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Lungfish fingers and bird fingers

An interesting paper came out last year in a journal that probably not many paleontologists read:

Zerina Johanson, Jean Joss, Catherine A. Boisvert, Rolf Ericsson, Margareta Sutija & Per E. Ahlberg (2007): Fish Fingers: Digit Homologues in Sarcopterygian Fish Fins, Journal of Experimental Biology (Mol Dev Evol) 308B, 757 -- 768 (published online 11 September 2007)

A defining feature of tetrapod evolutionary origins is the transition from fish fins to tetrapod limbs. A major change during this transition is the appearance of the autopod (hands, feet), which comprises two distinct regions, the wrist/ankle and the digits. When the autopod first appeared in Late Devonian fossil tetrapods, it was incomplete: digits evolved before the full complement of wrist/ankle bones. Early tetrapod wrists/ankles, including those with a full complement of bones, also show a sharp pattern discontinuity between proximal elements and distal elements. This suggests the presence of a discontinuity in the proximal-distal sequence of development. Such a discontinuity occurs in living urodeles, where digits form before completion of the wrist/ankle, implying developmental independence of the digits from wrist/ankle elements. We have observed comparable independent development of pectoral fin radials in the lungfish *Neoceratodus* (Osteichthyes: Sarcopterygii), relative to homologues of the tetrapod limb and proximal wrist elements in the main fin axis. Moreover, in the *Neoceratodus* fin, expression of Hoxd13 closely matches late expression patterns observed in the tetrapod autopod. This evidence suggests that *Neoceratodus* fin radials and tetrapod digits may be patterned by shared mechanisms distinct from those patterning the proximal fin/limb elements, and in that sense are homologous. The presence of independently developing radials in the distal part of the pectoral (and pelvic) fin may be a general feature of the Sarcopterygii.

The core of the paper is development genetics of the pectoral fins of the Australian lungfish.

One conclusion is that three parts can be distinguished in the cartilaginous/endochondral skeleton of the pectoral extremities of any sarcopterygian (the dermal skeleton being fin rays and scales):
- the (metapterygial) axis: humerus, ulna, ulnare, and 0 to at least 11 more elements (0 in rhizodonts and tetrapods, 1 in *Latimeria* and *Eusthenopteron*, 2 in *Tiktaalik*, 11 in the figured specimen of *Neoceratodus*). This forms as a continuous spike of prechondral (pre-cartilage) cells in *Neoceratodus* that segments itself while it grows. In *Neoceratodus* (and, judging from morphology, other lungfishes and porolepiforms) it reaches all the way to the tip of the fin.
- 1 to 3 "nonindependent preaxial radials" (perhaps homologous to part of the mesopterygium of non-sarcopterygian gnathostomes, but that's just my guess): one articulating with the humerus and consisting of the radius, radiale, and presumably centrale 1, distal precarpal, and prepollex; one (absent in *Neoceratodus*) articulating with the ulna and consisting of the intermedium (and presumably centralia 4 and 2 & 3); and one that articulates with the ulnare and is only present in *Eusthenopteron* among the taxa Johanson et al. mention (unless it is centrale 3, but that's unlikely and was just my guess in the first place). One might think that the axis simply branches, so that radius and ulna are formed at the same time, but that's not how it works in *Neoceratodus*: the radius appears, without a connection to humerus or ulna, after the humerus, ulna, and ulnare have already started to form. The only "nonindependent preaxial radial" *Neoceratodus* has (radius and so on) forms, like the axis, as a continuous spike that segments while it grows.
- the "independently developing radials" which are found on both sides of the axis in taxa with a long axis (lungfishes, porolepiforms, to a lesser extent *Tiktaalik*) and mostly distal to the axis in taxa with a short axis (coelacanths, rhizodonts, *Eusthenopteron*, tetrapods). They are very variable (between taxa) in number, size, shape, and arrangement and do not have a consistent relationship with elements of the axis of the nonindependent preaxial radials. In *Neoceratodus* they start forming after the axis has grown to at least 6 elements beyond the ulnare, and, just before they appear, Hoxd13 is expressed, which is exactly what happens just before the appearance of digits in tetrapods; it is not expressed in the axis. After appearing, they grow and segment (like the axis and the nonindependent preaxial radials), and then they form joints with the nearest elements of the axis.

