piece of DNA to generate thousands to millions of copies of a specific sequence of DNA (Mullis, 1986). I PCR amplified, cloned, and sequenced the HoxD11 bipartite enhancer in multiple primate species including catarrhines: human, gibbon (Hylobates lar) and rhesus macaque

(Macaca mulatta); platyrrhines: titi monkey (Callicebus moloch), saki monkey (Pithecia pithecia), spider monkey (Ateles paniscus), and capuchin monkey (Cebus apella); and a strepsirhine: lemur (Lemur catta). PCR was conducted using a program refined for optimum results for my specific primers. DNA was denatured, amplified, and extended according to the following protocols:

PCR Program 4:

Step 1: Denaturation: 95°C for 5 minutes Step 2: Denaturation: 95°C for 1 minute Step 3: Primer Annealing: 52°C for 1 minute Step 4: Extension: 72°C for 1 minute Step 5: Repeat steps 2 - 4 thirty times Step 6: Final Extension: 72°C for 10 minutes Step 7: 10°C indefinitely

PCR Program 5 (used for P. pithecia):

Step 1: Denaturation: 95°C for 5 minutes Step 2: Denaturation: 95°C for 1 minute Step 3: Primer Annealing: 50°C for 1 minute Step 4: Extension: 72°C for 1 minute Step 5: Repeat steps 2 - 4 thirty times Step 6: Final Extension: 72°C for 10 minutes Step 7: 10°C indefinitely

PCR was conducted using the following amounts of reagents:

A B C C D Samples: H2O 10x MgCl dNTPs PF PR gDNA absTaq

1. (+): 31 µL 5 µL 7 µL 4 µL 1 µL 1 µL 0.5 µL 0.5 µL

2. (-): 31.5 µL 5 µL 7 µL 4 µL 1 µL 1 µL 0 µL 0.5 µL

3. Primate 28.5 µL 5 µL 7 µL 4 µL 1 µL 1 µL 3 µL* 0.5 µL

A10x buffer with no Mg was used, B25mmol MgCl was used, C20ng/ µL primers were used, DEst 50ng/ µL, *More or less primate gDNA was used to optimize the sample during experimentation and the amount of H2O was adjusted accordingly.

PCR products, including positive (human DNA) and negative (no DNA) controls were visualized using agarose gel-electrophoresis in 1X TAE buffer and a 2% agarose gel in order to confirm identity as HoxD11. PCR products of the correct size were purified using Qiagen MinElute™ Gel Extraction Kit. Step 13 of this protocol was amended to elute the DNA in 10 L of sterile water. A ligation was conducted overnight at 16oC using the following 20 µL reaction: 10 µL Ligase Buffer, 8 µL purified PCR product, 1

µL T4 Ligase, 1 µL pGem-T vector. The ligation product was transformed into JM109 competent cells using the following protocol:

Step 1: Spread 30 µL XGAL on an LB + 50 mg/mL ampicillin plate Step 2: Place JM109 cells (Promega) on ice for 5 minutes Step 3: Add 2 µL of ligation to the JM109 cells Step 4: Place on ice for 20 minutes Step 5: Heat shock for 45 seconds in a water bath at 42°C and remove immediately Step 6: Place on ice for 2 minutes Step 7: Add 240 µL of LB (amended from manual protocol) Step 8: Shake at 37°C at 220RPM for 1.5 hours Step 9: Spread 240 µL on a plate and allow to dry for 1 - 2 hours Step 10: Incubate overnight at 37°C

The next morning, white colonies (containing insert) were selected in sets of seven for colony PCR (Chambers et al. 2009) under the PCR conditions originally used to amplify the inserts.

Colony PCR Protocol:

1x 9x (+) H2O: 34.5 µL 310.5 µL 34 µL 10x 5 µL 45 µL 5 µL MgCl 4 µL 36 µL 4 µL dNTPs 4 µL 36 µL 4 µL PF 1 µL 9 µL 1 µL PR 1 µL 9 µL 1 µL gDNA 0 µL 0 µL 0.5 µL absTaq 0.5 µL 4.5 µL 0.5 µL

To prevent contamination, an ultraviolet (UV) crosslinker was used for 600 seconds for

P. pithecia, L. catta, and C. apella.

Colony PCR confirmed clones were grown in media and purified using the

Qiagen QIAprep® Spin Miniprep Kit. Ten µL of each purified clone was then sent for sequencing (at Ohio University Genomics Facility) using the T7 and Sp6 primers, which have a binding site in the pGEM®-T vector. The results of sequencing were positively sequence identified using BLAST sequencing with the NCBI database. Newly generated

and confirmed sequences were analyzed and aligned with publicly available orthologs of

chimpanzee, orangutan, baboon, marmoset, mouse, and zebrafish (Appendix A).

In this thesis, I generated new sequence data of four human clones (H.sapiens),

three spider monkey clones (A.paniscus), four titi monkey clones (C.moloch), six gibbon

clones (H.lar), five rhesus macaque clones (M.mulatta), four saki monkey clones

(P.pithecia), two lemur clones (L.catta), and one capuchin monkey clone (C.apella).

RESULTS

The length of the HoxD11 enhancer orthologues generated was 506 base pairs

(bps) in human. The consensus sequence for all taxa is shown in Appendix A.

% Conservation % Conservation % Conservation with H sapiens with P hamadryas with M musculus

94%94 A. paniscus94%94 94%94%94 94% 82%

C. apella 94% 95% 82%

94 C. moloch 94%82% 94% 82%

98H. lar 98% 98% 84%

L. catta 91% 92% 84%

M. mulatta 96% 99% 83%

P. pithecia 92% 93% 81%

Table 2: The observed conservation of each species

Coding regions are the portion of a gene which code for a protein, whereas non-

coding regions are the portion of a gene which do not produce a protein but may be

occasionally transcribed (Carroll, 2001). Coding regions are highly conserved and most

often evolved under strong purifying selection, whereas non-coding regions are freer to vary because they can have a more limited impact on the gene’s role in the morphotype

(Carroll, 2001). Transcription factors control gene expression in cellular processes by interaction with specific DNA regulatory sequences (Berger, 2006). Transcription factors, which interact with coding and non-coding regions and control the identity of body segments, have been shown to be involved in tissue patterning (Pilbeam, 2004).

I next used MATCH™ (http://www.gene-regulation.com/pub/ regulation.com/ pub/ programs. html), a program that predicts transcription factor binding sites and phylogenetic analysis, to investigate whether there is variation in transcription factor binding sites in the HoxD11 enhancers throughout primate phylogeny (Figure 6, Table 3).

As illustrated in Figure 6, I found that New World monkeys exhibit unique variability in predicted transcription factor binding sites. This finding is congruent with the fact that

New World monkeys have the most variability in the length of their lumbar vertebral column. I found that humans and great apes share transcription factor binding sites. I found that all primate species sequenced share Nkx2-5 and Kr. Based on the results of this study, I showed that Nkx2-5 is most likely the ancestral transcription factor binding site. Nkx2-5 is a marker for precardiac cells and is essential for heart formation

(Durocher, 1987).

