DOI: 10.25338/b8zp83
title: The cichlid pharyngeal jaw novelty enhances evolutionary integration in the feeding apparatus

description: The modified pharyngeal jaw system of cichlid fishes is widely viewed as a
 key innovation that substantially facilitated the evolutionary exu- 1.70
 berance of this iconic evolutionary radiation. We conduct comparative
 phylogenetic analyses of integration, disparity, and rate of
 evolution among feeding-related, skeletal structures in
 Neotropical cichlids and North American centrarchids, which lack the
 specialized pharyngeal jaw. Contrasting evolutionary patterns in these two
 continental radiations, we test a classic decoupling hypothesis.
 Specifically, we ask whether the modified pharyngeal jaws in
 cichlids resulted in enhanced evolutionary independence of the oral and
 pharyngeal jaws, leading to increased diversity of trophic structures.
 Contrary to this prediction, we find significantly stronger evolutionary
 integration between the oral and pharyngeal jaws in cichlids compared to
 centrarchids, although the two groups do not differ in patterns of
 integration within each jaw system. Further, though we find no significant
 differences in disparity, centrarchids show faster rates of morphological
 evolution. Our results suggest that the modified pharyngeal jaw resulted
 in less evolutionary independence and slower rates of evolution within the
 feeding system. Thus, we raise the possibility that the cichlid novelty
 enhances feeding performance, but does not prompt increased morphological
 diversification within the feeding apparatus, as has long been thought.
description: <strong><em>Study specimens and anatomical
 measurements</em></strong> We examined 85
 Neotropical cichlid species (Family Cichlidae) and 30 North American
 sunfish species (Family Centrarchidae) with an average of 5 specimens per
 species. Cichlidae, being one of seven families where modified pharyngeal
 jaws are an independently derived synapomorphy for all known species,
 represents fishes with MPJs. Centrarchids were used to represent fishes
 lacking the MPJ, as all species exhibit a generalized pharyngeal jaw
 system that lacks all traits defining an MPJ (Fig. 1b). We used published
 morphological data for 218 cichlid specimens (Burress et al. 2020;
 Supplemental Table 1) and obtained an additional 170 and 129 adult cichlid
 and centrarchid specimens, respectively, from museum collections,
 collection trips, and the pet trade (Supplemental Table 1) in an effort to
 maximize the morphological diversity and generic coverage in both
 families. Within our dataset, we have 50% and 100% sampling of genera for
 Neotropical cichlids and centrarchids, respectively. Though Neotropical
 and African cichlids both exhibit trophic morphologies and functions
 unique to their radiations, these two cichlid sister groups show extensive
 convergent evolution and shared ecomorphs (Burress 2014; Burress et al.
 2017). Thus, we restricted our study to the continental radiation of
 Central and South American river and lake-dwelling cichlid species and we
 aimed to include species that would showcase the full breadth of
 ecomorphological variation exhibited by this spectacularly diverse clade
 (Burress 2014; Arbour et al. 2020). By excluding African cichlids, we also
 reduce the number of confounding factors that contribute to this group’s
 ecomorphological diversity, including lake effects (Seehausen 2006) and
 hybridization (Joyce et al. 2011; Meier et al. 2017, 2019). All specimens
 were fixed in formalin and stored in 70% ethanol prior to this study. Each
 specimen was then cleared and stained using procedures adapted from
 Dingerkus and Uhler (1977) and Taylor and Van Dyke (1985), and stained
 specimens were stored in 90% glycerin. Upper and lower pharyngeal jaws
 were dissected from each stained specimen and photographed. The left side
 of each stained specimen was also photographed in a relaxed posture where
 oral jaws were mostly protracted, according to supplemental procedures
 outlined in Roberts et al. 2021. Using digital calipers
 and NIH ImageJ software (Schneider et al. 2012), we measured anatomical
 traits to characterize the size, shape, and functionality of the oral jaw
 and pharyngeal jaw systems. Traits were separated into oral and pharyngeal
 jaw systems based on their contributions to prey capture or processing
 functions, respectively (Alexander 1967; Lauder 1983; Wainwright 2005). We
 measured and computed 13 oral jaw traits including premaxillary
 dentigerous arm length, premaxillary ascending process length, gape, oral
 jaw protrusion, buccal cavity length, the horizontal and vertical
 positions of the lower jaw joint, the horizontal and vertical positions of
 the maxilla-nasal joint, lower oral jaw length, opening and closing
 mechanical advantage, and kinesis (Figs. 1c and 3b; Supplemental Tables 1
 and 2). Kinesis, calculated according to procedures in Burress et al.
 2020, describes the amount of shape change in the oral, four-bar linkage
 following a fixed rotation of the mandible (Martinez et al. 2018).
