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Proc Natl Acad Sci U S A. Nov 4, 2008; 105(44): 16982–16987.
Published online Oct 28, 2008. doi:  10.1073/pnas.0805308105
PMCID: PMC2579364
Developmental Biology

A second wave of Sonic hedgehog expression during the development of the bat limb

Abstract

Sonic hedgehog (Shh) plays an integral role in both the anterior-posterior (A-P) patterning and expansion of developing vertebrate limbs through a feedback loop involving Fgfs, Bmps, and Gremlin. In bat limbs A-P patterning and the size of the digital field are unique. The posterior digits of the forelimb are elongated and joined by tissue, whereas the thumb is short. The hindlimb digits often are uniform in length. Here, we reveal novel expression patterns for Shh and its target, Patched 1 (Ptc1), during limb development in two bat species. Early Shh expression in the zone of polarizing activity is wider in the bat forelimb than in the mouse forelimb, correlating with the reported expansion of Fgf8 expression in the apical ectodermal ridge and the early loss of symmetry in the bat forelimb. Later in limb development, Shh and Ptc1 expression is reinitiated in the interdigital tissue. Shh is graded along the A-P axis in forelimb and is expressed uniformly at a lower level across the hindlimb interdigital tissue. We also show that the reported Fgf8 expression in the interdigital tissue precedes the expression of Shh. We propose that the reinitiation of Shh and Fgf8 expression in bat limbs reactivates the Shh-Fgf feedback loop in the interdigital tissue of stage 16 bat embryos. The cell survival and proliferation signals provided by the Shh-Fgf signaling loop probably contribute to the lengthening of the posterior forelimb digits, the survival of the forelimb interdigital webbing, and the extension of the hindlimb digits to a uniform length.

Keywords: Miniopterus natalensis, Carollia perspicillata, Patched1, Fgf8, evo-devo

The hypothesis that evolutionary changes in anatomy are brought about by alterations in the regulation of key developmental “toolkit genes” is central to the field of evolutionary developmental biology. In particular, changes in the spatial and temporal regulation of the Sonic hedgehog (Shh) pathway have been implicated in the diversification of limb morphology among the vertebrates. During limb development Shh expression in the zone of polarizing activity (ZPA) is essential for both the growth of the digital field (1, 2, 3) and patterning of the anterior-posterior (A-P) axis of the limb bud (4). The absence of this growth signal has been implicated in the termination of hindlimb development in the dolphin (5), whereas temporal shifts in Shh expression lead to variations in digit number in the limbs of Hemiergis lizards (6).

Changes in the spatial and temporal regulation of the Shh pathway may be responsible for the unique skeletal structure of the bat limb, because the processes of A-P patterning and limb bud growth are dramatically altered during bat limb development. Whereas the early mouse limb buds and the bat hindlimb bud initially are symmetrical across the A-P axis, the bat forelimb autopod begins to lose this symmetry as early as stage 15 of development (CS 15), because of the expansion of the posterior autopod relative to the anterior autopod (Fig. 1 B and C compared with A, and F and G compared with E) (7). Following this initial expansion, the chondrocytes in the posterior digits of the bat forelimb autopod undergo accelerated proliferation and differentiation when compared with developing digits of the bat hindlimb and the mouse (8). As a result of these developmental dynamics, digits 2 to 5 of the bat forelimb are elongated in comparison to digit 1 (thumb) (Fig. 1L). In contrast, the digits of the bat hindlimb are not drastically elongated, and in many bat species the hindlimb digits are identical in length (Fig. 1X). This limb morphology is distinct from that of the mouse, in which the forelimb and hindlimb digits 1 and 5 are shorter than the remaining digits (Fig. 1U). The foundation for the unique skeletal structure of the bat hindlimb is laid down at CS 16 when the most proximal anterior and posterior edges of the hindlimb autopod expand, lengthening the primordia of digits 1 and 5 (Fig. 1 R and S).

Fig. 1.
Differential limb development in morphologically equivalent mouse (M. musculus), M. natalensis, and C. perspicillata embryos. (A and M) E12.0 mouse forelimb and hindlimb. (E and Q) E13.0 mouse forelimb and hindlimb. (I and U) E13.5 mouse forelimb and ...