Figure 7: A phylogenetic comparison of transcription factor binding sites in sequenced specimens. Phylogenetic tree based on Wildman et al. 2009. In this figure, solid colored boxes represent the gain of a transcription factor binding site and hollow boxes with crosses represent the loss of a transcription factor binding site.

TABLE 3: Transcription Factor Binding Site Definitions

NKX 2-5 NKX205 is a marker for precardiac cells. It is essential for heart formation (Durocher, 1997).

KR Kruppel (KR) is expressed in early born deep layer neurons, it is a candidate for regulating temporal identity in neuroblast lineage, and it is needed for second born cell fates (Carroll, 2001; Isshiki, 2001). AG Angiotensinogen (AG) is the precursor for vasoactive octapeptide angiotension11. AG plays a role in the regulation of blood pressure and in electrolyte homeostasis (Yanai, 1997). SRF SRF binds to serum response element. Found in muscle promoters such as cardiac and skeletal actin genes. These sites act as growth factor regulated promoter elements in non-muscle cells (Pollock, 1991). PAX-4 Pax genes play regulatory roles in development as tissue specific regulators. Pax-4 is part of Group IV, which also includes Pax-6 and eyeless. (Balczarek, 1997). HAND1 Hand1 is needed for differentiation of trophoblast giant cells and for proper placental development (Scott, 2000). CRP CRP is the major acute-phase reactant in humans and provides a defense function by eliminating pathogens. It also promotes necrotic and apoptotic cells (Szalai et al., 2001). HNF-1 HNF-1 controls the expression of liver specific genes and binds to CRP (Toniatti, 1990). COMP-1 Human Cartilage Oligomeric Matrix Protein (COMP-1) is synthesized by chondrocytes, osteoblasts, tenocytes, and ligament cells. It may function to stabilize the extracellular matrix of articular cartilage by interactions with matrix components like collage types II and IX and fibronectin (Liu, 2004). BR-CZ1 BR-CZ1 is involved in determining tissue specific outcomes (Emery, 1994). MIG-1 MIG-1 is involved in yeast and glucose repression. It is an early growth response factor in mammals (Nehlin, 1990). V-Myb V-Myb encodes the transcription factor MIM-1. MIM-1 and MYB are correlated in the spleen (Burke, 1993; Queva, 1992).

DISCUSSION

New World monkeys had the most plasticity in their transcription factor binding sites. It would be interesting to continue this study further, comparing the transcription factor binding sites against those of humans using either gel electrophoresis mobility shift assay (EMSA), which is used to detect protein complexes with nucleic acids or transgenic analysis (Hellman, 2007).

Interestingly, New World monkeys’ variability in predicted transcription factor binding at the HoxD11 enhancer is correlated with the observed variation in lumbosacral osteological anatomy. As shown by Gerard et al., the replacement of this enhancer region of HoxD11 in mice activated transcription early and resulted in a rostral shift of the expression boundary and an anterior transposition of the sacrum, creating an additional free vertebra with a lumbar identity (1997). The result of an additional lumbar vertebra supports that minor time differences in Hox gene activation may have contributed to morphological changes throughout evolution and may have been a contributing factor to changes in the number and placement of lumbar and sacral vertebrae.

CHAPTER V

SUMMARY AND FINAL CONCLUSIONS

This thesis sought to explore convergent evolution and subsequent impacts on locomotion in primates, with a focus on atelines, using anatomical and genetic analysis.

Overall, data were collected from both anatomical and genetic studies that support the convergent evolution of pelvic morphology in hominids and atelines. These groups showed similar adaptations of reduced iliac height and free lumbar vertebrae at the lumbosacral junction with ligamentous tissue sufficiently lax to permit lordosis at the joint. In order for the terminal lumbar vertebra to be freed from the ilia, changes to the shape and size of the ilium and changes in the lumbars may be necessary. New World monkeys showed variability in lumbosacral morphology between atelines and non- atelines that was reflected in genetic variation in the HoxD11 enhancer sequence.

Changes in Hox gene activation, such as the enhancer studied in this thesis, may have contributed to alterations in morphology and may have been a contributing factor to anatomical changes in the lumbosacral column, allowing freeing of a trapped terminal lumbar vertebra. Furthermore, because hominids and atelines are convergent in their ability to lordose, the ateline HoxD11 enhancer could be different from that of human

(hominid), but still result in the same characteristic

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APPENDIX A

Full alignment of HoxD11: This sequence is the complete alignment generated by the genetic portion of this study. This alignment was used to generate forward and reverse primers and includes the primate sequences obtained by the use of these primers.

HSA = Homo sapiens (Human, A. Machnicki), HSA2 = Homo sapiens (Human, NCBI Data Base), PTROG = Pan troglodytes (Chimpanzee), PPYG = Pongo pygmaeus (Orangutan), HLAR = Hylobates lar (Gibbon), MMUL = Macaca mulatta (Rhesus Macaque), RMAC = Macaca mulatta (Rhesus Macque, NCBI), PHAM = Papio hamadryas (Baboon), APAN = Ateles paniscus (Spider monkey), CMOL = Callicebus moloch (Titi monkey), PPITH = Pithecia pithecia (Saki monkey), CJAC = Callithrix jacchus (Marmoset), CAPEL = Cebus apella (Capuchin monkey), LCAT = Lemur catta (Lemur), MMUS = Mus musculus (Mouse), GGAL = Gallus gallus (Chicken), AMEL = Ailuropoda melanoleuca (Giant Panda), ACAR = Anolis carolinensis (Lizard) , BTAU = Bos taurus (Cow), CFAM = Canis familiaris (Dog), CPOR = Cavia porcellus (Guinea Pig), ECAB = Equus ferus caballus (Horse), FCAT = Felis catus (Cat), LAFR = Loxodonta africana (African Elephant), MDOM = Monodelphis domestica (Opossum), OANA = Ornithorhynchus anatinus (Platypus), OCUN = Oryctolagus cuniculus (Rabbit), RNOR = Rattus norvegicus (Rat), SSCR = Sus scrofa (Pig), TGUT = Taeniopygia guttata (Zebra Finch), XTRO = Xenopus tropicalis (Frog), DRER = Danio rerio (Zebrafish).

