Genetic and developmental origins of a uniqueforaging adaptation in a Lake Malawi cichlid genusMoira R. Conitha, Yinan Hub,c, Andrew J. Conithd, Maura A. Maginnisd, Jacqueline F. Webbb, and R. Craig Albertsond,1

aGraduate Program in Organismic and Evolutionary Biology, University of Massachusetts, Amherst, MA 01003; bDepartment of Biological Sciences,University of Rhode Island, Kingston, RI 02881; cBiology Department, Boston College, Chestnut Hill, MA 02467; and dDepartment of Biology, University ofMassachusetts, Amherst, MA 01003

Edited by Hopi E. Hoekstra, HHMI and Harvard University, Cambridge, MA, and approved May 29, 2018 (received for review November 13, 2017)

Phenotypic novelties are an important but poorly understoodcategory of morphological diversity. They can provide insights intothe origins of phenotypic variation, but we know relatively littleabout their genetic origins. Cichlid fishes display remarkable diversityin craniofacial anatomy, including several novelties. One aspect of thisvariation is a conspicuous, exaggerated snout that has evolved ina single Malawi cichlid lineage and is associated with foragingspecialization and increased ecological success. We examined thedevelopmental and genetic origins for this phenotype and found thatthe snout is composed of two hypertrophied tissues: the intermax-illary ligament (IML), which connects the right and left sides of theupper jaw, and the overlying loose connective tissue. The IML ispresent in all cichlids, but in its exaggerated form it interdigitates withthe more superficial connective tissue and anchors to the epithelium,forming a unique ligament–epithelial complex. We examined theTransforming growth factor β (Tgfβ)→ Scleraxis (Scx) candidate path-way and confirmed a role for these factors in snout development. Wedemonstrate further that experimental up-regulation of Tgfβ is suffi-cient to produce an expansion of scx expression and concomitantchanges in snout morphology. Genetic and genomic mapping showthat core members of canonical Tgfβ signaling segregate with quan-titative trait loci (QTL) for snout variation. These data also implicate acandidate for ligament development, adam12, which we confirm us-ing the zebrafish model. Collectively, these data provide insights intoligament morphogenesis, as well as how an ecologically relevantnovelty can arise at the molecular level.

evo-devo | craniofacial | ligament | TGFβ | novelty

Species with novel phenotypes, which define the extremes of acollective morphospace, are a valuable resource for researchacross disciplines. In evolutionary biology, this phenotypic class canprovide insights into the origins and constraints on morphologicalevolution (1–4). Engineers have long used novel phenotypes as in-spiration for biology-based design and technology (5, 6). Suchphenotypes can also serve as “evolutionary mutant models” ofdisease states, whereby the adaptive trait mimics a maladaptivecondition in humans, and potentially provides insights into the ge-netic factors relevant for disease prediction and management (7).Finally, bizarre phenotypes serve to captivate the public and engagethem in the context of education (8).Novel phenotypes can arise as a dramatic reconfiguration of

the body plan (e.g., seahorses), from the elaboration of existingstructures (e.g., ref. 9) or as de novo structures with no obviousprecursor (e.g., ref. 10). Likewise, regulation at the molecularlevel can occur as the recruitment of existing genes and signalingpathways (e.g., through changes in timing, location, or amount ofexpression; refs. 9, 11, and 12) or by the evolution of new genes(e.g., following gene/genome duplication events; refs. 13 and 14).Unlike continuous phenotypic variation, novel morphologieshighlight the opportunistic and flexible nature of evolution to actas both a tinkerer and an innovator (15). A major pursuit inevolutionary developmental biology is to characterize the prox-imate genetic and developmental basis that can explain the ori-gin of novel phenotypes. However, our understanding remainslimited because most of our knowledge of development comes

from model organisms, which are selected in part because of theirconserved phenotypes (16, 17). Additionally, novel phenotypes of-ten have ancient origins and/or occur in lineages that are notamenable to laboratory investigations. Here, we address the originof a novel phenotype by identifying the genetic mechanisms un-derlying tissue-level changes of a unique craniofacial morphology ina genus of cichlid from Lake Malawi, Africa.Cichlids are an iconic model system for the study of rapid and

