peerj_json_files/PeerJ_Json_142.json

{
    "v1_Abstract": "A new species of an archaic primate (Pleisadapiformes) is described based on a maxilla containing the first and second upper molars from the Fort Union Formation, Atwell Gulch Member in northwestern Colorado. The preserved teeth show the unusual dental characteristics of members of the rare and poorly documented Picrodontidae family, including an elongated centrocrista and wide occlusal surface. The new species is placed within the genus Zanycteris (represented by a single specimen from southern Colorado). This placement is based on similarities in regard to the parastyle, curvilinear centrocrista, and wider anterior stylar shelf on the upper molars. However, the new species differs from the only known species of Zanycteris in exhibiting an upper first molar that is 30% larger in area, while retaining a similarly sized upper second molar. Phylogenetic analysis supports the separation of the Picrodontidae family from the Paromomyidae, while still recognizing picrodontids position within Pleisadapiformes. The unusual dental features of the upper molars likely functioned in life as an enhanced shearing surface between the centrocrista and cristid obliqua crests for a specialized diet of fruit. A similar arrangement is found in the living bat Ariteus (Jamaican fig-eating bat), which feeds on fleshy fruit. The new species showcases the rapid diversification of archaic primates shortly after the extinction of the dinosaurs during the Paleocene, and the unusual dental anatomy of picrodontids to exploit new dietary specializations.",
    "v1_col_introduction": "introduction  : The family Picrodontidae consists of rare fossil mammals known only in the late Paleocene (Torrejonian and Tiffanian North American Land Mammal Ages (NALMA)) of North America. Upon the initial discovery of the Picrodontidae Zanycteris in 1917 paleontologists placed the fossil within the Order Chiroptera (Matthew, 1917; Simpson, 1935; Simpson, 1937). Indeed, there is a close resemblance between Zanycteris and some of the fruit-eating bats of the New World, such as the living genus Ariteus (Jamaican fig-eating bat). This morphological similarity is exhibited in the upper first molar which is broadly shaped and greatly expanded. The expansion of the occlusal surface of the upper first molar is likely a reflection of similar diet, rather than of any similar phylogenetic relationship to fruit eating bats, since such specializations are absent in early fossil bat lineages (Simmons and Geisler, 1998). More recently studies have positioned the enigmatic Picrodontidae as aberrant members of archaic primates (Szalay, 1968). Researchers have viewed picrodontids as stemming from a Purgatorius-like ancestor (Tomida, 1982), a Palaechthon-like ancestor (Szalay, 1968) or more derived members of the Paromomyoidea (Szalay, 1968; Silcox, 2001; Silcox & Gunnell, 2008).\nThe relationship to archaic primates is strengthened by the presence of an enlarged incisor in a lower jaw of the picrodontid Picrodus recovered from Swain Quarry of the Fort Union Formation, middle Paleocene (Torrejonian) in Carbon County, Wyoming (Szalay, 1968; Williams, 1985). The enlarged incisor is often considered a synapomorphy of Plesiadapiformes, and is also found in the early Paleocene genus Purgatorius (Clemens, 2004), although this trait is also found in groups outside of Plesiadapiformes, such as the Apatemyidae (West, 1973).\nPicrodontids remain very elusive fossil mammals, with only a handful of specimens known from a narrow span of time during the middle to late Paleocene in North America (Silcox & Gunnell, 2008); in fact this is only the second known specimen of the rare genus Zanycteris.\n10\n11\n12\n13\n14\n15\n16\n17\n18\n19\n20\n21\n22\n23\n24\n25\n26\n27\n28\n29\n30\n31\n32\n33\nPeerJ reviewing PDF | (v2013:06:580:2:0:NEW 7 Oct 2013)\nR ev ie w in g M an\nus cr ip t\nThis paper reports on the occurrence of a new species of Zanycteris discovered in the late Paleocene (Tiffanian) deposits of the Fort Union Formation, Atwell Gulch Member in northwestern Colorado, and also discusses the phylogenetic relationship of picrodontids among various groups of archaic primates living in North America during the Paleocene.",
    "v2_Abstract": "A new species of an archaic primate (Pleisadapiformes) is described based on a maxilla containing the first and second upper molars from the Fort Union Formation, Atwell Gulch Member in northwestern Colorado. The preserved teeth show the unusual dental characteristics of members of the rare and poorly documented Picrodontidae family, including an elongated centrocrista and wide occlusal surface. The new species is placed within the genus Zanycteris (represented by a single specimen from southern Colorado). This placement is based on similarities in regard to the parastyle, curvilinear centrocrista, and wider anterior stylar shelf on the upper molars. However, the new species differs from the only known species of Zanycteris in exhibiting an upper first molar that is 30% larger in area, while retaining a similarly sized upper second molar. Phylogenetic analysis supports the separation of the Picrodontidae family from the Paromomyidae, while still recognizing picrodontids position within Pleisadapiformes. The unusual dental features of the upper molars likely functioned in life as an enhanced shearing surface between the centrocrista and cristid obliqua crests for a specialized diet of fruit. A similar arrangement is found in the living bat Ariteus (Jamaican fig-eating bat), which feeds on fleshy fruit. The new species showcases the rapid diversification of archaic primates shortly after the extinction of the dinosaurs during the Paleocene, and the unusual dental anatomy of picrodontids to exploit new dietary specializations.",
    "v2_col_introduction": "introduction  : The family Picrodontidae consists of rare fossil mammals known only in the late Paleocene (Torrejonian and Tiffanian North American Land Mammal Ages (NALMA)) of North America. Upon the initial discovery of the Picrodontidae Zanycteris in 1917 paleontologists placed the fossil within the Order Chiroptera (Matthew, 1917; Simpson, 1935; Simpson, 1937). Indeed, there is a close resemblance between Zanycteris and some of the fruit-eating bats of the New World, such as the living genus Ariteus (Jamaican fig-eating bat). This morphological similarity is exhibited in the upper first molar which is broadly shaped and greatly expanded. The expansion of the occlusal surface of the upper first molar is likely a reflection of similar diet, rather than of any similar phylogenetic relationship to fruit eating bats, since such specializations are absent in early fossil bat lineages (Simmons and Geisler, 1998). More recently studies have positioned the enigmatic Picrodontidae as aberrant members of archaic primates (Szalay, 1968). Researchers have viewed picrodontids as stemming from a Purgatorius-like ancestor (Tomida, 1982), a Palaechthon-like ancestor (Szalay, 1968) or more derived members of the Paromomyoidea (Szalay, 1968; Silcox, 2001; Silcox & Gunnell, 2008).\nThe relationship to archaic primates is strengthened by the presence of an enlarged incisor in a lower jaw of the picrodontid Picrodus recovered from Swain Quarry of the Fort Union Formation, middle Paleocene (Torrejonian) in Carbon County, Wyoming (Szalay, 1968; Williams, 1985). The enlarged incisor is often considered a synapomorphy of Plesiadapiformes, and is also found in the early Paleocene genus Purgatorius (Clemens, 2004), although this trait is also found in groups outside of Plesiadapiformes, such as the Apatemyidae (West, 1973).\nPicrodontids remain very elusive fossil mammals, with only a handful of specimens known from a narrow span of time during the middle to late Paleocene in North America (Silcox & Gunnell, 2008); in fact this is only the second known specimen of the rare genus Zanycteris.\n10\n11\n12\n13\n14\n15\n16\n17\n18\n19\n20\n21\n22\n23\n24\n25\n26\n27\n28\n29\n30\n31\n32\n33\nPeerJ reviewing PDF | (v2013:06:580:1:0:NEW 30 Aug 2013)\nR ev ie w in g M an\nus cr ip t\nThis paper reports on the occurrence of a new species of Zanycteris discovered in the late Paleocene (Tiffanian) deposits of the Fort Union Formation, Atwell Gulch Member in northwestern Colorado, and also discusses the phylogenetic relationship of picrodontids among various groups of archaic primates living in North America during the Paleocene.",
    "v3_Abstract": "A new species of an archaic primate (Pleisadapiformes) is described based on a maxilla containing the first and second upper molars from the Fort Union Formation, Atwell Gulch Member in northwestern Colorado. The preserved teeth show the unusual dental characteristics of members of the rare and poorly documented Picrodontidae family, including an elongated centrocrista and wide occlusal surface. The new species is placed within the genus Zanycteris (represented from a single specimen from southern Colorado). This placement is based on similarities in regard to a shorten parastyle, curvilinear centrocrista, and wider anterior stylar shelf on the upper molars. However, the new species differs from the only known species of Zanycteris in exhibiting an upper first molar that is 30% larger in area, while retaining a similar sized upper second molar. Phylogenetic analysis supports the separation of the Picrodontidae family from the Paromomyidae, while still recognizing picrodontids position within Pleisadapiformes. The unusual dental features of the upper molars likely functioned in life as an enhanced shearing surface between the centrocrista and cristid oblique crests for a specialized diet of fruit. A similar arrangement is found in the living bat Artiteus (Jamaican fig-eating bat), which feeds on fleshy fruit. The new species showcases the rapid diversification of archaic primates shortly after the extinction of the dinosaurs during the Paleocene, and the unusual dental anatomy of picrodontids to exploit new dietary specializations.",
    "v3_col_introduction": "introduction  : The family Picrodontidae consists of rare fossil mammals known only in the late Paleocene (Torrejonian and Tiffanian North American Land Mammal Ages (NALMA)) of North America. Upon the initial discovery of the Picrodontidae Zanycteris in 1917 paleontologists placed the fossil within the Order Chiroptera (Matthew, 1917; Simpson, 1935; Simpson, 1937). Indeed, there is a close resemblance between Zanycteris and some of the fruit-eating bats of the New World, such as the living genus Ariteus (Jamaican fig-eating bat). This morphological similarity is exhibited in the upper first molar which is broadly shaped and greatly expanded. The expansion of the occlusal surface of the upper first molar is likely a reflection of similar diet, rather than of any similar phylogenetic relationship to fruit eating bats, since such specializations are absent in early fossil bat lineages (Simmons and Geisler, 1998). More recently studies have positioned the enigmatic Picrodontidae as aberrant members of archaic primates. Researchers have viewed picrodontids as stemming from a Purgatorius-like ancestor (Tomida, 1982), a Palaechthon-like ancestor (Szalay, 1968) or more derived members of the Paromomyoidea (Szalay, 1968; Silcox, 2001; Silcox & Gunnell, 2008).\nThe relationship to archaic primates is strengthen by the presence of an enlarged incisor in a lower jaw of the picrodontid Picrodus recovered from Swain Quarry of the Fort Union Formation, middle Paleocene (Torrejonian) in Carbon County, Wyoming (Szalay, 1968; Williams, 1985). The enlarged incisor is considered a synapomorphy of Plesiadapiformes, and is also found in the early Paleocene genus Purgatorius (Clemens, 2004).\n10\n11\n12\n13\n14\n15\n16\n17\n18\n19\n20\n21\n22\n23\n24\n25\n26\n27\n28\n29\nPeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013)\nR ev ie w in g M an\nus cr ip t\nPicrodontids remain very elusive fossil mammals, with only a handful of specimens known from a narrow span of time during the middle to late Paleocene in North America; in fact this is only the second known specimen of the rare genus Zanycteris.\nThis paper reports on the occurrence of a new species of Zanycteris discovered in the late Paleocene (Tiffanian) deposits of the Fort Union Formation, Atwell Gulch Member in northwestern Colorado, and also discusses the phylogenetic relationship of picrodontids among various groups of archaic primates living in North America during the Paleocene.",