In the current model for growth of the digital field in vertebrate limbs, Shh in the ZPA interacts with Fgfs in the apical ectodermal ridge (AER) through a positive feedback loop, involving the bone morphogenic protein (BMP) inhibitor, Gremlin, as an intermediary (reviewed in 9). This signaling loop is integral to the regulation of limb size, with cell proliferation and limb outgrowth continuing as long as it is maintained (1). Recent observations of gene-expression patterns point toward a possible alteration in the regulation of the Shh-Fgf positive feedback loop during bat limb development. The domain of Fgf8 expression in the AER is significantly wider in the early (CS 14) bat embryo than in the mouse (10). Later in development (CS 16), Fgf8 and Gremlin acquire novel expression domains within the interdigital mesenchyme of the bat forelimb and hindlimb autopods (11). These observations suggest that spatial and temporal changes in the activation of the Shh-Fgf signaling loop during bat limb development underlie the unique A-P patterning of the bat limbs and the elongation of the forelimb digits.

To test this hypothesis, we compared the spatial and temporal patterns of Shh expression during limb development in two species of bat, Miniopterus natalensis and Carollia perspicillata, with those in the mouse, Mus musculus, at morphologically matched developmental stages. We also examined the expression of Patched1 (Ptc1), a downstream target of Shh (12), as an indicator of active Shh signaling. Consistent with the observed expansion of early Fgf8 expression in the AER (10), we found that Shh expression in the forelimb ZPA is likewise expanded. Later in limb development, Shh and Ptc1 acquire novel domains of expression within the interdigital tissue; again consistent with the observed novel expression domains of Fgf8 and Gremlin (11). We show that the novel expression of Fgf8 in the interdigital tissue precedes that of Shh and Ptc1.

Based on these findings, we propose that early enhancement of the Shh-Fgf feedback loop underpins the early loss of symmetry in the bat forelimb autopod. In addition, we suggest that the reinitiation of the Shh-Fgf feedback loop later in limb development, with different spatial dynamics in the forelimb and hindlimb, contributes to the elongation of the posterior forelimb digits, the retention of the webbing between these digits, and the uniform length of all of the digits in the hindlimb.

Results

The Initiation of Shh Expression in the ZPA Is Delayed and the Domain of Expression Is Expanded in the Developing Bat Limb.

Shh expression in both M. natalensis and mouse limbs is detected first in a posteriorly restricted domain corresponding to cells of the ZPA (Fig. 2 A–H). Shh is readily detectable from E10.0 (data not shown) to E11.5 in the mouse forelimb and hindlimb (Fig. 2A, C, E, and G). The corresponding Ptc1 expression pattern is clearly graded from posterior to anterior in response to the SHH morphogenic gradient across the A-P axis (Fig. 3 A, C, E, and G). The initiation of Shh expression seems to be delayed in the M. natalensis limbs. Shh expression in the forelimb is apparent only at CS 13L, corresponding to approximately E10.5 of mouse development (data not shown), and at a further stage later in the hindlimb (CS 14E) (Fig. 2D). The appearance of a corresponding graded Ptc1 expression pattern in the M. natalensis limbs also is delayed by an additional stage to CS 14E in the forelimb (Fig. 3B) and CS 14 in the hindlimb (Fig. 3H).

Fig. 2.
Shh expression in morphologically equivalent mouse (M. musculus) and bat (M. natalensis) forelimbs and hindlimbs. (A, E, I, O, S, and W) Mouse forelimbs. (C, G, K, Q, U, and Y) Mouse hindlimbs. (B, F, J, M, P, T, and X) M. natalensis forelimbs. (D, H ...
Fig. 3.
Ptc1 expression in morphologically equivalent mouse (M. musculus) and bat (M. natalensis) forelimbs and hindlimbs. (A, E, I, M, and Q) Mouse forelimbs. (C, G, K, O, and S) Mouse hindlimbs. (B, F, J, N, and R) M. natalensis forelimbs. (D, H, L, P, and ...

Following the delay in Shh signal initiation, the domain of Shh expression is wider in the M. natalensis forelimb at CS 14E and CS 14 than in the mouse at E11.0 and E11.5 (Fig. 2A, B, E, and F; arrowheads). In the mouse the area of Shh expression hugs the posterior edge of the limb bud, whereas in M. natalensis the corresponding region of expression is expanded toward the centre of the limb bud. This expansion in the Shh expression domain at CS 14 mirrors the reported expansion in the Fgf8 expression domain at the same stage in the AER (10) and occurs just before the initial posterior expansion of the forelimb autopod (Fig. 1B). In contrast, the region of Shh expression is not expanded in the M. natalensis hindlimb when compared with the mouse hindlimb (Fig. 2 G and H).

