A second nearly complete, articulated specimen of the basal troodontid Mei long (DNHM D2154) is reported from the Early Cretaceous (Hauterivian-Valanginian) lower Yixian Formation, Liaoning Province, China. New diagnostic features of Mei long are identified, including: a uniquely shaped maxilla, low with small, low maxillary fenestra; sacrum with an extremely wide caudal portion and elongate 4 th and 5 th sacral processes; and a large distal articular surface on the tibiotarsus which continues caudally on the tibia. A phylogenetic analysis including new data from the second specimen recovered Mei as a basal troodontid, in keeping with previous analyses. Although the skeleton exhibits several juvenile-like features including free cervical ribs, unfused frontals and nasals, and a short snouted skull, other attributes, full fusion of all neurocentral synostoses and the sacrum, and dense exteriors to cortical bone, suggest a small, mature individual. Microscopic examination of tibia and fibula histology confirms maturity and suggests an individual greater than two years old with slowed growth. Despite being one of the smallest dinosaurs, Mei long exhibits multi-year growth and cortical bone consisting largely of fibro-lamellar tissue marked by lines of arrested growth as in much larger and more basal theropods. This Mei long specimen lies in a similar but mirrored sleeping position to that of the holotype, strengthening the hypothesis that both specimens were preserved in a stereotypical life position. Like many Liaoning specimens, the new specimen also lacks extensive taphonomic and stratigraphic data, making further behavioral inference problematic.

Copyright: © Gao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

A , interpretive line drawing; B , photograph. Abbreviations : aofe , antorbital fenestra; c , cervical vertebra(e), unnumbered; c6 , sixth cervical vertebra; c7 , seventh cervical vertebra; ca1–ca18 , caudal vertebrae (one through eighteen); co , coracoid; d , dentary; dv , dorsal vertebra(e); f , frontal; fm , femur; fl , fibula; h , humerus; il , ilium; l , lacrimal; mt-II , mt-III , second metatarsal, third metatarsal etc.; mx , maxilla; mxfl? , maxillary fenestra; n? , nasal; nc , neural canal; ns , neural spine; o , orbit; p , parietal; pu , pubis; pmx , premaxilla; q , quadrate; r , radius; s1 , s2 , etc., first sacral vertebra, second sacral vertebra etc.; sa , surangular; sc ,scapula; sp , fused neural spine of sacrum; t , tibia; u? , possible manual ungual; ul , ulna; II-2 , second phalanx of digit II of pes; II-3 , third phalanx of digit II of pes; III-1 , first phalanx of digit III of pes; IV-1 – IV-4 , first through fourth phalanges of digit IV. Note: Abbreviations follow the convention of Weishampel et al., 2004. Scale bar equals 1 cm.

One of these three-dimensionally preserved specimens is a small troodontid, named Mei long by Xu and Norell [16] . It was preserved in a position that closely matched the stereotypical sleeping posture of modern birds [16] . Here we report on a second specimen of Mei long, found in a nearly identical sleeping position ( Figure 1 ), detailing its anatomy and aspects of its histology.

The Jehol Group is composed of the stratigraphically conformable Yixian and Jiufotang Formations and is exposed in western portions of Liaoning Province, China. These formations are dominated by laminated and finely bedded siliciclastic sediments interspersed with extrusive basalts and tuffs [1] . They have produced a widely acclaimed fossilized fauna that includes a wide diversity of fish, invertebrates, plants [1] , mammals [2] , [3] , diapsids [4] , [5] including dinosaurs [6] – [8] with many specimens displaying exceptional soft-tissue and integument preservation [9] – [11] . These spectacular fossils are predominantly strongly compressed [12] and come from the siliciclastic sediments thought to have been deposited by a series of inland, freshwater lacustrine environments [1] . In the lower Yixian Formation, a fossiliferous tuff layer, the Lujiatun beds, crops out near Beipiao City [1] . The Lujiatun beds lack obvious bedding planes [1] , and have been interpreted as volcaniclastic in origin, possibly representing volcanic mudflow events [13] . This results in beautiful three-dimensional preservation [13] , [14] . However, see Meng et al. [15] for alternative scenarios. Several specimens have emerged from the lower beds with three-dimensional preservation provides the opportunity for behavioral inferences [13] , [15] , [16] .

The analysis was run in T.N.T. 1.1 [28] , [29] . A seed of ten Wagner trees was used with 10000 replications of a traditional Tree-Bisection-Reconnection (TBR) search. A second round of TBR searching was conducted beginning with trees held in memory. There was no overflow with the second round of searching. Bremer support was calculated by sequentially increasing the suboptimal trees retained and examining the resulting strict consensus trees.

