I've got a crush on you...

Sharks are an incredibly diverse group of fish.  Most live in the ocean, some live in freshwater, and some move back a forth between both.  "Jaws" perpetuated the image of the shark as an ambush predator, tearing big pieces out of large prey and chewing it up.  Many sharks do eat like this, but more of them swim up onto a school of fish, open their mouths and swallow whatever goes in whole.  Some strain algae and other plankton out of the water, while others eat hard shelled critters like mussels, clams, lobsters, crabs...etc.

The Cretaceous Western Interior Seaway was inhabited by many different kinds of shark.  One of the most peculiar was the durophagous (eats hard-shelled animals) shark Ptychodus.  These sharks had jaws with robust teeth with low roots and massive crowns that could apply three point forces to hard material to break it.  The crowns have transverse ridges and the margin of the crowns are decorated with a number of ridges and bumps (tubercles).  Their mouths were filled with pavement dentitions composed of hundreds of teeth.  Collections of these teeth is seen at right and below (pictures by Mike Everhart).  Note the flattened surfaces caused by wear of the teeth from grinding hard materials.

Although teeth from these sharks are relatively common in the Cretaceous Greenhorn and Niobrara Formations of Kansas, little is truly known about the shark.  It is estimated some species of this shark were up to 11 meters in length.  Since there was abundant hard-shelled prey and little competition, this is entirely possible.  Included in this diet were likely mollusks such as these small inoceramid clams (left).  Nautiloids (think squid with shells) and small fish would have also been important food sources.  The general body shape has been inferred to be fusiform, since the vertebral centra are round.  The fact that these centra are calcified suggest that these are modern sharks (neoselachians).  The only semi-well known skeletal elements of these sharks are the jaws.  No well articulated skeleton of Ptychodus has ever been found, so all attempts at classification of this fish are based on circumstantial evidence.

While I was examining the enameloid of teeth of a 305 million year old shark that I had collected from the Farley Limestone as Park University, I decided that I should examine the enameloid of a more recent shark to understand the difference between primitive and modern sharks.  I did a couple of quick surface digests of Ptychodus teeth with 10% HCl.  After 30 seconds, I was able to see the single crystallite enameloid (SCE) on the surface (figure at right: Panels 1,3, 5 are before digestion and 2, 4, 6 are of single crystallites).  After 3 minutes, I could easily see parallel-bundled enameloid (PBE) crystals on the surface (figure below).  After a couple of days sectioning a tooth, I could see a triple-layered enameloid.  A pretty good week's work I thought.  Then I made the mistake of searching the literature for what was known about Ptychodus tooth ultrastructure.  Turns out, the answer is very little.  But what is accepted says that these teeth do not exhibit a triple-layered enameloid, but rather an SCE.  Based on this observation and ignoring a lot of other evidence, the experts placed this shark among the hybodonts, an ancestral group of modern sharks.

I puzzled over this for quite a while, because my results had seemed so clear-cut.  I repeated these observations on several teeth and in several planes of section, but kept coming up with the same result:  the enameloid of these teeth had a triple-layered structure.  There was a superficial SCE/SLE, PBE on the crown, especially at the level of the transverse ridge, and tangled-bundled enameloid (TBE) next to the dentine.  More careful examination of the literature revealed a couple of other studies that documented a triple-layered enameloid in Ptychodus.  One of the reports was in an obscure journal and the Ptychodus teeth were a side study and only shown in a couple of pictures.  The other report was a Masters thesis which was unpublished.  What had started as an attempt to gain a proper control for one study turned into the main focus of another.  I would have to prove that what I was seeing was a real phenomenon.

The figure at the right shows the PBE adjoining the TBE, and the TBE next to the dentine in sectioned teeth.  Getting just the right images with the correct brightness and contrast took about 6 months.  The enameloid of Ptychodus had a lot of similarities to that of Squalicorax curvatus, including having a TBE that became single crystallite in structure at the enameloid-dentine junction.  Dentinal tubules rise high into the crown, penetrating into the enameloid, much like those seen earlier in my post on the hybodontiform shark.  Preservation of the teeth is amazing and casts of the odontoblasts (tooth-building cells) can be seen below.

The figure at right is my recreation of the one experiment that is cited the most often.  A whole Ptychodus tooth (1, 2, 3 below) was soaked in 10% HCl for 23 minutes, 35 seconds (4, 5, 6) it is easy to see the great degree of erosion in the surface decoration of the tooth.  In 7 and 8 you can see that the enameloid has been eroded to the level of the dentinal tubules, which show up as divots in the surface of the tooth.  The enameloid in this area (9) is single crystallite enameloid in appearance.  The previous studies are correct in interpretation of the results of the experience.  The problem is in the preparation.  The tooth was soaked in acid way too long, and the bundled enameloid layers were destroyed.