extensive craniofacial diversification (18–20). Within Lake Malawi,the genus Labeotropheus exhibits extreme craniofacial anatomy thatdefines the boundary of cichlid morphospace (20). Among otherextreme phenotypes, the two species in this genus possess an ex-aggerated fleshy snout (e.g., Fig. 1A vs. Fig. 1B). We have previouslyshown that this elaborated snout folds in on itself, forming a flexibleflap that extends over the upper lip, sometimes reaching the distaltip of the upper jaw dentition (21). It has been postulated that thisprotuberance enhances foraging efficiency by acting as a fulcrum tocrop algae off rocks by leverage as an alternative to the energeticallycostly bite-and-twist mode of benthic feeding common in othercichlid species (22). In addition, prior genetic work suggests that thistrait is under directional selection (21). Labeotropheus is also one ofthe most ecologically successful genera in the lake, as it has a cos-mopolitan distribution and dominates the near shore rocky habitat(23). Indeed, although this trait is rare relative to the number ofspecies that possess it (i.e., 2 of over 800 cichlid species in LakeMalawi), it is well represented in the lake given the ubiquity ofLabeotropheus. Thus, as with novelties in general, the evolution ofthis putatively adaptive structure is coincident with a unique for-aging mode and expanded ecological success.

Significance

Biologists have long been captivated by novel traits because theyprovide insights into both the origin of and constraints on mor-phological variation. The iconic adaptive radiations of cichlid fisheshave led to incredible diversity of form, including some specieswith an exaggerated snout. This novelty is mechanically in-tegrated with the upper jaw, appears to be under directional se-lection, and is found in one of the most ecologically successfulcichlid lineages. We used protein manipulation, gene expression,and genetic mapping to implicate the Tgfβ pathway in the de-velopment of this unusual trait. Given the functions of Tgfβ sig-naling in tissue proliferation, migration, invasion, and organfibrosis, this represents an example of the cooption of existingpathways in the evolution of novelty.

Author contributions: M.R.C. and R.C.A. designed research; M.R.C., Y.H., A.J.C., M.A.M.,J.F.W., and R.C.A. performed research; M.R.C., Y.H., A.J.C., J.F.W., and R.C.A. analyzeddata; and M.R.C., A.J.C., and R.C.A. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).1To whom correspondence should be addressed. Email: albertson@bio.umass.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1719798115/-/DCSupplemental.

Published online June 18, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1719798115 PNAS | July 3, 2018 | vol. 115 | no. 27 | 7063–7068

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We hypothesized that this unique structure develops as an ex-pansion of the intermaxillary ligament, which runs laterally across theupper jaw and connects the left and right maxillary heads (24). Giventhe dynamic movement in fish skulls (25, 26), this class of tissue (e.g.,ligaments) is likely to play important roles in foraging adaptations.However, relative to the bony skeleton, the evolution of ligaments isgrossly understudied. Here, we employ a combination of genetic anddevelopmental approaches to test the hypothesis that elaboration ofthe intermaxillary ligament in Labeotropheus occurs via expansion ofthe Tgfβ → Scx candidate pathway (27–31). Through these analyses,we also identified a previously unknown candidate for ligamenthypertrophy, adam12, and verified its roles in ligament developmentvia functional analyses using the zebrafish model. Together these datasupport our main hypothesis and contribute to an understanding ofthe genetic regulation of complex soft tissue morphologies and,more generally, of how phenotypic novelties evolve.

Results and DiscussionAnatomy and Development of the Snout.The snout in Labeotropheusis a complex and dynamic soft tissue structure compared withTropheops, a closely related near ecological competitor that lacks anexaggerated snout (Fig. 1 A and B). Histological data showed twodistinct, but interdigitating, tissue types within the snout—a DirectRed-positive organized connective tissue high in collagen (pink, Fig.1 C and D) and an Alcian Blue-positive loose connective tissue(blue, Fig. 1 C and D). In both species, this basic configuration ismaintained across the medio-lateral expanse of the snout, but forconsistency we focused our comparisons between species andtreatments on midline sections of this structure. The anatomy andorganization of the Direct Red-positive tissue is consistent withligamentous tissue. To confirm this, we performed immunohisto-chemistry with an anti-Scleraxis antibody, which exhibited strongand consistent signal specific to this tissue (Fig. 1 E and F). Further,this ligament appears to be the intermaxillary ligament, as it insertson the left and right maxillary heads on either side of the ascendingarm of the premaxilla (Fig. 1 H and I), stretching medio-laterallyacross the upper jaw. Notably, this tissue differs from ordinary lig-aments by invading the surrounding loose connective tissue (Fig.1D) and anchoring to the overlying epithelium (Fig. 1D). Qualita-tively, the degree of invasion and anchoring to the epitheliumincreases during ontogeny in Labeotropheus (SI Appendix, Fig. S1A–C), whereas the interface between ligament and loose connectivetissue is much less complex in Tropheops (SI Appendix, Fig. S1D–F).Ligaments and tendons are defined by their stereotypical con-

