ZOOLOGY Zoology 111 (2008) 442–454 Morphology of a picky eater: A novel mechanism underlies premaxillary protrusion and retraction within cyprinodontiforms L. Patricia Hernandez a, , Lara A. Ferry-Graham b , Alice C. Gibb c a Department of Biological Sciences, The George Washington University, 2023 G. Street NW, Washington, DC 20052, USA b Moss Landing Marine Labs, California State Universities, 8272 Moss Landing Road, Moss Landing, CA 95039, USA c Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011-5640, USA Received 5 September 2007; received in revised form 4 December 2007; accepted 5 December 2007 Abstract Upper jaw protrusion is hypothesized to improve feeding performance in teleost fishes by enhancing suction production and stealth of the feeding event. However, many cyprinodontiform fishes (mid-water feeders, such as mosquitofish, killifish, swordtails, mollies and pupfish) use upper jaw protrusion for ‘‘picking’’ prey out of the water column or off the substrate; this feeding mode may require improved jaw dexterity, but does not necessarily require increased stealth and/or suction production. We describe functional aspects of the bones, muscles and ligaments of the anterior jaws in three cyprinodontiform genera: Fundulus (Fundulidae), Gambusia and Poecilia (Poeciliidae). All three genera possess a premaxillomandibular ligament that connects the premaxilla of the upper jaw to the mandible. The architecture of this ligament is markedly different from the upper–lower jaw connections previously described for basal atherinomorphs or other teleosts, and this loose ligamentous connection allows for more pronounced premaxillary protrusion in this group relative to closely related outgroup taxa. Within poeciliids, a novel insertion of the second division of the adductor mandibulae (A2) onto the premaxilla has also evolved, which allows this jaw adductor to actively retract the premaxilla during mouth closing. This movement is in contrast with most other teleosts, where the upper jaw is retracted passively via pressure applied by the adduction of the lower jaw. We postulate that this mechanism of premaxillary protrusion mediates the cyprinodontiforms’ ability to selectively pick specific food items from the water column, surface or bottom, as a picking-based feeding mechanism requires controlled and coordinated ‘‘forceps-like’’ movements of the upper and lower jaws. This mechanism is further refined in some poeciliids, where direct muscular control of the premaxillae may facilitate picking and/or scraping material from the substrate. r 2008 Elsevier GmbH. All rights reserved. Keywords: Poeciliidae; Cyprinodontiformes; Adductor mandibulae; Feeding mechanism; Premaxilla Introduction Teleosts have demonstrated an enormous capacity to evolve new feeding morphologies, mechanisms and behaviors. Trophic strategies in this group include: (1) biting tough, encrusting algae (e.g. robust-jawed scarids; Bellwood and Choat, 1990); (2) sucking miniscule crustaceans from the water column (e.g. gracile-jawed syngnathids; Bergert and Wainwright, 1997); (3) scraping epiphytic algae from the substrate (e.g. loricarids; Schaefer and Lauder, 1986); (4) suspension feeding on zooplankton and phytoplankton from the water column ARTICLE IN PRESS www.elsevier.de/zool 0944-2006/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.zool.2007.12.002 Corresponding author. Tel.: +1 202 994 6930; fax: +1 202 994 6100. E-mail address: [email protected] (L.P. Hernandez).
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Zoology 111 (2008) 442–454www.elsevier.de/zool
Morphology of a picky eater: A novel mechanism underlies premaxillary
protrusion and retraction within cyprinodontiforms
L. Patricia Hernandeza,�, Lara A. Ferry-Grahamb, Alice C. Gibbc
aDepartment of Biological Sciences, The George Washington University, 2023 G. Street NW, Washington, DC 20052, USAbMoss Landing Marine Labs, California State Universities, 8272 Moss Landing Road, Moss Landing, CA 95039, USAcDepartment of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011-5640, USA
Received 5 September 2007; received in revised form 4 December 2007; accepted 5 December 2007
Abstract
Upper jaw protrusion is hypothesized to improve feeding performance in teleost fishes by enhancing suctionproduction and stealth of the feeding event. However, many cyprinodontiform fishes (mid-water feeders, such asmosquitofish, killifish, swordtails, mollies and pupfish) use upper jaw protrusion for ‘‘picking’’ prey out of the watercolumn or off the substrate; this feeding mode may require improved jaw dexterity, but does not necessarily requireincreased stealth and/or suction production. We describe functional aspects of the bones, muscles and ligaments of theanterior jaws in three cyprinodontiform genera: Fundulus (Fundulidae), Gambusia and Poecilia (Poeciliidae). All threegenera possess a premaxillomandibular ligament that connects the premaxilla of the upper jaw to the mandible. Thearchitecture of this ligament is markedly different from the upper–lower jaw connections previously described for basalatherinomorphs or other teleosts, and this loose ligamentous connection allows for more pronounced premaxillaryprotrusion in this group relative to closely related outgroup taxa. Within poeciliids, a novel insertion of the seconddivision of the adductor mandibulae (A2) onto the premaxilla has also evolved, which allows this jaw adductor toactively retract the premaxilla during mouth closing. This movement is in contrast with most other teleosts, where theupper jaw is retracted passively via pressure applied by the adduction of the lower jaw. We postulate that thismechanism of premaxillary protrusion mediates the cyprinodontiforms’ ability to selectively pick specific food itemsfrom the water column, surface or bottom, as a picking-based feeding mechanism requires controlled and coordinated‘‘forceps-like’’ movements of the upper and lower jaws. This mechanism is further refined in some poeciliids, wheredirect muscular control of the premaxillae may facilitate picking and/or scraping material from the substrate.r 2008 Elsevier GmbH. All rights reserved.