Before this, in the lengthy introduction, Johanson et al. compare the ankles of *Acanthostega*, *Ichthyostega*, and the Carboniferous anthracosaur *Proterogyrinus* (which already has the usual 5 toes). Attention is directed to the fact that digits + metapodials seem to have evolved before the centralia were complete: *Acanthostega* has 3 metatarsals articulating directly with the fibulare; it has an intermedium, a tibiale, and distal tarsals for 4 or 5 of the remaining 5 metatarsals, and a hole in between that may have held 1 or 2 unossified centralia, but not 4. *Ichthyostega* has a completely ossified ankle with a single centrale (and 2 metatarsals articulating directly with the fibulare, while the remaining 5 metatarsals have distal tarsals). *Proterogyrinus* has 3 small centralia and no metatarsals articulating directly with the fibulare. They also point out that in salamanders "digits develop as individual buds, before the condensation of the mesopodium [carpus/tarsus]. For example, in *Desmognathus aenus*, metacarpal II is the first element of the autopodium [metapodials + phalanges] to condense and chondrify [become cartilaginous] and is well separated from the only other limb condensations [that exist at that time], the radius and ulna" (p. 759); in *Ambystoma mexicanum*, metacarpal I and digit I appear first. Johanson et al. propose that -- see above -- the digital rays (digits, metapodials, distal carpals/tarsals) are, in ontogeny and phylogeny, independent of the more proximal elements, that the distal carpals/tarsals are (in salamanders and stem-tetrapods) proximal outgrowths of the metapodials, and that the centralia are evolutionarily more recent outgrowths of the intermedium and radiale/tibiale.

In amniotes and frogs, the limb chondrifies strictly from proximal to distal, while in salamanders this is not the case (see above). The traditional assumption is therefore that the salamander condition is derived. Johanson et al. turn this on its head, based on the evidence from the development of lungfish fins and from the morphology of tetrapods in general and *Acanthostega* and *Ichthyostega* in particular: the salamander condition is plesiomorphic, and the frog + amniote condition is (twice independently) derived. I wonder if that happened because most frogs and amniotes use their limbs for terrestrial locomotion soon after the limbs are formed, so there's no point in regulating a complicated sequence of chondrification, while salamander larvae grow limbs early and leave the water (if at all) late...
From pp. 765f.: "The earliest phase of tetrapod evolution shows a gradual elaboration of the mesopodium after the establishment of the digital arch [...]. Furthermore, this elaboration involves the addition of new centrals and distal carpals/tarsals, elements that lie immediately on either side of the transverse 'discontinuity line' [...] across which it is impossible to trace one-to-one relationships between elements. This suggests that the elaboration may have comprised two mirror-image processes of terminal addition of new elements, one at the distal end of the limb proper, producing centrals, and one at the proximal ends of the digits [or rather metapodials], producing distal carpals/tarsals [...]. The fossil evidence thus implies a degree of digit independence in early tetrapods. We argue that the early establishment of digits during urodele limb development is a retained primitive character that has been lost independently in anurans and amniotes. We note, however, that Cohn et al. [...] questioned the occurrence of branching mechanisms in the more distal elements of the amniote limb skeleton. A piece of foil placed within a developing chick wing directly in front of the most recently condensing element failed to prevent more distal elements from forming, suggesting some degree of independence of the latter from the former. Independence of the digits may thus be a general feature, masked by the proximal-to-distal chondrification sequence within the amniote paddle." (In amniotes and frogs the limb bud assumes a paddle shape while the hand/foot forms; that's not the case in salamanders.)
Of course, as Johanson et al. point out, this moreover fits the well-known fact that the digits ossify before the carpus/tarsus in a wide variety of tetrapods, including many amniotes as well as fossil temnospondyls, seymouriamorphs and lepospondyls.
Johanson et al. go on to cite Fröbisch et al. (2007), who report that the digits of the temnospondyl *Apateon* ossified in the salamander sequence: first 1 and 2, then 3 and 4, and then (in the foot) 5. This was originally taken as evidence of close relationship; it may be plesiomorphic instead.
"These observations imply that the autopod [digits + metapodials] evolved before the origin of tetrapods, represented by the more distal region of the sarcopterygian fin, with the 'origin of digits' simply representing a modest repatterning of this region rather than the origin of a new structure." (p. 766) The authors also mention the correlation between late-phase Hoxd13 expression and "radial formation" in the basal actinopterygian *Polyodon* (Davis et al. 2007); that would mean that, rather than us gaining digits, the teleosts have lost them.