    "v1_text": "methods & materials : The fossil reported in this paper was recovered during geological mapping of the Citadel Plateau Quadrangle during the 1970s. The site has also produced a diverse fauna of mammals from the Fort Union Formation in western Colorado\u2019s Piceance Creek Basin (Burger & Honey, 2008). The site is referred to as University of Colorado Museum (UCM) Locality number 92177. Continued collection at the site over the years has produced additional fossil mammals (Table 1). However, the new species is represented by a single recovered specimen, despite 30 years of sporadic collection at the site. In the Piceance Creek Basin the Fort Union Formation is synonymous with the Atwell Gulch Member, which has been included as a member of the Wasatch Formation (Donnell, 1969) or DeBeque Formation (Kihm, 1984). In this paper I refer the Atwell Gulch Member, as the sole member of the Fort Union Formation, as it has been mapped elsewhere within the Piceance Creek Basin in western Colorado (Hail & Pipiringos, 1990; Hail & Smith, 1994). The Fort Union Formation (Atwell Gulch Mbr.) varies in thickness from 350 meters in the northeast to 196 meters in the south, and is divided into upper and lower informal units (Hail & Pipiringos, 1990). The fossil site UCM 92177 is 261 meters below the upper contact of the Late Paleocene Fort Union Formation (Atwell Gulch Member), with the Early Eocene Wasatch Formation (Molina Member). The lower unit of the Fort Union Formation consists of light-grey to light-brown sandstones; olive, purple, dark-reddish-brown claystone; and mudstones that are 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 PeerJ reviewing PDF | (v2013:06:580:2:0:NEW 7 Oct 2013) R ev ie w in g M an us cr ip t highly variegated. Large ribbon and sheet sandstone bodies are common in the north of the basin, where they can form massive sandstone cliffs measuring upwards of 25 meters thick, although most are 5-10 meters thick. Pebbles are exclusively composed of sedimentary rocks, including claystone and mudstones. The upper unit of the Fort Union Formation (Atwell Gulch Mbr.) is composed of carbonaceous shales; thin coals; and thin, but persistent highly calcareous sandstones. Mudstones and claystones are less common in the upper unit. Selenite is common, especially in the carbonaceous shales and coals. Fossil mammals are abundant in the lower unit of the Fort Union Formation, especially in the variegated beds found in association with UCM locality 92177. Fossil invertebrates, such as the bivalve Unio and a variety of gastropods are common in the upper unit, indicating a progression over time toward a more lacustrine environment. Large accumulations of gastropods are common in the upper unit, which have been interpreted as lakeshore accumulations (Hanley, 1974). Order PRIMATES Linnaeus, 1758 Family PICRODONITDAE Simpson, 1937 Genus ZANYCTERIS Matthew, 1917 ZANYCTERIS HONEYI new species Holotype\u2014 UCM 87378 right maxilla with upper first and second molars. Etymology\u2014 Posthumously named in honor of James G. Honey for his discovery of the holotype, type locality and for his kindness in allowing me to study this collection. 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 PeerJ reviewing PDF | (v2013:06:580:2:0:NEW 7 Oct 2013) R ev ie w in g M an us cr ip t Horizon\u2014 Fort Union Formation, Atwell Gulch Member, 261 meters below the top contact with the Wasatch Formation. Localities\u2014 Only known from UMC locality number 92177. Diagnosis\u2014 Z. honeyi exhibits an anteriorly protruding parastylar lobe on M1/. Differs from Z. paleocenus by having a 30% large M1/ area, while retaining a similar sized M2/ area to Z. paleocenus. Differs from Picrodus in lacking an extended parastyle on the M2/, having a better developed anterior stylar shelf, and postprotocrista on the M1/. Furthermore, the M1/ centrocrista is more curvilinear than Picrodus. Unlike Draconodus, crenulations occur in the trigon basin of M1/. Description\u2014 The holotype (UCM 87378) is the only specimen known from the Piceance Creek Basin. However, this specimen preserves morphology to indicate that it differs from Z. paleocenus from southwestern Colorado. The enlarged metastyle on the M1/ projects buccally from the posterior edge, and the stylar shelf bulges from the mid-point of the tooth. The paracingulum (the shelf formed by the paraconule) extends anteriorly, yet the stylar shelf encircling the paracone is poorly formed and resembles Picrodus rather than Zanycteris in shape. The strong postprotocrista of UCM 87378 extends directly toward the vestigial metaconule. This direct course of the postprotocrista results in a more enclosed trigon basin that closely resembles Z. paleocenus. The M2/ exhibits a wide stylar shelf, featuring a large parastyle. However, UCM 87378 typifies Zanycteris in lacking the greatly extended parastyle on the M2/ that is found in specimens of Picrodus. Despite wear, the molar paracone and metacone form a W-shaped crest across the midline on the M2/, while the broad protocone expands along the postprotocrista, a typical plesiadapiform trait. The M1/ measures 2.54 mm in length and 2.46 mm in width. The M2/ measures 1.26 mm in length and 1.60 in width. 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 PeerJ reviewing PDF | (v2013:06:580:2:0:NEW 7 Oct 2013) R ev ie w in g M an us cr ip t Comparison\u2014 Previous measurements of the holotype of Z. paleocenus (Simpson, 1935, Szalay, 1968) report a length of 2.05-2.20mm in length and 1.87-2.00 mm in width for the M1/, indicating that the new species (UCM 87378) is both larger and broader. However, reported measurements of the M2/ (1.25-1.30mm in length and 1.60-1.70mm in width) are similar in dimensions to UCM 87378. This indicates that while the first molar is enlarged, the second molar is of equal size between the two species of Zanycteris. This enlargement of the first molar likely served a functional role in providing a larger surface area for slicing between the centrocrista and cristid obliqua (Szalay, 1968). In some ways, UCM 87378 resembles Picrodus, such as the reduced anterior stylar shelf on the M1/ (Scott and Fox, 2005). However, other features more closely resemble Z. paleocenus, including the arrangement of the postprotocrista on the M1/ and smaller parastyle on the M2/. These features support inclusion of UCM 87378 within the genus Zanycteris rather than Picrodus. UCM 87378 and the holotype of Z. paleocenus (AMNH 17180) are the only two specimens of Zanycteris currently documented (Simpson, 1937; Scott and Fox, 2005). Zanycteris appears to be restricted to Colorado, while Picrodus has been found in Wyoming, Montana, Alberta and recently New Mexico (Simpson, 1937; Williams, 1985; Silcox & Gunnell, 2008; Scott & Fox, 2005; Silcox & Williamson, 2012). a new species of an archaic primate (pleisadapiformes) is described based on a maxilla : containing the first and second upper molars from the Fort Union Formation, Atwell Gulch Member in northwestern Colorado. The preserved teeth show the unusual dental characteristics of members of the rare and poorly documented Picrodontidae family, including an elongated centrocrista and wide occlusal surface. The new species is placed within the genus Zanycteris (represented by a single specimen from southern Colorado). This placement is based on similarities in regard to the parastyle, curvilinear centrocrista, and wider anterior stylar shelf on the upper molars. However, the new species differs from the only known species of Zanycteris in exhibiting an upper first molar that is 30% larger in area, while retaining a similarly sized upper second molar. Phylogenetic analysis supports the separation of the Picrodontidae family from the Paromomyidae, while still recognizing picrodontids position within Pleisadapiformes. The unusual dental features of the upper molars likely functioned in life as an enhanced shearing surface between the centrocrista and cristid obliqua crests for a specialized diet of fruit. A similar arrangement is found in the living bat Ariteus (Jamaican fig-eating bat), which feeds on fleshy fruit. The new species showcases the rapid diversification of archaic primates shortly after the extinction of the dinosaurs during the Paleocene, and the unusual dental anatomy of picrodontids to exploit new dietary specializations. PeerJ reviewing PDF | (v2013:06:580:2:0:NEW 7 Oct 2013) R ev ie w in g M an us cr ip t Benjamin John Burger* *Corresponding author: Department of Geology Utah State University Uintah Basin Regional Campus 320 North Aggie Blvd. Vernal, UT 84078 U.S.A. Phone 1-435-722-1778 benjamin.burger@usu.edu 1 2 3 4 5 6 7 8 9 PeerJ reviewing PDF | (v2013:06:580:2:0:NEW 7 Oct 2013) R ev ie w in g M an us cr ip t conclusions : In summary, the new species Zanycteris honeyi typifies the unique characteristics that set apart the Picrodontidae from other archaic primates known from the Paleocene of North America. Phylogenetic analysis supports the separation of the Picrodontidae family from the Paromomyidae family, while still recognizing their position within Pleisadapiformes. Further fossil discoveries, particularly cranial and postcranial remains will likely enable more confident placement of this unusual group of archaic primates among the evolutional tree during this pivotal time of primate diversification shortly after the extinction of the dinosaurs. acknowledgments : Special thanks to Jaelyn Eberle at the University of Colorado at Boulder for her support in my graduate studies which included this research. I would like to thank my committee Herbert H. Covert, Mary Kraus, Matthew Pranter, (University of Colorado) and Henry Fricke (Colorado College); Toni Culver, the collection manager at the University of Colorado Museum, as well as various members of the field crew including Alex Dutchak, Karen Lloyd, Pat Monaco, Andrea R. Bair, Ian J. Sweeney, Lou Taylor and Lea Ann Jolley. Fieldwork was conducted under BLM permit #C-60170 issued to the University of Colorado Museum. phylogenetic analysis : The acquisition of the highly specialized dentition found within Zanycteris and other members of the Picrodontidae remains a mystery. For example, how quickly did the specialized dentition evolve during the Paleocene? Among the known Plesiadapiformes, which one is most closely related to the family Picrodontidae and could possibly represent the ancestral condition for the specialized dentation exhibited by Zanycteris? To evaluate these questions and to work toward reconstructing the evolution of the specialized upper dentition