1 10 20 30 40 50 60 | | | | | | | Consensus ------HSA ------HSA2 ------CTT-TATGTTGCAGGGCCAGGCCA-GGCTGTTTACAAAACCTT PTROG ------CTT-TATGTTGCAGGGCCAGGCCA-GGCTGTTTACAAAACCTT PPYG ------CTT-TATGTTGCAGGGCCAGGCCA-GGCTGTTTACAAAACCTT HLAR ------MMUL ------RMAC ------CTT-TATGTTGCAGGGCCAGGC-A-GGCTGTTTACAAAACCTT PHAM ------CTT-TATGTTGCAGGGCCAGGC-A-GGCTGTTTACAAAACCTT APAN ------CMOL ------PPITH ------CJAC ------CTGCACTGCACTT-TATGTTTCAGGGCCAGGCCA-GGCTGTTTACAAAACCTT CAPEL ------LCAT ------MMUS ------CTGCAGTGCCCTT-TATGTCACAGGACCAAGCCA-GGCTGTTTACAAAACCTT GGAL ------GGCTGTTGGCGAAACCTT AMEL ------CTT-TATGTTGCAGGGCCAGGCCA-GGCTGTTTACAAAACCTT ACAR ------GCTGTTTACAAAACCTT BTAU ------CTT-TATGTTGCAGGGCCAGGCCA-GGCTGTTTACAAAACCTT CFAM ------CACTT-TATGTTGCAGGGCCAGGCCA-GGCTGTTTACAAAACCTT CPOR ------CTT-TATGTTGCAGGGCCAGGCCA-GGCTGTTTACAAAACCTT ECAB ------CTT-TATGTTGCTGGGCCAGGCCA-GGCTGTTTACAAAACCTT FCAT ------CTT-TATGTTGCAGGGCCAGGCCA-GGCTGTTTACAAAACCTT

LAFR ------CTT-TATGTTGCAGGGCCAGGCCA-GGCTGTTTACAAAACCTT MDOM ------GCTGTTTACAAAACCTT OANA ------GCTGTTTACAAAACCTT OCUN ------GCCA-GGCTGTTTACAAAACCTT RNOR ------TGCAGTGCACTT-TATGTCACAGGACCAAGCCA-GGCTGTTTACAAAACCTT SSCR ------CTT-TATGTTGCAGGGCCAGGCCA-GGCTGTTTACAAAACCTT TGUT ------GGCTGTTTACAAAACCTT XTRO ------GGCTGTTTAGAAAACCTT DRER ----AATCT----TTCCCTTGTATGTT---GTTTTACACTATGGCTATTTACTAAACCTT

Consensus ------TAGACAACACCATAAAAGCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT HSA ------TAGACAACACCATAAAAGCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT HSA2 GAACTGTCTAGACAACACCATAAAAGCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT PTROG GAACTGTCTAGACAACACCATAAAAGCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT PPYG GAACTGTCTAGACAACACCATAAAAGCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT HLAR ------TAGACAACACCATAAAAGCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT MMUL ------TAGACAACACCATAAAAGCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT RMAC GAACTGTCTAGACAACACCATAAAAGCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT PHAM GAACTGTCTAGACAACACCATAAAAGCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT APAN ------TAGACAATACCATAAAAGTCAAAGTTC--TAAACAGCTTAATTGGGTTATAT CMOL ------TAGACAATACCATAAAAGTCAAAGTTC--TAAACAGCTTAATTGGGTTATAT PPITH ------TAGACAATACCATAAAAGTCAAAGTTC--TAAACAGCTTAATTGGGTTATAT CJAC GAACTGTCTAGACAATACCATAAAAGTCAAAGTTC--TAAACAGCTTAATTGGGTTATAT CAPEL ------TAGACAATACCATAAAAGTCAAAGTTC--TAAACAGCTTAATTGGGTTATAT LCAT ------TAGACAACACCATAAAAGCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT MMUS GAACTGTCTAGACAACACCATAAAACCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT GGAL GAACTGTCTAGACAACACCATAAAAGCCGAGGTTC--TAAACAGCTTAATTGGGTTATAT AMEL GAACTGTCTAGACAACACCATAAAAGCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT ACAR GAACTGTCTAGACAACACCATAAAAGCTCAGGGTC--TAAATAGCTTAATTGGGTCATAT BTAU GAACTGTCTAGACAACACCATAAAAGCCAAAGTTC--TAAATAGCTTAATTGGGTTATAT CFAM GAACTGTCTAGACAACACCATAAAAGCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT CPOR GAACTGTCTAGACAACACCATAAAAGCCAAAGTTC--TAAATAGCTTAATTGGGTTATAT ECAB GAACTGTCTAGACAACACCATAAAAGCCTAAGTTC--TAAATAGCTTAATTGGGTTATAT FCAT GAACTGTCTAGACAACACCATAAAAGCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT LAFR GAACTGTCTAGACAACACCATAAAAGCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT MDOM GAACTGTCTAGACAACACCATAAAAACCGAAGTTC--TAAACAGCTTAATTGGGTTATAT OANA GAACTGTCTAGACAACACCATAAAAGCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT OCUN GAACTGTCTAGACAACACCATAAAAGCCAGAGTTC--TAAACAGCTTAATTGGGTTATAT RN4 GAACTGTCTAGACAACACCATAAAACCCAAAGTTC--TAAACAGCTTAATTGGGTTATAT SSCR GAACTGTCTAGACAATACCATAAAACCCAAAGTTC--TAAACAGCTTAATTAGGTTATAT TGUT GAACTGTCTAGACAACACCATAAAACCCGAGGTTC--TAAACAGCTTAATTGGGTTATAT XTRO GAACTGTCTAGACAACACTATAAAAGTATGGCTTC--CAAGCAGGATAATTGCCTTATAT DRER GAACAGTCTAGACAGTACAATAAA---CTTTGGGCAGTAAA----TTAAATTGGGGCTAT

Consensus TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGAAGT HSA TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGAAGT HSA2 TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGAAGT PTROG TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGAAGT PPYG TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGAAGT HLAR TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGAAGT MMUL TATACACCTTCTGGTGGACCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGGAGT RMAC TATACACCTTCTGGTGGACCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGGAGT PHAM TATACACCTTCTGGTGGACCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGGAGT APAN TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGAAGT CMOL TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGAAGT PPITH TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGAAGA CJAC TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGAAGT CAPEL TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAAC--GAGAAGT LCAT TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGAAGT MMUS TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGACGT GGAL TATACACCTTCTGCCGGGTGAAAAAGAGA-TTTCCGCAGTGTGCCATAAAGGCGAGGAGT AMEL TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGTAGGAGT ACAR TGTACACCTTCTGGTGGGCCGAAAAGAGA-TTTCCGCAATGTGCAATAAAGGGGATGAGT 41