Our analysis used the matrix from Xu et al. [23] with coding modifications to Mei based on new information available from this specimen. The coding modifications can be found in the phylogenetic analysis section. Two troodontids published after Xu et al. [23] were added to the matrix. The coding for Philovenator curriei was obtained from Xu et al [24] . Talos sampsoni was conservatively coded from the literature [25] . This matrix was chosen because, while far more extensive matrices exist for understanding interrelationships across Coelurosauria (e.g. [26] ) or the interrelationships of dromeosaurids [27] , these matrices do not have as wide a sampling of published troodontid species as the Xu et al. [23] matrix.

To assess the ontogenetic age of DNHM D2154, we removed, molded and cast a section of the mid-diaphysis of the right tibia and fibula. A cast of the removed section was then inserted into the original specimen to retain the overall morphology. Cross-sections were prepared for each element using standard paleohistologic techniques [18] – [20] , and examined with light microscopy. Descriptive terminology follows Francilion-Vieillot et al. [21] , and age at death was assessed from annual lines of arrested growth (LAGs) [22] .

For anatomical positioning, the terminology will follow the conventions of the Nomina Anatomica Avium [17] where feasible. In describing the orientation of the elements relative to each other as preserved, the terms “front” and “back” or “behind” are used to define directions along an axis roughly parallel to the dorsal series of the vertebral column, where moving from the sacrum towards the sharp curvature in the neck is defined as “forward” or the front. Consequently, moving from the neck towards the sacrum in any line roughly parallel to the dorsal vertebral column is defined as the rear, with terms indicating that direction including “rearward,” “back” or “behind.”

Results

Systematic Paleontology Theropoda Marsh, 1881 [30]. Maniraptora Gauthier, 1986 [31]. Troodontidae Gilmore, 1924 [32] (Currie, 1987 [33]). Mei long Xu and Norell, 2004 [16]. Holotype. IVPP V12733, a nearly complete, fully articulated skeleton. Locality and horizon. Lujiatun, Shangyuan, Beipiao City, western Liaoning, China; lowest more fluvial and volcaniclastic beds of Yixian Formation, older than 128 and younger than 139 Ma [16]. Referred Specimen. DNHM D2154, a nearly complete, articulated skeleton including skull, complete cervical series, partial dorsal series, complete sacrum and ilium, a partial pubis, complete hind limbs, fairly complete caudal series and complete forelimbs, except for the furculum and much of the left manus. The missing forelimb elements may still be present in the matrix, but are not currently exposed. Locality and horizon. Lujiatun beds, Kaoshangtun, Shangyuan, Beipiao City, western Liaoning, China. Revised Diagnosis. Mei long is a troodontid with the following autapomorphies: Extremely large naris extending caudally over one-half of the maxillary tooth row [16]; closely packed middle maxillary teeth [16]; maxillary tooth row extending caudally to the level of the preorbital bar [16]; caudal portion of sacrum extremely wide with elongate 4th and 5th sacral processes; ilium strongly sigmoid in dorsal view, with a stronger lateral curve than in Velociraptor and Anchiornis; presence of a caudolateral flange on the midshaft of metatarsal IV [16]; the most proximal end of the pubic shaft significantly craniocaudally compressed relative to other troodontids [16]. Additionally, Mei long possesses the following combination of characters with a wider distribution: a bulbous and fully convex caudal portion of frontal and a short and steeply angled snout, features shared with Anchiornis and the Ukhaa Tolgod perinates (IGM 100/972 and 100/974) described as Byronosaurus [34], but perhaps representing a new taxon [35]; premaxilla with steeply inclined front and long maxillary process (shared with Ukhaa Tolgod perinates); maxilla, low with small, low maxillary fenestra and dorsoventrally short rostral process but thinner than in the Ukhaa Tolgod perinates; unserrated teeth as in Anchiornis and Ukhaa Tolgod perinates with straight distal margins as in Ukhaa Tolgod perinates, and Sinovenator but not Anchiornis; robust, sub-‘U’-shaped furculum more similar to those of oviraptorids; long ulna and radius approximately equal to or >90% of humeral length; metacarpal III longer than metacarpal II; unguals only moderately recurved compared to other maniraptorans; and distal articular surface of tibiotarsus large, continuing caudally on the tibia.

