Updating and Recoding Enamel Microstructure in Mesozoic Mammals ...

C 2005) Journal of Mammalian Evolution, Vol. 12, Nos. 3/4, December 2005 ( DOI: 10.1007/s10914-005-6971-0

Updating and Recoding Enamel Microstructure in Mesozoic Mammals: In Search of Discrete Characters for Phylogenetic Reconstruction Craig B. Wood1,3 and Guillermo W. Rougier2

The previously unknown enamel microstructure of a variety of Mesozoic and Paleogene mammals ranging from monotremes and docodonts to therians is described and characterized here. The novel information is used to explore the structural diversity of enamel in early mammals and to explore the impact of the new information for systematics. It is presently unclear whether enamel prisms arose several times during mammalian evolution or arose only once with several reversals to prismless structure. At least two undisputed reversions or simplifications are known—in the monotreme clade from Obdurodon to Ornithorhynchus (via Monotrematum?), and (perhaps more than once) within the clade from archaeocete to a variety of odontocete whales. Similarly, both prismatic and nonprismatic enamel is present among docodonts. Seven discrete characters showing enough morphological diversity to be of potential importance in phylogenetic reconstructions may be identified as a more appropriate summary of enamel microstructural diversity among mammaliaforms than the single character “prismatic enamel-present/absent” employed in recent matrices. Inclusion of five of these characters in the matrix of Luo et al. (2002) modifies the original topology by collapsing several nodes involving triconodonts and other nontribosphenic taxa. There is considerable support for prismatic enamel as a synapomorphy of trithelodonts plus Mammaliamorpha, and multituberculates appear to have small or “normal” sized prisms as the ancestral condition, with some (as yet) enigmatic changes to nonprismatic structure in some basal members of the group and the appearance of “gigantoprismatic” structure as an autapomorphic state of less inclusive clades. Other potential qualitative characters and the need for attaining appropriate methods to incorporate quantitative features may be important for future analyses. KEY WORDS: Enamel microstructure, Mesozoic mammals, phylogenetic analysis.

INTRODUCTION Professor Clemens (1979) eloquently summarized the major points of mammalian tooth enamel research before 1980. He concluded with the following statement (Clemens (1979), pp. 199–200):

1 Department

of Biology, Providence College, Providence, Rhode Island, USA. of Anatomical Sciences and Neurobiology, University of Louisville, Louisville, Kentucky, USA. 3 To whom correspondence should be addressed at Department of Biology, Providence College, Providence, Rhode Island, USA. E-mail: [email protected] 2 Department

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The possibility of establishing phylogenetic affinity on the basis of enamel histology is particularly inviting. However, such analyses must await the completion of much more descriptive and analytical research. Although description and analysis have not yet ended, significant progress has occurred since 1979. Building upon the foundational research of Boyde (e.g., 1967, 1971, 1976) and many others after the advent of electron microscopy, descriptive and interpretational standards advanced to current levels by works such as those of Sahni (1979, 1987), Lester and Koenigswald (1989), Carlson (1990), Koenigswald and Clemens (1992), Stern and Crompton (1995), and finally in the benchmark provided by Koenigswald and Sander (1997a). This essay is not meant as a complete review of enamel studies. It is rather an opportunity to provide novel information on enamel microstructure, to revisit the major elements composing mammalian enamel (Table I) in search of characters with potential phylogenetic bearing, and finally to evaluate the distribution of these characters—and their impact—on a phylogenetic framework. It is in essence a brief summary and update of ongoing studies on the origin and evolution of prismatic enamel in mammals during the Mesozoic Era. Ultimately the question is whether enamel studies can contribute useful information to the broader studies on the origin of Mammalia. The main reason to attempt additional analysis here is in the new specimens that we have been able to analyze since the last broad reviews of earlier mammalian enamel (Koenigswald and Sander, 1997a; Wood, 2000). With the exception of some multituberculates (Krause and Carlson, 1986) and mesungulatid dryolestoids (Crompton et al., 1994), all Mesozoic mammals examined to date, which have prismatic enamel, have variations of Plesiomorphic Prismatic Enamel (PPE) (Wood and Stern, 1997; also see descriptions and definitions below). Previous work by Grine et al. (1979a,b),Grine and Vrba (1980), Lester and Koenigswald (1989), Koenigswald and Clemens (1992), Wood (1992, 2000), Stern and Crompton (1995), Sander (1997, 1999), Wood and Stern (1997), and Wood et al. (1999a), among others has established a reasonable hypothesis or framework for the origin and polarity of prismatic structural characters in synapsid tooth enamel. Figure 1 is a diagrammatic summary of our proposed transition from Synapsid Columnar Enamel (SCE) (Sander, 1997) to PPE, essentially by adding a semicylindrical prism sheath to a planar discontinuity (the “prism seam”—see Lester, 1989) already present in SCE. Since the presence or absence of a sheath is the result of a relatively small change in the Tomes process of a single ameloblast, which can be observed ontogenetically as well as phylogenetically, Lester and Koenigswald (1989) and Lester (1989) had already pointed out the relatively simple changes involved in the origin of prismatic enamel, as well as the probable ease of evolutionary reversal to SCE from PPE by losing the sheath once attained. Rowe (1988), in his landmark phylogenetic analysis of early mammals, scored enamel as (1) prismatic or (0) nonprismatic. Wible (1991) provisionally dismissed enamel characters because of poor taxonomic sampling and contradictory information about the condition of the enamel in certain taxa. Wood and Stern (1997) and Clemens (1997) mapped enamel characters inconclusively upon trees based on other characters, which to some degree updated a more fundamental or comprehensive mapping by Carlson (1990). In a more recent phylogenetic analysis of basal mammals and/or mammaliaforms, Luo et al. (2002) incorporated a single enamel character in their database. In summary, we provide here new microstructural characters and data on taxa scored as “?,” and for other

Recoding Mesozoic Mammal Enamel

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Table I. Scoring for Seven Microstructure Characters of the Mammalian Enamela

Probainognathus Tritylodontids Tritheledontids Adelobasileous Sinoconodon Morganucodon Megazostrodon Dinnetherium Haldanodon Hadrocodium Kuehneotherium Shuotherium Ambondro Ausktribosphenos Bishops Stereopodon Teinolophos Obdurodon Ornithorhynchus Gobiconiodon Amphilestes Jeholodens Priacodon Trioracodon Haramiyavia Plagiaulacidans Cimolodontans Tinodon Zhangheotherium Peramus Amphitherium Dryolestes Henkelotherium Vincelestes Kielantherium Aegialodon Deltatheridium Asiatherium Kokopellia Pucadelphys Didelphis Pappotherium Erinaceus Asioryctes Prokennalestes Montanalestes

P. shab

Seams

Pack

IPM

OAPZ

P. size

P. orient

0 0 1 ? 1 1 1 1 0 ? 0 ? ? ? ? ? ? 2 0 1 ? ? 1 ? 0 0 1 ? ? ? ? 1 1 1 ? ? 1 ? ? ? 2 ? 2 ? 1 ?