nections: bone to bone in the case of ligaments and muscle to bonefor tendons. The few examples in the literature that describe de-partures from this anatomy highlight the functional significance ofsuch novel arrangements. For instance, tendons in gecko feet insertdirectly onto toe pad integument, which increases stiffness and is vitalfor adhesion to surfaces during climbing (32). Likewise, stiffness ofshark skin, which enhances hydrodynamics during swimming, is dueto direct muscle to skin attachments (33). We speculate that theconnection between the intermaxillary ligament and overlying skinmay help to stiffen the exaggerated snout of Labeotropheus. A stiffersnout would provide a more robust fulcrum allowing Labeotropheusto pry algae from rocks in a manner that requires less energy com-pared with the bite-and-twist mode of feeding employed by mostTropheops species. The role of the integument during feeding islargely unexplored, and more work is needed to determine thebiomechanical implications of the interaction between the inter-maxillary ligament and the epithelium in Labeotropheus. However,given the unique configuration of this tissue, we suggest that thiswould be a fruitful line of future research.We also found that the snout exhibits dynamic growth. His-

tological data at three time points during juvenile developmentrevealed marked differences between Tropheops and Labeo-tropheus in the pace and pattern of growth of both the inter-maxillary ligament and loose connective tissue (Fig. 1J and SIAppendix, Fig. S1). In general, growth of both tissues is relativelymodest in Tropheops with consistently more ligament than looseconnective tissue at every stage, whereas in Labeotropheus

Fig. 1. Morphology and development of the snout. An unremarkable, flatsnout of most cichlids (and most fishes; represented here by TRC) (A) com-pared with the unique, exaggerated snout of Labeotropheus (representedhere by L. fueleborni) (B). Images courtesy of Ad Konings. (C) Sagittal sectionof the snout in Labeotropheus. (D) Close-up of the black box in C shows theintermaxillary ligament (iml, pink) invading the surrounding loose connec-tive tissue (lct, blue) and anchoring to the overlying epithelium (epi). (E, F,and I) Immunohistochemical staining in Labeotropheus using anti-Scleraxisantibody (green) and cell nuclei counterstained with DAPI (blue). (E) Close-up of the upper black box in B shows the iml invading the surrounding lct.(F) Close-up of the lower black box in B shows the iml anchoring to the epi.Schematic (G) and corresponding histology (H) and immunohistochemistry (I)show the iml inserting onto the maxilla (mx). (J) Amount of lct and iml atthree developmental time points in Labeotropheus and Tropheops. Eachtime point is represented by one individual with tissue measured in two tothree sections close to the midline where the snout reaches its maximumsize. Representative histological sections of both species at the 2.5-cm SLtime point are overlaid on the graph. Sections of the other time points areshown in SI Appendix, Fig. S1. pmx, premaxilla.

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growth is more vigorous, and at every stage there is more looseconnective tissue than ligament. In addition, the pattern of snoutgrowth is notable in Labeotropheus in that accelerated growth of theoverlying connective tissue occurs early [i.e., 1.5–2.5 cm standardlength (SL)] and precedes accelerated growth of the ligament inlarger fish (i.e., 2.5–4.0 cm SL). The decoupling of growth ratesbetween these tissues in Labeotropheus reveals a complex pattern ofdevelopment. In addition, the accelerated growth of the looseconnective tissue relative to the ligamentous tissue suggests that theinductive cues for ligament hypertrophy in Labeotropheus may arisefrom the loose connective tissue. Conversely, modest ligamentgrowth in Tropheops may be due to more limited inductive signalfrom the growth-restricted loose connective tissue.