Anatomical Description The specimen (DNHM D2154) is encased in green mudstone with up to fine sand-size clasts. The individual is small, with a total length of approximately 325 mm and not more than 400 mm. This is some 130 to 200 mm shorter than the type specimen of Mei long (IVPP V12733). However, individual elements may provide a better estimate of the comparative size of these two specimens (Table S1). DNHM D2154 is consistently smaller than the original Mei long with individual linear measurements averaging 83% those of the holotype. The skeleton is oriented dorsal side up from the base of the neck through the first caudals. The hind limbs are folded on either side of the torso (Figure 1). The forelimbs are both oriented with the elbows pointing up and slightly rearwards with the hands ventral, lying horizontally and directed rearwards. The only elements visible from the left manus are a large ungual, possibly phalanx I-2, and an elongate element. The latter cannot be clearly identified and may not be part of the manus. The right manus is better exposed, running rearward just beneath the skull. All elements are visible and in place except for metacarpal-I and possibly phalanx III-1. Although missing, an outline remains for metacarpal-I and an estimate can be made of its size and shape. No carpals are observable. The neck arches up and back so that the skull lies on the right side of the body, above the right knee and hand, behind the right elbow (Figure 1). The skull is resting on its right side and facing rearward. The tail also curls to the right and travels forward under the skull. Most of the tail is lying on its left side. Lying in this position the greatest front-rear and transverse dimensions of the specimen are 120 mm and 83 mm respectively. This posture is nearly identical to the avian sleeping posture of the type specimen of Mei long [16] and also bears some similarities to the curled pose seen in the type specimen of Sinornithoides youngi, IVPP V9612 [36]. Further basic measurements of the specimen are provided in Table S1.

Skull The skull (Figure 2A) is an estimated 5.0 cm long and has a steep anterior profile more reminiscent of Archaeopteryx [37]–[39] than other troodontids [40]–[42]. The skull to femur length ratio of Mei long is approximately 0.75, which is similar to the condition in Sinusonasus magnodens [16] and some other basal troodontids and dromeosaurs [36], [43], [44]. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Photos and interpretive drawings for Mei long, DNHM D2514. A. Skull of Mei long in left lateral to left dorsolateral view. B, sacrum and ilia in dorsal view. C, pelvis in right lateral view. Abbreviations as in Figure 1. Scale bars equal 0.5 cm. https://doi.org/10.1371/journal.pone.0045203.g002 The relatively small premaxilla possesses a dorsal border that angles steeply back and a fairly stout caudal process that extends over the rostral most portion of the maxilla. It is impossible to determine whether this process excludes the maxilla from contributing to the nares or not. This subnarial process to the premaxilla is shared with Sinusonasus [44]; DNHM D2154 lacks the slit-like premaxillary fenestra seen in this taxon. The external naris cannot be delineated in DNHM D2154, however, it is elongate in the holotype. Byronosaurus is referred to as having a similarly extensive naris [34], but the left side of the skull appears to clearly show the naris ending at approximately 1/3 the total length of the toothrow [42]. The maxilla is 16.5 mm long and unusually low (approximately 1.4 mm deep), particularly over its rostral portion. The rostral process is lower relative to its length than any other troodontid. The maxillary fenestra is small and narrow dorsoventrally. The antorbital fenestra is larger than the maxillary fenestra and appears to be broadly triangular (Figure 2A). The interfenestral bar sits flush with the rim of the antorbital fossa in a condition reminiscent of Byronosaurus jaffei [42] and Linhevenator [23]. On the caudoventral portion of the bone a prominent slit-like groove occurs. A similar feature occurs in Troodon formosus and may accommodate the jugal. The T-shaped lacrimal has a slightly concave dorsal surface. The full length of the rostral process is obscured by matrix. A small lateral extension sits just above the mediolaterally broad descending process. A matrix-filled recess occurs on the rostral face of this process, suggesting the possibility of a pneumatic fossa (recessus pneumaticus lacrimalis, Witmer [45]), as in most troodontids [34], [41], [46], or the opening of the lacrimal duct [42]. The nasals are unfused thin broad plates of bone. Both left and right frontals are nearly fully exposed in dorsal view (Figure 2A). They appear to be completely unfused along their entire lengths, as is the condition of the holotype. The caudal margin of each frontal is relatively straight, with a slight triangular enlargement at the midline where the frontals meet. The frontal is unusually broad and convex over its posterior half. This convexity continues to the lateral margin of the bone, unlike the condition seen in other troodontids (e.g. Troodon, Zanabazar) [47] and extends forward to the beginning of the rostral extensions of the frontal. In dorsal view, the frontal narrows rapidly at the level of the orbit. The lateral margin of the rostral portion of the frontal is dorsally upturned and thickened, presenting a slim vertical lateral face abutting the lacrimal. The rostral portion of each frontal is dorsally concave across a transverse transect. The frontal lamina on DNHM D2154 is similar to that reported for Sinovenator [14]. The caudal portion of the skull is not well preserved. Remnants of the parietal suggest it was broadly convex, confluent with the convexity of the frontals. A small portion of the supraoccipital is visible and suggests a broad central raised portion with shallow, caudal-facing depressions located laterally. The left quadrate is displaced rearwards revealing the moderately concave caudal aspect of the element. A sizable pneumatic foramen opens caudally at midheight (Figure 2B). The proximal portion tapers dorsally to a presumably narrow head, while the distal end flares for the articular surface. A slight ridge marks the caudomedial aspect of the element. The dentary is long and narrows dorsoventrally toward the rostral end. It is 27.7 mm long along the ventral margin. At its caudal end, it is 3.6 mm tall, and it is about 1.5 mm tall at its rostral end. The dentary conforms to that of other troodontids in tapering rostrally and possessing a groove containing nutrient foramina along much of its length [33], [42], [46], [47], [48], [49]. The ramus of the dentary appears straight in ventral view, not curving towards the midline. This character is plesiomorphic for Troodontidae [42] being found in basal taxa [36], [42] and in the fragmentary Urbacodon itemirensis [50]. Middle and caudal maxillary teeth are laterally compressed and recurved. Teeth reach the greatest mesial-distal basal length (∼0.5 mm) near the mid length of the maxilla. Smaller teeth sit rostral and caudal. The teeth of the rostral dentary are narrow and tightly spaced. They appear similar to embryonic Troodon teeth [51] and are reminiscent of small, unidentified teeth of the Jurassic of Utah [52]. The first alveolus on the dentary is not exposed, so it cannot be determined if this specimen shares the small, rounded rostral dentary alveolus of Archaeornithoides [53]. DNHM D2154 appears to lack denticles on the teeth, as does Byronosaurus [42] and Urbacodon [50].