0 0 0 ? 0 0 0 0 0 ? 0 ? ? ? ? ? ? 1 1 0 ? ? 0 ? 0 0 1 ? ? ? ? 0 0 0 ? ? 0 ? ? ? 1 ? 1 ? 0 ?

? ? 0 ? 1 1 0 1 ? ? ? ? ? ? ? ? ? 2 ? 1 ? ? 1 ? ? ? 0 ? ? ? ? 0 0 0 ? ? 0 ? ? ? 2 ? 2 ? 0 ?

? ? 0 ? 0 0 0 0 ? ? ? ? ? ? ? ? ? 1 ? 0 ? ? 0 ? ? ? 0 ? ? ? ? 0 0 0 ? ? 0 ? ? ? 1 ? 1 ? 0 ?

0 0 1 ? 0 0 1 0 0 ? ? ? ? ? ? ? ? ? ? 0 ? ? 0 ? ? ? ? ? ? ? ? 0 ? 0 ? ? 0 ? ? ? 1 ? 1 ? 0 ?

? ? 0 ? 0 0 0 0 ? ? ? ? ? ? ? ? ? 0 ? 0 ? ? 0 ? ? ? 0/1 ? ? ? ? 0 0 ? ? ? 0 ? ? ? 0 ? 0 ? 0 ?

0 0 0 ? 0 0 0 0 0 ? ? ? ? ? ? ? ? 0 ? 0 ? ? 0 ? ? 0 0 ? ? ? ? 0 0 0 ? ? 0 ? ? ? 0 ? 0 ? 0 0

a The

first five were included in the matrix as characters 276–280, following continuous numeration of the matrix in Luo et al. (2002). b Abbreviations: IPM, Interprismatic matrix; OAPZ, outer a-prismatic zone; Or, Orientation; P, Prism; Pack, packing; Sha, shape.

relevant taxa not previously included in the matrix that served as the basis for analysis by Luo et al. (2002). Our purpose is to test the effect of these additional characters upon the published tree topology, and to improve our view of the distributional pattern of prismatic (versus nonprismatic) enamel microstructure amongst early mammals.

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Fig. 1. Hypothesis for transformation of Synapsid Columnar Enamel (SCE, Sander, 1997, 1999) to Plesiomorphic Prismatic Enamel (PPE, Wood and Stern, 1997). A. SCE has discontinuous and irregular columns defined by planar discontinuities (after Sander, 1997; the top of Sander’s illustration depicts ameloblasts with uncertain or unknown (?) relationship to the maturing enamel pattern below). The ameloblast epithelium would have been located beyond the left-facing surfaces of the blocks in B and C. B. A “transitional” stage (after Wood and Stern, 1997) with incipient sheath discontinuities caused by thin bundles of crystallites, at less than ◦ 45 to the other (radial or interprismatic) crystallites, on the dentine–enamel junction (DEJ) side of some planar discontinuities, which are now incipient “prism seams.” This transitional structure appears to be present in Sinoconodon, Morganucodon, and Dinnetherium (Wood, 2000), as well as in Gobiconodon and some other “triconodonts” (Wood and Stern, 1997). C. Fully formed PPE (after Wood and Stern, 1997), such as found in Pachygenelus and Megazostrodon (Stern and Crompton, 1995), and in virtually all Mesozoic “tribotheres” examined to ◦ date (Wood, 2000). Here the larger prism bundle of crystallites is at a higher angle (at least 45 ) to the radial, interprismatic crystallites, and thus a clear, arc-shaped sheath discontinuity results on the DEJ side where radial crystallites abut prism crystallites. Additional abbreviations are as follows: sh, prism sheath; sm, prism seam; ip, interprismatic matrix.

METHODS AND MATERIALS Preparation R Whenever possible, teeth are embedded in laboratory resin (usually Epofix (Nielsen R and Maiboe, 2000) or Araldite ), then ground and polished in three standard planes of section (horizontal, parasagittal or radial, and transverse or radial; see Wood (2000) and Koenigswald and Sander (1997b)). At final stages of preparation, a maximally flat, polished surface may be obtained with polishing cloth (on a rotary wheel) and either diamond or alumina grit. Our standard is 0.05-µm alumina in suspension. Finally, polishing 15–20 s with the Dentsply ProphyjetTM device (Boyde, 1984) is also vital to bring out details of individual crystallites and to see enamel tubules clearly, when tubules are present. The last step of acid etching is easier to control from 50 to 70 s (for example) with a milder acid, than


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to try to control between 3 and 6 s while using a more aggressive reagent. Our standard is to etch with 1% phosphoric acid, from 30 to 90 s depending on the degree of surface relief desired. Re-deposition of (relatively insoluble) salts, when it occurs, seems to be removed effectively by 20–40 s under ultrasonication in a water or ethanol bath prior to sputter coating of the specimens. Further review of preparation methods including experimental data on a variety of etchants may be found in Carlson and Krause (1985) and Grine (1986). As the micrographs in Fig. 2 demonstrate, proper etch combined with polish to a very smooth, flat surface is of primary importance in being able to “see” the subtle details of structure (mainly at the crystallite and prism levels—see Koenigswald and Clemens, 1992) that are present in the PPE. Definition of PPE As stated above most taxa included in the phylogenetic analysis have plesiomorphic prismatic enamel. It is, therefore, appropriate to characterize or to define PPE as a combination of the following (assumed to be independent) states: a) b) c) d) e) f) g)

Small prisms (2.5–6.5 µm on average). Prisms bounded by arc-shaped sheath, open toward the outer enamel surface (OES). Prisms bisected by linear (actually planar) seam. Prisms hexagonally packed. Prisms separated by interprismatic matrix on all sides. Prisms with no decussating or abrupt simultaneous change of direction. Schmeltzmuster consists of inner radial enamel with a relatively thick outer aprismatic layer. Phylogenetic Analysis and Methods