BTAU TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAAGAGT CFAM TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGTAGGAGT CPOR TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTAGAGAAGT ECAB TATACACCTTCTGGTGGGCCCAGAAGAGA-TTTCCGCAATGTGCAATAAACTGCAGGAGT FCAT TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGTAGGAGT LAFR TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAACGTGCAATAAACTGGAAGAGT MDOM TATACACCTTCTGGTGGGGGAAAAAAAGA-TTTCCGCAATGTGCAATAAAGTGGA---GT OANA TATCCACCTTCTGGTGGGCGCAGAAGAGA-TTTCCGCAATGTGCAATAAAGGGAAGGAGT OCUN TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGAAGT RN4 TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAGACGT SSCR TATACACCTTCTGGTGGGCCCAAAAGAGA-TTTCCGCAATGTGCAATAAACTGGAAGAGT TGUT TATACACCTTCTGGTGGGCGAAAAAGAGA-TTTCCGCAATGTGCAATAAAGGCGACGAGT XTRO TATACACCTTCTGCTGGGCT-AAGGGAGA-TTTCCGCACAGTGCAATAAA-TGGACAAGT DRER --TGGACCCTCTATCCACTTCAACTGCGACTTTGCCC----TGAAACACAAAGG----GT

Consensus G-AGAAAAGCTTCTCTTTCTCT------G-AAAACAGTTAA-GT HSA G-AGAAAAGCT-CTCTCTCT------GAAAAGAGTGAA-GT HSA2 G-AGAAAAGCT-CTCTCTCT------GAAAAGAGTGAA-GT PTROG G-AGAAAAGCT-CTCTCTCT------GAAAAGAGTGAA-GT PPYG G-AGAAAAGCT-CTCTCTCT------GAAAAGAGTGAA-GT HLAR G-AGAAAAGCT-CTCTCTCT------GAAAAGAGTGAA-GT MMUL G-AGAAAAGCTTCTCTCTCTCT------GAAAAGAGTGAA-GT RMAC G-AGAAAAGCTTCTCTCTCTCT------GAAAAGAGTGAA-GT PHAM G-AGAAAAGCTTCTCTCTCTCT------GAAAAGAGTGAA-GT APAN G-AGAAAAGCTTCTCTCTCTCTCTCT------GAAAAGAGTGAA-GT CMOL G-AGAAAAGCTTCTCTCTCTCTCTCT------GAAAAGAGTGAA-GT PPITH G-AGAAAAGCTTCTCTCTCTCTCT------GAAAAGAGTGAA-GT CJAC G-AGAAAAGCTTCTCTCTCTCTCT------GAAAAGAGTGA--GT CAPEL G-AGAAAAGCTTCTCTCTCTCTCT------GAAAAGAGGGAA-GT LCAT G-AGAAAAGCTTCTCT-T-TCTCT------GAAAACAGTTAA-GTG MMUS G-AGAAAACCTCCTCTTGATCT------GACCAGAGTGAA-GT GGAL G-AGAAAAGAATTTGGCTTTTTTTTTTT------GAATAGGACGGAGCG AMEL G-AGAAAAGCATCTCTTTCTCT------GAAAAAAAAAGTAAA-GT ACAR G-AGAAAAGAATTTGGCTTTTTTTTTTTCAAAGGGAGGGGGAGACTAAAACATTAAGGAA BTAU G-AGAAAAGCTTCTCTTTCTCT------GAAAAGAGTAAA-GT CFAM G-AGAAAAGCATCTCTCCCTCT------G---AAAAAAGTAAA-GT CPOR G-AGAAAAG--TTTCTTTCTCT------GAAGAGAGTGAA-AT ECAB G-AGAAAAGCTTCTCTTTCTCT------GAAAAGAGTAAA-GT FCAT G-AGAAAAGCATCTCTTTCTCT------G---AAAAAAGTAAA-GT LAFR G-AGAAAAGCTTTTCTTTCCCT------G---AAAAGAGTAAA-GT MDOM G-AGAAAGGGTTTTCTTTTTCTTTTT------TTTTTTAAGAC-AAA-GT OANA G-AGAAAAGATTCTCTCTCTCTCCCTCCCTCTCCCCCCCCCCTTTTTAAAGACTAAAGTA OCUN G-AGAAAATCT--TCTTTCTCT------GAAAAGAGTGAA-GT RN4 G-AGAAAACCTCCTCTTGATCT------GACAAGAGTGAA-GT SSCR G-AGAAAAGCTTTTCTTTCTCT------GAAAAGAGTAAA-GT TGUT G-AGAAAAGAATTTGGCTTTTT------TGAATAG-GACGAGGT XTRO A-AGAATGGGGGCTGGTT-TCT------TCAATG- DRER GCAGA------TGTGT-TTTTTCTCA------GAAAAATATGA--GT

Consensus GAA------TCTTAT-GACTCT-TAACCTCTCAT-AAGCCCAAAGTCAAAA----- HSA GAA------TCTTAT-GAGTCT-TAACCTCTCCT-AAGCCCAAAGACAAAA----- HSA2 GAA------TCTTAT-GAGTCT-TAACCTCTCCT-AAGCCCAAAGACAAAA----- PTROG GAA------TCTTAT-GACTCT-TAACCTCTCCT-AAGCCCAAAGACAAAA----- PPYG GAA------TCTTAT-GACTCT-TAACCTCTCCT-AAGCCCAAAGACAAAA----- HLAR GAA------TCTTAT-GACTCT-TAACCTCTCCT-AAGCCCAAAGACAAAA----- MMUL GAA------TCTTAT-GACTCT-TAACCTCTCCT-AAGCCCAAAGACAAAA----- RMAC GAA------TCTTAT-GACTCT-TAACCTCTCCT-AAGCCCAAAGACAAAA----- PHAM GAA------TCTTAT-GACTCT-TAACCTCTCCT-AAGCCCAAAGACAAAA----- APAN GAA------TCTTAT-GACTC---AACCTCTCCT-AAGCCCAAAGACAAAA----- CMOL GAA------TCTTAT-GACTC---AACCTCTCCT-AAGCCCAAAGACAAAA----- PPITH CAA------TCTTAT-GACTC---AACCTCTCCT-AACCCCATAGACAAAA----- CJAC GAA------TCTTAT-GACTC---AACCTATCCT-AAGTCCAAAGACAAAA----- 42