Shh Expression Is Reinitiated in the Interdigital Tissue of the Developing Bat Limb.

Shh expression in the ZPA ceases in the mouse forelimb and hindlimb by E12.0 and E12.5, respectively (Fig. 2 I and Q). Shh also is absent from M. natalensis limbs by CS 15 (Fig. 2 J and L). Surprisingly, during CS 16 Shh expression is reinitiated in both the forelimb and hindlimb of M. natalensis (Fig. 2M, P, R, T, and V) and C. perspicillata (Fig. 4A). Shh is not detected in mouse limbs of comparable stage (Fig. 2O, Q, S, U, W, and Y). This novel expression domain is detected first in the forelimb of M. natalensis at very early stage 16 (CS 16VE) and is confined to the most distal region in the tissue between digits 3 and 4 (Fig. 2M; arrow). At CS 16E Shh expression is graded from posterior to anterior across the interdigital tissue of the forelimb, with the highest expression between digits 4 and 5 (Fig. 2P). Shh expression is reinitiated in the M. natalensis hindlimb during this stage and also is localized to the interdigital tissue; however, this signal is uniform along the A-P axis and is not as strong as the forelimb expression (Fig. 2R). Interdigital Shh expression persists, although at lower levels, to CS 16. At this stage, forelimb expression is highest in the tissue between digits 3 and 4 and along the borders of the condensations of all of the digits (Fig. 2T), whereas expression is uniformly low across the hindlimb interdigital tissue (Fig. 2V). By CS 17 Shh expression is absent from both the forelimb and hindlimb (Fig. 2 X and Z).

Fig. 4.
(A) Shh and Ptc1 expression in CS 16 C. perspicillata forelimbs (A and B) and hindlimbs (C and D). Anterior is up in all images. Both Shh and Ptc1 are expressed in the interdigital tissue of both the forelimbs and the hindlimbs. Scale bars show 0.5 mm. ...

From CS 16E to 16, Ptc1 expression is visible in the interdigital tissue of M. natalensis in response to the novel Shh expression (Fig. 3J, L, N, and P), and similar expression is visible in C. perspicillata limbs at CS 16 (Fig. 4A). Ptc1 is absent from this region in equivalently staged mouse limbs (Fig. 3I, K, M, and O). In both M. natalensis and mouse limbs, high levels of Ptc1 expression are visible in the perichondrium of the developing digits, most likely in direct response to Indian hedgehog in the developing cartilage condensations (Fig. 3 I–T) (13). This latter Ptc1 expression is noticeably longer along the proximal-distal axis of the developing digits in the M. natalensis forelimb than in the M. natalensis hindlimb or in equivalently staged mouse limbs (Fig. 3 M–P), providing evidence that the primordia of the M. natalensis forelimb digits are longer than those of the hindlimb or the digits of the mouse limbs. In addition, the Ptc1 expression pattern in the CS 17 M. natalensis hindlimb gives evidence of the symmetry of the hindlimb digits. The pattern of Ptc1 expression is identical in each of the M. natalensis digit primordia, which all are of equal length (Fig. 3T). In the mouse hindlimb, on the other hand, the pattern of Ptc1 expression in the short digits 1 and 5 is noticeably different from that in the longer digits (Fig. 3S).

Novel Fgf8 Expression in the Interdigital Tissue of the Bat Forelimb and Hindlimb Precedes Expression of Shh and Ptc1.

Fgf8 has been shown to be expressed in a novel domain in the interdigital tissue of developing bat limbs at CS 16 and CS 17 (11). To determine whether novel Shh or Fgf8 expression appears first in the interdigital tissue of developing bat limbs, we examined Shh and Fgf8 expression in the contralateral limbs of bisected embryos. Fgf8 expression becomes visible in the forelimb interdigital tissue as early as CS 15E, when Shh expression is absent (Fig. 4B and data not shown). At this stage, Fgf8 expression is absent from the hindlimb interdigital tissue but is present in the both the hindlimb and forelimb AER (Fig. 4B). From CS 16VE to CS 17, Fgf8 expression is maintained in the forelimb interdigital tissue and is graded from posterior to anterior (Fig. 4B) (11). Fgf8 becomes visible in the hindlimb mesenchyme at CS 16VE, before the appearance of the equivalent Shh expression (Fig. 4B and data not shown). Fgf8 expression in the hindlimb becomes confined to the tissue between the digits at CS 16E (Fig. 4B) and is absent from the interdigital tissue by CS 17 (11). The novel interdigital expression of Fgf8 was observed in both C. perspicillata (Fig. 4B) and M. natalensis (data not shown).