Molecular Basis of Exaggerated Snout Development. Tgfβ signaling iscritical to ligament development via the transcriptional regulation ofscleraxis (scx) (27–31), and we hypothesized that this pathway wouldbe associated with snout development. Consistent with this, im-munohistochemistry confirmed that Tgfβ1 is localized to the looseconnective tissue of the snout (Fig. 2A), the putative source of theinductive signal for ligament overgrowth. We confirmed andquantified mRNA expression of tgfβ1 and scx in the snout usingquantitative real-time PCR in Labeotropheus and Tropheops. Wefound that (i) both transcripts were expressed in the snout in bothgenera, (ii) tgfβ1 was expressed at higher levels than scx in bothgenera, and (iii) Labeotropheus had higher levels of tgfβ1 (t test, n =6 Labeotropheus, n = 5 Tropheops, df = 8.7, t = 2.2, P = 0.05) andscx (t test, n = 6 Labeotropheus, n = 5 Tropheops, df = 6.9, t = 3.7,P = 0.008) compared with Tropheops (Fig. 2B).The differential expression of these genes led us to experimen-

tally manipulate this pathway to confirm its causal role in snoutdevelopment. Specifically, we implanted either Tgfβ1-soaked beadsor negative control beads into the snouts of juvenile Labeotropheusand Tropheops. We measured gene expression of the downstreamtarget, scx, 12 h after implantation (based on a time series analysis,SI Appendix, Fig. S3) and found that exogenous Tgfβ1 is indeedsufficient to increase levels of scx expression in both species (Tro-pheops, t test, n = 5 control, n = 8 Tgfβ1-treated, df = 7.3, t = −4.4,P = 0.003; Labeotropheus, t test, n = 6 control, n = 6 Tgfβ1-treated,df = 6.5, t = −4.4, P = 0.004; Fig. 3E). Additionally, both speciesresponded with the same amount of increased scx expression, sug-gesting a conserved capacity to respond to Tgfβ1 signaling. Notably,this increase in scx expression also resulted in a tissue level response.Specifically, we assessed ligament morphology 7 d after bead im-plantation using histology and found that ligament size increased inTgfβ1-treated animals in both species (Tropheops, t test, n = 3control, n = 3 Tgfβ1-treated, df = 16.0, t = −2.5, P = 0.02;Labeotropheus, t test, n = 3 control, n = 3 Tgfβ1-treated, df = 14.1,

t = −2.1, P = 0.05), whereas the amount of loose connective tissuedid not change (Fig. 3 A–D, F, and G). In addition, the response ofthe ligament was dependent on the placement of the bead; therewas more ligament growth and epithelial connections when beadswere localized to the loose connective tissue overlaying the ligamentcompared with bead placement below the ligament (SI Appendix,Fig. S2). These data are consistent with our developmental timeseries and immunohistochemistry data and collectively demonstratethat (i) morphogenesis of the two tissues are molecularly decou-pled, and (ii) the inductive cue for ligament growth likely includesTgfβ emanating from the overlying loose connective tissue.

Genetic Basis for Variation in Snout Size.Given the complexity of thesnout phenotype and the wide range of interacting partners of Tgfβ,we wanted to know whether the causative genetic variants for snoutsize are associated with canonical members or known interactors ofthis pathway. To this end, we reanalyzed our published quantitativetrait locus (QTL) data for snout morphology (21, 34) and per-formed additional analyses that capture different aspects of snoutshape. We identified a total of 11 loci that contribute to either snoutlength or depth (Fig. 4 and SI Appendix, Table S1). Each of theseQTL map to distinct regions of the genome, suggesting that snoutlength and depth are under independent genetic control. Likewise,separate loci were identified when fish were reared under alternatebenthic and limnetic foraging environments, which supports theassertion that the environment can dictate regulation of develop-ment at the molecular level (34).By anchoring our genetic linkage map to physical genomic

sequence, we could determine whether QTL colocalize withmembers of the Tgfβ → Scx pathway or with other candidategenes for snout development. To this end, we mapped membersof the canonical Tgfβ pathway (i.e., ligands, receptors, and in-tracellular Smads) and the transcriptional output for ligamentdevelopment (i.e., scx). Notably, our QTL overlap with the in-tracellular transcriptional partner smad2 on linkage group 7, twoparalogs of smad4 on linkage groups 7 and 11, and a scx paralogon linkage group 21 (Fig. 4 and SI Appendix, Table S1). All fourof these loci are associated with SNPs with outlier FST valueswhen Labeotropheus are compared with either Tropheops spe-cifically, or a more general subset of rock-dwelling cichlids (i.e.,

Fig. 2. Protein and gene expression in the snout. (A) Tgfβ1 protein ex-pression (green) in the lct of the snout of Labeotropheus via immunolabel-ing using anti-Tgfβ1 antibody and cell nuclei counterstained with DAPI(blue). (B) qPCR of tgfβ1 and scx expressed in Labeotropheus and Tropheopssnouts. Asterisks indicate significant differences between species, P ≤ 0.05.epi, epithelium; iml, intermaxillary ligament; lct, loose connective tissue.