CAPEL GAA------CCTTAT-GACTC---AACCTCTCCT-AAGCCCAAAGACAAAA----- LCAT AA------TCTTAT-GACTCT-TAACCTCTCAT-AAGCCCAAAGTCAAAA----- MMUS GGA------TCTCAT-GACTCT-TAACCTCTCAG-AAGCCCAAAGTCTAAA----- GGAL GC------TCTCGC-TACGCT-CG-CGTTCGCGGAAGGCAGAAGTGCGCAAACGA AMEL GAA------TCAT-GACTCT-TAACCTCTCCT-AAGCCCAAAGTAAAGAA---- ACAR GAAGAAATCCTTCTCTCAT-T-CTCT-TTGCATCCCTTAAAGGCCAAACTAAGAG----- BTAU GAA------TCTTAT-GACTCT-TAACCTCTTAT-AAGTCCAAAGTCAAAA----- CFAM GAA------TCGT-GACTCT-TAACCTCTTCT-AAGCCCAAAGTAAAAAAAAAA CPOR GAA------TCTTAT-GATTCT-TAGCATCTTAT-AAACCCAAAGTCAAAA----- ECAB GAA------TCTTAT-GACTCT-TAACCTCTCAT-CAGCCCAAAGTCAAAA----- FCAT GAA------TCTTAT-GACTCT-TAACCTCTCCT-AAGCCCAAAGTCAAAAACA-- LAFR GGA------TCTTAT-GAGTC---AACTTCTCAT-AAGCTCAAAGTAAAATAAA-- MDOM AGA------TCTTAT-GGCTCT--GGCTTCTTAT-AAGCCTAAAGTCGAAA----- OANA GT------TCTTAT-GATTCT-T-GCTTCTAGT-AAGCTCAAAGTAGGAA----- OCUN GAA------TCTTAT-GACTCT-TAACCTCTCCT-AAGTCTAAAGTAAAAA----- RN4 GGA------TCTTAT-GACTCT-TAACCTCTCAG-AAGCCCAAAGTCTAAA----- SSCR GAG------TCTTAT-GACTCT-TAACCTCTCAT-AAGCCCAAAGTAAAAAATA-A TGUT GGC------CCTCGC-TACTCT-CT-CAGGCAGGGAAGGCAAAAGTGCCAA----- XTRO GAA------CTTGG-TGGGCG-TTATTTCACTG---GCATGAAGTAAACT----- DRER GAACATA----TATATTACAGACTATGTTACCCCCCC---GGCCAAGTGTCTGGCTGGAA

Consensus -TATAAAT--CCATAAA------TGCAC---AG-C---ACCCCATGGATCT- HSA ------T----ATAAATCTA------TAA-ATGCAC--AG----TACCCCATGCATC-- HSA2 ------T----ATAAATCTA------TAA-ATGCAC--AG----TACCCCATGCATC-- PTROG ------T----ATAAATCTA------TAA-ATGCACG--G----TACCCCATGCATC-- PPYG ------T----ATAAATCTA------TAA-ATGCAC--AG----TACCCCATGCATC-- HLAR ------T----ATAAATCTA------TAA-ATGCAC--AG----TACTCCATGCATC-- MMUL ------T----ATAAATCTG------TAA-ATGCAC--AG----TATCCTATGCATC-- RMAC ------T----ATAAATCTG------TAA-ATGCAC--AG----TATCCTATGCATC-- PHAM ------T----ATAAATCTG------TAA-ATGCAC--AG----TATCCCATGCATC-- APAN ------T----ATAAATCTA------TAA-ATGCGGG--G----TACCCCATGCATC-- CMOL ------T----ATAAATCTA------TAA-ATGCGGG--G----TACCCCATGCATC-- PPITH ------T----ATAAATCTG------TAA-AAAAGGG--G----TACCCCATGCGTC-- CJAC ------T----ATAAATCTA------TAAG-TGCAG--AG----TACCCCATGCATC-- CAPEL ------T----ATAAATCTA------TAAA-TGCAGG--G----TACCCCATGCATC-- LCAT ------T----ATAAATCCA------TAAA-TGCAC--AGC---ACCCCATGGATCT-- MMUS ------A----ATAAATCCA------TAAGATGCAC--AG----CAGCTCATGCCTC-- GGAL GTCGGGAA----C--GGCCCC-GGAATTAAGAAAAAAAAA------CGGAGAGAAAGA AMEL -----AAA----AAAAATCCA------TAAGATGTAC--AG----TACCCCGTGCACC-- ACAR ------GA----ATAAAACGA-AGGGGAAAGCTACCAAAAAAAGAAAACCCCTAAACC-- BTAU ------A----ATAAATCCA------TGAGCTATAC--AGC----ACCCCATGCATC-- CFAM --ATAAAA----AAAAATCCA------TAAGATGTAC--AG----TACTCCAAGCATC-- CPOR ------T----ATAAATCTG------TGAGGTGCAT--AGC----AGTTTATGCATC-- ECAB ------AA----ATAAATTCA------TAAGATGTGC--AGC----ACTCCATACATCCA FCAT ------AA----ACAAAACAA-AACCATAAGGTGTAC--AG----GACCCCATGCATC-- LAFR -----GAA----ATACACCCA------TAAGATGCGCA------CCATCCCACGCATC-- MDOM ------A----ATAAATCCA------CAAAAGCACTAA------AACATACTAAAAGA OANA ------A----ATTAGTCCA------TAAAAAGTACAAAA---TAAAAACCTGAAACGA OCUN ------T----ATAAATCCA------TGAGATTCAC--AG----CACTCCATGCATC-- RN4 ------A----ATAACTCCA------TAAGATGCAC--AG----CGCCTCACGCCTC-- SSCR ATAAATAA----ATAAATCCA------TAAGATGTGC--AG----CACCCCAGGCATC-- TGUT ------CTAAGTTAG------GAAAAAAACCGGAGAATTCAAAAAATAGAGAAA XTRO ------T----AGACAACGG-----CTCAGAAACAG--AGT---CACCAAAGGGAT--- DRER TTACACATCACCTCATTTCTACAAGAGTCTCTGGTTCTAGGCTGTT------TACGTCAA

Consensus TTCCRA--GAGCTGTGCATTTTGAAATG--CAA------TTTTWAAAAATTGCTAAGGC HSA TTCCAA-GGAGTCATGCACTTCAAAATG--CAA-----TTAA-AAA--AATTGCTAAGCC HSA2 TTCCAA-GGAGTCATGCACTTCAAAATG--CAA-----TTAA-AAA--AATTGCTAAGCC PTROG TTCCAA-GGAGTCATGCACTTCAAAATG--CAA-----TTTT-AAA--AATTGCTAAGCC PPYG TTTCAA-GGAGTCATGCATTTCAAAATG--CAA-----TTTT-AAA--AATTGCTAAGCC HLAR TTCCAA-GGAGTCATGCATTTCAAAATG--CAA-----TTTA-AAA--AATTGCTAAGCC MMUL TTCCAA-GGAGTCATGCATTTCAAAATG--CAA-----TTTT-AAA--AATTGCTAAGCC RMAC TTCCAA-GGAGTCATGCATTTCAAAATG--CAA-----TTTT-AAA--AATTGCTAAGCC 43