of Picrodontidae, a phylogenetic analysis was undertaken using the 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 PeerJ reviewing PDF | (v2013:06:580:2:0:NEW 7 Oct 2013) R ev ie w in g M an us cr ip t morphological characteristics of the dentition of known Plesiadapiformes and outgroups (Paradectes, Cimolestes, and Leptacodon), which lived during the Paleocene in North America. The character matrix consisted of 113 dental characters, 97 of which were adopted from Silcox (2001). The analysis included 58 fossil taxa of contemporary North American Paleocene primates. A heuristic search using Mesquite version 2.75 (Maddison & Maddison, 2011) produced 6,579 most parsimonious trees (597 steps, consistency index [CI] = 0.36, retention index [RI] = 0.74). The strict consensus tree shows Zanycteris honeyi as closely related to Zanycteris paleocenus within a monophyletic clade of Picrodontidae (Picrodus, Draconodus, and Zanycteris). The family Picrodontidae was found to be within a clade consisting of Plesidapidae and Carpolestidae, rather than a placement within Paromomyoidea (Silcox & Gunnell, 2008). This phylogenetic position postulates that the expansion of the occlusal surface seen in the upper molars of both paromomyids and picrodontids is convergent, having evolved independently during the Paleocene. Possible ancestors of picrodontids are the early Paleocene taxa Plesiolestes, Torrejonia, Phoxomylus, and Talpohenach, while paromomyids appear to have arisen from the early Paleocene with a Palaechthon or Anasazia or the poorly known Premnoides like ancestor. Although there is ambiguity concerning the phylogenetic position of these primitive taxa, there is strong support for monophyletic clades of Paromomyidae, Picrodontidae, Plesiadapidae, and Carpolestidae, as well as a monophyletic clade of Plesiadapiformes, with the addition of Micromomyidae and Microsyopidae. Overall the resulting phylogenetic tree supports a position of Zanycteris honeyi within the Picrodontidae family, and that this new species is closely related to other archaic primates from the Paleocene of North America. Discussion 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 PeerJ reviewing PDF | (v2013:06:580:2:0:NEW 7 Oct 2013) R ev ie w in g M an us cr ip t The members of the Picrodontidae are exceptional in the development of a dentition that maximizes the shear forces along the long contact between the centrocrista on the upper molar (composed of a tall crest between the paracone and metacone) and the cristid obliqua that spans much of the length of the elongated talonid basin of the lower molar (Szalay, 1968). The molar teeth were thus probably specialized for cutting through hard outer husks of fruits and nuts. This was accomplished by positioning the fruit along the outer (buccal) shearing surface, which was greatly expanded (anteriorly and posteriorly) to maximize the amount of contact, much like a pair of long sharp scissors (Shaw, 1917). A similar expansion of the centrocrista is found in the upper molars of the Jamaican fig-eating bat (Ariteus), which feeds on the native Jamaican naseberry also known as sapodilla (Manilkara zapota), a fruit with a fleshy but firm texture (Sherwin & Gannon, 2005). Thus in both Ariteus and Zanycteris the major shearing surface is between the crests of the centrocrista above and the cristid obliqua below, demonstrating a similar specialized diet on fruit. This arrangement differs substantially from paromomyids, which retain distinct paracone and metacone cusps on the upper molars, with no development of a long and tall centrocrista between the two cusps. Rather, paromomyids expand the upper molars by broadening the postcingulum to form a wide talon basin on the lingual edge of the tooth. This broadening of the tooth functioned to expand the surface area particularly for holding food during mastication (Shaw, 1917). Thus paromomyids, such as Phenacolemur, broaden the upper molars to allow increased surface area for a larger and a more varied diet, while picrodontids, such as Zanycteris, expanded the upper molars to increase the shearing surface for a more specialized diet of a particular style of fleshy fruit. Further research on dental specialization within these groups might reveal why picrodontids have a limited stratigraphic range (Torrejonian to Tiffanian), when compared to the closely related, but dentally distinct paromomyids which ranged from the early Paleocene (Puercan) until the late Eocene (Duchesnean) (Silcox & Gunnell, 2008). 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 PeerJ reviewing PDF | (v2013:06:580:2:0:NEW 7 Oct 2013) R ev ie w in g M an us cr ip t image of fossil specimen. : Figure 1: Buccal and occlusal views of the holotype specimen, a maxilla containing the upper first and second molars. UCM 87378. PeerJ reviewing PDF | (v2013:06:580:2:0:NEW 7 Oct 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:580:2:0:NEW 7 Oct 2013) R ev ie w in g M an us cr ip t Figure 2 Strict consensus tree of the most parsimonious trees generated from the phylogenetic analysis. Figure 2: Strict consensus tree of the most parsimonious trees generated from the phylogenetic analysis of 113 upper dental characters scored against the 58 North American archaic primates known from the Paleocene. Temporal ranges during the Paleocene are shown for each species by blackened line. PeerJ reviewing PDF | (v2013:06:580:2:0:NEW 7 Oct 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:580:2:0:NEW 7 Oct 2013) R ev ie w in g M an us cr ip t Table 1(on next page) Mammal faunal list for fossil locality 92177. Table 1. Mammal faunal list for UCM locality 92177. PeerJ reviewing PDF | (v2013:06:580:2:0:NEW 7 Oct 2013) R ev ie w in g M an us cr ip t Table 1. Mammal Faunal List for UCM locality 92177. Mammalia Allotheria Multituberculata Ptilodus kummae Ectypodus musculus Theria Erinaceomorpha (\u201cApheliscidae\u201d) Haplaletes serior Litomylus ishami Phenacodaptes sabulosus Haplomylus simpsoni Lipotyphla Leptacodon tener Mesonychia cf. Sinonyx sp. Procreodi Thryptacodon australis Arctocyonides mumak Carnivoramorpha Protictis proteus Protictis cf. schaffi Primates Nannodectes gazini Plesiadapis fodinatus Chiromyoides gigas Zanycteris honeyi new species Ignacius frugivorus Ignacius sp. Carpodaptes cygneus Condylarthra Ectocion medituber Phenacodus grangeri Phenacodus magnus Pholidota Propalaeanodon sp. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 PeerJ reviewing PDF | (v2013:06:580:2:0:NEW 7 Oct 2013) R ev ie w in g M an us cr ip t",
    "v2_text": "a new species of an archaic primate (pleisadapiformes) is described based on a maxilla : containing the first and second upper molars from the Fort Union Formation, Atwell Gulch Member in northwestern Colorado. The preserved teeth show the unusual dental characteristics of members of the rare and poorly documented Picrodontidae family, including an elongated centrocrista and wide occlusal surface. The new species is placed within the genus Zanycteris (represented by a single specimen from southern Colorado). This placement is based on similarities in regard to the parastyle, curvilinear centrocrista, and wider anterior stylar shelf on the upper molars. However, the new species differs from the only known species of Zanycteris in exhibiting an upper first molar that is 30% larger in area, while retaining a similarly sized upper second molar. Phylogenetic analysis supports the separation of the Picrodontidae family from the Paromomyidae, while still recognizing picrodontids position within Pleisadapiformes. The unusual dental features of the upper molars likely functioned in life as an enhanced shearing surface between the centrocrista and cristid obliqua crests for a specialized diet of fruit. A similar arrangement is found in the living bat Ariteus (Jamaican fig-eating bat), which feeds on fleshy fruit. The new species showcases the rapid diversification of archaic primates shortly after the extinction of the dinosaurs during the Paleocene, and the unusual dental anatomy of picrodontids to exploit new dietary specializations. PeerJ reviewing PDF | (v2013:06:580:1:0:NEW 30 Aug 2013) R ev ie w in g M an us cr ip t Benjamin John Burger* *Corresponding author: Department of Geology Utah State University Uintah Basin Regional Campus 320 North Aggie Blvd. Vernal, UT 84078 U.S.A. Phone 1-435-722-1778 benjamin.burger@usu.edu 1 2 3 4 5 6 7 8 9 PeerJ reviewing PDF | (v2013:06:580:1:0:NEW 30 Aug 2013) R ev ie w in g M an us cr ip t methods & materials : The fossil reported in this paper was recovered during geological mapping of the Citadel Plateau Quadrangle during the 1970s. The site has also produced a diverse fauna of mammals from the Fort Union Formation in western Colorado\u2019s Piceance Creek Basin (Burger & Honey, 2008). The site is referred to as University of Colorado Museum (UCM) Locality number 92177. Continued collection at the site over the years has produced additional fossil mammals (Table 1). However, the new species is represented by a single recovered specimen, despite 30 years of sporadic collection at the site. In the Piceance Creek Basin the Fort Union Formation is synonymous with the Atwell Gulch Member, which has been included as a member of the Wasatch Formation (Donnell, 1969) or DeBeque Formation (Kihm, 1984). In this paper I refer the Atwell Gulch Member, as the sole member of the Fort Union Formation, as it has been mapped elsewhere within the Piceance Creek Basin in western Colorado (Hail & Pipiringos, 1990; Hail & Smith, 1994). The Fort Union Formation (Atwell Gulch Mbr.) varies in thickness from 350 meters in the northeast to 196 meters in the south, and is divided into upper and lower informal units (Hail & Pipiringos, 1990). The fossil site UCM 92177 is 261 meters below the upper contact of the Late Paleocene Fort Union Formation (Atwell Gulch Member), with the Early Eocene Wasatch Formation (Molina Member). The lower unit of the Fort Union Formation consists of light-grey to light-brown sandstones; olive, purple, dark-reddish-brown claystone; and mudstones that are 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 PeerJ reviewing PDF | (v2013:06:580:1:0:NEW 30 Aug 2013) R ev ie w in g M an us cr ip t highly variegated. Large ribbon and sheet sandstone bodies are common in the north of the basin, where they can form massive sandstone cliffs measuring upwards of 25 meters thick, although most are 5-10 meters thick. Pebbles are exclusively composed of sedimentary rocks, including claystone and mudstones. The upper unit of the Fort Union Formation (Atwell Gulch Mbr.) is composed of carbonaceous shales; thin coals; and thin, but persistent highly calcareous sandstones. Mudstones and claystones are less common in the upper unit. Selenite is common, especially in the carbonaceous shales and coals. Fossil mammals are abundant in the lower unit of the Fort Union Formation, especially in the variegated beds found in association with UCM locality 92177. Fossil invertebrates, such as the bivalve Unio and a variety of gastropods are common in the upper unit, indicating a progression over time toward a more lacustrine environment. Large accumulations of gastropods are common in the upper unit, which have been interpreted as lakeshore accumulations (Hanley, 1974). Order PRIMATES Linnaeus, 1758 Family PICRODONITDAE Simpson, 1937 Genus ZANYCTERIS Matthew, 1917 ZANYCTERIS HONEYI new species Holotype\u2014 UCM 87378 right maxilla with upper first and second molars. Etymology\u2014 Posthumously named in honor of James G. Honey for his discovery of the holotype, type locality and for his kindness in allowing me to study this collection. 