Fig. 3. Morphological and genetic consequences of Tgfβ1 manipulation inthe snout. Representative sagittal sections of the snout in Tropheops neg-ative control (A) and Tgfβ1-treated animals (B), and Labeotropheus negativecontrol (C) and Tgfβ1-treated animals (D). (E) Relative scx expression 12 hafter bead implantation in Tropheops and Labeotropheus. Intermaxillaryligament area (iml, pink in A–D) (F), and loose connective tissue area (lct,blue in A–D) (G) in Tropheops and Labeotropheus 7 d after bead implan-tation. Significant differences between negative control and Tgfβ1 treat-ments indicated by *P ≤ 0.05 or **P < 0.005.

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mbuna; SI Appendix, Table S2). Consistent with the Tgfβ path-way being up-regulated in Labeotropheus, expression data alsoshow that the smad4 paralog on linkage group 7 (but not linkagegroup 11) is expressed at higher levels in Labeotropheus com-pared with Tropheops (t test, n = 6 Labeotropheus, n = 5 Tro-pheops, df = 9.0, t = 2.4, P = 0.04; SI Appendix, Fig. S4). Thesedata provide additional genetic evidence of a role for Tgfβ → Scxin snout development and identify specific members of thispathway where causative mutations may be found.Despite these cases of colocalization, many QTL for snout size do

not overlap with Tgfβ pathway members, which provides an op-portunity to identity other regulators of this pathway during liga-ment development. To this end, we focused on a robust QTL onlinkage group 13.1 with allele effects that maximize the difference insnout phenotype between parental genotypes (SI Appendix, Fig. S5and Table S1). We used a fine-mapping approach to narrow thisQTL interval and FST data to identify fixed SNPs between Labeo-tropheus and Tropheops (Fig. 5A). Fine-mapping implicated tworegions of peak association between genotype and phenotype (Fig.5A, shaded regions), but only one of these regions contained vari-ants with FST values of 1 (Fig. 5A, black circle). Notably, both ofthese SNPs fell within an intron of the gene ADAM metal-loproteinase domain 12 (adam12), a known regulator of Tgfβ sig-naling (35). Indeed, when Labeotropheus was compared with otherrock-dwelling species, 21 additional intronic high FST SNPs and onedownstream SNP (SI Appendix, Table S2) were identified, sug-gesting high potential for differential regulation in Labeotropheus.Besides its role in Tgfβ signaling, Adam12 is known to mediate a

host of complex cell/tissue behaviors, including proliferation, mi-gration, hypertrophy, and invasion (36–38). These are consistentwith the distribution and development of tissues in the snout ofLabeotropheus. We used immunohistochemistry and an anti-Adam12antibody to show that Adam12 is in fact expressed in the exag-gerated snout of Labeotropheus, and is localized to the looseconnective tissue, similar to the pattern of Tgfβ1 expression (Fig.5B). We also quantified adam12 expression in the snout of juvenileLabeotropheus and Tropheops using qPCR and found that it isexpressed at relatively low levels compared with tgfβ1 or scx, and atequal levels in Labeotropheus (i.e., 2.28e−4 ± 4.68e−5) and Tropheops(i.e., 2.31e−4 ± 1.05e−4) at this stage. Given the dynamic nature ofthis tissue as well as the complex regulatory capacity of Adam12 (35–38), we speculate that differences in expression may still occur, butat an earlier stage and/or in a transient manner.