 Finally, we measured seven pharyngeal jaw traits including lower
 pharyngeal jaw length, width, depth, and tooth width (averaged across the
 three largest teeth), as well as upper pharyngeal jaw length, width, and
 depth (Figs. 1c and 3a; Supplemental Tables 1 and 2). Pharyngeal jaws from
 especially small species
 (e.g., <em>Apistogramma</em> and <em>Enneacanthus</em>) were measured with calipers under a dissecting microscope. All pharyngeal jaw measurements for the 218 cichlid specimens from Burress et al. 2020, excluding average tooth width, were retaken, as original trait data collected were not comparable to fishes with generalized pharyngeal jaws.  Morphological measurements for each specimen were converted to shape ratios where each trait value was divided by the cubed root of (head length x head width x head depth). By size correcting each specimen using the geometric mean of these three main size dimensions, we account for the complexity of body size and our data capture shape variation due to allometry (Claude 2013; Klingenberg 2016; Price et al. 2019). Shape ratios were averaged across specimens, log transformed to achieve normal distributions, and used in all subsequent analyses in R v. 4.2.0 (R Core Team 2022). For select multivariate analyses, each species’ log shape ratios were converted to standard normal deviates (i.e., z-scores) using the scale function (Becker et al. 1988) to ensure that all 20 measurements were in commensurate units and scales (Huttegger and Mitteroecker 2011; Adams and Collyer 2019b). These standardized values were used for analyses where noted. <strong><em>Statistical analyses</em></strong> To conduct our analyses in a phylogenetic context, we used a time-calibrated molecular phylogeny of ray-finned fishes (Rabosky, Chang, Title, and Alfaro et al. 2018). This phylogeny, which includes a dense sampling of species in both focal groups, was trimmed to include fishes represented by molecular sequence data and then to match the species included our study. For species that were not present on the phylogeny, we used the closest related species within the same genus as proxy. Four substitutions were made, including <em>Apistogramma hoignei </em>for <em>Apistogramma cacatuoides</em>, <em>Cichlasoma dimerus</em> for <em>Cichlasoma orientale</em>, <em>Pterophyllum leopoldi </em>for <em>Pterophyllum scalare</em>, and <em>Amatitlania nigrofasciata</em> for <em>Amatitlania siquia</em>*. To visualize the multivariate morphological data in shape space, we performed Principal Component Analyses (PCA) on the correlation matrix of unstandardized, log shape ratios using the prcomp function (Mardia et al. 1979; Becker et al. 1988; Venables and Ripley 2002) in STATS v. 4.2.0 (R Core Team 2022). Analyses were run on all 20 morphological traits for cichlids and centrarchids separately, as well as in one combined analysis. We ran additional PCAs on the correlation matrices of 13 oral jaw traits and the seven pharyngeal jaw traits separately to visualize how cichlids and centrarchids fall in shape space based on each jaw unit. To test whether the cichlids and centrarchids have statistically different oral and pharyngeal jaw morphology, we performed a phylogenetic multivariate analysis of variance (phylogenetic MANOVA) on the z-scores of all 20 morphological traits over 10,000 iterations under a Brownian Motion model. This same method, implemented with the procD.pgls function (Adams 2014a; Adams and Collyer 2015, 2016, 2018; Collyer et al. 2015) in GEOMORPH v. 4.0.4 (Collyer and Adams 2018; Baken et al. 2021; Adams and Collyer 2022; Adams et al. 2023), was used to perform individual phylogenetic ANOVAs on each of the 20 traits in our dataset. To quantify and compare the magnitude of interspecific variation among cichlid and centrarchid morphologies, we used morphol.disparity (Zelditch et al. 2012; Collyer and Adams 2021)* in GEOMORPH. Using unstandardized, log shape ratios, we estimated overall disparity for all 20 morphological traits, two subsets of traits representing each jaw unit, and for each individual trait. We also estimated and contrasted the Brownian multivariate rate parameter for all 20 traits,  two subsets of traits representing each jaw unit, and for each individual trait using compare.evol.rates (Adams 2014b; Denton and Adams 2015; Adams and Collyer 2018, 2019a) in GEOMORPH. We used the phylo.integration function (Adams and Felice 2014; Collyer et al. 2015; Adams and Collyer 2016, 2018, 2019a) in GEOMORPH to quantify the degree of morphological covariance between oral jaw and pharyngeal jaw traits in an evolutionary context over 10,000 iterations. As this was done separately on the z-scores from cichlids and centrarchids, we then compared the strength of between-jaw-unit integration between families with the compare.pls function (Collyer et al. 2015; Adams and Collyer 2016) in GEOMORPH. For each family, we used standard normal deviates to calculate the degree of evolutionary integration within each jaw system using the integration.Vrel function (Pavlicev et al. 2009; Conaway and Adams 2022) and compared the results using the compare.ZVrel (Conaway and Adams 2022) in GEOMORPH. Further, using standard normal deviates and the phylo.modularity and compare.CR functions (Adams and Felice 2014; Adams 2016; Adams and Collyer 2019a) in GEOMORPH, we quantified and contrasted the degree of evolutionary modularity in each family. Finally, we produced the evolutionary correlation matrices for each family using code modified from integration.Vrel and the cor function (Kendall 1938, 1945; Becker et al. 1988) in STATS. We then used the corrplot function (Murdoch and Chow 1996; Friendly 2002) in CORRPLOT v. 0.92 (Wei and Simko 2021) to visualize these matrices. <strong>References</strong> Adams, D. C. 2014a. 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description: Version 3 (12/23/24) -- Updated trait data
 (ComboFin2_new.csv, Supplemental_Table_1_data.xlsx,
 SR_data_mean.csv) Version 2 (7/21/23) -- Supplemental
 materials now include an R markdown file that outlines all analyses
 completed in R studios as well as two dataframes and a tree file to be
 uploaded when running analyses. This is in addition to the three
 supplemental files that were previously published. Please note that .csv
 files include species mean data for each trait. Also note that
 Supplemental Table 2 now includes information on which data columns in
 each .csv file correspond to which traits. Please see the README file for
 additional details about each published file. Version
 1 (6/21/23) -- Supplemental files include trait data for each specimen,
 definitions for all measurements taken and calculated, and PCA
 loadings.