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 PeerJ reviewing PDF | (v2013:06:580:1:0:NEW 30 Aug 2013) R ev ie w in g M an us cr ip t Horizon\u2014 Fort Union Formation, Atwell Gulch Member, 261 meters below the top contact with the Wasatch Formation. Localities\u2014 Only known from UMC locality number 92177. Diagnosis\u2014 Differs from Z. paleocenus by having a 30% large M1/ area, while retaining a similar sized M2/ area to Z. paleocenus. Differs from Picrodus in lacking an extended parastyle on the M2/, having a better developed anterior stylar shelf, and postprotocrista on the M1/. Furthermore, the M1/ centrocrista is more curvilinear than Picrodus. Unlike Draconodus, crenulations occur in the trigon basin of M1/. Description\u2014 The holotype (UCM 87378) is the only specimen known from the Piceance Creek Basin. However, this specimen preserves morphology to indicate that it differs from Z. paleocenus from southwestern Colorado. The enlarged metastyle on the M1/ projects buccally from the posterior edge, and the stylar shelf bulges from the mid-point of the tooth. The paracingulum (the shelf formed by the paraconule) extends anteriorly, yet the stylar shelf encircling the paracone is poorly formed and resembles Picrodus rather than Zanycteris in shape. The strong postprotocrista of UCM 87378 extends directly toward the vestigial metaconule. This direct course of the postprotocrista results in a more enclosed trigon basin that closely resembles Z. paleocenus. The M2/ exhibits a wide stylar shelf, featuring a large parastyle. However, UCM 87378 typifies Zanycteris in lacking the greatly extended parastyle on the M2/ that is found in specimens of Picrodus. Despite wear, the molar paracone and metacone form a W-shaped crest across the midline on the M2/, while the broad protocone expands along the postprotocrista, a typical plesiadapiform trait. The M1/ measures 2.54 mm in length and 2.46 mm in width. The M2/ measures 1.26 mm in length and 1.60 in width. 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 PeerJ reviewing PDF | (v2013:06:580:1:0:NEW 30 Aug 2013) R ev ie w in g M an us cr ip t Comparison\u2014 Previous measurements of the holotype of Z. paleocenus (Simpson, 1935, Szalay, 1968) report a length of 2.05-2.20mm in length and 1.87-2.00 mm in width for the M1/, indicating that the new species (UCM 87378) is both larger and broader. However, reported measurements of the M2/ (1.25-1.30mm in length and 1.60-1.70mm in width) are similar in dimensions to UCM 87378. This indicates that while the first molar is enlarged, the second molar is of equal size between the two species of Zanycteris. This enlargement of the first molar likely served a functional role in providing a larger surface area for slicing between the centrocrista and cristid obliqua (Szalay, 1968). In some ways, UCM 87378 resembles Picrodus, such as the reduced anterior stylar shelf on the M1/ (Scott and Fox, 2005). However, other features more closely resemble Z. paleocenus, including the arrangement of the postprotocrista on the M1/ and smaller parastyle on the M2/. These features support inclusion of UCM 87378 within the genus Zanycteris rather than Picrodus. UCM 87378 and the holotype of Z. paleocenus (AMNH 17180) are the only two specimens of Zanycteris currently documented (Simpson, 1937; Scott and Fox, 2005). Zanycteris appears to be restricted to Colorado, while Picrodus has been found in Wyoming, Montana, Alberta and recently New Mexico (Simpson, 1937; Williams, 1985; Silcox & Gunnell, 2008; Scott & Fox, 2005; Silcox & Williamson, 2012). conclusions : In summary, the new species Zanycteris honeyi typifies the unique characteristics that set apart the Picrodontidae from other archaic primates known from the Paleocene of North America. Phylogenetic analysis supports the separation of the Picrodontidae family from the Paromomyidae family, while still recognizing their position within Pleisadapiformes. Further fossil discoveries, particularly cranial and postcranial remains will likely enable more confident placement of this unusual group of archaic primates among the evolutional tree during this pivotal time of primate diversification shortly after the extinction of the dinosaurs. acknowledgments : Special thanks to Jaelyn Eberle at the University of Colorado at Boulder for her support in my graduate studies which included this research. I would like to thank my committee Herbert H. Covert, Mary Kraus, Matthew Pranter, (University of Colorado) and Henry Fricke (Colorado College); Toni Culver, the collection manager at the University of Colorado Museum, as well as various members of the field crew including Alex Dutchak, Karen Lloyd, Pat Monaco, Andrea R. Bair, Ian J. Sweeney, Lou Taylor and Lea Ann Jolley. Fieldwork was conducted under BLM permit #C-60170 issued to the University of Colorado Museum. phylogenetic analysis : The acquisition of the highly specialized dentition found within Zanycteris and other members of the Picrodontidae remains a mystery. For example, how quickly did the specialized dentition evolve during the Paleocene? Among the known Plesiadapiformes, which one is most closely related to the family Picrodontidae and could possibly represent the ancestral condition for the specialized dentation exhibited by Zanycteris? To evaluate these questions and to work toward reconstructing the evolution of the specialized upper dentition of Picrodontidae, a phylogenetic analysis was undertaken using the 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 PeerJ reviewing PDF | (v2013:06:580:1:0:NEW 30 Aug 2013) R ev ie w in g M an us cr ip t morphological characteristics of the dentition of known Plesiadapiformes and outgroups (Paradectes, Cimolestes, and Leptacodon), which lived during the Paleocene in North America. The character matrix consisted of 113 dental characters, 97 of which were adopted from Silcox (2001). The analysis included 58 fossil taxa of contemporary North American Paleocene primates. A heuristic search using Mesquite version 2.75 (Maddison & Maddison, 2011) produced 6,579 most parsimonious trees (597 steps, consistency index [CI] = 0.36, retention index [RI] = 0.74). The strict consensus tree shows Zanycteris honeyi as closely related to Zanycteris paleocenus within a monophyletic clade of Picrodontidae (Picrodus, Draconodus, and Zanycteris). The family Picrodontidae was found to be within a clade consisting of Plesidapidae and Carpolestidae, rather than a placement within Paromomyoidea (Silcox & Gunnell, 2008). This phylogenetic position postulates that the expansion of the occlusal surface seen in the upper molars of both paromomyids and picrodontids is convergent, having evolved independently during the Paleocene. Possible ancestors of picrodontids are the early Paleocene taxa Plesiolestes, Torrejonia, Phoxomylus, and Talpohenach, while paromomyids appear to have arisen from the early Paleocene with a Palaechthon or Anasazia or the poorly known Premnoides like ancestor. Although there is ambiguity concerning the phylogenetic position of these primitive taxa, there is strong support for monophyletic clades of Paromomyidae, Picrodontidae, Plesiadapidae, and Carpolestidae, as well as a monophyletic clade of Plesiadapiformes, with the addition of Micromomyidae and Microsyopidae. Overall the resulting phylogenetic tree supports a position of Zanycteris honeyi within the Picrodontidae family, and that this new species is closely related to other archaic primates from the Paleocene of North America. Discussion 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 PeerJ reviewing PDF | (v2013:06:580:1:0:NEW 30 Aug 2013) R ev ie w in g M an us cr ip t The members of the Picrodontidae are exceptional in the development of a dentition that maximizes the shear forces along the long contact between the centrocrista on the upper molar (composed of a tall crest between the paracone and metacone) and the cristid obliqua that spans much of the length of the elongated talonid basin of the lower molar (Szalay, 1968). The molar teeth were thus probably specialized for cutting through hard outer husks of fruits and nuts. This was accomplished by positioning the fruit along the outer (buccal) shearing surface, which was greatly expanded (anteriorly and posteriorly) to maximize the amount of contact, much like a pair of long sharp scissors (Shaw, 1917). A similar expansion of the centrocrista is found in the upper molars of the Jamaican fig-eating bat (Ariteus), which feeds on the native Jamaican naseberry also known as sapodilla (Manilkara zapota), a fruit with a fleshy but firm texture (Sherwin & Gannon, 2005). Thus in both Ariteus and Zanycteris the major shearing surface is between the crests of the centrocrista above and the cristid obliqua below, demonstrating a similar specialized diet on fruit. This arrangement differs substantially from paromomyids, which retain distinct paracone and metacone cusps on the upper molars, with no development of a long and tall centrocrista between the two cusps. Rather, paromomyids expand the upper molars by broadening the postcingulum to form a wide talon basin on the lingual edge of the tooth. This broadening of the tooth functioned to expand the surface area particularly for holding food during mastication (Shaw, 1917). Thus paromomyids, such as Phenacolemur, broaden the upper molars to allow increased surface area for a larger and a more varied diet, while picrodontids, such as Zanycteris, expanded the upper molars to increase the shearing surface for a more specialized diet of a particular style of fleshy fruit. Further research on dental specialization within these groups might reveal why picrodontids have a limited stratigraphic range (Torrejonian to Tiffanian), when compared to the closely related, but dentally distinct paromomyids which ranged from the early Paleocene (Puercan) until the late Eocene (Duchesnean) (Silcox & Gunnell, 2008). 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 PeerJ reviewing PDF | (v2013:06:580:1:0:NEW 30 Aug 2013) R ev ie w in g M an us cr ip t image of fossil specimen. : Figure 1: Buccal and occlusal views of the holotype specimen, a maxilla containing the upper first and second molars. UCM 87378. PeerJ reviewing PDF | (v2013:06:580:1:0:NEW 30 Aug 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:580:1:0:NEW 30 Aug 2013) R ev ie w in g M an us cr ip t Figure 2 Strict consensus tree of the most parsimonious trees generated from the phylogenetic analysis. Figure 2: Strict consensus tree of the most parsimonious trees generated from the phylogenetic analysis of 113 upper dental characters scored against the 58 North American archaic primates known from the Paleocene. Temporal ranges during the Paleocene are shown for each species by blackened line. PeerJ reviewing PDF | (v2013:06:580:1:0:NEW 30 Aug 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:580:1:0:NEW 30 Aug 2013) R ev ie w in g M an us cr ip t Table 1(on next page) Mammal faunal list for fossil locality 92177. Table 1. Mammal faunal list for UCM locality 92177. PeerJ reviewing PDF | (v2013:06:580:1:0:NEW 30 Aug 2013) R ev ie w in g M an us cr ip t Table 1. Mammal Faunal List for UCM locality 92177. Mammalia Allotheria Multituberculata Ptilodus kummae Ectypodus musculus Theria Erinaceomorpha (\u201cApheliscidae\u201d) Haplaletes serior Litomylus ishami Phenacodaptes sabulosus Haplomylus simpsoni Lipotyphla Leptacodon tener Mesonychia cf. Sinonyx sp. Procreodi Thryptacodon australis Arctocyonides mumak Carnivoramorpha Protictis proteus Protictis cf. schaffi Primates Nannodectes gazini Plesiadapis fodinatus Chiromyoides gigas Zanycteris honeyi new species Ignacius frugivorus Ignacius sp. Carpodaptes cygneus Condylarthra Ectocion medituber Phenacodus grangeri Phenacodus magnus Pholidota Propalaeanodon sp. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 PeerJ reviewing PDF | (v2013:06:580:1:0:NEW 30 Aug 2013) R ev ie w in g M an us cr ip t",