We attempted two approaches to re-coding enamel characters in order to test the effects upon the trees generated by Luo et al. (2002). First, we simply filled in the matrix with new data unavailable to Luo et al. (2002), or with corrected enamel character states for taxa such as (for example) Gobiconodon or Ornithorhynchus. The second approach involved running the matrix with more enamel characters added, other than simple absence or presence of prisms. In doing so, we have effectively deleted character 118 (enamel microstructure) and replaced it by characters B, C, D, E and G (see Discussion). Characters A and F are relevant for studies at a different phylogenetic level than the one attempted here. Character A presents different conditions within Multituberculata, but based on the distribution of the character states on this tree it seems more parsimonious to conclude that small prism size, and not gigantoprismatic, is the ancestral condition for multituberculates. Heuristic searches of the modified matrices were performed on Nona (v. 2.0) and Pee-Wee with a minimum of 400 repetitions and the use of the “ratchet” function to maximize the chances of finding a global optimum. Only unambiguous synapomorphies were used to diagnose nodes. Upper and lower molar teeth are denoted with M and m, respectively.

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Fig. 2. Three views of the same specimen of Pediomys (uncatalogued lower molar, Late Cretaceous metatherian from Bug Creek Anthills, Montana; see Clemens, 1966) in horizontal section, to illustrate difficulty in viewing PPE structure with incomplete preparation of the cross-sectional surface. All micrographs in figure at approximately same scale (bar in C. = 6.25 µm). A. Naturally broken surface, etched with phosphoric acid but not polished prior to etching (after Wood and Stern, 1997). A thick outer aprismatic zone (AP) with incremental lines is evident, but structure is unclear in the inner zone. B. A section polished with 600 grit paper but not with finer abrasive or with the ProphyjetTM device; acid etching has revealed some problematic structure, but re-deposition from the acid and a relatively uneven surface make it difficult to see prisms clearly (after Wood, 2000). C. Although acid has been applied for a relatively short time and the structure therefore does not stand out in high relief, it is easy to see clear prisms, seams, tubules, and differences between and inner vs. outer layers in the enamel. This view demonstrates a highly polished surface that has been through all the steps of preparation described in the text (after Wood, 2000). OES, outer enamel surface; other abbreviations are given in the legend to Fig. 1.


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RESULTS New Enamel Description Vincelestes Specimens: right M3, V. neuquenianus, La Amarga, Patagonia, Argentina; left I 4, V. neuquenianus, La Amarga Fm. (Early Cretaceous, Bonaparte, 1986; Rougier et al., 1992), Argentina. Isolated teeth, found in association with the hypodigm; no number. Qualitatively, Vincelestes has enamel in some ways reminiscent of certain therian stem taxa (Lester and Koenigswald, 1989; Wood, 2000) yet in other ways unique or uniquely in combination with structure observed in some specimens of the basal taxa, Morganucodon or Dinnetherium (Wood, 2000). As seen in Fig. 3, the prism sheaths in this specimen of Vincelestes are somewhat flatter arcs (sometimes virtually straight lines in the plane of section) than usual in PPE, bisected by very prominent prism seams; in these features Vincelestes most resembles the “eupantothere” Laolestes (Wood et al., 1999a) and the undetermined Guimarota pantothere described by Lester and Koenigswald (1989). The structure of Vincelestes is unique in that the prisms are erratically spaced, but intimately associated with very strong incremental growth lines in the enamel. A similar appearance is evident in limited views of Dinnetherium and especially Morganucodon (Figs. 24 and 27 of Wood, 2000), yet the sheaths in Vincelestes are much more distinct as well as larger than in these earlier genera. The occasionally convoluted course of some of the incremental lines in several of the Vincelestes photomicrographs (see Fig. 3(A)) is unique to our experience (but see Sander, 1999), and raises potentially interesting ontogenetic questions as well, such as the cause of such prominent lines and how or why the course of the lines is so irregular. Finally, it is interesting to note that (in comparison to the descriptions of dryolestoids and Monotrematum below) enamel tubules are not obvious in the Vincelestes micrographs, unless indistinctly but rarely present in the first few µm after the dentine-enamel junction (DEJ). It is also interesting to note that relatively flat sheaths, prominent seams, and absent or inconspicuous tubules are uniquely (among metatheres observed) present in Didelphodon from the Late Cretaceous of North America (Wood, 2000). Other than in these particular features, however, there is little resemblance between Didelphodon and Vincelestes. La Colonia Dryolestoid Specimens: left M1 (#1), La Colonia Fm (Maastrichtian, Pascual et al., 1999a), Patagonia, Argentina; paracone of M1 (#2), La Colonia, Patagonia; protoconid, undetermined lower molar (#3), La Colonia, Patagonia. The enamel structure of this species is very similar, if not identical to that already described from Mesungulatum (Crompton et al., 1994). The close resemblance agrees with the attribution of the La Colonia dryolestoids to Mesungulatidae (Rougier et al., 2000, 2001, 2003a,b). The enamel is relatively thick (>100 µm in most views), as expected from a relatively large animal. In all characteristics, the enamel of this species exhibits the PPE morphology. The prisms are normal in size (∼2–5 µm), well separated, hexagonally packed, and not erratic in occurrence, and they display seams especially toward the outer enamel surface. In most views (see Fig. 4), there is a thick outer aprismatic (AP) zone (20–25% of total thickness) with incremental growth lines that rapidly fade toward the outer surface. Prominent enamel tubules are, however, an especially notable feature in

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Fig. 3. Enamel structure in Vincelestes. A. A single, combined sheath and seam are visible in this view, with several other linear features that probably are seams alone; the more interesting feature here is, however, the convoluted and discontinuous incremental growth lines that are otherwise roughly parallel to DEJ and OES surfaces (1500×). B. Strong incremental lines combined with erratically spaced sheaths and seams (micrograph @750×; compared to Figs. 24 and 27 in Wood, 2000). C. Thinner area of enamel (@750×) with distinct outer AP zone, sheaths, and seams; note the very flat or straight appearance of many sheaths. Abbreviations as in Figs. 1 and 2.