behaviors. Trophic strategies in this group include:(1) biting tough, encrusting algae (e.g. robust-jawedscarids; Bellwood and Choat, 1990); (2) sucking minisculecrustaceans from the water column (e.g. gracile-jawedsyngnathids; Bergert and Wainwright, 1997); (3) scrapingepiphytic algae from the substrate (e.g. loricarids;Schaefer and Lauder, 1986); (4) suspension feeding onzooplankton and phytoplankton from the water column
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(e.g. tilapia; Goodrich et al., 2000); (5) ambush attackson mobile prey (e.g. Luciocephalus; Lauder and Liem,1981); (6) rotational feeding to tear chunks from largerprey (e.g. eels; Helfman and Clark, 1986); and (7)suction feeding on highly elusive prey (e.g. largemouthbass; Sanford and Wainwright, 2002). Although thesefeeding mechanisms are diverse, they can generally bedivided into one of two broad categories: biting orsuction feeding. While there is a continuum betweenthese two end points, there is also a functional trade-offassociated with specialization for one category versusthe other (Bouton et al., 1998). Effective suction-feedingperformance is associated with a relatively small mouthaperture, well-developed hyoid musculature and asso-ciated skeletal elements, lateral occlusion of the gapeduring a feeding event, and rapid movements of cranialelements. In contrast, effective biting is associated with alarge mouth aperture to maximize contact with the prey,well-developed adductor muscles to generate a forcefulbite, as well as robust cranial bones to withstand forcesgenerated by the jaws.
Another example of trophic diversity in teleosts isdemonstrated by ‘‘picking’’-based prey capture; this is asubcategory of biting where individual prey items aregrasped from the water column and/or substrate(Motta, 1982, 1985; Ferry-Graham et al., 2008). Incontrast to most forms of biting, however, fish with thiscapture mechanism may procure individual nutritiveprey items from the substrate or water column, whileleaving non-nutritive items behind (Horn and Ferry-Graham, 2006). Because fish first select and then graspspecific individual food items, fine control of the jawapparatus is key. Thus, a picking-based feeding mechan-ism should be most effective when performed usingprecisely controlled and coordinated ‘‘forceps-like’’movements of the upper and lower jaws (Motta, 1988).
Anyone who has used a pair of fine forceps knowsthat brute force is not the preferred mode of operation;rather, more delicate control is key. Moreover, forceps/tweezers are often curved to concentrate forces at theanterior tips. This analogy, coupled with the functionaldemands of picking, suggests that pickers possess a suiteof morphological features distinct from those seen ineither suction feeders or strong biters. Effective pickersshould possess: (1) fine control of the oral jaws, (2) jawsin which force application has been shifted anteriorlyand (3) a biomechanical coupling that enables synchro-nized movements of the upper and lower jaws.
Many species in the teleost order Cyprinodontiformesare characterized by a picking-based feeding behaviorthat employs a beak-like, open mouth to grasp preyfrom the substrate, water column or surface (Alexander,1967b; Ferry-Graham et al., 2008). This order, whichincludes mosquitofish, killifish, swordtails, mollies andpupfish, is often considered a large group of picking,mid-water and substrate feeders (Weisberg, 1986;
Taylor, 1992; Mansfield and McArdle, 1998; Hargrave,2006). However, while some basal members are omni-vorous mid-water feeders (e.g., Hargrave, 2006), morederived members of Poeciliidae appear to be specializedfor herbivory (e.g., Fares Alkahem et al., 2007). To date,most research on feeding behavior within this group hasfocused on the role of the premaxilla and maxilla (upperjaw bones) in effecting upper jaw protrusion (Eaton,1935; Alexander, 1967a, b). In contrast, the muscles andligaments that produce and coordinate movement of theanterior cranial elements have been given less attention,but may greatly inform our understanding of the feedingmechanism and behavior.
Here, we assess the underlying mechanism of pre-maxillary protrusion and retraction within Cyprinodon-tiformes in two ways: (1) through a morphologicaldescription of the musculoskeletal and ligamentousarchitecture of the upper jaws and (2) with simplemanipulative tests of competing models of jaw protru-sion. To this end, we describe an unusual jaw ligament(a greatly modified premaxillomandibular ligament)within three cyprinodontiform genera, and evaluate itspotential role in upper jaw protrusion. A ligament tyingtogether the premaxilla and dentary is commonly foundwithin teleosts, including within the sister group ofcyprinodontiforms, the atherinomorphs; however, wepropose that the distinct architecture of the premax-illomandibular ligament in cyprinodontiforms mediatesa heretofore unrecognized mechanism of premaxillaryprotrusion. We also describe the unusual insertion of akey jaw-closing muscle (A2, a division of the adductormandibulae, the primary jaw adductor) within Poeciliidae.We discuss the relationship between these anatomicalfeatures and previously proposed mechanisms ofpremaxillary protrusion for cyprinodontiform fishes,and use simple manipulations on one species to assessthe validity of these proposed mechanisms. Moreover,we hypothesize that this suite of morphological modi-fications constitutes an evolutionary innovation thatenables cyprinodontiform fishes to use fine-scale mani-pulations of the anterior jaws in a forceps-like mannerto procure specific prey items via carefully controlledjaw opening and closing movements. Finally, we notethat within more derived poeciliids these morphologicalfeatures have undergone secondary changes that resultin a jaw apparatus well suited for algal scraping and thatother unrelated teleostean families show ecomorpho-logical convergence in these features.
Material and methods
Morphology of the oral jaws
We initially hypothesized that members of thePoeciliidae are highly specialized for a picking habit,
L.P. Hernandez et al. / Zoology 111 (2008) 442–454444
relative to other Cyprinidontiformes. The following liveadult specimens of three target species were obtainedfrom commercial suppliers and sacrificed according tothe Northern Arizona University IACUC approvedprotocols (NAU IACUC ] 04-007): Fundulus rubrifrons
(n ¼ 5, 20–28mm SL) and Poecilia sphenops (n ¼ 5,27–43mm SL). Additional specimens preserved in 10%formalin and stored in 70% ethanol were obtained fromteaching collections from the University of SouthFlorida; these specimens were used for assessment ofintra-clade variation in feeding morphology. Theseadditional species were Fundulus similis, Fundulus
chrysotus, Fundulus grandis, Gambusia affinis, G. hol-
brooki, Poecilia latipinna and P. sphenops. Fresh andpreserved specimens were dissected and stained withiodine to facilitate identification of muscle fiber orienta-tion. These specimens were used to examine basicfeatures associated with feeding – features likely toaffect both jaw protrusion and retraction. Between fiveand ten specimens of each species were dissected todetermine musculoskeletal and ligamentous architec-ture, as well as to assess inter-individual variation infeeding morphology for a given species.
