RESEARCH ARTICLE

A peculiar mechanism of bite-force enhancement in lunglesssalamanders revealed by a new geometric method for modelingmuscle momentsStephen M Deban and Jason C Richardson

ABSTRACTDesmognathine salamanders possess unusual morphologicalfeatures for lungless salamanders that have been proposed to aid inburrowingandbiting includingwell-ossified jawsandskull andapair ofrobust ligaments connecting the atlas to the mandible We evaluatedthe function of these and other peculiar desmognathine cranialfeatures in biting by examining the morphology mechanics and invivo biting performance of the largeDesmognathus quadramaculatusWe estimated theoretical biting force using a novel geometric methodthat we describe Results provide quantitative evidence to bolsterearlier conclusions that the unusual atlanto-mandibular ligamentscouple ventral head flexion a unique desmognathine behavior withbiting performance Our analysis also reveals that the ligaments notonly transmit but also amplify the force of head flexion when actingtogether with the unusual stalked occipital condyles enlarged atlasand massive quadratopectoralis muscles The geometric modelpredicts that this mechanism contributes five times the biting force ofthe three jaw levator muscles combined and predicts that maximumbiting force inD quadramaculatusmatchesorexceeds forces reportedfor similarly sized lizards The in vivo biting performancewemeasuredwas several times greater in D quadramaculatus than anotherplethodontid salamander Pseudotriton ruber which lacks theunusual morphology and mechanism of desmognathines Theeffective biting mechanism of D quadramaculatus we describe is anemergent property of many of the distinguishing morphologicalfeatures of desmognathine salamanders and likely plays animportant role in their natural history given that desmognathines usebiting in feeding defense and even courtship

KEY WORDS Amphibian Biomechanics Feeding

INTRODUCTIONDesmognathine salamanders of the Plethodontidae possess a uniquesuite of morphological features that has long been used to establishthe common ancestry of the group (Soler 1950 Wake 1966) Therobust wedge-shaped skull and mandible enlarged anteriorvertebrae and axial muscles larger hindlimbs compared withforelimbs and other unusual features have been interpreted asadaptations for burrowing or biting or both (Dunn 1926 Wake1966 Hinderstein 1971 Schwenk and Wake 1993)Desmognathines are known to burrow in soil and wedge

themselves beneath rocks they are also atypical amongsalamanders in their propensity for biting strongly in defense andduring feeding displaying a characteristic behavior while biting inwhich the head is strongly flexed directing the snout downward(Baldauf 1947 Brodie 1978 Dalrymple et al 1985 Bakkegard2005) The long history of interest in the morphology and behaviorof desmognathines has generated numerous propositions regardingthe function and biological roles of their unique morphology Weassess one proposed function ndash the generation of elevated bitingforces ndash that is an emergent property of several morphologicalfeatures interacting during biting and head flexion by examining themorphology and performance of a large desmognathineDesmognathus quadramaculatus (Holbrook 1840)

A distinctive feature of desmognathine morphology the robustatlanto-mandibular ligament on each side of the skull that originateson the atlas and inserts on the lower jaw (Figs 1 and 2) has longbeen a puzzle regarding its function and biological role (Dunn1926 Wake 1966 Hinderstein 1971 Dalrymple et al 1985reviewed by Schwenk and Wake 1993) The ligament restrictsventral movement of the mandible relative to the trunk and therebycouples head movements with mandible movements Its proposedfunctions are to prevent or limit ventral head flexion and requirehead lifting to open the mouth (Dunn 1926 Wake 1966) to holdthe mandible against the quadrate (Hinderstein 1971) to recoilelastically to close the jaws and hold them firmly closed in biting(Dalrymple et al 1985) and to increase biting force (Larsen andBeneski 1988 Schwenk and Wake 1993) The atlanto-mandibularligament may contribute to biting force only if it is placed in tensionsuch as when the cranium is flexed ventrally relative to the atlas orthe jaws are open to a degree Desmognathines are known to engagein head flexion during feeding but the flexion must be forceful tocontribute substantially to biting pressure