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Fig. 4. Enamel structure in the La Colonia mesungulatid. A. Relatively thick enamel (@250×) from DEJ to OES, with relatively broad area of outer AP enamel below OES. B. Prisms and tubules near the DEJ in another area of the section (@1500×); note an abundance of tubules toward the DEJ, many associated with the open ends of arc-shaped sheaths, and a decrease (to almost total absence) of tubules toward the lower right (in the direction of the OES). C. Near the outer AP zone and closer to the OES (@2500×); note relatively smaller prism widths, absence of tubules, and more easily seen prism seams. D. Prisms near the DEJ (also @2500×); due to lower angle of intersection with plane of section, some tubules appear to be more obviously tube-shaped features. Some prism seams are also visible in this view; it is not clear, however, to what degree some tubules may (or may not be) continuous over the DEJ. t = tubule; other abbreviations as in Figs. 1 and 2.

these photomicrographs. The tubules are abundant in the inner 20–25% of the enamel layer (near the DEJ) but rapidly become sparse or entirely absent beyond the inner zone. A few of the tubules seem to have random distribution within the interprismatic areas, but the majority of them have regular positions at the open ends of prisms, slightly toward the OES from the ends of the sheath arcs. Tubules do not accompany every prism, but the position

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of the tubules is the same as that of prism seams that are visible in prisms closer to the outer AP zone. In specimen #1, none of the inner prisms (accompanied by tubules or not) has detectable seams; in specimen #3, however, seams and a few tubules occur together. Most intriguingly, this distinct association of tubules with the open sides of prisms has also been observed in Mesungulatum and Groebertherium (Crompton et al., 1994), as well as in Monotrematum and reigitherids, as described below. It has furthermore been noted (Wood, 1992; Wood and Stern, 1997) that a similar occurrence of tubules appears in illustrations of enamel from the enigmatic genus, Gondwanatherium (Sigogneau-Russell et al., 1991, Figs. 3(E) and 4(A)). The phylogenetic significance of these observations, if there is any, has yet to be ascertained, although the geographic and chronological association of the taxa provides additional interest that has not escaped our attention. Reigitheridae Indet Specimen: upper molar, La Colonia Fm (Maastrichtian, Pascual et al., 1999), Patagonia, Argentina. In addition to itscontroversial taxonomic position (Gondwanan docodont versus dryolestoid, recently clarified as the latter by Rougier, 2003a,b) this South American, Cretaceous taxon is worthy of note because its enamel (Fig. 5) shares the peculiar tubule characteristics described above for Mesungulatum, the La Colonia dryolestoids, and Monotrematum. If the microstructure of the molar studied here is representative, Reigitherium, from the Maastrichtian Los Alamitos Formation (Andreis 1987; Andreis et al., 1990) and its relative from La Colonia are similar to Monotrematum in that, from available views, no clear prism seams have as yet been detected. Reigitherids seem to be unique, compared to these other genera, in the degree to which the prisms are packed close together. In some of the micrographs the sheaths are almost “shoulder-to-shoulder” similar to Boyde’s (1967, 1976) “type 3” enamel pattern. It is our interpretation, as discussed below, that lack of prism seams and close packing (with minimal interprismatic area) are both derived states compared to PPE. The reigitherid specimen does, however, retain a varyingly thick outer AP zone in all micrographs obtained to date. Although there are only partial similarities between Reigitherium and the South American dryolestoids, the difference compared to docodonts is more specific; the enamel microstructure therefore partially makes docodont affinities for Reigitherium (Pascual et al., 1999b) unlikely, yet partially supports links with dryolestoids (Rougier et al., 2003a,b). Monotrematum Specimen: upper molar (M1?), Salamanca Formation “ Banco negro inferior,” Lower Paleocene (Pascual et al., 1992a,b; Bond et al., 1995; Gelfo and Pascual, 2001), Patagonia Argentina. Monotrematum has relatively thick enamel (Fig. 6) that, qualitatively, most resembles that of the Los Alamitos and other South American Cretaceous (i.e., la Colonia) dryolestoids. The enamel in Fig. 6 is not deeply etched, but a number of prominent tubules are located on the open sides of prisms (i.e., toward the OES). As in dryolestoids, not all prisms are associated with a tubule, and tubules are not visible among prisms closer to the OES. The main difference in the available micrographs of Monotrematum, compared


Fig. 5. Enamel structure in the La Colonia regitherid. A. Crown view of upper molar. B. Horizontal section of enamel from DEJ to OES; note abundance of tubules in inner zone, substantial outer AP zone, and relatively close packing of prisms throughout. A few isolated tubules extending beyond prisms into the AP zone appear to be real and not artifactual. Prisms are 3–5 µm in width. C. Higher magnification of prisms near the DEJ; note that the prisms and tubules are at a lower angle to the plane of section than in B, but that the tubules here are not as abundant nor as definitely associated with the open ends of prisms. D. Prisms and tubules in a thicker region of the enamel layer on the specimen, OES toward lower right corner of micrograph; note that the more abundant tubules toward the upper left appear to be most often associated with the open ends of prisms. Abbreviations as in Figs. 1 and 2.

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Fig. 6. Enamel structure in Monotrematum. A. Un-embedded upper molar, hand-polished facet on enamel band toward right side of micrograph, on the unbroken enamel loop. B. Prisms and tubules toward the middle of the polished and etched surface (OES to right); some prisms (which are about 3–5 µm wide) have been more completely etched into view than others. C. Prisms and tubules at higher magnification; note that although not all prisms are associated with tubules, the majority of tubules are associated with the open ends of prisms; note also that in this view (as well as in B) there is no clear indication of prism seams. Abbreviations as in Figs. 1, 2, and 4.

to the dryolestoids, is that no prism seams have been observed at any level or location within the enamel layer, which would therefore be regarded as derived in this feature unless contradicted by later observation. Otherwise, with hexagonal packing, substantial interprismatic area, and a wide outer AP zone, this enamel has all the remaining attributes of generalized PPE.