Additional specimens of the above species werecleared and stained using a protocol presented inDingerkus and Uhler (1977) with modifications byPotthoff (1984). Cleared and stained specimens wereused to assess the osteology of the anterior jaws. Line-drawn illustrations of specimens were made fromphotographs taken using an Olympus DP12 digitalcamera attached to an Olympus SZX12 dissectingmicroscope, or using an Olympus SZX-DA cameralucida attached to the same dissecting microscope.Digital illustrations of skeletal and muscle morphologywere constructed by creating vector drawings from thecamera lucida drawings using a technical illustrationprogram (Canvas X and/or Adobe Illustrator CS3).Images captured using an Olympus DP12 digital cameraattached to an Olympus SZX12 dissecting microscopewere imported into Adobe Photoshop (CS3) andbrightness, contrast and color of the images wereadjusted as necessary to facilitate the visualization offine detail.
Fig. 1. Premaxillary protrusion mediated by abduction of the
mandible. (A) Resting state, (B) protruded state. The jaw is
lowered and via tension placed on the ligament tying the
premaxilla to the mandible the premaxilla is pushed forward.
An elongated ascending process of the premaxilla slides along
the neurocranium. Monocirrhus sp. modified from Liem
(1970). Abbrev.: asc pmx, ascending process of the premaxilla;
dent pmx, dentigerous process of the premaxilla; mand,
As multiple hypotheses have been proposed regardinghow the premaxilla is protruded within certain membersof Cyprinodontiformes (Eaton, 1935; Alexander, 1967a;Gosline, 1981; Motta, 1984), we used ligament ablationand subsequent manipulations of fresh specimens ofFundulus to evaluate the validity of each hypothesis andto determine how our morphological data mightinfluence these models. We also performed simple
manipulations of cleared and stained Gambusia andPoecilia. After first describing the more conventionalmechanism of premaxillary protrusion within teleosts, abrief overview of the various hypotheses is given toprovide the rationale behind the specific ligamentablations performed on Fundulus.
Within many acanthopterygians, depression of thelower jaw enables premaxillary protrusion; lower jawdepression is generated via the opercular linkage, whichconnects the neurocranium to the lower jaw (Fig. 1A). Ataut ligament tying the premaxilla to the lower jaw pullsthe premaxilla forward as the jaw is lowered. During jawprotrusion the maxilla also swings dorsally and ante-riorly to partially occlude the lateral gape (Fig. 1B).
Within cyprinodontiforms, two different mechanismsof premaxillary protrusion have been suggested. Gosline(1981) proposed that the ‘‘lip membrane’’ that links thepaired premaxillae to the dentary bones of the lower jawis the primary agent that effects protrusion in this group
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(Fig. 2A). In this model, ventral rotation of the lowerjaw distends the lip membrane and places tension on thepremaxillae, which are then pulled anteriorly (Fig. 2B).Alexander (1967b) and Eaton (1935) also describe theprotrusion exhibited by cyprinodontiform fishes andconsider this pattern of protrusion as functionallydistinct from the mechanisms characterizing otheracanthopterygians. However, Eaton (1935) and Alexander(1967b) both suggest that lateral twisting of the pairedmaxillae along their dorsoventral axes, brought aboutby contraction of the first branch of the adductormandibulae (AM) (A1, which inserts on the ventralaspect of the maxilla), causes movement of the pairedpremaxillae. In this scenario, the ascending processes ofthe premaxillae are affected by movements of themaxillae such that they are either forced or pulledanteriorly, which creates upper jaw protrusion(Fig. 2C–F). While Eaton (1935) suggests that theascending processes are ‘‘squeezed’’, Alexander (1967a)
max
pmx
pal
rc
Lip membrane mediated protrusion
pal
pmxmax
dent
Twisting maxilla mediated protrusion
Fig. 2. Two hypotheses explaining protrusion within Fundulus,
lip membrane- and twisting maxilla-mediated premaxillary
suggests that this twisting pulls the ascending processesanteriorly via a thick rostral cartilage.
These different mechanisms have been suggestedbased on morphology; however, ablations of individualligaments to test these hypotheses have not beenperformed. We performed a number of experiments inwhich key ligaments were severed and the subsequentdegree of protrusion was examined following theapplication of an input force. Thus, we performed thefollowing sequential ablations on fresh specimens ofF. rubifrons and F. stellifer, as all previous workers inthis area also used this genus as a model: (1) removal ofthe lip membrane, followed by (2) decoupling thepremaxilla from the lower jaw by severing the ligamen-tous attachment (described in detail below) connectingthe descending process of the premaxilla to the lowerjaw, but leaving the maxilla and premaxilla attached toone another at their ventral ends; (3) decoupling themaxilla and premaxilla by severing the ligament thatconnects the maxilla to the premaxilla, but leaving thepremaxilla attached to the lower jaw; (4) freeing thepremaxilla and maxilla from one another and the lowerjaw; (5) on an additional set of specimens, freeing thedescending process of the premaxilla and maxilla (as instep 4), while leaving the lip membrane intact. Aftereach ablation, the lower jaw of the specimen wasmanually rotated to approximately 301 (previouslydetermined to represent a typical feeding event; Gibband Ferry-Graham 2005), and resultant movements ofthe upper jaw were recorded.
Results
Morphology
Musculoskeletal architecture of Fundulus spp.
Within F. rubrifrons, a species chosen to represent themore primitive cyprinodontiform condition that ispresent in the Fundulidae, the bony elements comprisingthe anterior jaws are similar to those described for manyother teleosts. The descending (or alveolar) arms ofthe paired premaxillae have a strongly recurved shape(Fig. 3A, B). The ascending arms of the premaxillaeare relatively long and diverge from one another attheir postero-dorsal tips (Fig. 3B). The space betweenthe dorsal ends of the ascending processes is filled withthick connective tissue within which two rostralcartilages are located (Fig. 3B). This connective tissueties the ascending processes of the premaxillae to themedial arms/hooks of the paired maxillae, such thatanterior movement of the medial hooks of the maxillaepulls on this ligament, which pulls the premaxillaeforward. The ventral end of each maxilla is relativelythin. However, the dorsal end of each maxilla is more
L.P. Hernandez et al. / Zoology 111 (2008) 442–454446
complex, with a relatively small dorsal knob, but a large,twisted medial hook (Fig. 3B). Prominent ventral anddorsal processes are present on each dentary where theyarticulate with the anguloarticular. The process on theventral aspect of the anguloarticular is quite long andinserts into the ‘‘V’’ formed by the two processes of thedentary; as a result, the anguloarticular and dentary aretightly articulated, relative to the other species examinedhere.