This study solidified (for me, at least) the idea that "If it isn't published, it isn't known.  If it is published, ask if it is truly good science in technique and interpretation."  The work done here shows that Ptychodus is not a hybodont (primitive shark or proto-shark), but rather is a selachimorph neoselachian fish (modern shark).  Reviews of the work have been very positive and the paper is cited in the second edition of "Oceans of Kansas" by Mike Everhart, which should be published this Fall.

Oh, the shark has pretty teeth, dear...

In the last post, I talked about the triple-layer enameloid that is one of the defining characteristics of a modern shark.  That enameloid has an outer single crystallite enameloid/shiny-layered enameloid (SCE/SLE) that resists the spread of cracks in the teeth; a middle parallel-bundled enameloid (PBE) that resists compressional (straight down) force; and an inner tangled-bundled enameloid (TBE) that resists rotational (twisting) forces.  These are tremendous properties for a shark that bites big chunks out of something else and needs to chew them up.  A Cretaceous predator, Squalicorax curvatus from the Western Interior Seaway of Kansas (about 90 million years ago), has teeth that everyone would recognize as shark's teeth.  Squalicorax has a labiolingually compressed (flat) shape, with a strong triangular cusp and a shoulder with serrated edges (like a steak knife).  These teeth are perfect for running up on something even bigger than the shark, slicing out a chunk of prey and chewing it up.

Squalicorax curvatus teeth coated with ammonium chloride to show detail

Squalicorax curvatus would be very recognizable to the casual observer as a shark.  It just LOOKS like a shark.  Mouth behind and below snout, large dorsal fin, fusiform body, mouth full of nasty teeth and big (up to about 10 feet long).  A few extremely well preserved specimens have been found, including a nearly complete skeleton (very rare for cartilage - even calcified cartilage) recently sold by PaleoSearch, Inc. in Hays, KS from the Smoky Hill Chalk of Kansas (about 85 million years ago).

Reconstruction of Squalicorax curvatus  by Dmitry Bogdanov

Exceptional Squalicorax skeleton - absolute once-in-a-lifetime find

Squalicorax skull detail

Squalicorax curvatus SLE and PBE - surface etch.  Size bars: (1,2,6)
1 micron, (3) 100 microns, (4) 50 microns, (5) 5 microns.

There is no argument in the literature that this particular shark is a selachimorph neoselachian (read SHARK).  The external anatomy of Squalicorax curvatus teeth was examined first using teeth that had not been exposed to 10% hydrochloric acid (HCl) (panel 1), treated with HCl for 30 seconds (panel 2) or 3 minutes (panel 3-6).  There is not much relief on the surface of the untreated teeth, but you can easily see randomly oriented enameloid crystallites on the surface of a tooth treated for 30 seconds with 10% HCl.  Longer treatments remove all of this SCE/SLE from the surface and expose the parallel bundled enameloid of the Squalicorax tooth.  Panel three shows the parallel bundles at the level of the serrations in the tooth.  As the parallel bundles approach the serration, they turn direction, so that they point towards the edge of the serration, instead of towards the apex of the tooth.  As you zoom in on the parallel bundles, it is possible to see that there are two populations of bundles; one that runs parallel to the long axis of the tooth, and one that runs perpendicular to the long axis.

Cross section of Squalicorax teeth.  Size bars: (1) 500 microns,
(2,3,6) 50 microns, (4) 20 microns, (5) 5 microns

It is possible to see all three enameloid layers simultaneously in sectioned teeth.  In panel 1, the lighter area is the enameloid and the darker area inside is the dentine of the tooth.  The tooth is embedded in plastic.  In the upper right and lower left of the picture, you can see the serrations (cutting edges) of the teeth.  In panel 2, we are looking at the interface between the enameloid upper right and the dentine (left).  The arrows show where the dentine and enameloid meet.  The TBE (T) is seen next to the dentine (left) in panel 3, and the PBE (P) is to the right.  Panels 4-5 show the interface between the TBE and dentine.  As the dentine is approached, the enameloid becomes more like an SCE/SLE.  The dentine has a structure that looks a lot like bone, with channels for odontoblasts that are surrounded by concentric rings of dentinal material.  Shark teeth have not changed a whole lot in appearance in the past 90 million years.  There are small changes, but those mostly reflect stress introduced onto the tooth what the shark eats.

Almost to the pay-off for the paper.....

Is you is or is you ain't my sharky?

Life was going swell, then work showed up and shot everything to hell.  My writing got in the way of my writing anyway.  So this is what I have been doing instead of writing blog entries.  One line of my research is examining the value of tooth enameloid characteristics in sharks as a way of determining whether they are a "shark ancestor/primitive shark" or a "modern shark".  "Sharks" are generally thought of as fish with a skeleton made out of cartilage (chondrichthyan), a mess of teeth in the mouth that are replaced over time, tooth like scales (denticles) in their skin, and a torpedo- shaped (fusiform) body.  As such, shark remains can be identified from the Devonian Period, over 400 million years ago.