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Prokennalestes (and Procerberus) Specimens: talonid, left m2, Prokennalestes major, Khoboor, Mongolia; talonid, left m3, Prokennalestes major, Khoboor, Mongolia; trigonid, left m3, Prokennalestes major, Khoboor, Mongolia. Uncatalogued m1 or m2, Procerberus, Bug Creek Anthills, Montana, Museum of Comparative Zoology collections. The well-developed plesiomorphic prismatic enamel (Fig. 7) from these specimens of eutherian Prokennalestes is indistinguishable from that in other early therian taxa such as metatherian Deltatheridium (Wood, 2000), or eutherian Procerberus (Fig. 8; also see, Lester, 1989). Deltatheridium qualitatively appears to have slightly more erratic prism distribution in some areas of the crown, and the prism diameters may be slightly smaller than in the other taxa (thus appearing to be more similar to metatherian Alphadon than to metatherian Pediomys (Wood, 2000)), but these differences have yet to be quantified effectively. In essence, however, all of these taxa including Prokennalestes exhibit the full set of character states that define PPE. In the micrographs obtained, a few tubules may be indicated penetrating the enamel near the DEJ, but there are not any visible signs that they would extend more than a few µm toward the OES. In some areas of the crown enamel prisms become smaller and more tilted to the plane of section (with more prominent prism seams) as sometimes observed toward the OES of PPE, yet there is little or no zone of outer AP enamel before the OES. In other areas, however, in addition to the usual changes in prisms there is also a relatively thick outer zone of AP enamel toward the OES. Although Lester (1989) has more effectively illustrated the structure of Procerberus, the views in Fig. 8 are notable, not only for their overall resemblance to Prokennalestes, etc., but also because they depict prisms, prism sheaths, and prism seams three-dimensionally from DEJ to OES. As in other views (see Figs. 3(C) and 7(B)) of enamel in other taxa, Fig. 8(A) and (B) also illustrates the ontogenetic “reversal” of prismatic structure to (effectively) SCE where prism sheaths and prismatic crystallites drop out but prism seams continue for an additional distance into the outer AP zone.

Glirodon grandis Specimen: DINO 10822, Lm1, Morrison Formation (Late Jurassic), Fruita, Colorado (Simmons, 1993; Engelmann and Callison, 1999). A single horizontal section through several cusps on this plesiomorphic multituberculate molar (Fig. 9) indicates abundant tubules in the inner zone of the enamel, but virtually no sign of prism sheaths or planar seams that would appear to divide the enamel into radial columns typical of SCE (Sander, 1997). It would therefore be correct to say that this taxon has prismless enamel, but incorrect to say that it has the ancestral condition of SCE that is typical of nonmammaliaform cynodonts (except the trithelodont, Pachygenelus, which has PPE) or other nonmammaliaform synapsids (Sander, 1999; Luo et al., 2002). This observation is in conflict with reports of SCE in other basal multituberculates from the Jurassic (Fosse et al., 1985; Simmons, 1993; Kielan-Jaworowska and Hurum 2001). In fact, this species is what Simmons (1993) calls “Morrison multituberculate,” scoring it as having gigantoprismatic enamel (Simmons, 1993, p. 155, who refers to an abstract by Engelmann et al., 1990, in which she is a coauthor). Kielan-Jaworowska and Hurum (2001) follow Simmons (1993) scoring for Glirodon. We are unaware of the material basis for such a

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Fig. 7. Enamel structure in Prokennalestes. A. Horizontal section from DEJ to OES (@1500×); note that in this section, although there is less outer AP zone than elsewhere on the crown (not illustrated), prisms nevertheless become smaller toward the OES until in some cases the sheath has all but dropped out, leaving only some extension of the planar seam into the AP zone. B. Another view of horizontal section (also @1500×), in which the prisms intersect the plane of section at slightly higher angles than in A (thus the full extent of the prismatic crystallite bundle is visible); very faint incremental lines are visible in the relatively narrow outer AP zone. Abbreviations as in Figs. 1 and 2.

scoring in Glirodon because no data have ever been published. The strong contrast of our observation on the same species raises some doubts about the original interpretation. Character Analysis Dentine, cementum, mineralogical and protein composition have been surveyed among living and fossil mammals (for instance Fong et al., 2003; Botha et al., 2004) with, in most


Fig. 8. Enamel structure in Procerberus. A. Broken, etched, but nonpolished oblique section from DEJ to OES (800×), in which prisms are at a very low angle (almost parallel) to the plane of section. Although not so obvious from this view, the structure is in every way very comparable to that seen from Prokennalestes (Fig. 7). B. Area from upper left portion of A (box), near OES, at higher magnification; a smaller prism, sheath, and prism seam are pointed out (arrows) where they come to an end in the narrow outer AP zone. C. Area from lower portion of A (box), near DEJ, at higher magnification. Prismatic crystallites, prism sheaths, and prism seams are all visible together in this plane of section. Abbreviations as in Figs. 1 and 2.

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Fig. 9. Enamel structure in the Morrison multituberculate, Glirodon grandis. A. Crown view of molar specimen before embedding in resin (44×). B. Embedded specimen after polishing and etching (42×). C. Horizontal section of enamel (OES toward lower left) from B, at higher magnification (700×); incremental lines are visible through most of the layer, especially toward the OES, and some minor convergences and divergences of crystallite orientation are visible, but there do not appear to be any distinct discontinuities to divide the enamel into columns of any type. D. Higher magnification within C (1500×), DEJ to upper right. Tubules and tubule fillings of some kind are abundant in the dentine, and many appear to cross the DEJ and continue into the enamel for at least a third of the thickness toward the OES. E. View of enamel layer in another part of the plane of section, DEJ to lower right (930×). Again, although tubules and tubule fillings are abundant in both dentine and enamel (and here a few tubules extend a much greater distance toward the OES), no obvious prisms or seams occur; the same pertains to all areas of enamel, on all cusps visible in the plane of section. Abbreviations as in Figs. 1 and 2.

cases, ultimately clinical purposes, but are mostly absent from wide ranging phylogenetic studies. Edentates, because of their almost complete absence of enamel (Simpson, 1932; unpublished information), are one of the few groups where dentine and cementum have been used in a phylogenetic context (Ferigolo, 1985; Kalthoff, 2004). Although we recognize


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the potential phylogenetic importance of dentine and cementum, these tissues lay outside our area of expertise and were not studied in the specimens examined here. We recognize the following enamel characters and propose the following character states: a) b) c) d) e)

Prism size: (0) small (i.e., “normal”), 2.5–6.5 µm; (1) large, >7.5 µm Sheath: (0) absent; (1) arc-shaped; (2) fully enclosed Prism seam: (0) present; (1) absent Prism packing: (0) hexagonal; (1) erratic; (2) stacked in rows Interprismatic matrix: (0) on all sides, widely separates prisms; (1) distinct inter-row sheets; (2) prisms “shoulder to shoulder,” IPM therefore not abundant f) Prism orientation: (0) parallel (radial); (1) simultaneous change of direction; (2) decussating Hunter–Schreger bands g) Thick outer AP layer: (0) present; (1) absent