Within F. rubrifrons, the musculature that controlsmovements of the anterior jaws is similar to that seen inmany generalized teleosts, thus the nomenclature usedhere is taken from Winterbottom (1974). There are twodiscrete branches of the first division of the AM (A1):A1a and A1b (Fig. 4A). A1a is substantially larger thanA1b, and the fibers of A1a proceed horizontally fromtheir origin on the hyomandibula and preopercle to theirinsertion on the ventral third of the maxillae (Fig. 4A).A1b originates from the ventral edge of the suspensor-ium and proceeds anterodorsally to insert on the ventralthird of the maxilla. Each of the species examined here
also has a very small branch of A1 going to the ventraledge of the lacrimal (as has been previously noted byParenti, 2005; data not shown here).
The fibers of the second and third divisions of the AM(A2 and A3) are medial to A1 and closely associated,although they insert on discrete regions of the lower jawor mandible (Fig. 4B). Most of the fibers of A3 are‘‘deep’’ (medial) to A2, and insert on the medial face ofthe anguloarticular via a discrete tendon. A2, largelycombined with A3, originates from the hyomandibulaand symplectic (medial to A1) and inserts muscularly(i.e., without a distinct tendon) on the dorsocaudal edgeof the dentary and anguloarticular. As in most teleosts,the Ao originates from the medial surface of theventralmost portion of the quadrate and inserts on themedial side of the dentary, filling most of the Meckelianfossa (Winterbottom, 1974). We noted a lack ofvariation in Ao among all three genera examined here,thus this division will not be discussed further. Compa-risons among Fundulus spp. revealed that interspecificvariation within this genus consists predominantly of
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A2
A3
A2
A1α
A1β
Fundulus
Poecilia
A1α
A1β
A2
A3
Fig. 4. Adductor mandibulae complex of Fundulus heteroclitus (A, B) and Poecilia latipinna (C, D) from a superficial (A, C) and
deep aspect after removal of branches of adductor mandibulae A1 (B, D). Drawn from fresh and preserved specimens.
L.P. Hernandez et al. / Zoology 111 (2008) 442–454 447
subtle changes in the size of distinct divisions of the AMcomplex, but not in origin or insertion site.
Musculoskeletal architecture of Gambusia spp.
Within G. affinis, a basal poeciliid, the bony elementscomprising the anterior jaws are generally similar tothose described for Fundulus (Fig. 3C, D). Thepremaxilla has the same characteristic shape, with thedescending arm strongly recurved, although less so thanin Fundulus (Fig. 3C). The ascending arm of thepremaxilla is relatively long, though not as long as seenin Fundulus. Moreover, from a dorsal aspect, theascending processes of the paired premaxillae ofGambusia are more triangular in shape and quite broadwhere they connect with the descending arms (Fig. 3D).The maxilla in Gambusia is more robust than that ofFundulus. In contrast to Fundulus, where the dorsal endof the maxilla had a large twisted medial hook with asimple dorsal knob, in Gambusia the maxilla has anelaborate dorsolateral process, in addition to the medialhook. This process consists of a large dorsal knob and astrongly curved, flattened medial hook. In a non-protruded state, the medial hooks of the maxilla weremore medially directed, whereas those of Fundulus weremore anteriorly directed (Fig. 3D vs. B). Moreimportantly, the ligament connecting the ascendingarms of the premaxilla to the medial hooks of themaxillae has been greatly reduced and is composed ofvery slack connective tissue. Rostral cartilages have beenlost. In addition, the ventral and dorsal processes of
each dentary are not as long in Gambusia as they are inFundulus (Fig. 3C).
In G. affinis, the musculature controlling movementsof the anterior jaws is similar to that of Fundulus, thus isnot figured here. There are two branches of A1 (A1a andA1b); again, as in Fundulus, A1a is significantly largerthan A1b. The fibers of A1a are directed horizontallyfrom their origin on the hyomandibula and preopercleto their insertion on the ventral third of the maxilla. A1boriginates from the ventral edge of the suspensoriumand its fibers proceed anterodorsally to insert on themaxilla. The maxilla, slightly wider in Gambusia than inFundulus (Fig. 3A, B), allows for a greater separationbetween the insertion of A1a and A1b. As in Fundulus,A2 and A3 are somewhat interconnected; however, thebulk of A2 originates from the suspensorium (deep toA1) and inserts directly on the dorsocaudal edge of thedentary with few fibers inserting on the anguloarticular.A very small dorsal portion of A2 appears to insert onligamentous tissue that invests the premaxilla. The bulkof A3 is found deep to A2 and inserts on the medial faceof the anguloarticular via a discrete tendon. Examina-tion of G. holbrooki suggests that, as in Fundulus,variation within the group consists predominantly ofchanges in the size of distinct divisions of the AMcomplex, but not in origination or insertion sites.
Musculoskeletal architecture of Poecilia spp.
Within P. sphenops, a more derived poeciliid (Ghe-dotti, 2000; Parenti, 2005), the general cyprinodontiform
Fig. 5. Premaxillomandibular ligament linking the premaxilla
to the lower jaw in Fundulus rubrifrons (A, lateral; B, medial)
and Poecilia latipinna (C, lateral; D, medial). Within Fundulus
a simple ligament ties the ventrolateral aspect of the premaxilla
(A) to the medial aspect of the dentary (B). Within Poecilia a
larger and more complex pair of ligaments ties the ventro-
lateral aspect of the premaxilla (C) to the medial aspect of the
dentary and anguloarticular (D). In (C–D) ligaments are
drawn such that the lighter portion represents typical
ligamentous tissue, while the darker portion represents the
more cellular portion of this ligament (hyaline cell cartilage,
sensu Benjamin, 1989). (E, F) Atherinops californiensis show-
ing the presumed ancestral ligament tying together the
premaxilla and mandible, in resting lateral view (E), as well
as with the premaxilla folded over dorsally to show the extent
of taut ligamentous attachment (F). Abbrev.: ang, anguloarti-
cular; dent, dentary; pmx, premaxilla.