PHAM TTCCAA-GGAGTCATGCATTTCAAAATG--CAA-----TTTT-AAA--AATTGCTAAGCC APAN TTCCAA-GGAGTCGTGCATTTTGAAATG--CAA-----TTTT-TAAAAAATTGCTAAGGC CMOL TTCCAA-GGAGTCGTGCATTTTGAAATG--CAA-----TTTT-TAAAAAATTGCTAAGGC PPITH TTCCAA-GGAGTCATGCATTTTGAAATG--CAA-----TTTT-TAAA-AATTGCTAAG-C CJAC TTTCAA-GGAGTCATGCAATTTGAAATG--CAA-----TTTT-TAAA-AATTGCTAAGCC CAPEL TTCCAA-GGAGTCGTGCATTTTGAAATG--CAA-----TTTT--TAAAAATTGTTAAGCC LCAT TTCCAA--GAGCTGTGCATTTTGAAATG--CAA------TTT-TTAAAAATTGCTAAGGC MMUS TTCTGA-GGAAGTATGCTTTTTAAAATA--T----GAATTTT-TTA--AATTGCCAAAGC GGAL AGCGGAGGGAGGC--GCGGCTGAGAAGG--GAAAAAA----T-TTAAAAACC------C AMEL TTCAGA-GGGGGTATGCATTTCCAAATG--CA---CAATTCT-TTT--AATTGCCAAAG- ACAR -----AGGGAGAT-TGTAGGTAATTAGG--GACAAAAATCAA------AATTTGAAACTC BTAU TTCCAA-GGAGGCATGCATTTCCAAATG--CA---GAATTTT-TTTTTAATTATCAAAGC CFAM TGCAGA-GGAGGTATGCATTTCCAAATG--CAA---AATTTT-TTT--AATTACCAAAG- CPOR TTTTGA-GGAAGTGTATATTTTGAAATG--CT---AGATTTT-TTA--AATTGCTAAGGC ECAB TTCCAA-GGAGGCATGCACTTCCAAATG--CAA---AATTTT-TTT--AATTGCCAAAGC FCAT TTCAGA-GGACGTATGCATTTCCAAATG--CAA---AATTTT-TTT--AATTGCCAAAG- LAFR TTCTGA-AGAGGCATGCGTTTTCAAATG--GAAAAAAAAATT-TTT--AATTGCCAAGTC MDOM TACC---CGAGAAAGCTATCTCTAAAGG--AAGAAATAAAAT-TAA--AATGGCAAACTC OANA TTGCGATAGTGGTAGGCACCTCTAAA------AAATAAA------AATTTCCAAGTC OCUN TTTTGA-ATAGGTGTGCACTTCGAAATG--T-----AATTTT-TAAA-AATT---AAGGC RN4 TTCTGAGGAAGTTCGCTTTTTTAAAATA--T----GATTTTT-CTTTAAATTGACAAAGC SSCR TTCCAA-GGAGGCATGCATTTCCAAATG--C---AAAATTTT-TTT--AATTACCAAAGC TGUT TAGAAGAGGAGGGAGGCATGACTAAA-----AAAAAAAA------AATTTAAAACAC XTRO TTGAGAACAAAGGGTGGGGGGAGAAAAA--T----ATATTGT-TTAGAGA-AGAAAAAAC DRER GTGTAAC--ACGTGAGCACT-AACAGTGACCAGCGTAATTTTGTTGGATGTTGTT--GAC

Consensus T-TCTGTGG--AGAA---GGAAAAAA-ATAAGTGCATAAGTGTRTGCCTTTGAACTTCCA HSA T-TATTTGG---GAA---GGAAAAA--ATAAGTGCATAAATGTATGCCTTTGAACTTCCA HSA2 T-TATTTGG---GAA---GGAAAAA--ATAAGTGCATAAATGTATGCCTTTGAACTTCCA PTROG T-TATTTGG---GAA---GGAAAA---ATAAGTGCATAAATGTATGCCTTTGAACTTCCA PPYG T-TATTTGG---GAA---GGAAAAA--ATAAGTGCATAAATGTATGCCTTTGAACTTCCG HLAR T-TATTTGG---GAA---GGAAAAA--ATAAGTGCATAAATGTATGCCTTTGAACTTCCG MMUL T-TATTTGG---GAA---GGAAAAA--ATAAGTGCATAAATGTATGCCTTTGAACTTCCG RMAC T-TATTTGG---GAA---GGAAAAA--ATAAGTGCATAAATGTATGCCTTTGAACTTCCA PHAM T-TATTTGG---GAA---GGAAAAA--ATAAGTGCATAAATGTATGCCTTTGAACTTCCG APAN T-TATTTGG---GAA---GGAAAAA--ATAAGTGCATAAATGTATGCCTTTGAACTTCCA CMOL T-TATTTGG---GAA---GGAAAAA--ATAAGTGCATAAATGTATGCCTTTGAACTTCCA PPITH TTTATTTAG---GAA---GGAAAAA--ATAAGTGCATAAATGTATGCCTTTGAACTTCCA CJAC T-TATTTGG---GAA---GGAAAAA--ATAAGTGCATAAATGTATGCCTTTGAACTTCCA CAPEL T-TATTTGG------GAAGGAAAAAA--TAAGTGCATAAATGTATGCCTTTGAACTTCCA LCAT T-TCTGTGG-----AGAAGGAAAAAA-ATAAGTGCATAAGTGTATGCCTTTGAACTTCCA MMUS ----TTTGGGGGGGGTAAAGAAAAA-CATAAGTGTATAAGTGTATGCCTTTGAACTTCCA GGAL CTTA------GCA---AGAAAAA------CATAAGTGTATGCCTTTGAACTTCCA AMEL ----TTTGGTGGGGG--GGGGGGAAACATAAGTGCATAAGTGTATGCCTTTGAACTTCCA ACAR ----TTTGG---GAA----GCAAAACCATACT--CCT-----TATGCCTTTGAACTTCCA BTAU ---TTTTGGAAGGAA---GGAAAAA-CATAAGTGCATAAGTGTATGCCTTTGAACTTCCA CFAM ---TTTAGGGGGGGGGGAGGAAAAA-CATAAGTGCATAAGTGTATGCCTTTGAACTTCCA CPOR ----TTTGGAGGAAA---GGAAAAA-CATAAGTGCATAAGTGTATGCCTTTGAACTTCCA ECAB T-TTTTTGGG--GAA---AGAAAAA-CATAAGTGCATAAGTGTATGCCTTTGAACTTCCA FCAT ----TTTGGGGGGGGGAAGGAAAAA-CATAAGTGCATAAGTGTATGCCTTTGAACTTCCA LAFR TTTTTTTTTTTTGAG--AAGGAAAAACATAAGTGCATAAGTGTATGCCTTTGAACTTCCG MDOM ----TTTTGCAAGAAG------CAAAAA------CATAAGTGTATGCCTTTGAACTTCCA OANA ----TCTTGCAAG------AAACAAAAA--CATAAGTGTATGCCTTTGAACTTCCA OCUN T-TCTTTGG--GGAA---GGA-AAAACATAAGTGCATAAGTGTATGCCTTTGAACTTCCA RN4 --TTTTTGGGGGTAA---AGA-AAAACATAAGTGCATAAGTGTATGCCTTTGAACTTCCA SSCR T---TTTGGAGGGAA----GGAAAAACATAAGTGCATAAGTGTATGCCTTTGAACTTCCA TGUT ----TTTAGCAAGA------AAAA------CATAAGTGTATGCCTTTGAACTTCCA XTRO AGAAATTTGCTTCAATTTGGACAAAAAAAAACTCCA--AGCCTTTGCCTTTGAACTTCCG DRER TATCTT------AAAATGTCCAATCAAA------TAA-TGTAT----TTGAACTTCTG