    "v3_text": "methods & materials : The fossil reported in this paper was recovered during geological mapping of the Citadel Plateau Quadrangle during the 1970s. The site has also produced a diverse fauna of mammals from the Fort Union Formation in western Colorado\u2019s Piceance Creek Basin (Burger & Honey, 2008). The site is referred to as University of Colorado Museum (UCM) Locality number 92177. Continued collection at the site over the years has produced a diverse fauna of fossil mammals (Table 1). However, the new species is represented by a single recovered specimen, despite 30 years of sporadic collection at the site. In the Piceance Creek Basin the Fort Union Formation is synonymous with the Atwell Gulch Member, which has been included as a member of the Wasatch Formation (Donnell, 1969) or DeBeque Formation (Kihm, 1984). In this paper I refer the Atwell Gulch Member, as the sole member of the Fort Union Formation, as it has been mapped elsewhere within the Piceance Creek Basin in western Colorado (Hail & Pipiringos, 1990; Hail & Smith, 1994). The Fort Union Formation (Atwell Gulch Mbr.) varies in thickness from 350 meters in the northeast to 196 meters in the south, and is divided into upper and lower informal units (Hail & 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Pipiringos, 1990). The fossil site UCM 92177 is 261 meters below the upper contact of the Late Paleocene Fort Union Formation (Atwell Gulch Member), with the Early Eocene Wasatch Formation (Molina Member). The lower unit of the Fort Union Formation consists of light-grey to light-brown sandstones; olive, purple, dark-reddish-brown claystone; and mudstones that are highly variegated. Large ribbon and sheet sandstone bodies are common in the north of the basin, where they can form massive sandstone cliffs measuring upwards of 25 meters thick, although most are 5-10 meters thick. Pebbles are exclusively composed of sedimentary rocks, including claystone and mudstones. The upper unit of the Fort Union Formation (Atwell Gulch Mbr.) is composed of carbonaceous shales; thin coals; and thin, but persistent highly calcareous sandstones. Mudstones and claystones are less common in the upper unit. Selenite is common, especially in the carbonaceous shales and coals. Fossil mammals are abundant in the lower unit of the Fort Union Formation, especially in the variegated beds found in association with UCM locality 92177. Fossil invertebrates, such as the bivalve Unio and a variety of gastropods are common in the upper unit, indicating a progression over time toward a more lacustrine environment. Large accumulations of gastropods are common in the upper unit, which have been interpreted as lakeshore accumulations (Hanley, 1974) Genus ZANYCTERIS Matthew, 1917 ZANYCTERIS HONEYI new species Holotype\u2014 UCM 87378 right maxilla with upper first and second molars. 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Etymology\u2014 Posthumously named in honor of James G. Honey for his discovery of the holotype, type locality and for his kindness in allowing me to study this collection. Horizon\u2014 Fort Union Formation, Atwell Gulch Member, 261 meters below the top contact with the Wasatch Formation. Localities\u2014 Only known from UMC locality number 92177. Diagnosis\u2014 Differs from Z. paleocenus by having a 30% large M1/ area, while retaining a similar sized M2/ area to Z. paleocenus. Differs from Picrodus in lacking an extended parastyle on the M2/, having a better developed anterior stylar shelf, and postprotocrista on the M1/. Furthermore, the M1/ centrocrista is more curvilinear than Picrodus. Unlike Draconodus, crenulations occur in the trigon basin of M1/. Description\u2014 The holotype (UCM 87378) is the only specimen known from the Piceance Creek Basin. However, this specimen preserves morphology to indicate that it differs from Z. paleocenus from southwestern Colorado. The enlarged metastyle on the M1/ projects buccally from the posterior edge, and the stylar shelf bulges from the mid-point of the tooth. The paracingulum (the shelf formed by the paraconule) extends anteriorly, yet the stylar shelf encircling the paracone is poorly formed and resembles Picrodus rather than Zanycteris in shape. The strong postprotocrista of UCM 87378 extends directly toward the vestigial metaconule. This direct course of the postprotocrista results in a more enclosed trigon basin that closely resembles Z. paleocenus. The M2/ exhibits a wide stylar shelf, featuring a large parastyle. However, UCM 87378 typifies Zanycteris in lacking the greatly extended parastyle on the M2/ that is found in specimens of Picrodus. Despite wear, the molar paracone and metacone form a W-shaped crest across the midline on the M2/, while the broad protocone spreads into the postprotocrista, a 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t typical plesiadapiform trait. The M1/ measures 2.54 mm in length and 2.46 mm in width. The M2/ measures 1.26 mm in length and 1.60 in width. Comparison\u2014 Previous measurements of the holotype of Z. paleocenus (Simpson, 1935, Szalay, 1968) report a length of 2.05-2.20mm in length and 1.87-2.00 mm in width for the M1/, indicating that the new species (UCM 87378) is both larger and broader. However, reported measurements of the M2/ (1.25-1.30mm in length and 1.60-1.70mm in width) are similar in dimensions to UCM 87378. This indicates that while the first molar is enlarged, the second molar is of equal size between the two species of Zanycteris. This enlargement of the first molar likely served a functional role in providing a larger surface area for slicing between the centrocrista and cristid obliqua (Szalay, 1968). In some ways, UCM 87378 resembles Picrodus, such as the reduced anterior stylar shelf on the M1/. However, other features more closely resemble Z. paleocenus, including the arrangement of the postprotocrista on the M1/ and smaller parastyle on the M2/. These features support inclusion of UCM 87378 within the genus Zanycteris rather than Picrodus. UCM 87378 and the holotype of Z. paleocenus (AMNH 17180) are the only two specimens of Zanycteris currently documented (Simpson, 1937; Scott and Fox, 2005). Zanycteris appears to be restricted to Colorado, while Picrodus has been found in Wyoming, Montana, Alberta and recently New Mexico (Simpson, 1937; Williams, 1985; Silcox & Gunnell, 2008; Scott & Fox, 2005; Silcox & Williamson, 2012). discussion : The members of the Picrodontidae are exceptional in the development of a dentition that maximizes the shear forces along the long contact between the centrocrista on the upper molar (composed of a tall crest between the paracone and metacone) and the cristid obliqua that spans much of the length of the elongated talonid basin of the lower molar (Szalay, 1968). The molar teeth were thus specialized for cutting through hard outer husks of fruits and nuts. This was accomplished by positioning the fruit along the outer (buccal) shearing surface, which was greatly expanded (anteriorly and posteriorly) to maximize the amount of contact, much like a pair of long sharp scissors (Shaw, 1917). A similar expansion of the centrocrista is found in the upper molars of the Jamaican fig-eating bat (Artiteus), which feeds on the native Jamaican naseberry also known as sapodilla (Manilkara zapota), a fruit with a fleshy but firm texture (Sherwin & Gannon, 2005). Thus in both Ariteus and Zanycteris the major shearing surface is between the crests of the centrocrista above and the cristid obliqua below, demonstrating a similar specialized diet on fruit. This arrangement differs substantially from paromomyids, which retain distinct paracones and metacones cusps on the upper molars, with no development of a long and tall centrocrista between the two cusps. Rather, paromomyids expand the upper molars by broadening the postcingulum to form a wide talon basin on the lingual edge of the tooth. This broadening of the tooth functioned to expand the surface area particularly for holding food during mastication (Shaw, 1917). Thus paromomyids, such as Phenacolemur, broaden the upper molars to allow increased surface area for a larger and a more varied diet, while picrodontids, such as Zanycteris, expanded the upper molars to increase the shearing surface for a more specialized diet of a particular style of fleshy fruit. This specialization may also explain the 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t limited stratigraphic range (Torrejonian and Tiffanian) of Picrodontids, when compared to Paromomyids which ranged from the early Paleocene (Puercan) to late Eocene (Duchesnean) (Silcox & Gunnell, 2008). a new species of an archaic primate (pleisadapiformes) is described based on a maxilla containing the : first and second upper molars from the Fort Union Formation, Atwell Gulch Member in northwestern Colorado. The preserved teeth show the unusual dental characteristics of members of the rare and poorly documented Picrodontidae family, including an elongated centrocrista and wide occlusal surface. The new species is placed within the genus Zanycteris (represented from a single specimen from southern Colorado). This placement is based on similarities in regard to a shorten parastyle, curvilinear centrocrista, and wider anterior stylar shelf on the upper molars. However, the new species differs from the only known species of Zanycteris in exhibiting an upper first molar that is 30% larger in area, while retaining a similar sized upper second molar. Phylogenetic analysis supports the separation of the Picrodontidae family from the Paromomyidae, while still recognizing picrodontids position within Pleisadapiformes. The unusual dental features of the upper molars likely functioned in life as an enhanced shearing surface between the centrocrista and cristid oblique crests for a specialized diet of fruit. A similar arrangement is found in the living bat Artiteus (Jamaican fig-eating bat), which feeds on fleshy fruit. The new species showcases the rapid diversification of archaic primates shortly after the extinction of the dinosaurs during the Paleocene, and the unusual dental anatomy of picrodontids to exploit new dietary specializations. PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Benjamin John Burger* *Corresponding author: Department of Geology Utah State University Uintah Basin Regional Campus 320 North Aggie Blvd. Vernal, UT 84078 U.S.A. Phone 1-435-722-1778 benjamin.burger@usu.edu 1 2 3 4 5 6 7 8 9 PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t acknowledgments : Special thanks to Jaelyn Eberle at the University of Colorado at Boulder for her support in my graduate studies which included this research. I would like to thank my committee Herbert H. Covert, Mary Kraus, Matthew Pranter, (University of Colorado) and Henry Fricke (Colorado College); Toni Culver, the collection manager at the University of Colorado Museum, as well as various members of the field crew including Alex Dutchak, Karen Lloyd, Pat Monaco, Andrea R. Bair, Ian J. Sweeney, Lou Taylor and Lea Ann Jolley. Fieldwork was conducted under BLM permit #C-60170 issued to the University of Colorado Museum. References 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Burger BJ, Honey JG. 2008. Plesiadapidae (Mammalia, Primates) from the late Paleocene Fort Union Formation of the Piceance Creek Basin, Colorado. Journal of Vertebrate Paleontology 28(3):816-825. Clemens WA. 2004. Purgatorius (Plesiadapiformes, Primates?, Mammalia), a Paleocene immigrant into northeastern Montana: stratigraphic occurrences and incisor proportions. Bulletin of Carnegie Museum of Natural History 36:3-13. Donnell JR. 1969. Paleocene and lower Eocene units in the southern part of the Piceance Creek Basin, Colorado. U.S. Geological Survey Bulletin 1274-M:1-18. Hail WJ, Pipiringos GN. 1990. Geologic map of the lower Piceance Creek area, northwestern Colorado. U.S. Geological Survey IMAP 1936 Scale 1:100,000. Hail WJ, Smith MC. 1994. Geologic map of the northern part of the Piceance Creek basin, northwestern Colorado. U.S. Geological Survey IMAP 2400 Scale 1:100,000. Hanley JH. 1974. Systematics, paleoecology, and biostratigraphy of nonmarine Mollusca from the Green River and Wasatch Formations (Eocene), southwestern Wyoming and northwestern Colorado. PhD. thesis, University of Wyoming, Laramie, Wyoming, 1-285. Kihm AJ. 1984. Early Eocene mammalian faunas of the Piceance Creek Basin, northwestern Colorado. D. Phil. Thesis. University of Colorado at Boulder, Boulder Colorado. Maddison WP, Maddison DR. 2011. Mesquite: a modular system for evolutionary analysis. Version 2.75 http://mesquiteproject.org Matthew WD. 1917. A Paleocene bat. Bulletin of the American Museum of Natural History 37:569-571. 