L.P. Hernandez et al. / Zoology 111 (2008) 442–454448
osteology of the anterior jaws is retained; however, wesee several substantial morphological changes withlikely functional implications (Fig. 3E). As in Gambusia,the strongly recurved descending arm of the premaxillais obvious, although the relative length of the ascendingarm is reduced. As in Gambusia, the ascending processesof the paired premaxillae have broad bases where theyconnect with the wide descending arms (Fig. 3F); thiscondition is more pronounced in Poecilia than inGambusia. The posterodorsal portion of the maxilla,which articulates with the palatine bone, has becomemore elaborate in Poecilia (characterized by a longmedial hook of the maxilla), relative to the conditionseen in Gambusia. More importantly, the thick ligamentconnecting the ascending arms of the premaxilla to themedial hooks of the maxillae has been lost. Althoughthe maxilla is thinner in Fundulus, within Poecilia thiselement is somewhat wider (Fig. 3E). The medial hooksof the paired maxillae, already complex in Gambusia, aresignificantly elongated and even more complex in shapein Poecilia (given the twisted appearance of the bones,they resemble corkscrews). Moreover, the complexmedial hook of each maxilla has become slightly moremediolaterally directed (Fig. 3F). In Poecilia, as inGambusia, there is little overlap between the medialhook of the maxilla and the ascending process of thepremaxilla in the resting state (Fig. 3F). In addition, theretroarticular process on the ventral aspect of eachdentary is considerably foreshortened. The coronoidprocess of the dentary is widely separated from thecoronoid process on the corresponding anguloarticular,potentially allowing for some intramandibular bending.
In P. sphenops, as in Fundulus and Gambusia, A1 issplit into two subdivisions, both of which insert on theventral third of the maxillae (Fig. 4C). However, themorphology of A2 and A3 is significantly different fromthat seen in either Fundulus or Gambusia. A2 andA3originate largely from the preopercle and symplectic,forming a cylindrical mass found deep to A1. As in allspecies examined, A3 inserts on the medial face of theanguloarticular. However, the insertion and origin of A2is markedly different from that described previously: A2constitutes a fairly thin, cylindrical muscle that origi-nates both from the suspensorium and from part of themuscular belly of A3, then bifurcates to insert on boththe lower jaw (dentary) and, notably, the premaxilla.Insertions of A2 on the premaxilla and dentary areequally robust (Fig. 4D). However, the insertion of A2on the lower jaw is via a relatively thick, discrete tendonto the caudal edge of the dentary, the insertion of A2 onthe upper jaw is via a more diffuse tendinous connectionto the anterior aspect of the premaxilla. Given theforeshortened nature of the lower jaw elements, theinsertion of A2 on the dentary is more anterior thanthat of other species examined here. Examination ofP. latipinna suggests that variation within this genus also
consists predominantly of changes in the size of distinctdivisions of the AM complex, but not in insertion site.
Ligaments of the anterior jaws
For all three genera examined (Fundulus, Gambusia
and Poecilia), a premaxillomandibular ligament ties thelateral face of the premaxilla to the medial edge ofthe dentary or anguloarticular (Fig. 5). Although thehomology of this ligament is uncertain, we refer to it
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here as the premaxillomandibular ligament, because itties the premaxilla to the mandible. Whereas others havenoted generalized connective tissue uniting the premax-illa to the dentary in this group, the complex morphol-ogy outlined below has not been previously described.For example, Alexander (1967a) recognizes what heterms ‘‘tracts of thickened dermis’’ that attach thepremaxilla to the lower jaw; however, this descriptiondoes not capture the complex biomechanical architec-ture of this coupling.
Within Fundulus, this ligament originates from theventral aspect of the lateral descending arm of thepremaxilla (Fig. 5A). It wraps around the posterior edgeof the premaxilla and turns anteriorly to insert on themedial side of the dentary or anguloarticular. Thisventral attachment is often adjacent to the insertion ofA2 on the dentary (Fig. 5B). Importantly, this ligamentis not attached to the posterior margin of the premaxilla,but rather slides freely across this region of the bonewhen the jaws are manipulated.
There is some variation in the architecture of thisligament among the cyprinodontiform species examinedhere. In both Fundulus and Gambusia, the connectivetissue across the lateral face of the premaxilla constitutesa small and discrete ligament, which is localized on thecaudal face of the premaxilla (Fig. 5A; data not shown).After wrapping around the posterior aspect of thepremaxilla, it then attaches to the medial aspect of thedentary (Fig. 5B). In Gambusia, a bit of tissue inserts onthe anguloarticular as well. Within Poecilia, thisligament has become increasingly complex, and is splitinto two discrete elements: the ligament bifurcatesdistally and attaches to both the coronoid process ofthe dentary (adjacent to the insertion to the A2) and tothe anguloarticular (Fig. 5C, D). Moreover, this‘‘ligament’’ has an unusual morphology in that thepoints of attachment are composed of what appears tobe typical ligamentous, fibrous tissue, but the freelymoving, central portion of this connective tissue appearsto be composed of more cellular, and less fibrous,material (see Fig. 5C, D).
In comparison, Atherinops californiensis (Atherini-formes; sister group to Cyprinodontiformes+Beloni-formes) has a small, taut ligament that ties the medialedge of the descending arm of the premaxillae to thelateral surface of the anguloarticular (Fig. 5E, F).Within Atherinops, the bulk of this ligament is foundbetween these two bones and serves to connect thepremaxilla firmly to the lower jaw. Unlike in thecyprinodontiforms, in Atherinops this taut connectiondoes not appear to allow significant premaxillaryprotrusion.
Thus, in an evolutionary cross-section that encom-passes basal atherinomorphs and derived poeciliids, wesee increasing complexity of this premaxillomandibularligament. Within Atherinops it is a small and very taut
ligament, made of typical collagenous material. Withincyprinodontiforms, it has become a much looser andlonger ligament. Finally, within derived poeciliids it hasincreased in size, complexity and cellular structure (seeFig. 5C, D).