Discussion

Early in limb development, Shh in the ZPA interacts in a positive feedback loop with Fgfs in the AER to ensure the outgrowth of the limb bud (14, 15). Here, together with data from Cretekos et al. (10), we provide preliminary evidence to suggest that this early signaling loop has been enhanced in the bat forelimb, leading to an expansion in the Shh and Fgf8 expression domains at CS 14 of bat development. A similar phenomenon has been described in the limb buds of Bmp mutant mice, which display expanded Fgf8 expression in the AER resulting from a lack of antagonism from BMPs (16). These mice also exhibit an expanded Shh expression domain and display posteriorly expanded limb buds. This phenotype is explained in terms of an enhanced Shh-Fgf interaction (16). A similar enhancement occurring naturally during bat limb development may result in a relative increase in cell proliferation and cell survival in the posterior as compared with the anterior autopod. This enhancement of the early Shh-Fgf signalling loop may explain the posterior expansion and resulting loss of symmetry across the A-P axis in the bat forelimb autopod at CS 15 when compared with the bat hindlimb or mouse E12.0 forelimb.

The observed expansion of the Shh signal in the early bat forelimb may be triggered by an autoregulatory mechanism linked to Shh signaling. In the developing chick limb, Shh has been shown to buffer its own expression, with more cells being induced to produce Shh if a loss of signal is induced by removing ZPA cells or by inhibiting the Shh signaling pathway (17, 18). The initial delay in Shh expression in the bat forelimb may induce this buffering mechanism, stimulating a subsequent expansion of the population of Shh-producing cells at CS 14E and CS 14 when compared with equivalently staged mouse limbs. Further research involving real-time analysis of Shh expression levels during early bat limb development compared with expression levels in the mouse may confirm this hypothesis.

At CS 16 of bat development, Shh and Ptc1 are recruited to new domains of expression within the interdigital tissue of the forelimb and hindlimb (summarized in Fig. 4B). Interestingly, Gremlin and Fgf8 also are expressed in novel domains in the interdigital tissue of C. perspicillata limbs at CS 16 and CS 17 (11). The observed up-regulation of all four of these genes in the interdigital tissue suggests that the Shh-Fgf feedback loop is initiated for a second time during bat limb development (summarized in Fig. 5). Early in limb development, Fgf8 expression in the AER precedes the formation of the ZPA and is required to initiate and maintain Shh expression (15, 19). It is likely that the same is true for the interdigital expression of these genes during bat limb development. Fgf8 is first detected in the bat forelimb and hindlimb interdigital tissue at CS 15E and CS 16VE, respectively, preceding the reinitiation of Shh expression at CS 16VE and CS 16E.

Fig. 5.
A model for the reinitiation of the Shh-Fgf feedback loop in the interdigital tissue of the CS 16E bat forelimb. Fgf8 (gold) is expressed in a novel domain within interdigital tissue of the CS 15E forelimb in a gradient from posterior to anterior as well ...

In the mouse and chick, the Shh-Fgf signaling loop involves the activation of Gremlin in the limb mesenchyme by SHH from the ZPA (20, 21). The Shh-expressing cells themselves, however, are not able to turn on Gremlin (22, 23). The complementary expression domains of these genes in the stage 16E forelimb suggest that the same is true when these genes are up-regulated for the second time during bat limb development. Shh, which is highest in the tissue between digits 3 to 5, may activate Gremlin in the tissue between digits 1 to 3 (11).

Bmp2 is suggested to be the link between Shh and Gremlin expression in the Shh-Fgf feedback loop, activating the expression of its own antagonist (23). In developing C. perspicillata limbs Bmp2 expression is detected in regions corresponding to Shh in this study (11). Thus, it is possible that in the bat forelimb between CS 16 and CS 17, Shh activates Bmp2, which in turn activates Gremlin expression (Fig. 5).