To functionally explore the role of adam12 in ligament de-velopment, we took advantage of the recently described zebra-fish adam12(−/−) mutant (39). Homozygous recessive mutants areviable and were reported as having no obvious morphologicaldefects aside from reduced body size (39). Given our geneticmapping data, we hypothesized that animals lacking functionalAdam12 would exhibit subtle differences in ligament develop-ment, and that defects would be apparent at both the transcriptand phenotypic level. To this end, we measured mRNA ex-pression of the two scleraxis paralogs in zebrafish, scxa and scxb,in the pharyngeal skeleton of mutant and WT adult (∼1 y)zebrafish. We found that scxa mRNA levels are lower in mutantscompared with wild-type fish (t test, n = 5 wild-type, n = 3 mu-tants, df = 5.9, t = −2.3, P = 0.058). Scxb expression is lower thanscxa in both mutant and wild-type animals and shows similar (i.e.,lower in mutants) but nonsignificant trends (t test, n = 5 wild-type, n = 3 mutants, df = 4.0, t = −1.5, P = 0.22) (SI Appendix,Fig. S6). We also measured the volume of a relatively large,functionally salient ligament that connects the mandible to theceratohyal (cerato-mandibular ligament, Fig. 6 and SI Appendix,Fig. S7) and found that fish lacking adam12 have smaller liga-ments (t test, n = 4 wild-type, n = 4 mutants, df = 4.4, t = −4.2,P = 0.012; Fig. 6B and SI Appendix, Fig. S8). In addition, adam12mutants exhibit smaller tendons, and the bony processes onwhich tendons, ligament, and other soft-tissues insert are moregracile and slender in mutants relative to those in wild types (i.e.,arrowheads in Fig. 6 and SI Appendix, Fig. S9). Given that nor-mal bone development and growth relies on mechanical input

Fig. 4. Summary of QTL results for snout size in hybrids between LF and TRC. Agenetic map is shown summarizing locations of QTL from the F2 analyses (pink),F3 benthic analysis (green), and F3 limnetic analyses (blue). Vertical bars to theright of linkage groups represent the 95% confidence interval for each QTL, andarrowheads represent QTL LOD peaks. In addition, the positions of canonicalmembers of the Tgfβ pathway (i.e., ligands, receptors, and intracellular smads),interactors (i.e., adam12), and scleraxis paralogs are indicated on the linkagemap. Raw data can be found in SI Appendix, Table S1.

Fig. 5. QTL and gene expression data implicate adam12 as another candidatefor snout size. (A) Fine-mapping analysis of the QTL peak that maximizes alleleeffects (SI Appendix, Fig. S5) showing average difference in snout size (i.e., av-erage phenotypic effect, black line) between hybrids with homozygous geno-types at markers across scaffold 26 on linkage group 13.1 (i.e., across the QTLinterval). Peaks (highlighted in gray) are regions where hybrids with theLabeotropheus genotype have the largest snouts and those with the Tropheopsgenotype have the smallest snouts. FST data are also mapped onto the scaffold(blue dots). SNPs that fall above the blue dashed line exceed an empiricalthreshold for divergence between cichlid genera. There are two SNPs with FSTvalues of 1.0 that fall within a peak (black circle). Both of these markers areintronic in the gene adam12. (B) Adam12 protein expression is similar to Tgfβ inthat it localizes to the lct of the snout in Labeotropheus. (C) Model of a proposedmolecular pathway of exaggerated snout development. In the lct (blue) Adam12may activate Tgfβ1, which in turn up-regulates Scx in the iml (pink), leadingto the expansion of this tissue. epi, epithelium; iml, intermaxillary ligament;lct, loose connective tissue.

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from the local soft tissue environment, these data suggest thatmore slender ligaments and tendons in adam12 mutants result inweaker mechanical input and the development of more gracilebone. Taken together, these results support our hypothesis thatadam12 is necessary for normal ligament growth and suggestbroad roles for this gene in craniofacial development.Based on these data as well as the concordance between tissue

anatomy in the snout and the known functions of Adam12, wehypothesize that Adam12 interacts with the Tgfβ → Scx pathway tohelp mediate snout development and exaggeration in Labeo-tropheus. Specifically, we predict that Adam12 increases Scx activityin the intermaxillary ligament, likely through Tgfβ1 signaling (35)(Fig. 5C).