Basal members of Cyprinodontiformes also have ataut ligament that ties the lateral face of the maxilla tothe lacrimal (data not shown). This ligament ties eachlacrimal to a discrete lateral portion of the correspond-ing maxilla halfway along its shaft (Parenti, 2005).Alexander (1967b) briefly mentions a ligament unitingthe maxilla and lacrimal and notes its importance for thetwisting maxilla model. This ligament is quite thick inFundulus, is greatly reduced in Gambusia and is presentin Poecilia.
Mechanism
Manipulation of both fresh, intact specimens andcleared and stained specimens of Fundulus confirmedthat depression of the lower jaw consistently generatesanteriorly directed protrusion of the premaxillae. Rota-tion or twisting of the paired maxillae (along theirdorsoventral axes) was also observed when viewed fromthe dorsal aspect, both when the lower jaw wasdepressed and when the paired premaxillae were graspedand pulled anteriorly. Rotation or twisting was suchthat the medial hooks on the dorsal ends of the maxillaerotated outward, away from both the midline and oneanother. The maxillae rotated along their long axesnearly 901 in response to lower jaw depression of 30–401.Induced rotation of the medial hooks of the maxillaepulls on the thick connective tissue that connects themedial hooks to the ascending processes of the pairedpremaxillae; this action appears to pull the ascendingprocesses forward (Fig. 2C–F).
When the lip membrane was removed in Fundulus
spp., protrusion of the premaxilla occurred with noobservable differences in protrusion direction or dis-tance (relative to intact specimens); this result suggeststhat Gosline’s (1981) hypothesis is incorrect (Fig. 2A,B). It also appeared that the lower jaw could rotate to alarger degree (i.e., greater jaw depression) when thismembrane was removed, which suggests that the lipmembrane may constrain the degree of jaw opening.When each maxilla was detached from the correspond-ing premaxilla, but each premaxilla left connected to thelower jaw, lower jaw depression resulted in anteriorlydirected protrusion of the paired premaxillae, and itagain appeared that the lower jaw could rotate to alarger degree in the absence of the attachments to themaxillae. When the descending process of each pre-maxilla was subsequently freed from its attachment tothe lower jaw (but remained attached to the maxilla,which is lateral to the premaxilla at the ventral tip), it
L.P. Hernandez et al. / Zoology 111 (2008) 442–454450
tended to slide under (i.e., medial to) the maxilla.Depression of the lower jaw, however, still generatedanteriorly directed protrusion of the paired premaxillae,presumably due to the action of the lower jaw throughthe remaining connection between the maxillae and thepremaxillae. It is noteworthy, however, that the protru-sion distance observed under these circumstances wasapproximately half of that observed when the connec-tions between the premaxillae and the lower jawremained intact. When both maxillae and premaxillaewere detached from one another and from the lower jaw,then no protrusion occurred. If these ligaments weretransected, but the lip membrane kept intact, protrusiononly occurred when the lower jaw was depressed toextreme and biologically unrealistic angles (41801);even in this scenario, protrusion was minimal.
pmxmandligament
Fig. 6. New models of premaxillary protrusion for Fundulus
and Poecilia. (A, B) Schematic indicating dual mechanisms of
premaxillary protrusion in Fundulus. The twisting maxilla
indirectly transfers force to the premaxilla from the lower jaw
via the thick ligament connecting the premaxilla and maxilla,
while the premaxillomandibular ligament directly transfers
force from the lower jaw to the premaxilla. Within Poecilia
(C, D), taut ligamentous attachments between the premaxilla
and maxilla have been lost, thus premaxillary protrusion is
largely caused by the lowering of the jaw. Since the maxilla
would obscure these ligaments, it has been removed from C
and D. Abbrev.: pmxmand, premaxillomandibular.
Discussion
Based on our morphological investigations, wepropose that cyprinodontiform fishes employ a novelmechanism of premaxillary protrusion, which wesuggest is largely mediated by a previously undescribedconformation of the premaxillomandibular ligamentthat connects the premaxilla to the lower jaw. We positthat, within Cyprinidontiformes, this ligament mayincrease premaxillary protrusion distance (relative tooutgroup taxa), and enhance the use of the anterior jawsas forceps for picking individual prey items by enablingthe upper and lower jaws to move in a highlycoordinated fashion (see also Ferry-Graham et al.,2008). We also note that the novel insertion of AM A2division on the premaxillae (Fig. 4) – an unusualcondition in teleosts – may allow for fine control ofthe closing of the anterior tips of the jaws when prey areindividually ‘‘picked’’ and fine control of retraction ofthe upper jaws once prey are caught. Relative to theanterior jaws of teleosts with more ‘‘typical’’ mechan-isms of jaw protrusion (Motta, 1984), these twomechanisms – an unusual ligament and a novel insertionof a major jaw adductor – appear to have transformedthe anterior jaws of derived poeciliids into a mechanismthat is extremely well suited for picking- and scraping-based feeding behaviors.
Refining previous models of premaxillary protrusion
In our manipulations, protrusion of the pairedpremaxillae could be initiated via depression of thelower jaw, without transfer of force through themaxillae. Thus, a ‘‘Type A’’ protrusion mechanism(sensu Motta, 1984), in which force is transferreddirectly from the lower jaw to the premaxillae, existsin this group (Fig. 6C, D). We posit that this mechanism
is mediated in cyprinodontiform fishes by the aforemen-tioned premaxillomandibular ligament that connectseach premaxilla to the lower jaw, and not by the lipmembrane (as postulated by Gosline, 1981). However,we also note that premaxillary protrusion still occurredwhen the ligament connecting the lower jaw to thepremaxilla was severed (bilaterally), although it wasmuch less pronounced. This suggests that the connectionbetween the maxilla and the premaxilla also plays a role,albeit a smaller one, in jaw protrusion. Apparently, noneof the previous models of jaw protrusion for cyprino-dontiform fishes is entirely correct, as none accounts forthe role of the premaxillomandibular ligament in forcetransfer. We suggest that members of this groupdemonstrate a distinct mechanism of premaxillaryprotrusion that, at least within Fundulus, works inconcert with the twisting maxilla model outlined byAlexander (1967b) and Eaton (1935).