Gremlin promotes Fgf expression in the AER, through the suppression of BMPs (20, 21). Fgf8 in the AER then activates Shh expression in the ZPA, completing the Shh-Fgf feedback loop (15). In the CS 16 bat forelimb, Shh and Fgf8 (11) are both expressed at high levels in the posterior interdigital tissue (i.e., outside the ZPA and AER, respectively) (Fig. 5). Thus, this reinitiation of the Shh-Fgf feedback loop differs from the earlier signaling loop in that Shh and Fgf8 are able to promote each other's expression in the same domain rather than being confined to the ZPA and AER, respectively.

It is possible that the cell-proliferation and survival signals provided by the Shh-Fgf signaling loop are co-opted to perform the novel dual functions of lengthening the posterior forelimb digits and promoting the survival of the interdigital tissue. This model is supported by studies in the chick. These studies found that the application of SHH to the interdigital tissue of developing limbs after Shh expression in the ZPA has ceased prolongs Fgf8 expression in the AER and facilitates the lengthening of the last phalange of the digits or the formation of an additional phalange and the survival of the interdigital tissue (17, 24). The extended Fgf8 signal is described as an anti-differentiation signal, promoting the proliferation of the mesenchyme cells in the digital rays while inhibiting their differentiation into cartilage (25). Shh also has been shown to promote cell survival and proliferation, because a loss of Shh signal during limb development results in an increase in the proportion of cells in G1 phase and a decreased proportion of cells entering S phase (26). The widespread elongation effect evident in the metacarpals and phalanges of the posterior digits of the bat forelimb may be caused by the activation of Fgf8 and Shh throughout the posterior interdigital digit tissue rather than just in the AER and ZPA, respectively, exposing the metacarpals and each phalanx to the Fgf8 and Shh proliferation signals.

The lack of extra phalanges in the bat forelimb digits could result from the high levels of Shh surrounding the digits. Retroviral-induced misexpression of Shh at high concentrations within the digital rays of chicken limbs blocks the formation of joints (27). In addition, Shh misexpression in the chondrocytes of developing mouse digits under the control of a procollagen gene promoter blocks joint formation and promotes cell proliferation (28). Thus, the high Shh concentration surrounding the forelimb digits in the bat may have the same effect, allowing the cells of the digital rays to proliferate and suppressing the joint-formation pathway until after Shh expression ceases.

Although the Shh and Fgf8 signals are recruited to the interdigital tissue of the hindlimb, the duration of expression is shorter and the level is lower than in the forelimb (Fig. 4B) (11). In addition, the Shh and Ptc1 signals are expressed uniformly across the hindlimb interdigital tissue, rather than in a gradient, as in the forelimb (Fig. 4B). It is possible that the short exposure to the cell-survival and proliferation signals of the Shh-Fgf feedback loop lengthens the primordia of digits 1 and 5 of the hindlimb but is insufficient to lengthen the remaining digits extensively or to suppress the apoptosis of the interdigital tissue. Thus, despite the early asymmetrical expression of Shh in the hindlimb ZPA, the late symmetrical expression of Shh across the hindlimb bud may contribute to the proximal expansion of the hindlimb autopod at CS 16 (Fig. 1R) and the growth of digits 1 and 5 to the same length as the remaining digits.

The analysis of Shh, Ptc1, and Fgf8 expression in both M. natalensis and C. perspicillata has revealed that the novel expression domains of these genes are common within the chiropteran suborder Verspertilioniformes (29, 30). This observation suggests that the mode of wing development is constant within this taxon and supports the monophyly of the group (30, 31). Given the recent fossil find that suggests flight evolved only once in the Chiroptera (32), the mode of wing development may be common for the entire chiropteran order. If so, subtle differences in the spatial extent and timing of Shh expression in the forelimbs of different bat species may allow variation in the lengthening of the digits and lead to differences in adult wing shape. The analysis of Shh and Ptc1 expression in the developing limbs of a species with a wing shape very different from that of M. natalensis and C. perspicillata, such as the long, narrow wings of the mollosid bats, may provide further support for this hypothesis. Analysis of the expression patterns of these genes in a species from the Pteropodiformes, the second chiropteran suborder, will reveal whether the mechanism of wing development is constant within the Chiroptera. A positive finding would provide additional support for the hypothesis that wings evolved once within this order.

It is possible that an ancient change in the highly conserved Shh limb-specific cis-regulatory region, known as the ZPA regulatory sequence (9), led to the altered Shh expression reported here. If indeed wings evolved once within the Chiroptera, this sequence change should be conserved across diverse bat species.

Methods

Collection and Staging of Embryos.