The possible phylogenetic impact of enamel characters is currently hindered by the less than comprehensive understanding we have of factors such as function, diagenetic process, planes of section, and possibly a multitude of other biological properties; however, the poor taxonomic sampling density is undoubtedly the biggest obstacle for a full appreciation of the importance, or lack thereof, of enamel traits for phylogeny. The most complete characters (276, 277) are scored for 50% of the taxa while the remaining are known only in about 30% of the taxa. To isolate the impact of the enamel characters here recognized and the information of taxa previously scored as “?,” all the original characters by Luo et al. (2002) were retained exactly as originally proposed except character 118, which was effectively deleted by scoring all its entries as “?” and adding five new microstructure characters at the end of the character list. Maintenance of the matrix in this way retains the original numeration of the characters with numbers above 118 in Luo et al. (2002), and also facilitates cross-reference. Of the seven enamel characters discussed above, only five showed enough taxonomic variation at the level considered to be included in this matrix. The two excluded are (1) prism size and (2) prism orientation; these characters are part of our quest for a broader standard in the characterization of mammalian enamel. Prism Size (0) small, or “normal” (2.5–6.5 µm); (1) large, “gigantoprismatic” (>7.5 µm). Division of the two groups by size is based on Krause and Carlson (1986), who found that averages grouped into a distinctly bimodal distribution by taxon with statistically little or no overlap. Among the Mammaliaformes for which this character can be studied only taeniolabidoid cimolodontans show the derived condition (Fosse, 2003). Fosse (2003) may have used the term “normal” for other than “gigantoprismatic” prisms because KielanJaworowska and Hurum (2001) adopted a new term, “microprismatic” for smaller prisms in multituberculates; but in fact all prisms known in all mammals (other than those multituberculates with gigantoprisms) have average diameters (per species) that range from about 2.5 to about 6.5 µm. Therefore, we suggest here that the term “microprismatic” should be suppressed. Dumont (1996) has tabulated data from a remarkably extensive survey of dentally plesiomorphic eutherian taxa in which statistical variability of prism diameters and center-to-center distances is intriguing yet not very large.

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Accepting prior phylogenetic hypotheses of multituberculate relationships (KielanJaworowska and Hurum, 1997, 2001; Rougier et al., 1997) taeniolabidoids would be nested among other cimolodontan groups that show conditions called “normal” by Fosse (2003). For example, Carlson (1990) illustrated the djadochtatherian Kryptobaatar with normal-sized prisms. Therefore, the gigantoprismatic condition would be nested amidst plesiomorphic conditions that are present among the successive “plagiaulacidan” outgroups, ptilodontoids and djadochtatherians. The multituberculate taxa included in Luo et al. (2002) are supra-generic groups represented by cimolodontans and plagiaulacidans. Therefore, even under lax optimization the condition for cimolodontans would be at least polymorphic, resulting in a character without enough variability to carry a phylogenetic signal within the taxonomic framework considered here. Multituberculates as a group would have normalsized prisms as the primitive condition, merging into the common microstructure (PPE) present in generalized Mesozoic mammals. The reported presence of gigantoprismatic enamel in some Early Cretaceous multituberculates (Fosse et al., 1985) complicates the historical mapping of this character. Regardless of the condition considered ancestral for multituberculates, a problem ultimately out of the scope of this contribution, this character may be plagued by homoplasy. Prism Orientation All the taxa included here have parallel, radial prism orientation, which in premolars and molars does not abruptly change through the thickness of the enamel. The derived states for this character, i.e., (1) simultaneous direction change and (2) Hunter–Schreger bands, occur only among therians (defined as in Rougier et al., 1998) more deeply nested than those employed here to represent the ancestral conditions for the group. Therefore, although the presence and characteristics of HSB have been used inside Placentalia (for example Glires, carnivores, primates, artiodactyls, etc. (Meng and Wyss, 1994; Koenigswald, 1997; Martin, 1997)) they are not informative at the level of the exploratory analysis that we perform here. Phylogenetic Analysis Our analysis was performed in two ways. First, the original 275 characters from Luo et al. (2002) were employed with corrections to the scoring of character 118, either based on a different interpretation of the observable structures or by completing cells coded as “?,” based on new data. The corrections were: A new state was created for Ornithorhynchus, because the condition in the platypus (Lester and Boyde, 1986) is unlike SCE and likely results from the reduction of the prismatic enamel probably present in the common ancestor of all monotremes as indicated by the presence of fully prismatic enamel in Monotrematum (this study) and Obdurodon (Lester and Archer, 1986; Lester et al., 1987). Probainognathus was changed from ? to 0 (SCE, based on Sander, 1997), and Vincelestes and Prokennalestes were scored as 2, that is PPE based on the specimens described here. Gobiconodon was changed from condition 1 (intermediate) to 2, because the prisms are fully formed but the prisms are arranged in an erratic, loosely spaced packing pattern. The matrix was run with NONA (version 9.3, Golloboff, 1993) and TNT (Golloboff, 2003) and latter modifications, following different heuristic searches, which included various combinations of mult∗ n; max∗ ; nixwts, etc. These searches resulted in 20 trees with consensus far less resolved than


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that of Fig. 1 of Luo et al. (2002). The statistics for the individual trees are length = 890, ci = .47, and ri = .76. The consensus has a length of 946, collapsing four nodes at the same levels as in the original tree by Luo et al. (2002). In some of the most parsimonious trees (MPT) the presence of PPE is diagnostic of node 3 (Fig. 1 of Luo et al., 2002): that is crown-group Mammalia, expressed as a major dichotomy between the australosphenidans and the traditional tribosphenic lineage. This distribution of prismatic enamel is similar to the results of Luo et al. (2002); Priacodon, with a condition 1 (intermediate) would represent an autapomorphic state if the characters are treated as unordered, or a reversion if they were ordered. However, in most of the MPT, especially in those that place Tinodon, Kuehneotherium, or both at the base of Australosphenida, this character loses all diagnostic value and becomes equivocal. The second analysis resulted from the compilation of 279 characters (275 from Luo et al. (2002), minus character 118, plus five introduced here [B–E, G]). This new iteration resulted in 22 trees (see Fig. 10) with a length of 906 steps (ci = .47; ri = .75). The strict consensus collapsed 11 nodes, resulting in a topology essentially identical to that produced by our previous iteration. A multichotomy involving seven branches is located at the base of Mammalia; included in this multichotomy are Triconodontidae, australosphenidans, Tinodon, Gobiconodon, Kuehneotherium, Amphilestes, and the stem lineage leading to

Fig. 10. Strict consensus of 22 equally most parsimonious trees resulting from the replacement of Character 118 of Luo et al. (2002) for the characters discussed in this study. The length of the individual trees was 906 steps. The topology of the consensus shows a multichotomy formed by several distinct groups, Theria, Triconodonta, Australosphenida, and some isolated taxa. The alternative resolutions of this consensus correspond mostly to different positions for Amphilestes and Gobiconodon, which are alternatively allied to different members of the polytomy and the permutation among the terminal eutherians. The lack of resolution in the cladogram at the level traditionally considered Mammalia (crown-group definition) underscores the fragility of our current phylogenetic understanding.