This mechanism is distinct in that the premaxillo-mandibular ligament is structurally different from anyligaments previously proposed to be of importanceduring premaxillary protrusion. Because this ligament
ARTICLE IN PRESSL.P. Hernandez et al. / Zoology 111 (2008) 442–454 451
wraps around the posterior edge of the premaxilla, itwill allow for a much wider gape within species withsuch a ligament. Given the architecture of this ligamen-tous attachment, the only thing constraining maximumgape production is the degree of jaw depression.
During jaw opening in Fundulus spp., the medial hookof each maxilla rotates outward, pulling the thickligament (associated with the rostral cartilages) ante-riorly (Fig. 6A). As suggested by Alexander (1967b), thethick ligament attaching each maxilla to the lacrimalbone is important for stabilizing the maxilla along itsposterior margin, such that the bone can pivot outwardalong its long axis, rotating the medial hook by as muchas 901. Eaton (1935) proposed that the medial hooks ofthe paired maxillae ‘‘pinched’’ the ascending processesof the premaxillae, forcing them to slide anteriorly.However, we hypothesize that the ascending processesof the premaxillae are pulled forward by the rotationalforce imparted by movement of the medial hooks of themaxillae. The medial hooks are attached to theascending processes of the paired premaxillae via athick, Y-shaped ligament, which Alexander (1967a)described as the ‘‘rostral cartilage’’. Two ligamentousarms of the Y attach to the medial hooks of themaxillae, whereas a single ligament attaches to both tipsof the ascending processes of the premaxillae. ThisY-shaped structure appears to be a thickened ligamentin which two rostral cartilages are embedded. Twisting/rotation of the maxillae causes the premaxillae to pivotforward through a rotational motion (Fig. 2).
premaxillomandibular ligament with twomodified premaxillomandibular ligamen
A2 inserts on dentary
Cyprinodontoidei
Poeciliidae
Atherinomorpha
A2 inserts on dentary and premaxilla
twisting maxilla mediated premaxillary pprimarily premaxillomandibular ligamenloss of rostral cartilage and associated
12
34
567
1 3 5
6 7
Fig. 7. Phylogenetic position of species examined in this study. C
cyprinodontiform relationships are after Costa (1998), Ghedotti (20
In this sense, this constitutes a ‘‘Type B’’ mechanism(sensu Motta, 1984), in which twisting of the pairedmaxillae effects anterior movement of the premaxillae.However, given that rotation of the lower jaw is alsolikely required to generate such twisting of the maxilla,Fundulus may show a combination of Types A and Bmechanisms. Interestingly, the ligaments as well as theascending processes of the premaxilla are all less welldeveloped in more derived species (i.e., Gambusia andPoecilia). Indeed, rostral cartilages have been lost inthese more derived species (Fig. 7). We suggest thatderived cyprinodontiform species rely more heavily ontransfer of force through the premaxillomandibularligament (Fig. 6C, D). The fact that this ligament ismuch more complex within derived species supports thiscontention.
Alexander (1967b) refers to the ligament that ties eachpremaxilla to the lower jaw in Fundulus as a ‘‘thicken-ing’’, but does not assign it further functional signifi-cance. However, our manipulations confirm that thisligament mediates jaw protrusion by transferring forcedirectly to the paired premaxillae from the lower jaw.Interestingly, this ligament also limits the anteriorexcursion of the descending process of the premaxillaeduring mouth opening; in other teleosts, the descendingarms of the premaxillae rotate anteriorly and laterallyocclude the gape. This restriction of the ventral ends ofthe premaxillae gives the cyprinodontiforms their chara-cteristic ‘‘beak-like’’ appearance during mouth opening.The lip membrane creates some lateral occlusion,
Fundulus
Heterandria
Gambusia
Poecilia
divisionst
rotrusiont mediated premax illary protrusion
Atherinops
Kryptolebias
ligaments
2 4
haracter states are as noted on the figure. Atheriniform and
ARTICLE IN PRESSL.P. Hernandez et al. / Zoology 111 (2008) 442–454452
although occlusion is much less pronounced than thatseen in other teleosts where the descending processes ofthe premaxillae laterally occlude gape.
The distinct architecture of the premaxillomandibularligament (Fig. 5A–D) may be a synapomorphy ofCyprinodontiformes, as this conformation is not presentin Atheriniformes. As previously mentioned, withinA. californiensis there is a ligament that attaches thepremaxilla to the dentary; however, it is a short, taut,fibrous ligament that attaches the medial edge of thedescending arm of each premaxilla to the lateral surfaceof the corresponding anguloarticular (Fig. 5E, F). Itdoes not curve around the caudal edge of thedentigerous arm of the premaxilla. Curiously, thepoeciliid premaxillomandibular ligament also differshistologically from typical ligaments within teleosteanheads. Benjamin (1989) described tissue surrounding thejaws of Poecilia sphenops as hyaline cell cartilage, whichis characterized by closely packed cells within anextracellular matrix. Based on Benjamin’s histologicalsections (Fig. 1 in Benjamin, 1989), it is clear that thepremaxillomandibular ligament in Poecilia is at leastpartially composed of hyaline cell cartilage.
While the architecture of the cyprinodontiformligament that connects the premaxilla to the lower jawis certainly distinct as compared to basal atherino-morphs, the presence of a ligament connecting thepremaxilla to the lower jaw is not. Indeed, it is likelythat many teleosts have such a ligament. A ‘‘premax-illomandibular’’ ligament is described in the Nandidae(Liem, 1970). However, these ligaments appear to besimilar to the simple, taut ligaments that we haveidentified in Atherinops californiensis (Fig. 5E, F); thusimportant functional aspects of these ligaments eitherare not present in these other species or have goneunnoted. Until a more thorough phylogenetic compar-ison can be performed, we cannot determine if theligament described herein is homologous with thepremaxillomandibular ligament previously describedwithin other fish groups (e.g., Nandidae), or representsan evolutionary novelty.