description: # The cichlid pharyngeal jaw novelty enhances evolutionary integration in
 the feeding apparatus
 [https://doi.org/10.25338/B8ZP83](https://doi.org/10.25338/B8ZP83) ##
 Description of the data and file structure #### File:
 Supplemental\_Table\_1\_data.xlsx **Description:** Anatomical measurements
 made on cichlid and centrarchid feeding structures. We examined 388
 specimens representing 85 Neotropical cichlid species and 129 specimens
 representing 30 North American sunfish species. Accession numbers are
 provided for specimens obtained from the following museums: Auburn
 University Museum of Natural History (AUM), the California Academy of
 Sciences (CAS/CAS-ICH), the Academy of Natural Sciences of Philadelphia
 (ANSP), the Field Museum of Natural History (FMNH), the Florida Museum of
 Natural History (UF/FLMNH), the Illinois Natural History Survey (INHS),
 the Louisiana State University Museum of Natural Science (LSUMZ/LSU), the
 Smithsonian National Museum of Natural History (NMNH/USNM), the
 Biodiversity and Research Teaching Collections at Texas A&M (TCWC),
 and the Tulane University Museum of Natural History (TU). Shape ratios for
 each specimen are provided for all traits, excluding kinesis which is a
 provided as a mean value for each species. Before completing analyses,
 shape ratios were averaged across specimens to calculate a mean trait
 value and log-transformed for each species. For some analyses, log shape
 ratios were converted to standard normal deviates. Definitions for all
 traits can be found in Supplemental Table 2. Components of kinesis,
 opening and closings in-lever measurements, tooth measurements and trait
 measurements for *Micropterus warriorensis*, *Micropterus tallapoosae*,
 *Micropterus chattahoochae*, *Micropterus cahabae*, and *Lepomis
 peltastes* are also available upon request. #### File:
 Supplemental\_Table\_2\_definitions.xlsx **Description:** Anatomical
 measurement definitions of all trait measurements included in this
 study. Descriptions of all measurements collected for this study including
 standard length, reference lines, craniofacial traits, oral jaw traits,
 components of the four-bar linkage (used to calculate kinesis), and
 pharyngeal jaw traits. Measurements were taken directly from cleared and
 stained specimens unless otherwise noted. Also, note that measurements
 taken relative to horizontal and vertical reference lines result in both
 positive (in front/above) and negative (behind/below line) values. Thus,
 we added a value of X mm to all measurements for a specified trait to make
 all values positive in preparation for log-transformations. We did not
 take the absolute value of these measurements because we wanted to see the
 positional difference between species. Contact the corresponding author
 for additional information regarding how to collect measurements. ####
 File: Supplemental\_Table\_3\_PCA.xlsx **Description:** Principal
 component loadings for principal component analyses in Figures 2 and 3.
 #### File: Rcode\_EVO-qpad109.Rmd **Description:** R markdown file
 detailing all quantitative analyses using the data. This file imports
 several .csv and .tre files as listed below. Please note that this code
 does not provide details on how we size corrected specimen data using the
 geometric mean, computed species average shape data, or log transformed
 the species averages. Please contact the corresponding author for
 questions about this portion of our methods. #### File: ComboTreeFin2.tre
 **Description:** Tree file trimmed from a published molecular phylogeny
 (Rabosky et al. 2018) to match the 115 species in this dataset. #### File:
 ComboFin2\_new\.csv **Description:** Excel file with log-transformed,
 average shape ratios per species. Here rows are also arranged to match the
 order of the tip labels in the tree. See Supplemental Table 2 for all
 variable information.  #### File: SR\_data\_mean.csv
 **Description:** Excel file with average shape ratios per species. See
 Supplemental Table 2 for all variable information.  #### File:
 README\_Cich\_v\_Cent\_Jaw\_Integration.docx **Description:** read me file
 with information included here ## Code/software * NIH ImageJ software
 (Schneider et al. 2012) * R (R Core Team 2022)