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Tribosphenida. The trees produced by the original matrix are congruent with those resulting from the extended matrix; however, a few of the tree topologies of the modified matrix cannot be found in the first iteration and account for the larger number of MPT and the less resolved strict consensus topology. The most substantial alternative resolutions involve a position for triconodonts basal to Mammalia (i.e., australosphenidans and Tribosphenida) and the relocation of Kuehneotherium, Tinodon, or either one of them to the australosphenidan lineage. These alternative resolutions may suggest tantalizing scenarios, for example some kind of validation of previous statements about distant relationship of Kuehneotherium to therians (Rougier et al., 1996a,b, 2001; Sigogneau-Russell and Ensom, 1998) by removing Kuehneotherium from the lineage leading to therians. However, it is still true, and perhaps comforting, that the tree topology continues to be governed by the remaining macro-morphological characters. By mapping the distribution of the enamel characters on the resulting topology it is apparent that in most cases there are not enough taxa known for the characters to be diagnostic at any meaningful level of the cladogram. This is due in large measure not solely to the lack of sufficient cell scores, but also to the apparently common reversions of some of the characters under the dominant topology. DISCUSSION There is considerable support for “PPE” as a synapomorphy of trithelodonts plus Mammalia (Mammaliamorpha, Rowe, 1988) with several early and later reversals to “SCE” or less organized structure. Despite the occurrence of enamel prisms in mammals only (the one exception being in the agamid lizard, Uromastyx, in which prisms are clearly nonhomologous to those in mammals (Cooper and Poole, 1973; Wood and Stern, 1997; Sander, 1999)) some question has lingered regarding a multiple versus a single origin of prismatic structure in mammals (Clemens, 1997; Sahni and Koenigswald, 1997; Pascual et al., 1999a). For example, does the prismatic enamel in multituberculates represent an independent origin of the structure (Krause and Carlson, 1986), or was there an early reversal from PPE to SCE or even less organized structure in some “plagiaulacidans,” with a later derivation of “gigantoprismatic” structure in certain clades (Wood and Stern, 1997)? As discussed below, the present analysis indicates that multituberculates as a group have normal-sized prisms as the primitive condition, merging into the common microstructure (PPE) present in generalized Mesozoic mammals. The taxonomic sample of this particular matrix is not designed to fully test multituberculate interrelationships, but the normal prismatic, or nonprismatic enamel results as a likely basal character for the group. “Gigantoprismatic” structure would therefore be a derived state autapomorphically nested within the larger multituberculate clade. With the exceptions of those multituberculates and mesungulatid “dryolestoids,” significant derivations from PPE structure do not occur until the Cenozoic. Ornithorhynchus, on the other hand, clearly does not seem to represent a reversion to SCE structure (see Lester et al., 1987) but is a simplification of structure probably related to loss of molar function in adults. Wood et al. (1999a) discussed some of the biological aspects of reversal in enamel patterns, but research in this area remains largely unexplored territory for the future. Fossil and extant whales are another documented case of evolutionary simplification of enamel structure, which has barely been tapped


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for biological and phylogenetic studies (Ishiyama, 1984, 1987; Werth and Stern, 1992; Thewissen, 1994; Maas and Thewissen, 1995; Sahni and Koenigswald, 1997; Wood et al., 1999b). Although not extremely numerous, microstructural characters provide enough variability to have an impact in general phylogenetic studies. Quantitative treatment of some of the morphological variables may provide additional characters for future analyses, but at present we are unsure how to treat them. The impact of function on enamel microstructure is still unclear, at least in terms of homoplasic lability within generic or familial clades of mammals. A recent discussion of the relationship of function to structure in nonprismatic enamel may be found in Donoghue (2001). Crompton et al. (1994) and Rensberger (1997) provide reviews of function and structure among mammals. Other potential microstructure characters, such as enamel (and dentine) tubules, incremental growth lines, relationship of tubules to prisms and prism seams, ratio of outer AP and prismatic zones, and of course quantitative characters in size and packing pattern (Fosse, 2003) provide additional potential for phylogenetic studies. Tubules, in particular, have seemed to be an attractive systematic feature (Tomes, 1897; Carter, 1920, 1922; Osborn, 1974; Sahni and Lester, 1988; Gilkeson, 1997); but difficulty in imaging tubules, as well as questions of ontogeny and homology of them (odontoblastic, ameloblastic, or both—see Lester et al., 1987) has led to less emphasis or no use of tubule characters in more recent phylogenetic studies. Discussing enamel evolution in the context of Luo’s et al. (2002) matrix provides a broad reference framework, but at the same time excludes from discussion some of the taxa studied here or elsewhere, namely Glirodon, Monotrematum, and Docodon. The inclusion of Monotrematum and Docodon increases the number of MPT by collapsing the cuspidal monotremes (this based only in the scoring of microstructural characters). These examples of excluded taxa are particularly appropriate because they do not show the same conditions as other putative members of the same groups for some of the microstructural characters, which highlights the variability of some of these features. Particular problems relating to these taxa are discussed below.

Character Optimization 276 Sheath poorly differentiated or no sheath (0), arc-shaped sheath (1), or fully enclosed prism (usually circular or ovate sheath) (2). Condition 0, that is a poorly differentiated arc, is present in the basal-most portions of the tree; from tritheledontids up to the multichotomy that includes the root of Mammalia (Monotremata) the dominant and primitive condition is to have condition 1, or well developed arcs. Haldanodon and Kuehneotherium would be reversals to condition 0 under this tree topology. The australosphenidans are scored mostly “?” with the exception of the monotremes, which show a problematic distribution: Ornithorhynchus has poorly differentiated prisms (0), while Obdurodon presents fully enclosed prisms (see below for discussion of monotremes). Condition 1 continues to be primitive among nontribosphenic cladotheres and basal tribosphenic mammals. The presence of enclosed prisms (2) in Didelphis and Erinaceus currently renders equivocal the primitive state for crown group Theria.