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Scott CS, Fox RC. 2005. Windows on the evolution of Picrodus (Plesiadapiformes: Primates): Morphology and relationships of a species complex from the Paleocene of Alberta. Journal of Paleontology 79(4):635-657. Shaw DM. 1917. Form and function of teeth: A theory of \u201cMaximum Shear.\u201d Journal of Anatomy 52:97-106. Sherwin RE, Gannon WL. 2005. Ariteus flavescens. Mammalian Species. 787:1-3. Silcox MT. 2001. A phylogenetic analysis of the Plesiadapiformes and their relationship to Euprimates and other archontans. D. Phil. Thesis, Johns Hopkins University, Baltimore, Maryland. Silcox MT, Gunnell GF. 2008. Plesiadapiformes. In: Janis CM, Gunnell GF, Uhen, MD, eds. Evolution of Tertiary Mammals of North America, Volume 2: Small Mammals, Xenarthrans, and Marine Mammals. Cambridge: Cambridge University Press, 207-238. Silcox MT, Williamson TE. 2012. New discovers of early Paleocene (Torrejonian) primates from the Nacimiento Formation, San Juan Basin, New Mexico. Journal of Human Evolution 63:805-833. Simmons NB, Geisler JH. 1998. Phylogenetic relationships of Icaronycteris, Archaeonycteris, Hassianycteris, and Palaeochiropteryx to extant bat lineages, with comments on the evolution of echolocation and foraging strategies in Microchiroptera. Bulletin of the American Museum of Natural History 235:1-182. Simpson GG. 1935. The Tiffany fauna, upper Paleocene I. Multituberculata, Marsupialia, Insectivora, and ?Chiroptera. American Museum Novitates. 795:1-19. Simpson GG. 1937. The Fort Union of the Crazy Mountain Field, Montana and its mammalian faunas. Smithsonian Institution United States National Museum Bulletin 169:1-277. 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Szalay FS. 1968. The Picrodontidae, a family of early primates. American Museum Novitates. 2329:1-55. Tomida Y. 1982. A new genus of picrodontid primate from the Paleocene of Utah. Folia Primatologica. 37:37-43. Williams JA. 1985. Morphology and variation in the posterior dentition of Picrodus silberlingi (Picrodontidae). Folia Primatologica. 45: 48-58. 227 228 229 230 231 232 PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Figure 1 Holotype specimen (UCM 87378) Buccal and occlusal views showing the maxilla containing the upper first and second molars. PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t Figure 2 Strict consensus tree of the most parsimonious trees generated from the phylogenetic analysis. Tree based on 113 dental characters scored against the 58 North American archaic primates known from the Paleocene. Upper dentitions are illustrated representing each fossil species examined. Temporal ranges during the Paleocene are shown for each species by blackened lines. PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t phylogenetic analysis : The acquisition of the highly specialized dentition found within Zanycteris and other members of the Picrodontidae remains a mystery. For example, how quickly did the specialized dentition evolve during the Paleocene? Among the known Plesiadapiformes, which one is most closely 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t related to the family Picrodontidae and could possibly represent the ancestral condition for the specialized dentation exhibited by Zanycteris? To evaluate these questions and to work toward reconstructing the evolution of the specialized upper dentition of Picrodontidae, a phylogenetic analysis was undertaken using the morphological characteristics of the dentition of known Plesiadapiformes and outgroups (Paradectes, Cimolestes, and Leptacodon), which lived during the Paleocene in North America. The character matrix consisted of 113 dental characters, 97 of which were adopted from Silcox (2001). The analysis included 58 fossil taxa of contemporary North American Paleocene primates. A heuristic search using Mesquite version 2.75 (Maddison & Maddison, 2011) produced 6,579 most parsimonious trees (597 steps, consistency index [CI] = 0.36, retention index [RI] = 0.74). The strict consensus tree shows Zanycteris honeyi as the sister-taxa to Zanycteris paleocenus within a monophyletic clade of Picrodontidae (Picrodus, Draconodus, and Zanycteris). The family Picrodontidae was found to be within a clade consisting of Plesidapidae and Carpolestidae, rather than the previous placement within Paromomyoidea (Silcox & Gunnell, 2008). This phylogenetic position implies that the expansion of the occlusal surface seen in the upper molars of both paromomyids and picrodontids is convergent, having evolved independently during the Paleocene. Possible ancestors of picrodontids are the early Paleocene taxa Plesiolestes, Torrejonia, Phoxomylus, and Talpohenach, while paromomyids appear to have arisen from the early Paleocene Palaechthon or Anasazia. Although there is some ambiguity concerning the phylogenetic position of these primitive taxa, there is support for a monophyletic clade of Paromomyidae, Picrodontidae, Plesiadapidae, and Carpolestidae, as well as a monophyletic clade of Plesiadapiformes, with the addition of Micromomyidae and Microsyopidae. 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 PeerJ reviewing PDF | (v2013:06:580:0:0:NEW 12 Jun 2013) R ev ie w in g M an us cr ip t conclusions : In summary, the new species Zanycteris honeyi typifies the unique characteristics that set apart the Picrodontidae from other archaic primates known from the Paleocene of North America. Phylogenetic analysis supports the separation of the Picrodontidae family from the Paromomyidae family, while still recognizing their position within Pleisadapiformes. Further fossil discoveries, particularly cranial and postcranial remains will likely enable more confident placement of this unusual group of archaic primates among the evolutional tree during this pivotal time of primate diversification shortly after the extinction of the dinosaurs.",
    "url": "https://peerj.com/articles/192/reviews/",
    "review_1": "Desmond Tobin \u00b7 Oct 8, 2013 \u00b7 Academic Editor\nACCEPT\nSome of the last remaining points have been addressed satisfactorially, and the paper overall now makes a nice contribution to the literature in this area.",
    "review_2": "Desmond Tobin \u00b7 Sep 30, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nThe authors are to be commended on a much improved manuscript, with the majority of reviewers\u2019 comments addressed. I believe final polishing of this manuscript could be undertaken by attending to a small number of minor issues before formal acceptance of the paper.\n\nWhile the biochemical nature of the protein factor, and of the tendon and bone cell cofactors, has been detailed in this manuscript, there appears to be no formal proof per se that either the protein/cofactor or cofactor alone control cell density. Thus, it would be prudent to alter the statement \u201cfurther proof of its role\u201d in the Abstract to something like \u201cin support of this proposed role\u2026\u201d.\n\nSimilarly, could the authors look again at those parts of the manuscript describing the biological role of the protein and cofactors, as the writing here needs to be consistent with the proposed model of controlling cell density. As current written this appears to state explicitly that cell density signaling is controlled by a protein plus cell-specific cofactor.\n\nAs the reviewer writes directly, greater prudence is required when ascribing alkaline phosphatase expression to bone production per se, rather than as a phenotypic marker of bone cells.\n\nPlease provide a clear reference from the literature in the your manuscript text of the origin, characterization and use of the U2OS cells. The current supporting statement is rather weak i.e.,\u201d from frozen stocks that have been in the laboratory for over a dozen years\".",
    "review_3": "Reviewer 1 \u00b7 Sep 29, 2013\nBasic reporting\nThe paper has been improved. The previous version had mislabeled figures, which very much complicated the previous review. The writing is still in places vague, but it has improved from the previous version, which was quite poor. Even the rebuttal letter is odd - here is a section of the response to Reviewer 2:\n\"One must remember that PAT cells were less than a week earlier in an embryo. They will not be polite like U2OS cells and wait for things to develop.\"\nPolite? How can a cell be polite? To be honest, in many many years of reviewing papers, I have never read anything quite as odd.\nMany of the points previously raised have not been answered. Here is just one example: I previously asked the simple question \"give a ref for U2OS\". This means to give a paper reference for these cells, such as \"Smith et al, 1967\", and then in the References give the authors, journal name, year, volume, and page numbers. The reference still in the paper is\n\"(from frozen stocks that have been in the laboratory for over a dozen years)\".\nExperimental design\nno comments\nValidity of the findings\nno comments\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Cells determine cell density using a small protein bound to a unique tissue-specific phospholipid (v0.2)\". PeerJ https://doi.org/10.7287/peerj.192v0.2/reviews/1",