M. natalensis embryos were collected from wild-caught, pregnant females in September 2006 from De Hoop Nature Reserve, Western Cape Province, South Africa (Western Cape Nature Conservation Board permit number: AAA004–00030–0035; University of Cape Town Faculty of Science Animal Experimentation Committee application number: 2006/V4/DJ). C. perspicillata embryos were collected from wild-caught, pregnant females on the island of Trinidad in either January or May of 2003 to 2007, as previously described (7, 8, 10, 11). The samples were collected and exported with the permission of the Wildlife Section, Forestry Division of the Ministry of Agriculture, Land and Marine Resources of the Republic of Trinidad and Tobago. All bat embryos were staged according to (7). Some of the embryos were placed in early- or late-stage categories (e.g., CS 16VE: very early; CS 16E: early; CS 16L: late) based on the progression of limb development. Mouse embryos (ICR strain) were obtained from timed matings conducted by the Animal Unit at the University of Cape Town Medical School (Animal Ethics Committee application number: 006/040).

Skeletal Imaging.

The developing skeleton of bat limbs was imaged using whole-mount alcian blue staining of cartilage. C. perspicillata embryos at the appropriate stages were fixed overnight in Bouin's fixative (Polysciences) at room temperature, washed several times in 70% ethanol, and stained with alcian blue 8GX (Sigma) as described previously (33). After clearing in a 1:2 ratio of benzyl alcohol:benzyl benzoate, limbs were dissected, mounted in glass depression slides, and imaged under a stereodissecting Leica (model MZ9) microscope equipped with digital capture.

Gene-Expression Analysis.

Whole-mount in situ hybridization was performed using digoxigenin-labeled RNA probes based on mouse and bat sequences. Ptc1 primers (5′-ACCTTTGGACTGCTTCTGGGAA-3′ and 5′-AAAIGGCAAAACCTGAGT TG-3′) were designed from regions of near identity in cDNA sequence alignments of the human, mouse, rat, and dog gene and were used to clone a region of 830 bp from exon 5 to 10 of Ptc1 from M. natalensis (CS 13L) and mouse (E13.5) cDNA. The M. natalensis Ptc1 sequence has been submitted to GenBank (accession no. EU562193).

The cloned Ptc1 sequences were used as templates in in vitro transcription reactions for the synthesis of bat- and mouse-specific RNA probes. A mouse Shh RNA probe provided by A. McMahon was used for expression analysis in both bat and mouse embryos. The sequence of this mouse Shh probe has been submitted to GenBank (accession no. EU664592). The Fgf8 RNA probe used is based on the C. perspicillata cDNA sequence and has been described previously (10). RNA probes were used at concentration of 0.5–1 μg/ml. For analysis of dual Shh and Ptc1 expression in all M. natalensis embryos and in E13.0 and E13.5 mouse embryos, specimens were cut in half along the midline to allow analysis of Shh expression on one side and Ptc1 expression on the other. For E11.0 to E12.5 mouse embryos and for CS 16 C. perspicillata embryos, separate specimens were used for analysis of Shh and Ptc1 expression. For analysis of dual Shh and Fgf8 expression, all M. natalensis and C. perspicillata embryos were cut in half along the midline to allow analysis of Shh expression on one side and Fgf8 expression on the other. One to four samples were used for each stage of development.

Acknowledgments.

We thank Robyn Verrinder for assistance in collecting bats and embryos at De Hoop Nature Reserve; John Rasweiler, Scott Weatherbee, and Simeon Williams for assistance in sample collection on Trinidad; the management of De Hoop Nature Reserve and the Department of Life Sciences, University of the West Indies, Trinidad for generous assistance and the use of research facilities; the Western Cape Nature Conservation Board and the Wildlife Section, Forestry Division, Agriculture Land and Marine Resources of the Republic of Trinidad and Tobago for providing collecting and export permits. Research completed in South Africa was supported by funding from a University of Cape Town Stimulation Grant and National Research Foundation Grant No 65525 (to D.S.J.). D.H. and M.K.M. were supported by National Research Foundation Prestigious Masters Scholarships. D.H. was supported by the Society for Integrative and Comparative Biology Grant-in-Aid-of-Research Award. R.R.B was supported by National Science Foundation Grant IBN 0220458. C.J.C. was supported by National Institutes of Health Training Grants CA09299 and HD07325.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition footnote: The sequences reported in this paper have been deposited in the GenBank database (accession nos. EU562193 and EU664592).

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