In their last paragraph Johanson et al. discuss the "primary axis" hypothesis, whose latest incarnation is Wagner & Larsson (2007). This hypothesis says that digit IV is the tip of the (metapterygial) axis which has assumed digit shape because it has grown into digit territory, and that it is repeated on both sides to initiate the other digits. Wagner & Larsson go so far as to suggest that digits I and/or II of salamanders, which (see above) appear first, are in fact digit IV, with the lower-numbered digits lost and the higher-numbered ones lacking homologues in frogs and amniotes -- a frameshift much more massive than the one they suggest for birds. Without mentioning this extreme, Johanson et al. disagree, "because it does not seem to fit well with the palaeontological or molecular data" (p. 767) explained above.

To recapitulate, there are as of 2007 three hypotheses on the origin of digits, and Johanson et al. consider the first two more or less falsified:
- The digital arch: the metapterygial axis runs (forelimb example) humerus-ulna-ulnare-distal carpal 4-dc3-dc2-dc1. The radius-radiale-who knows what else branches off preaxially, and so does the intermedium, which (if I understand it correctly) continues into centralia and digits I and II of salamanders; dcV is a postaxial branch, and so are all metacarpals + digits. Illustrated here http://www.press.uchicago.edu/books/gee/shubin3.jpeg (ignore *Panderichthys* and the gene expression patterns).
- The primary axis: the metapterygial axis runs humerus-ulna-ulnare-dc4-mcIV-digit IV; its distal part is repeated to form the other digits.
- Digits as "radials": the metapterygial axis runs humerus-ulna-ulnare and then stops in tetrapods and rhizodonts, while it continues in other sarcopterygians with elements that lack tetrapod homologs; the radius-radiale-unspecified centralia and the intermedium-unspecified centralia form independently; and so do the "radials".

Shubin (2002) discusses three modes of reducing the number of elements in the hands and feet of salamanders:
- Truncation by paedomorphosis: elements are lost because they never form. This applies to the absence of digits V (foot only), V + IV, or V + IV + III, and probably to the rare appearance of centrale 3 (traditionally called "mediale" in salamanders).
- "Fusion" by paedomorphosis: elements that form from a single cluster of prechondral cells never separate. This applies to the "fusion" of distal tarsals 4 and 5 (commonly seen in salamanders as inter- and intraspecific variation), the "fusion" of distal tarsals 3 + 4 + 5 (same, but rarer), to the rare separation of centrale 1 (traditionally called "element y" in salamanders) and the distal tarsal of the prehallux, and to the (outside of hynobiids) rare separation of the intermedium and centrale 4. One cannot help but suspect that this also accounts for the "basale commune", the fact that distal carpals/tarsals 1 and 2 are not known to ever separate in any salamander. "The phylogenetic outcome of this is that patterns of consolidation of elements involve neighboring elements that lie within one of the three series of the mesopodium: the preaxial column, the central series, and the digital arch. Condensations in these zones largely remain distinct from one another and rarely fuse" (p. 25). In the terms of Johanson et al., these are the first "nonindependent preaxial radial" (radius, radiale, etc.), the second "nonindependent preaxial radial" (intermedium etc.), and the "radials" -- with the only exception that Johanson et al. do not postulate a connection between the distal carpals/tarsals, but adding one does not seem to contradict the rest of their model.
- Actual fusion: elementsfirst form and then fuse. Apparently invariably, they are from different "series". Centrale 2 and dc4 apparently fuse fairly often, intermedium and ulnare rarely, and that basically was it.

In sum, more support for the model of Johanson et al..