    "review_4": "Reviewer 2 \u00b7 Sep 25, 2013\nBasic reporting\nNo comments\nExperimental design\nNo comments\nValidity of the findings\nThe majority of this reviewer\u2019s minor comments have been addressed in this revised manuscript. However, some general considerations remain. The biochemical natures of the protein factor, and of the tendon and bone cell cofactors, have been detailed in this manuscript; these findings are valid. Yet, there is no proof that either protein/cofactor or cofactor alone control cell density. Thus, statements such \u201cfurther proof of its role\u201d in the abstract should be modified to something like \u201cin support of this proposed role\u2026\u201d. Overall the parts of the manuscript describing the biological role of the protein and cofactors need to be rewritten to indicate the results are consistent with the proposed model of controlling cell density, rather than stating explicitly that cell density signaling is controlled by a protein plus cell-specific cofactor.\nAdditional comments\nLines 287-288: alkaline phosphatase is a marker of bone cells, but does not mean that bone is being produced. The sentence could read \u201cthe ridges produced alkaline phosphatase on the cell surface, indicating increased expression of the osteoblast phenotype.\u201d\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"Cells determine cell density using a small protein bound to a unique tissue-specific phospholipid (v0.2)\". PeerJ https://doi.org/10.7287/peerj.192v0.2/reviews/2",
    "pdf_1": "https://peerj.com/articles/192v0.3/submission",
    "pdf_2": "https://peerj.com/articles/192v0.2/submission",
    "review_5": "Desmond Tobin \u00b7 Jul 26, 2013 \u00b7 Academic Editor\nMAJOR REVISIONS\nPlease pay particular attention to addressing the very significant concerns of Reviewer 1, especially those pertaining to appropriate controls, activity assays and on how you determined the significance of your results.",
    "pdf_3": "https://peerj.com/articles/192v0.1/submission",
    "review 6": "Reviewer 1 \u00b7 Jul 18, 2013\nBasic reporting\nNo comments\nExperimental design\nnot rigorous - key controls missing, no description of repeating experiments\nmethods missing sufficient detail\nValidity of the findings\nno statistics\nno clear conclusion, other than the experiment didn't work\nAdditional comments\nThis manuscript examines a potentially interesting topic \u2013 how cells determine their density. Unfortunately, key experiments have not been done, key controls have not been done, and the manuscript appears to have many logic errors. In addition, the writing is very opaque, and much of the writing doesn\u2019t seem to make sense. Major points\nThere is no control for the immunofluorescence, and the antibody was used at 1:40. At this high concentration, any antibody will stain any cell or tissue. You need a preimmune control. Figure 1 shows that two different anti-myc antibodies cannot detect a myc-tagged version of \u201cthe protein\u201d made in E. coli. This suggests that you have a frameshift mutation somewhere in the cDNA, and are not making the correct fusion protein. The polyclonal can barely detect the protein used as an antigen- also indicating that something is wrong here. A variety of purification / modification steps are shown for some sort of protein. However, the critical thing when purifying/ modifying a factor is to show whether it has activity. There are no activity assays done for the various treatments of the factor. You need to do test the activity of a series of concentrations of the \u2018factor\u2019, or treated factor, since many signals show activity in a concentration range that has lower and upper limits. In the mass spectrometry, the lipase buffer control should look pretty much like the untreated material, whereas it actually looks almost exactly like the lipase treatment. Something is clearly wrong here. Two tiny peaks that are different between lipase and lipase buffer control were chosen for analysis, but there are mmany other peaks that could have been similarly chosen. Also, why is only a small region of the mass spectrum shown? Most importantly, there is no clear demonstration that you have purified or identified the factor affecting tendon cells. Minor points\nAbstract-\n\u201cCell density is the critical parameter controlling tendon morphogenesis\u201d \u2013 I imagine many other parameters are also just as critical. Also, what is the evidence for this opening statement? \u201cwith an affinity for the cell layer\u201d what cell layer? A cell layer in the tendon? \u201cthe band that best correlated with a cell proliferation assay\u201d How can a band (what kind of band?) \u2018correlate\u2019 with an assay? \u201cTo function as a SNZR would require that the\nfull length protein be cleaved to a smaller protein, then secreted\u201d Why is this a requirement? \u201cthe chicken cDNA\u201d The cDNA for what? \u201cto test whether the recombinant protein would exhibit the expected activity\u201d What activity? \u201cOutside the cell, a small band was detected\u201d What kind of band? Protein? How was it detected? \u201cSignal transduction is postulated to occur by an increased ordering\nof the plasma membrane\u201d Where does this come from? Aren\u2019t most signals sensed by receptors? Page, line\n2,28 \u201cbecause induction is slow from a single copy gene.\u201d What is the evidence for this? Lots of single copy genes can show a fast induction.\n2, 32 \u201cSo manipulating procollagen mRNA levels\nis not feasible when the cells are required to make high levels of procollagen\u201d This doesn\u2019t make sense.\n3,14 \u201ccaused a dramatic change\u201d increase? Decrease?\n3,23 \u201cThe one described and a second that is not diffusible but interacts with\nthe first and changes the cellular response(Schwarz 1996).\u201d This sentence doesn\u2019t make any sense.\n3,29 \u201cThis classic approach is complicated by the\nfinding that the cell type producing this protein binds a unique tissue-specific lipid cofactor\nand this composite molecule imparts a tissue-specific response\u201d This sentence is too vague\n4,7 give a ref for U2OS, also, why do we need to know \u201c(from frozen stocks that have been in the laboratory for over a dozen years)\u201d?\n4,12 if you put 12 ml into a 225 cm2 flask, there will be so little liquid therer the cells will dry up, yes?\n4,26 give the primers used\n\u201cThis yielded an expressed protein\nthat was 7 AA smaller in the linker between the protein and the tags\u201d where does this come from?\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Cells determine cell density using a small protein bound to a unique tissue-specific phospholipid (v0.1)\". PeerJ https://doi.org/10.7287/peerj.192v0.1/reviews/1 Reviewer 2 \u00b7 Jul 10, 2013\nBasic reporting\nSome of the introduction is too detailed and not relevant to the current research. For example, the discussion of collagen production and regulation of PAT cells could be consolidated.\nThe introduction should include a discussion of what is known about the gene from which the cell density factor is derived, such as its known cellular functions, cellular location, and other known cleavage forms if they exist.\nThe nature of the Schwartz (2002) reference should be specified (U.S. Patent?).\nExperimental design\nThe results section could be improved by simply stating what experiments had been conducted and why, rather than introducing each with an extensive discourse on the rationale.\nIt is not clear exactly what was expressed in E. coli, i.e. which portion of the gene was tagged and expressed. Therefore it is not convincing evidence that the observed larger-than-expected U2OS-expressed protein is aggregated, rather than a longer form with additional amino acid sequences.\nIt should be clarified why U2OS cells were used as the heterologous expression system, rather than a more standard eukaryotic expression system. Assuming the authors are correct that different cell types express different cofactors, a less committed/differentiated cell type may have been more appropriate.\nValidity of the findings\nThe reader deduces that the straightforward experiment of taking the U2OS conditioned medium, either straight or partially purified, to PAT cells failed to affect their growth. These experiments and results should be more completely described.\nAdditional comments\nThis manuscript describes a complex system which is involved in the cellular sensing of cell density. The factor involves a relatively small protein associated with a lipid-based cofactor, a unique situation which has greatly complicated the characterization of this activity.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"Cells determine cell density using a small protein bound to a unique tissue-specific phospholipid (v0.1)\". PeerJ https://doi.org/10.7287/peerj.192v0.1/reviews/2",
    "all_reviews": "Review 1: Desmond Tobin \u00b7 Oct 8, 2013 \u00b7 Academic Editor\nACCEPT\nSome of the last remaining points have been addressed satisfactorially, and the paper overall now makes a nice contribution to the literature in this area.\nReview 2: Desmond Tobin \u00b7 Sep 30, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nThe authors are to be commended on a much improved manuscript, with the majority of reviewers\u2019 comments addressed. I believe final polishing of this manuscript could be undertaken by attending to a small number of minor issues before formal acceptance of the paper.\n\nWhile the biochemical nature of the protein factor, and of the tendon and bone cell cofactors, has been detailed in this manuscript, there appears to be no formal proof per se that either the protein/cofactor or cofactor alone control cell density. Thus, it would be prudent to alter the statement \u201cfurther proof of its role\u201d in the Abstract to something like \u201cin support of this proposed role\u2026\u201d.\n\nSimilarly, could the authors look again at those parts of the manuscript describing the biological role of the protein and cofactors, as the writing here needs to be consistent with the proposed model of controlling cell density. As current written this appears to state explicitly that cell density signaling is controlled by a protein plus cell-specific cofactor.\n\nAs the reviewer writes directly, greater prudence is required when ascribing alkaline phosphatase expression to bone production per se, rather than as a phenotypic marker of bone cells.\n\nPlease provide a clear reference from the literature in the your manuscript text of the origin, characterization and use of the U2OS cells. The current supporting statement is rather weak i.e.,\u201d from frozen stocks that have been in the laboratory for over a dozen years\".\nReview 3: Reviewer 1 \u00b7 Sep 29, 2013\nBasic reporting\nThe paper has been improved. The previous version had mislabeled figures, which very much complicated the previous review. The writing is still in places vague, but it has improved from the previous version, which was quite poor. Even the rebuttal letter is odd - here is a section of the response to Reviewer 2:\n\"One must remember that PAT cells were less than a week earlier in an embryo. They will not be polite like U2OS cells and wait for things to develop.\"\nPolite? How can a cell be polite? To be honest, in many many years of reviewing papers, I have never read anything quite as odd.\nMany of the points previously raised have not been answered. Here is just one example: I previously asked the simple question \"give a ref for U2OS\". This means to give a paper reference for these cells, such as \"Smith et al, 1967\", and then in the References give the authors, journal name, year, volume, and page numbers. The reference still in the paper is\n\"(from frozen stocks that have been in the laboratory for over a dozen years)\".