ConclusionsEvolution acts as both a tinkerer and innovator. This metaphorunderscores the continuous and discontinuous nature of phenotypicvariation among organisms. While continuous variation may char-acterize the majority of existing biodiversity, many examples of in-novation (or saltation) also exist, especially in the fossil record (40).However, what exactly is a phenotypic novelty? At what point doescontinuous variation cross over to become discontinuous variation?There is no clear consensus in the literature on these points. Rather,much like the concepts of species or homology, the definition ofnovelty depends on context and level. Over 40 y ago, Jacob (15)suggested that evolution cannot produce novelties from scratch butmust work with what already exists. In other words, innovations atthe organ level must arise by tinkering at the molecular level (15),and as we delve deeper into the molecular origins of differentphenotypic novelties, the exact nature of this tinkering is revealingitself. For instance, evolutionary loss of certain structures can betraced to the loss of genetic elements (41, 42). Alternatively, phe-notypic gain has also been linked to the loss of genetic enhancers(43). Another recurrent theme in the study of novelties is the re-deployment (cooption) of genetic/developmental networks in noveltissues/locations (12, 44). Finally, gene/genome duplication can fa-cilitate the evolution of novelty by providing greater opportunitiesto tinker at the molecular level (13, 14). These examples suggestthat there are many molecular paths to morphological novelty, andthat saltatory evolution can arise from both large (e.g., genomeduplication) and small (e.g., local deletion) mutational events. Inthis way, developmental genetics is bringing new insights to an old

debate between gradualists and saltationists by showing that acontinuum of genetic changes can all lead to major shifts in mor-phology; however, more examples are needed.While we are gaining unprecedented insights into the devel-

opment and evolution of morphological variation, novelties arestill underrepresented in the literature. Here, we describe a softtissue novelty whose origins are associated with the recruitment ofan existing signaling pathway (e.g., Tgfβ-Scx). However, it is worthnoting that the evolution of the exaggerated snout may also havebeen facilitated by larger-scale mutational events. When searchingin public genomic databases [e.g., National Center for Bio-technology Information (NCBI)], teleost fishes appear to haveundergone an expansion of smad4, with up to four paralogs inmost teleosts (e.g., ref. 45). In theory, this ancestral gene dupli-cation could lead to divergence in Tgfβ functioning in differenttissues, which is consistent with recent evidence of selection onancient gene duplicates in Malawi cichlids (46). It is thereforenotable that our data show that three smad4 paralogs colocalize tosnout QTL, two of which define QTL peaks, and one that is dif-ferentially expressed in species with different snout sizes. Thus, itis possible that the evolution of the exaggerated snout in Labeo-tropheus is due, at least in part, to the molecular tinkering ofsmad4 duplicates. While many questions regarding the develop-ment and evolution of this structure remain (e.g., there are severalsnout QTL with no obvious candidate genes), this work contrib-utes to a growing body of literature by providing another exampleof the evolution of novelty by tinkering at the molecular level.

MethodsAnimals. Cichlids used for experiments were derived from wild-caught LakeMalawi fish. They were reared and euthanized following standard protocolsapproved by the University of Massachusetts Institutional Animal Care andUse Committee. Since both of the species in the Labeotropheus genus (i.e.,Labeotropheus fueleborni, LF, and Labeotropheus trewavasae, LT) haveexaggerated snouts, we used both species depending on availability. Like-wise, we used both Tropheops “red cheek” (TRC) and Tropheops tropheops(TT) in different experiments. Fish are thus referred to as simply Labeo-tropheus or Tropheops throughout.

Histology. Histology was used to visualize and quantify tissue-level anatomyof the snout. Animals for the developmental time series (LF, n = 3; TRC, n = 3)were collected at 1.5 cm, 2.5 cm, and 4.0 cm SL. We have shown previouslythat the snout scales with body size throughout the life of both LF and TRC;however, 1.5 cm represents the time point at which the snout in LF is firstapparent at a gross morphological level (21). By 4.0 cm, the snout is well-formed and clearly different in the two species (21). While both species canreach 8–9 cm in laboratory, ∼4.0 cm is also the size when animals first beginto reach sexual maturity. In addition, animals for the Tgfβ1 manipulationexperiment (LT, n = 3 Tgfβ1-treated, n = 3 control; TT, n = 3 Tgfβ1-treated,n = 3 control) were collected 7 d after bead implantation (see below) andwere 2.9–4.3 cm SL in size. In both experiments, serial sagittal sections werestained with the Hall-Brunt Quadruple connective tissue stain (47). Theamount of intermaxillary ligament and loose connective tissue in a repre-sentative section were quantified by measuring the area occupied by each ofthese tissues relative to a standardized portion of the snout.