Another distinctive desmognathine feature ndash largequadratopectoralis muscles ndash can indeed pull the craniumventrally and the large size of the muscles suggests they can doso forcefully These muscles originate on the pectoral fascia andinsert on the quadrate giving them a strong mechanical advantagefor head flexion about the atlas Acting in conjunction with theatlanto-mandibular ligaments the quadratopectoralis musclesappear to be capable of increasing biting force beyond what thejaw levator muscles that span the jaw joint can achieve (Dalrympleet al 1985 Larsen and Beneski 1988 Schwenk and Wake 1993)

The ecological and evolutionary significance of thedesmognathine biting mechanism is appreciated when oneconsiders the natural history of these salamanders and thepotential constraints on their morphology Desmognathinesburrow and wedge themselves beneath stones (Dunn 1926Wake 1966 Worthington and Wake 1972 Bakkegard 2005) abehavior that limits the shape and dimensions of the headReceived 23 June 2017 Accepted 3 August 2017

Department of Integrative Biology University of South Florida 4202 East FowlerAvenue Science Center 110 Tampa FL 33620 USA

Author for correspondence (sdebanusfedu)

SMD 0000-0002-7979-4346

3588

copy 2017 Published by The Company of Biologists Ltd | Journal of Experimental Biology (2017) 220 3588-3597 doi101242jeb165266

Journal

ofEx

perim

entalB

iology

Protruding jaw muscles would impede this behavior yet large jawlevator muscles are commonly observed in vertebrates that engagein vigorous biting for example in the salamanders of the genusAneides (Stebbins 1985 Staub 1993) A system that makes use of aventrolaterally located muscle such as the quadratopectoralis tocontribute to biting forces allows the skull to be flattened importantin a salamander that wedges under rocks (Larsen and Beneski 1988Schwenk and Wake 1993)We sought to evaluate the hypothesis that the atlanto-mandibular

ligaments and quadratopectoralis muscles allow desmognathines toproduce elevated biting forces when they interact with otherdistinctive morphological features of desmognathines We alsoaimed to assess the hypotheses that the atlanto-mandibularligaments limit mouth opening or engage in elastic recoil or bothTo achieve these aims we examined the cranial morphology ofD

quadramaculatus and quantified dimensions of its jaw apparatusWe derived a novel 3D geometric method for modeling moments inmusculoskeletal systems such as jaws that does not require 3Dcoordinate data We present a novel interpretation of the unusualdesmognathine morphology and biting mechanism revealing amechanism in which the atlanto-mandibular ligaments transmit the

amplified force of the quadratopectoralis muscles to the mandible tobe used as biting force We explore the performance of thismechanism quantitatively using the geometric model and comparethe predicted performance with in vivo bite-force measurementsAdditionally we compare biting performance in Dquadramaculatus with that of a plethodontid of similar sizePseudotriton ruber which lacks the unusual features ofdesmognathines (ie stalked occipital condyles reinforcedquadrates robust jaws enlarged quadratopectoralis muscles andatlanto-mandibular ligaments)

MATERIALS AND METHODSFunctional morphology of D quadramaculatus jawsFour adult D quadramaculatus were killed by immersion in a2 g lminus1 aqueous solution of MS-222 (tricaine methanesulfonateSigma-Aldrich St Louis MO USA) buffered with sodiumbicarbonate and specimens were massed (Ohaus CorporationScout Pro SP2001 balance Pine Brook NJ USA) Head width atthe jaw joints and snoutndashvent length (SVL) were measured usingNeiko 01408A digital calipers The head and neck of the freshspecimens were then skinned and tissue was dissected away toexpose the muscles and their origins and insertions the quadrate-prearticular joints (ie jaw joints) and the atlanto-occipital jointsThe dorsoventral movements of the jaws and head through theirrange of motion were examined bymanipulation before and after thespecimens were skinned paying attention to the relationshipbetween the angle formed by the jaws and the angle formed bythe head and trunk given that these were noted to be related inprevious studies (Dunn 1926 Wake 1966 Hinderstein 1971Dalrymple et al 1985 Schwenk and Wake 1993)