\nExperimental design\nno comments\nValidity of the findings\nno comments\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Cells determine cell density using a small protein bound to a unique tissue-specific phospholipid (v0.2)\". PeerJ https://doi.org/10.7287/peerj.192v0.2/reviews/1\nReview 4: Reviewer 2 \u00b7 Sep 25, 2013\nBasic reporting\nNo comments\nExperimental design\nNo comments\nValidity of the findings\nThe majority of this reviewer\u2019s minor comments have been addressed in this revised manuscript. However, some general considerations remain. The biochemical natures of the protein factor, and of the tendon and bone cell cofactors, have been detailed in this manuscript; these findings are valid. Yet, there is no proof that either protein/cofactor or cofactor alone control cell density. Thus, statements such \u201cfurther proof of its role\u201d in the abstract should be modified to something like \u201cin support of this proposed role\u2026\u201d. Overall the parts of the manuscript describing the biological role of the protein and cofactors need to be rewritten to indicate the results are consistent with the proposed model of controlling cell density, rather than stating explicitly that cell density signaling is controlled by a protein plus cell-specific cofactor.\nAdditional comments\nLines 287-288: alkaline phosphatase is a marker of bone cells, but does not mean that bone is being produced. The sentence could read \u201cthe ridges produced alkaline phosphatase on the cell surface, indicating increased expression of the osteoblast phenotype.\u201d\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"Cells determine cell density using a small protein bound to a unique tissue-specific phospholipid (v0.2)\". PeerJ https://doi.org/10.7287/peerj.192v0.2/reviews/2\nReview 5: Desmond Tobin \u00b7 Jul 26, 2013 \u00b7 Academic Editor\nMAJOR REVISIONS\nPlease pay particular attention to addressing the very significant concerns of Reviewer 1, especially those pertaining to appropriate controls, activity assays and on how you determined the significance of your results.\nReview 6: Reviewer 1 \u00b7 Jul 18, 2013\nBasic reporting\nNo comments\nExperimental design\nnot rigorous - key controls missing, no description of repeating experiments\nmethods missing sufficient detail\nValidity of the findings\nno statistics\nno clear conclusion, other than the experiment didn't work\nAdditional comments\nThis manuscript examines a potentially interesting topic \u2013 how cells determine their density. Unfortunately, key experiments have not been done, key controls have not been done, and the manuscript appears to have many logic errors. In addition, the writing is very opaque, and much of the writing doesn\u2019t seem to make sense. Major points\nThere is no control for the immunofluorescence, and the antibody was used at 1:40. At this high concentration, any antibody will stain any cell or tissue. You need a preimmune control. Figure 1 shows that two different anti-myc antibodies cannot detect a myc-tagged version of \u201cthe protein\u201d made in E. coli. This suggests that you have a frameshift mutation somewhere in the cDNA, and are not making the correct fusion protein. The polyclonal can barely detect the protein used as an antigen- also indicating that something is wrong here. A variety of purification / modification steps are shown for some sort of protein. However, the critical thing when purifying/ modifying a factor is to show whether it has activity. There are no activity assays done for the various treatments of the factor. You need to do test the activity of a series of concentrations of the \u2018factor\u2019, or treated factor, since many signals show activity in a concentration range that has lower and upper limits. In the mass spectrometry, the lipase buffer control should look pretty much like the untreated material, whereas it actually looks almost exactly like the lipase treatment. Something is clearly wrong here. Two tiny peaks that are different between lipase and lipase buffer control were chosen for analysis, but there are mmany other peaks that could have been similarly chosen. Also, why is only a small region of the mass spectrum shown? Most importantly, there is no clear demonstration that you have purified or identified the factor affecting tendon cells. Minor points\nAbstract-\n\u201cCell density is the critical parameter controlling tendon morphogenesis\u201d \u2013 I imagine many other parameters are also just as critical. Also, what is the evidence for this opening statement? \u201cwith an affinity for the cell layer\u201d what cell layer? A cell layer in the tendon? \u201cthe band that best correlated with a cell proliferation assay\u201d How can a band (what kind of band?) \u2018correlate\u2019 with an assay? \u201cTo function as a SNZR would require that the\nfull length protein be cleaved to a smaller protein, then secreted\u201d Why is this a requirement? \u201cthe chicken cDNA\u201d The cDNA for what? \u201cto test whether the recombinant protein would exhibit the expected activity\u201d What activity? \u201cOutside the cell, a small band was detected\u201d What kind of band? Protein? How was it detected? \u201cSignal transduction is postulated to occur by an increased ordering\nof the plasma membrane\u201d Where does this come from? Aren\u2019t most signals sensed by receptors? Page, line\n2,28 \u201cbecause induction is slow from a single copy gene.\u201d What is the evidence for this? Lots of single copy genes can show a fast induction.\n2, 32 \u201cSo manipulating procollagen mRNA levels\nis not feasible when the cells are required to make high levels of procollagen\u201d This doesn\u2019t make sense.\n3,14 \u201ccaused a dramatic change\u201d increase? Decrease?\n3,23 \u201cThe one described and a second that is not diffusible but interacts with\nthe first and changes the cellular response(Schwarz 1996).\u201d This sentence doesn\u2019t make any sense.\n3,29 \u201cThis classic approach is complicated by the\nfinding that the cell type producing this protein binds a unique tissue-specific lipid cofactor\nand this composite molecule imparts a tissue-specific response\u201d This sentence is too vague\n4,7 give a ref for U2OS, also, why do we need to know \u201c(from frozen stocks that have been in the laboratory for over a dozen years)\u201d?\n4,12 if you put 12 ml into a 225 cm2 flask, there will be so little liquid therer the cells will dry up, yes?\n4,26 give the primers used\n\u201cThis yielded an expressed protein\nthat was 7 AA smaller in the linker between the protein and the tags\u201d where does this come from?\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Cells determine cell density using a small protein bound to a unique tissue-specific phospholipid (v0.1)\". PeerJ https://doi.org/10.7287/peerj.192v0.1/reviews/1 Reviewer 2 \u00b7 Jul 10, 2013\nBasic reporting\nSome of the introduction is too detailed and not relevant to the current research. For example, the discussion of collagen production and regulation of PAT cells could be consolidated.\nThe introduction should include a discussion of what is known about the gene from which the cell density factor is derived, such as its known cellular functions, cellular location, and other known cleavage forms if they exist.\nThe nature of the Schwartz (2002) reference should be specified (U.S. Patent?).\nExperimental design\nThe results section could be improved by simply stating what experiments had been conducted and why, rather than introducing each with an extensive discourse on the rationale.\nIt is not clear exactly what was expressed in E. coli, i.e. which portion of the gene was tagged and expressed. Therefore it is not convincing evidence that the observed larger-than-expected U2OS-expressed protein is aggregated, rather than a longer form with additional amino acid sequences.\nIt should be clarified why U2OS cells were used as the heterologous expression system, rather than a more standard eukaryotic expression system. Assuming the authors are correct that different cell types express different cofactors, a less committed/differentiated cell type may have been more appropriate.\nValidity of the findings\nThe reader deduces that the straightforward experiment of taking the U2OS conditioned medium, either straight or partially purified, to PAT cells failed to affect their growth. These experiments and results should be more completely described.\nAdditional comments\nThis manuscript describes a complex system which is involved in the cellular sensing of cell density. The factor involves a relatively small protein associated with a lipid-based cofactor, a unique situation which has greatly complicated the characterization of this activity.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"Cells determine cell density using a small protein bound to a unique tissue-specific phospholipid (v0.1)\". PeerJ https://doi.org/10.7287/peerj.192v0.1/reviews/2\n",
    "LLM_extracted_review": "['Review 1: Some of the last remaining points have been addressed satisfactorily, and the paper overall now makes a nice contribution to the literature in this area.\\n\\nReview 2: The authors are to be commended on a much improved manuscript, with the majority of reviewers\u2019 comments addressed. However, there appears to be no formal proof that either the protein/cofactor or cofactor alone control cell density. It would be prudent to alter the statement \u201cfurther proof of its role\u201d in the Abstract to something like \u201cin support of this proposed role\u2026\u201d. The writing describing the biological role of the protein and cofactors needs to be consistent with the proposed model of controlling cell density. Greater prudence is required when ascribing alkaline phosphatase expression to bone production per se, rather than as a phenotypic marker of bone cells. A clear reference from the literature regarding the origin, characterization, and use of the U2OS cells is needed, as the current supporting statement is weak.\\n\\nReview 3: The paper has been improved, but the writing is still vague in places. Many points previously raised have not been answered, such as providing a reference for U2OS cells. The response to Reviewer 2 contains odd phrasing, such as referring to cells as \"polite.\"\\n\\nReview 4: The majority of this reviewer\u2019s minor comments have been addressed, but there are still general considerations. There is no proof that either protein/cofactor or cofactor alone control cell density. Statements like \u201cfurther proof of its role\u201d should be modified to \u201cin support of this proposed role\u2026\u201d. The manuscript needs to be rewritten to indicate that the results are consistent with the proposed model of controlling cell density. Alkaline phosphatase is a marker of bone cells, but does not mean that bone is being produced.\\n\\nReview 5: Please pay particular attention to addressing the very significant concerns of Reviewer 1, especially those pertaining to appropriate controls, activity assays, and how you determined the significance of your results.\\n\\nReview 6: Key controls are missing, and there is no description of repeating experiments. The methods lack sufficient detail, and there are no statistics provided. The manuscript appears to have many logic errors, and the writing is very opaque. Key experiments have not been done, and there is no clear demonstration that you have purified or identified the factor affecting tendon cells. The introduction contains too much irrelevant detail, and the results section could be improved by stating what experiments were conducted and why. It is unclear what was expressed in E. coli, and the choice of U2OS cells as the expression system should be clarified. The straightforward experiment of taking the U2OS conditioned medium to PAT cells failed to affect their growth, and these results should be more completely described.']"
}