Immunohistochemistry. Immunohistochemistry was used to visualize presenceand location of proteins in the snout of juvenile (∼3.0 cm SL) LF. After blocking fornonspecific staining, serial sagittal sections were incubated in primary antibodyat 4 °C overnight (Rabbit Anti-Scleraxis Polyclonal Antibody, Rabbit Anti-TGFβ1Polyclonal Antibody, Rabbit Anti-ADAM12 Polyclonal Antibody; Bioss Anti-bodies) followed by incubation in a fluorescent secondary antibody at 4 °Covernight (Goat anti-Rabbit IgG Alexa Fluor 488; Life Technologies). Finally, cellnuclei were counterstained with DAPI (Sigma).

Quantitative Real-Time PCR. qPCR was used to measure tgfβ1, scx, 2 smad4paralogs on linkage groups 7 and 11 (see below), and adam12 expression injuvenile (3.8–5.0 cm SL) LT (n = 6), TRC (n = 3), and TT (n = 2) as well as tocompare scx expression following the Tgfβ manipulation experiment (LT, n =6 Tgfβ1-treated, n = 6 control; TRC, n = 5 Tgfβ1-treated, n = 3 control; TT, n =3 Tgfβ1-treated, n = 2 control). qPCR was also used to measure scxa and scxbin Adam12(−/−) mutant zebrafish (n = 3) and several wild-type lines (caspern = 1, AB n = 2, EW n = 2). RNA was isolated from homogenized snout tissue

Fig. 6. μCT data demonstrate reduced ligament volume in adam12 zebrafishmutants. (A) Reconstructed 3D model in a wild-type zebrafish illustrating posi-tion of the cerato-mandibular ligament (cml) relative to mandible (den) andceratohyal (ch). Maxilla (mx), premaxilla (pmx), and interopercle (iop) are alsoshown for perspective. (B) Reconstructed 3D model of the same structures in anadam12 mutant zebrafish. Note the marked reduction in the size of the cml(blue) and the coronoid process of the mandible (arrowheads).

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by the phenol chloroform extraction technique and standardized beforereverse transcription. Levels of gene expression were measured using SYBRGreen chemistry (Power SYBR Green Master Mix), and relative quantificationwas analyzed using the comparative CT method (48).

Tgfβ Manipulation. Beads (Affi-Gel Blue Gel; Bio-Rad) soaked in eitherrecombinant human Tgfβ1 protein (R&D Systems) or control buffer wereimplanted into the snouts of juvenile (2.9–5.3 cm SL) LT (n = 9 Tgfβ1-treated,n = 9 control), TRC (n = 5 Tgfβ1-treated, n = 3 control), and TT (n = 6 Tgfβ1-treated, n = 5 control). Because of the nature of this experiment, we usedlarger juvenile fish, but they were still in the range of those used for thehistological analysis. A small incision was made in anesthetized fish parallel tothe snout, and a path was bored using a sewing needle to the tip of the snout.Four incubated beads were then pushed through the incision and guided tothe tip of the snout, taking care to leave tissue as undisturbed as possible. Allfish were recovered from the surgery and were collected either 12 h later forgene expression analysis or 7 d later for morphological analysis.

QTL Mapping.Generation of the hybrid pedigrees and mapping strategies aredescribed in Albertson et al. (49) and Parsons et al. (34). Further details maybe found there or in SI Appendix.

μCT Scanning. We quantified the volume of ligamentous tissue in adam12(−/−)

zebrafish mutants using X-ray microcomputed tomography (μCT). Zebrafishdo not possess an intermaxillary ligament. We instead focused on the cerato-mandibular ligament (CML), which runs from themedial surface of themandibleanteriorly to the medial surface of the ceratohyal posteriorly. We compared thevolume of the CML between adam12 mutant zebrafish (n = 4) and wild-typecasper zebrafish (n = 4), the line in which the mutation was made. All fish weresubmerged for 5 h in 2.5% Lugol iodine solution, which is absorbed into the softtissues and acts as a contrast agent (50). We scanned all specimens at 5-μmresolution using an X-Tek HMXST 225 μCT scanner (Nikon Corporation) at 90 kVand 75 μA. We segmented out the CML using Mimics v19 (Materialise NV) andexported the 3D models before calculating volumes. We corrected the vol-umes by head depth and statistically compared the adam12 to WT residualligament volumes using a t test in R (v3.4.0).

ACKNOWLEDGMENTS. We thank Joy Richman for supplying beads for theTGFβ manipulation, Atsuo Iida for generously providing adam12 mutantzebrafish, and members of the R.C.A. laboratory for their thoughtful com-ments on this project. Funding was provided by the National Science Foun-dation Grant CAREER IOS-1054909 (to R.C.A.).

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