Consensus AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT HSA AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAG-TTCTT 44

HSA2 AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAG-TTCTT PTROG AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT PPYG AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT HLAR AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT MMUL AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT RMAC AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT PHAM AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT APAN AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT CMOL AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT PPITH AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCT- CJAC AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT CAPEL AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT LCAT AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT MMUS AAATGTCAAGGTCATCACCTTTAACCTCTCTGAATAATT--AGGCGCC-TTAAAGTTCTT GGAL AAATGTCAAGGTCATCACCTTTAACCTTTCGGAATAATT--AGGCGCC-TTAAGGTTCCT AMEL AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT ACAR GGATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGACCC-TTAAAG----- BTAU AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT CFAM AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT CPOR AAATGTCAAGGTCATCACCTTTAACCTCTCTGAATAATT--AGGCGCT-TTAAGGTTCTT ECAB AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCACC-TTAAGGTTCTT FCAT AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTT LAFR AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCTC MDOM AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-CTAAGGTTCTT OANA AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAAGTTCTT OCUN AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCCT RN4 AAATGTCAAGGTCATCACCTTTAACCTCTCTGAATAATT--AGGCGCC-TTAAAGTTCTT SSCR AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TAAAGGTTCTT TGUT AAATGTCAAGGTCATCACCTTTAACCTTTCTGAATAATT--AGGCGCC-TTAAGGTTCCT XTRO AAATGTCAAGGTCATCACCCTTCACCTTGCAGAATAATT--AAATGCC-TTAA------DRER TAATGTCAAGGTCGTCACCCTTAACCTTTTTGAATAAGTAAAGGCGCCGTTTTTGCGCTT

Consensus CTGTGATTTTCAACTGCCACCCACTCTC------ATA-TATA-CATGCTCACA---- HSA CT-TGATTTTCAGCTGCCACCCACTCTCA------CACA-TACA---TGCTCACA---- HSA2 CT-TGATTTTCAGCTGCCACCCACTCTCA------CACA-TACA---TGCTCACA---- PTROG CT-TGATTTTCAGCTGCCACCCACTCTCA------CACA-TACA---TGCTTACA---- PPYG CT-TGATTTTCAGCTGCCACCCACTCTCA------CACA-TACA---TGCTCACA---- HLAR CT-TGATTTTCAGCTGCCACCCACTCTCA------CACA-TATA---TGCTCACA---- MMUL CT-TGATTTTCAGCTGCCACCCACTCTCA------CACA-TACA---TGCTCACA---- RMAC CT-TGATTTTCAGCAGCCACCCACTCTCA------CACA-TACA---TGCTCACA---- PHAM CT-TGATTTTCAGCTGCCACCCACTCTCA------CACA-TACA---TGCTCACA---- APAN CT-TGATTTTCAGCTGCCACCCACTCTCA------CACA-TACA---TGCTCACA---- CMOL CT-TGATTTTCAGCTGCCACCCACTCTCA------CACA-TACA---TGCTCACA---- PPITH ---TGATTTTCAGCTGCCACCCACTCTCA------CACA-TACA---TGCTCACA---- CJAC CT-TGATTTTCAGCTGCCACCCACTCTCA------CA---- CAPEL CT-TGATTTTCAGCTGCCACCCACTCTCA------CACA-TACA--TGCTCACA----- LCAT CTGTGATTTTCAACTGCCACCCACTCTCA------TATA-TACA--TGCTCACA----- MMUS CTGTGATTTCTAACCACCGCCCAGGCTC------A---- GGAL CTG------AMEL CTGTGATATTCAGCTGCCACCCACTCAC------A-CACA-CATGCTTACA---- ACAR ------BTAU CTGTGATATTCAACTGCCACCCACTCTCA------C--A-CACA-CATGCTCACA---- CFAM CTGTGATATTCAGCCGCCACCCACTCTCA------CA-CACA-CATGCTTACA---- CPOR CTGTGATTTTCCACCGCCACCCACTCTCA------T--G-CGTA-CATACT--CA---- ECAB CTGTGATATTCAACCACCACCCACTCTCA------C--A-CACA-CATGCTCACA---- FCAT CTGTGATATTCAGCCGCCACCCACTCTCA------CACA-CACA-CATGCTTACA---- LAFR CTGTGATTTTCAACTGCCACCCACTCTCA------C--A-CACA-CATGCT--CA---- MDOM CTGTTATTTTCAACCACCACCCACTATCA------CACA-CAT------OANA CTGTTATTTTCAACCACCACCCA------OCUN CTGTGATTTTCAGCCGCCACCCACCCTCA------C--A-CATG-CATGCTCACA---- RN4 CTGTGATTTCCAACCACCACCCAGTCTCA------TA----ACA-CAGGCT--CA---- SSCR CTGTGATATTCAACTGCCACCCACTCACA------CACA-CATGCTCACA---- TGUT CTGTTATTTTCAACCACCACCCA------45