Functional significance of the insertion of the
adductor mandibulae on the premaxilla
In the vast majority of teleosts, the lower jaw simplypushes the premaxilla back into place as the mandible isadducted; thus, no direct, muscle-mediated retraction ofthe premaxilla occurs. However, there are a fewteleostean taxa outside of the cyprinodontiforms inwhich a division of the AM inserts onto the premaxilla.Within the Siluriformes, two lineages have evolved aconnection between the AM and the upper jaw. Onegroup, the loricariids (comprised of approximately 1,100species), are characterized by a discrete insertion of A2
unto the premaxilla (Schaefer and Lauder, 1986, 1996).Indeed, Schaefer and Lauder (1986) document a gradualtransformation of the anterior jaws from minimalpremaxillary protrusion within primitive loricariids toa derived condition in which the paired premaxillae arecontrolled independent of the lower jaw via the AMcomplex. This morphological transformation appears tobe associated with functional specialization for algalscraping. Independent control of the premaxillae viadirect insertion of the AM may enable the increaseddexterity necessary for scraping algae from unevensurfaces. In addition, recent work has established thatmembers of another siluriform group, the Synodonti-dae, also have a distinct insertion of the AM on thepremaxilla (Celine Ide and Dominique Adriaens, pers.comm.). Interestingly, synodontids also use algal scrap-ing as their predominant feeding mode.
Although discrete branches of A2 insert on both thepremaxilla and dentary within derived poeciliids (i.e.,Poecilia), the overall size of the AM A2/3 is relativelysmall, which suggests that forceful biting is not beingproduced. Although the thin, largely muscular connec-tion of the A2 to the premaxilla is unlikely to generatelarge forces during biting, it may allow small amounts offorce to be applied directly to the anterior upper jaw –and thus to the prey items. This small addition of forceto the upper jaw may prevent small, but elusive preyitems from escaping, or allow a ‘‘nipping’’ behavior tobe produced. Given the insertion of A2 on both theupper and lower jaws, contraction of A2 will allow finecontrol of captured items as the jaws are retracted, asboth the upper and lower jaws can hold the prey item.Moreover, as is seen within loricariids, some poeciliidshave secondarily adopted a grazing habit (FaresAlkahem et al., 2007) – a trophic niche for which thisfeeding mechanism is well suited.
A small, mobile and independently controlled pre-maxilla, in association with the intramandibular jointthat characterizes Poecilia, should facilitate a shift to agrazing habit. Such a mechanism appears to allow forboth a much wider gape, due to the architecture of thepremaxillomandibular ligament, as well as the ability togenerate force at the anterior ends of the upper andlower jaws; both of these functional traits are likely to beimportant during scraping. During cyprinodontiformoral jaw evolution, we also see an anterior shift in theinsertion of A2 on the dentary; the insertion of A2 onthe premaxilla probably serves to increase and directforce to the tips of the jaws. In addition, synchronizedabduction of the upper and lower jaws, via thepremaxillomandibular ligament described herein,coupled with controlled adduction of both the lowerand upper jaws, via the novel insertion of A2 on thepremaxilla, apparently allows poeciliids to performprecise occlusal movements with their jaws, much as ispossible with forceps.
ARTICLE IN PRESSL.P. Hernandez et al. / Zoology 111 (2008) 442–454 453
Schaefer and Lauder (1986) suggest that an increasein the complexity of the AM complex in the Loricar-ioidea generates the refined jaw movements necessary toscrape material from irregular surfaces. Correspond-ingly, we suggest that the evolutionary changes in jawmechanism outlined here for cyprinodontiform fishesincrease jaw dexterity during feeding events (Fig. 7).During the early evolution of cyprinodontiforms(Fig. 7), increased dexterity and precision granted bythe ligament that directly connects the upper jaw to thelower jaw may have enhanced the ability of basal speciesto select individual prey items from the substrate orwater column using picking-based prey capture beha-vior. During the later evolution of this clade, the directcontrol of upper jaw movements during retractiongranted by the insertion of A2 on the upper jawenhanced the ability of derived species to removeencrusting material using a nipping or scraping-basedfeeding behavior.
Acknowledgments
We thank the following people for the loaning ofspecimens used in this study (and for specimensultimately not included): Lynne Parenti (SmithsonianInstitution), Philip Motta (University of South Florida),Dave Catania (California Academy of Sciences),Daphne Gehringer (Moss Landing Marine Labs). Wealso thank Nathan Bird and Katie Staab for assistancewith specimen preparations at GWU. LPH was sup-ported by NSF IOS-0615827, IOS-0726087 and DBI-0721662. LFG was supported by NSF MRI-0320972and IOS-0726081; ACG was supported by NSF IOS-0726001 and Science Foundation Arizona CAA 0057-07.
References
Alexander, R.M., 1967a. Mechanisms of the jaws of some
atheriniform fish. J. Zool. 151, 233–255.
Alexander, R.M., 1967b. The functions and mechanisms of the
protrusible upper jaws of some acanthopterygian fish. J.
Zool. 151, 43–64.
Bellwood, D.R., Choat, J.H., 1990. A functional analysis of
grazing in parrotfishes (family Scaridae): the ecological
implications. Environ. Biol. Fish. 28, 189–214.
Benjamin, M., 1989. The development of hyaline-cell cartilage
in the head of the black molly, Poecilia sphenops. Evidence
for secondary cartilage in a teleost. J. Anat. 164, 145–154.
Bergert, B.A., Wainwright, P.C., 1997. Morphology and
kinematics of prey capture in the syngnathid fishes
Hippocampus erectus and Syngnathus floridae. Mar. Biol.
127, 563–570.
Bouton, N., van Os, N., Witte, F., 1998. Feeding performance
of Lake Victoria rock cichlids: testing predictions from
morphology. J. Fish Biol., Suppl. A 53, 118–127.
Costa, W.J.E.M., 1998. Phylogeny and classification of
Rivulidae revisited: origin and evolution of annualism
and miniaturization in rivulid fishes (Cyprinodontiformes:
Aplocheilidae). J. Comp. Biol. 3, 33–92.
Dingerkus, G., Uhler, L.D., 1977. Enzyme clearing of alcian
blue stained whole small vertebrates for demonstration of
cartilage. Stain Technol. 52, 229–232.
Eaton, T.H., 1935. Evolution of the upper jaw mechanism in