And now I have finally waffled enough about lungfish and salamanders and can go on to the birds: to the implications of that model on the frameshift hypothesis (and, of course, at the same time on the BAND hypothesis).

If digit IV is not part of the metapterygial axis, we cannot safely use the spatial relations between any digits on the one hand and the radius, ulna, or any proximal or central carpals on the other hand as evidence for the identity of any digit. Nor can we safely assume that digit IV must be the first to chondrify; in amniotes that retain 5 digits as adults, it is in all of the few cases that have been checked, but in salamanders it isn't. In the absence of more convincing evidence, the most parsimonious option is of course that the first digit to chondrify in the bird wing is digit IV, but we can no longer assume that this alone, and/or its position vaguely opposite the ulna, is stronger evidence than morphology.

I conclude that the only evidence for the frameshift hypothesis so far is, surprisingly, fossil. It was presented at this year's SVP meeting; I'll post the abstract later (this e-mail is long enough, and I should go to bed for a change). But, while suggestive, this, too, is not a clincher (I think I'm allowed to mention that I talked to the presenter, Jim Clarke, and we agree it's all very confusing). If the digits really are independent of the metapterygial axis and the "primary axis" hypothesis is wrong, the frameshift hypothesis may simply be unnecessary, for birds and for salamanders, and we can stop feeling guilty about calling the fingers of birds I, II and III.

Ironically, however, Johanson et al. confirm that an axis can grow into "radial" territory and have its tip modified into the shape of a "radial": as mentioned way above, the most proximal preaxial radial of *Neoceratodus* really does form by segmentation of the radius-radiale-etc. axis (in other words, *Neoceratodus* has a prepollex...), unlike all other "radials". It's just not the metapterygial axis, but the first "nonindependent preaxial radial".

Unsurprisingly, questions remain. Shubin (2002) casually mentions (especially in fig. 5) that plenty of (at least cryptobranchoid) salamanders have an extra distal tarsal called the postminimus. What is that? More evidence that the digits don't care about the rest of the limb when deciding where to form? Or is it the fourth element of the metapterygial axis, homologous to the last one in *Latimeria* and *Eusthenopteron*? Shubin reproduces the only drawing of one of about two decently known fully ossified temnospondyl feet, that of *Acheloma* (...he uses the junior synonym *Trematops*). Dt 4 is very large, mt V articulates _between_ dt4 and dt5, and dt5 is a rounded isosceles triangle, with the tip lying postaxial to mtV. Isn't that strange? Both dt4 and dt5 lie distal to the fibulare. Perhaps "dt4" is actually the "fusion" product of dt4 and dt5, and "dt5" is actually the postminimus? And what is the pisiform (an extra proximal carpal widespread in mammals, but also captorhinids, diadectomorphs, and many others) -- an ancient sesamoid, the postminimus, or what? And what about the toes in *Acanthostega* and *Ichthyostega* that articulate directly with the fibulare? Not to mention the finger of *Tulerpeton* that articulates directly with the ulna -- not the ulnare, but the ulna? (Illustrated here http://www.press.uchicago.edu/books/gee/shubin3.jpeg and http://www.nature.com/nature/journal/v440/n7085/fig_tab/nature04637_F4.html here.) To what extent are digits of Devonian tetrapods homologous to digits of pentadactyl tetrapods or to those of other Devonian tetrapods -- is it all just an unspecified mess of "radials" that grew wherever there was space for them? And to what extent are _bird_ fingers homologous to anything, then...?

All that makes me tired. It's a quarter to three at night. I'm going to bed.

==== References ====

Davis C, Dahn RD, Shubin NH. 2007. An autopodial-like pattern of _Hox_ expression in the fins of a basal actinopterygian fish. Nature 447: 437 -- 476.
Fröbisch NB, Carroll RC, Schoch RR. 2007. Limb ossification in the Paleozoic branchiosaurid *Apateon* (Temnospondyli) and the early evolution of preaxial dominance in tetrapod limb development. Evolution & Development 9: 69 -- 75.
Shubin NH. 2002. Origin of evolutionary novelty: examples from limbs. Journal of Morphology 252: 15 -- 28.