XTRO ------DRER CT----CTTTGAAAAACTACGCCCTCTCAGAGGCGCCGCAGTGCAGCACG-TCCCAATCT

Consensus --CACATCCAGACTCACAC-----ACACAYTTACGAC--CACACAGAGTCATATCAGGAG HSA --CATATAGAGAGCCACAC-----ACACATTTATGAC--CACACAAAATCATACCAGGAG HSA2 --CATATAGAGAGCCACAC-----ACACATTTATGAC--CACACAAAATCATACCAGGAG PTROG --CATATAGAGAGCCACAC-----ACACATTTATGAC--CACACAAAATCATACCAGGAG PPYG --CGTAGCCAGAGCCACAC-----ACACATTTATGAC--CACACAAAATCATACCAGGAG HLAR --CATAGCCAGAGCCACAC-----ACACATTTATGAC--CACACAAAATCATACCAGGAG MMUL --CATAGCCAGAGCCACAC-----ACACACTTATGAC--CACACAAAATCATACCAGGAG RMAC --CATAGCCAGAGCCACAC-----ACACACTTATGAC--CACACAAAATCATACCAGGAG PHAM --CATAGCCAGAGCCACAC-----ACACACTTATGAC--CACACAAAATCATACCAGGAG APAN --CATAGCCAGACCCACAT-----ACACATTTATGAC--CACACAGAATAATACCAGGAG CMOL --CATAGCCAGACCCACAT-----ACACATTTATGAC--CACACAGAATAATACCAGGAG PPITH --CATAGCCAGACCCAGAC-----ACACATTTATGAC--CACACAGAATAATACCAGGAG CJAC --CGTATCCAGACCCACAC-----ATACATTTATGAC--CACACAGAATAATACCAGGAA CAPEL --CATAGCCAGACCCACAC-----ACACATTTATGAC--CACACAGAATAATACCAGGAG LCAT --CACATCCAGACTCACAC-----ACACACTTACGAC--CACACAGAGTCATATCAGGAG MMUS --TACATCCAGACCCACAC-----ACATATTTATGAC--CATACAGATTCATACTGGGGG GGAL ------AMEL --CACATCCAGACCCCCAC-----ATACATTTATGAC--CACACAGCATCATATCAGAGG ACAR ------BTAU --CACATCCAGAGCCCCAC-----ACACATTTATGAC--CACACAGAATCATATCGAGGG CFAM --CACCTCCAGACCCCCAC-----ATACATTTATGAC--CACACAGCATCATATCAGGGG CPOR --CACATCCATACT-ATGC-----ACACATTTATGAC--CACACAGAATCATATTGAGAG ECAB --CACATCCAGACCCCCAC-----ACACATTTATGAC--CACACAGAATCATATCTGGGC FCAT --CATATCCAGACCCCCAC-----ATACATTTATGAC--CACACAGAATCATAACAGGGG LAFR --CACATCCAGACCCCCAC-----ACACATTTATGAC--CACACAGAATCATATTGGGGC MDOM ---ATACCC---CTCACAC-----ACTCATTTATGACTTTGCAGAGAATCATATCAGGGG OANA ------OCUN --CACTTCCAGACTCTCCC-----CCACATTTATGAC--CACACAAAATCATATCAGGGA RN4 --TACATCCAGACCCACAC-----ACATATTTATGAC--CATACAGGATCATACTGGGGG SSCR --CATATCCAGACCCCCAC-----ACACGTTTATGAC--CACACAGAATCATATCGTGGC TGUT ------XTRO ------DRER GGCCGCTCGAGCCCCTCACTGGAAACACA---ATGG--TCGTCCTGCAA-ATACC--GA-

Consensus C-TCACCATAAATCT--AAGAAAATTCC-CAATTTC------HSA C-TCACCATAAATCT--AAGAAAATTCC-TAATTT------HSA2 C-TCACCATAAATCT--AAGAAAATTCC-TAATTTCAGGATCAATAACCTTAATTTCCCT PTROG C-TCACCATAAATCT--AAGAAAATTCC-TAATTTCAGGATCAATAACCTTAATTTCCCT PPYG C-TCACCATAAATCT--AAGAAAATTCC-TAATTTCAGGATCAATAACCTTAATTTCCCT HLAR C-TCACCATAAATCT--AAGAAAATTCC-TAATTT------MMUL C-TCACCATAAATCT--AAGAAAATTCC-TAATTT------RMAC C-TCACCATAAATCT--AAGAAAATTCC-TAATTTCAGGATCAATAACCTTAATTTCCCT PHAM C-TCACCATAAATCT--AAGAAAATTCC-TAATTTCAGGATCAATAACCTTAATTTCCCT APAN C-TCACCATAAATCT--GAGAAAATTCC-TAATTT------CMOL C-TCACCATAAATCT--GAGAAAATTCC-TAATTT------PPITH C-TCACCATAAATCT--AAGAAAATTCC-TAATTT------CJAC C-TCACCATAAATCT--AAGAAAATTCC-TAATTTCAGGATCAATAACCTTAATTTCCCT CAPEL C-TCACCATAAATCT--AAGAAAATTCC-TGATTT------LCAT C-TCACCATAAATCT--AAGAAAATTCC-CAATTTC------MMUS T-TCACCATAAATCT--AGGAAAATTCC-TAATTTCAGGATCAATAACTATAATTTCCCT GGAL ------AMEL C-TCACCATAAATCT--AAGCAAATGCC-TAATTTCAGGATCAATAACTTTAATTTCCCT ACAR ------BTAU C-TCACCATAAATCT--AAGCAAATTCC-TAATTTCAGGATCAATAACCTTAATTTCCCT CFAM C-TCACCATAAATCT--AAGCAAATGCCTTAATTTCAGGATCAATAACCTTAATTTCCCT CPOR CTT-GCCATAAATCT--AAGAAAATTCC-TAATTTCAGGATCAATAACCTTAATTTCCCT ECAB --TCATCATAAATCT--AAGCAAATTCC-TAATTTCAGGATCAATAACCTTAATTTCCCT FCAT C-TCACCATAAATCT--AAGCAAATTCC-TAATTTCAGGATCAATAACCTTAATTTCCCT LAFR --TCACCATAAATCT--AAGCAAATTCC-TAATTTCAGGATTAATAATCTTAATTTCCCT MDOM AATTGCCATAAATCTCTGAGCAAATTCC-TAATTTCATGATCAATACTCTTAATTTCCCT 46

OANA ------OCUN C-TCACCATAAATCT--AAGAAAATTCC-TAATTTCAGGATCAATAACCTTAATTTCCCT RN4 T-TCATCATAAATCT--AAGAAAATTCC-TAATTTCAGGATCAATAACCATAATTTCCCT SSCR --TCACCATAAATCT--AAGCAAATTTC-TAATTTCAGGATCAATAACCTTAATTTCACT TGUT ------XTRO ------DRER CAGCAACGGAATTC------

Consensus ------HSA ------HSA2 ------PTROG ------PPYG ------HLAR ------MMUL ------RMAC ------PHAM ------APAN ------CMOL ------PPITH ------CJAC GCAAACGTTAGTGTAATGCAAAAGCAGTCAACTTCTTTTTAAAA--- CAPEL ------LCAT ------MMUS CCCAACATTTGAGTAATTTAATAGCAACTGACTTCTTAGGAAAAAA- GGAL ------AMEL TCAAACATTTGTGTAATTCGATAGCAGTTGACTTCTT------ACAR ------BTAU TCAAACATTTGTGTAATTCAGTACCAATTGACTTCTT------CFAM TCAAACATTTGTGTAATTCAATAGCACTTGACTTCTT------CPOR TCAAACATTTGAGTAA------ECAB TCAAACATTTGTGTAATTCAATAGCAATTGACTTCTT------FCAT TCAAACATTTGTGTAATTCGATAGCAATTGACTTCTT------LAFR TCAAACATTTATGTAATTCAATAGCAA------MDOM CCAAACATTT------OANA ------OCUN CCAAACATTTGTGCAATTCAATAGCAATTGACTTCTTTGGAAAAA-- RN4 CCAAACATCTGAGTAATTTAATAGCCACTGACTTCTTAGGAAAAAA- SSCR TCAAACATTTGTGTAATTCAACAGCAATTGACTTCTT------TGUT ------XTRO ------DRER ------