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277 Prism seams: absent (0) present (1). Seams are present all the way up the tree to the base of Theria, where it becomes equivocal because of the acquisition of the derived state by Didelphis and Erinaceus, whereas Prokennalestes retains the primitive condition. The autralosphenidan branch is equivocal with the exception of the monotremes Obdurodon and Ornithorhynchus, which are diagnosed by the absence of seams (1), incidentally supported by absence of them in Monotrematum also. 278 Prism packing. Hexagonal (0), erratic (1), in rows (2). This character has a complex history under the dominating tree topology. Tritheledontids have prismatic enamel arranged in a hexagonal pattern, but it is erratic in morganucodontids and triconodonts (sensu lato). Obdurodon is the only monotreme included in the tree with prismatic enamel; surprisingly Obdurodon shows a highly organized enamel with prisms arranged in rows (see monotreme discussion below). Hexagonal packing diagnoses the stem members of the lineage leading to therians, including multituberculates, cladotheres and basal tribosphenic or quasi tribosphenic forms such as Vincelestes and Deltatheridium. The condition for Theria is equivocal because the opposing states shown by Prokennalestes (0) on one hand, and Erinaceus/Didelphis on the other (2). 279 Interprismatic matrix. On all sides (0), Distinct inter-row sheets (1), or Prisms “shoulder to shoulder” with therefore little IPM (2). This character is not informative with the present distribution. The primitive condition is present at the base of the tree and sporadic appearances of the derived condition are either autapomorphic or result in an equivocal attribution to any given node. There is, however, potential for this feature to become diagnostic at some level once there is a less incomplete record—condition (2), for example, is variably present within Hominidae and Elephantidae. The primitive condition for Theria is once more obscured by the different conditions present in Prokennalestes (0) and the living members of the crown-group (1). 280 Outer aprismatic zone. Present (0); absent (1). With the exception of the living members of Theria the remaining taxa included here show a thick outer aprismatic zone in their enamel. Prokennalestes (0) and Erinaceus (1) differ also in this feature, stressing the numerous differences between basal eutherians and the crown-group Placentalia. Monotreme and Docodont Enamel At the time of writing, three monotremes have been studied for microstructural characters of their enamel, Ornithorhynchus (Lester et al., 1987), Obdurodon (Lester et al., 1987), and Monotrematum (this paper). Monotrematum is known by little else beside teeth (Pascual et al., 1992a,b; Pascual et al., 2002; Forasiepi and Martinelli, 2003). The nature of the monotreme dentition has been the subject of intense research and debate


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(Simpson, 1929; Archer et al., 1985, 1992, 1993; Kielan-Jaworowska et al., 1987; Bonaparte, 1990; Musser and Archer, 1998; Rich et al., 2001, 2002; Woodburne, 2003; Woodburne et al., 2003). We did not include Monotrematum in this study because it was not one of the taxa originally included in the matrix by Luo et al. (2002). However, we did score the taxon as an exercise. The result is further collapse of the consensus tree. Monotremes are problematic with regard to their enamel microstructure. It is commonly accepted that the dentition of the living platypus is reduced and extremely autapomorphic (Luckett and Zeller, 1989; Simpson, 1929), which perhaps helps to explain the reduced enamel and relatively simplified structure. Obdurodon and Monotrematum have well developed and apparently functional dentitions that were likely retained throughout life. These two fossil monotremes differ in the shape of their prisms, their packing, and in the distribution of interprismatic matrix present. With regard to prism shape and packing Monotrematum shows a more generalized condition widely present among Mesozoic groups, while the later Obdurodon shows the same condition as Didelphis and Erinaceus. It is likely that the acquisition of the derived morphology is autapomorphic. With regard to interprismatic matrix, Monotrematum again shows a more generalized morphology, while Obdurodon represents a more derived condition. The rather dramatic variation and diversity of the monotreme enamel reflects the plasticity of mammalian enamel within a given lineage and should serve as a cautionary note against generalizing conditions for a large group based on a limited sample. Docodonts present a similar example of two closely related taxa with fundamental differences in enamel structure. Haldanodon, used in the matrix by Luo et al. (2002) because it is better known and it is believed to be a generalized and primitive docodont (Krusat, 1980; Lillegraven and Krusat, 1991; Martin and Averianov, 2004) does not have prismatic enamel, whereas the closely related Docodon does have it. Closing Comments Our understanding of the variation and significance of enamel microstructure of fossil mammals remains in its infancy but results are encouraging. Enamel provides a set of characters that can be regarded independently of more traditional morphological analysis, and which can be readily integrated into previous matrices. Mammals show enough variation and change to recognize trends and distinctions even among groups relatively small (such as monotremes); a better sampling of fossil and recent taxa is likely to result in an increased utility of enamel characters to diagnose clades, in particular, among the Theria. Continuous characters may add significantly to the study of enamel microstructure, because the nature of the discipline lends itself to mathematical description (Fosse, 2003, for example); however, at present we are reluctant to break a continuum arbitrarily into discrete character states until a better understanding of the genetic principles involved in the production and maintenance of a prismatic enamel is achieved. ACKNOWLEDGMENTS We thank our home institutions for continued support of all kinds. Much of this research has been supported by Providence College faculty support grants (C.A.F.R.—to CBW) and by grants from NSF (DEB 0129061) and the Antorchas Foundation (to GWR). We thank P. D. Polly, P. C. J. Donoghue, and an anonymous reviewer for comments that have improved

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the manuscript. F. Grine gave invaluable advice about preparation techniques early on. We are especially grateful to A. W. Crompton and W. A. Clemens for allowing all this to be started in the first place. Dr. Clemens’ generosity and great good humor during a sabbatical year (1988–1989, CBW) at U.C. Berkeley were particularly important to the earliest stages of this work. It is also our wish, finally, to dedicate a special part of this paper to the memory of our recently departed colleague, Dr. Doris N. Stern, whose pioneering efforts in enamel research have been so inspirational and foundational for the rest of us. Thank you, Doris.

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