Histology We removed a mid-length section of the right tibia and fibula of DNHM D2154 for histologic sampling. The small size of the individual and its articulated nature allowed for the tibia and fibula to be thin-sectioned together (Figure 7). The fibula cross-section measures 0.86 by 0.63 mm and lacks an open medullary cavity (Fig. 7B). Instead, the cross-section possesses two distinct histologic regions: an interior, elliptical region of fibro-lamellar bone with approximately 20 longitudinally oriented primary osteons; and multiple exterior chevron-shaped zones with diffuse banding and only a few, more circumferentially arranged osteons. The latter zones narrow both anteriorly and posteriorly and show a parallel extinction pattern under crossed-polars indicative of lamellar bone. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 7. Histology of the right tibia and fibula of Mei long, DNHM D2514. A, cross-section of tibia and fibula in articulation. B, partial cross-section of fibula. C, partial cross-section of tibia. Note two zones of fibro-lamellar bone separated by a LAG in the tibia and the largely avascular lamellar bone that marks the periphery of both elements. The latter can be interpreted as an external fundamental system. Abbrevaitions: el, endosteal lamellar bone; fl, fibro-lamellar bone; fl1, first and interior zone of fibro-lamellar bone; fl2, second and more exterior zone of fibro-lamellar bone; LAG, line of arrested growth; mc, medullary cavity; pl, peripheral lamellar bone. Scale bars equal 0.5 mm in A and 0.2 mm in B and C. https://doi.org/10.1371/journal.pone.0045203.g007 The elliptical cross-section of the tibia measures 3.4 deep by 4.85 mm medio-laterally and has a straighter anterior face and a more convex posterior face (Fig. 7C). The well-developed medullary cavity mirrors the overall cross-section in shape. The cavity lacks any cross-cutting trabeculae. Permineralization consists of orange to opaque mineral growth along the interior of the cortical bone from which emerge small lath-like crystals. Sparry calcite fills the central and a majority portion of the medullary cavity. Cortical bone thickness ranges from 0.54 to 0.93 mm. A thin, 0.001–0.05 mm thick band of circumferential lamellar bone, devoid of osteons marks the endosteal portion of the cortex. The orientation of the long axes of lacunae and of the crystals as visible under crossed polars concurs with a circumferential arrangement. The majority of the cortex consists of two zones of fibrolamellar tissue separated by a band of circumferential and largely avascular bone with a single line of arrested growth (LAG) (Fig. 7). The interior zone of fibrolamellar bone is 0.32 to 0.50 mm thick and possesses longitudinally aligned osteons. These osteons are regularly spaced but do not form discrete circumferential bands. The band separating the two zones possesses circumferentially oriented lacunae and crystals. The latter are evidenced by parallel extinction patterns under crossed polars. This band also bears a distinct LAG visible through the entire circumference and only a few osteons occur in proximity to the LAG. The second, more external zone of fibrolamellar tissue is largely similar to the interior one and measures 0.30–0.50 mm in thickness. It differs in that not all the osteons are longitudinally oriented and instead include some circumferentially arranged osteons primarily close to the separating band and others radially oriented. The periphery of the bone along the posterior aspect shows a decrease in osteon and vessel density vessels by as much as 0.2 mm. Additionally, there is a partial realignment of osteon orientation. In contrast, a circumferentially oriented band of avascular lamellar bone with circumferentially oriented lacunae and crystals comprises the remaining two-thirds of the periphery. This tissue reaches a maximum thickness of 0.15 mm on the anterior aspect of the cross-section and then tapers around the lateral and medial edges. LAGs or bands within this tissue are consequently not traceable through the entire bone’s circumference.