1) Enameloid layers in shark teeth; 2) possible directions of
parallel bundles; 3) sectioning directions of teeth

Shark biologists spend a bit of time arguing about what characteristics make a chondrichthyan a modern shark or a shark ancestor.  The one telling characteristic of the teeth seems to be the structure of the tooth enameloid.  A shark's tooth has a core of dentine, surrounded by fluoroapatite crystals.  Primitive sharks such as the ctenacanths, symmoriids and hybodonts had a single-layered enameloid composed of randomly oriented single crystals of fluroapatite (single crystallite enameloid - SCE).  A modern shark (selachimorph) has a triple-layered enameloid, with an outer layer of SCE (called shiny layered enameloid - SLE) plus two underlying layers of bundled crystals.  The middle layer is composed of bundles arranged in parallel (parallel bundeled enameloid - PBE), while the inner layer next to the dentine is composed of interwoven bundles (tangled-bundled enameloid - TBE).

Looking at these enameloid crystals takes some doing, since they are held in place with smaller "cement" molecules.  Fortunately the enameloid crystals are more acid resistant than are the cement molecules, so 5 sec to 3 minutes exposure of fossil shark teeth to 10% hydrochloric acid is usually enough to see the enameloid, depending on what layer you want to look at, and how you have prepared the teeth.   With whole teeth, 15-30 seconds is enough to see the randomly oriented crystals of the SCE/SLE; while seeing the PBE may take 1-3 minutes.  You can see all of the layers at once if you cut the tooth open (or embed it in plastic and sand it down) and then treat with acid for about 5 seconds.

Gold coated Ptychodus tooth, about 1 cm across

Enameloid crystals are really small, so you have to use a very powerful microscope to see them.  A regular light microscope will usually allow for a 1000X magnification, but you need to magnify these teeth about 5000X to see the individual crystallites of the SCE/SLE well at all.  This requires the use of an electron microscope, which uses a beam of electrons in a vacuum to image a specimen instead of light.  Most biomaterials are natural insulators, which means that they will absorb electrons and not reflect or re-emit them.  To solve this problem, we coat the specimen with a one atom thick layer of gold.  These sharks are all pimped out.  The gold will allow us to see the shape of the surface they are deposited on by reflecting electrons (backscatter) or by absorbing an electron and emitting one in its place (secondary electrons).

Tooth embedded in plastic, ground sandpaper, and coated in gold.
Copper tape is used as a pointer

The Hybodont Tooth

One group of primitive sharks that are seen as being ancestral to the modern shark are the hybodonts.  The first record of hybodontiform sharks is seen in the Mississippian of the Carboniferous Period, about 340 million years old.  The hybodonts were very successful, surviving late into the Cretaceous Period.  This is an evolutionary life of about 270 million years.  Remember that dinosaurs went extinct only 67 million years ago.  The hybodonts had mouths that were not overhung by their noses (rostrum), large cranial scales, barbed spines in front of their dorsal fins, a variety of different denticle types, and SCE on their teeth.

Reconstruction of Onychoselache

One of the early hybodontiforms was Onychoselache.  They were very small, about 10 inches long with teeth that were about 1 mm in maximum dimension.  Their teeth had low, flat crowns for crushing hard shelled organisms.  They had barbed dorsal fin spines, oval hooked denticles on their pectoral fins, and c-shaped denticles along their lateral line (sensory region on flanks of fish).  I have found very similar remains from the 307 million year old Farley Limestone of the Kansas City Group.

Hybodontiform parts:  scale bars(1-5) 1 mm, (6-9) 0.5 mm,
(10-11) 0.2 mm

The remains at the right are from an indeterminate hybodontiform.  There is not adequate material to describe a fish, and while the remains are associated, they are separate bits and pieces.  This is just not enough material to describe an organism down to the species level.  Views 1-5 are various views of a tooth: 1-top (occlusal) view; 2-front (labial); 3-side (lateral); 4-broken side (medial); 5-back (lingual) views.  View 6 is a piece of dorsal fin spine with hooked denticles on the posterior (back) side.  Views 7-9 are denticles from the pectoral (front) fins and 10-11 are denticles from the lateral line.  I sectioned several pieces of these teeth and looked at the structure to make sure that they were really hybodontifom and had an SCE.

Sectioned hybodontiform teeth:  Scale bars: (1)  200 microns;
(2) 10 microns ; (3,4,6) 1 micron; (5) 50 microns

The tooth crown is composed of dentine, which extends in columns up into the enameloid.  In the electron micrographs at left, the dentine is dark and the enameloid is light colored.  In panel 1 you can see columns of dentine approaching the surface of the tooth.  The enameloid has several channels for conducting odontoblast (tooth-building cells) processes through the enameloid (panel 2).  In panels 3-4, randomly distributed enameloid crystals make up the outer layer of the tooth.  Dentinal tubules are seen in section in panel 5.  The junction (arrows) between the dentine (D) and enameloid (E) is seen in panel 6.  The SCE is clearly visible in this section as well.  A channel carrying an arm of an odontoblast cell can be seen between the left two arrows.

That is about 3 months worth of work to get everything done just right.  Next is the last two years of my research life.




Reconstruction by Kahless28