Threemuscles that span the quadrate-prearticular joint and one thatspans the atlanto-occipital joint are in a position to contribute to biteforce levator mandibulae externus levator mandibulae internusanterior levatormandibulae internus posterior and quadratopectoralis(Fig 1) These muscles were examined with regard to their originsand insertions trajectory muscle fiber angles and mass

B

A LMIA LMIP IVE DT

GHM

DMAQP

DMPG

SARP

GHL

SARA

IH

QP

G

IMP

GG

LME

Fig 1 Musculature of Desmognathus quadramaculatus (A) Dorsolateraland (B) ventral views show the enlarged quadratopectoralis muscle (QPgreen) and the anterior portion of the levator mandibulae internus posteriormuscle (LMIP gold) under which lies the atlanto-mandibular ligament TheQPmyofibers originate broadly on the pectoral fascia and converge to insert on thequadrate and prearticular in a position to flex the cranium ventrocaudallyOther muscles shown include the levator mandibulae internus anterior (LMIA)intervetebral epaxial (IVE) dorsalis trunci (DT) levator mandibulae externus(LME) depressor mandibulae anterior (DMA) gularis (G) subarcualis rectusposterior (SARP) genioglossus (GG) geniohyoideus medialis (GHM)geniohyoideus lateralis (GHL) subarcualis rectus anterior (SARA)intermandibularis posterior (IMP) and interhyoideus (IH)

B

C

D

A

Fig 2 Morphology and position of the atlanto-mandibular ligaments andtheir associated myofibers (A) Robust atlanto-mandibular ligaments (whitearrowheads) in stained and cleared D quadramaculatus originate on the atlasand insert on the coronoid processes of the prearticular of the lower jawLigaments are displaced laterally more so on the left side to reveal trochlea inskull Note the subocular mineralization (black arrowhead) (BndashD) Levatormandibulae internus posterior muscle and ligament in dorsal (B) lateral (C)and ventral (D) views Myofibers (BC) originate and insert along the dorsalsurface ligament in a staggered fashion reducing muscle thickness andallowing tension placed on the myofibers to share the stress with the ligamentThe proximal surface of the ligament is smooth and devoid of myofibers

3589

RESEARCH ARTICLE Journal of Experimental Biology (2017) 220 3588-3597 doi101242jeb165266

Journal

ofEx

perim

entalB

iology

Straight-line distances between morphological landmarks weremeasured on specimens with the head in the resting position and themouth closed to calculate theoretical bite force via a staticmusculoskeletal model described below A total of 27 distanceswere measured on each specimen including six distances for each ofthe four muscles that can contribute to biting For each of the threecranial jaw-levator muscles on the left side of each specimen(levator mandibulae externus levator mandibulae internus anteriorand levator mandibulae internus posterior) six distances weremeasured muscle origin to insertion left jaw joint to muscle originleft jaw joint to muscle insertion right jaw joint to muscle originright jaw joint to muscle insertion and left to right jaw joint centerFor the left quadratopectoralis muscle which can contribute tobiting by flexing the cranium ventrally six distances weremeasured muscle origin to insertion left atlanto-occipital joint tomuscle origin right atlanto-occipital joint to muscle origin leftatlanto-occipital joint to muscle insertion right atlanto-occipitaljoint to muscle insertion and left to right atlanto-occipital jointcenter Six additional distances were measured for the model leftatlanto-occipital joint center to the closest point on the left atlanto-mandibular ligament right atlanto-occipital joint center to theclosest point on the left atlanto-mandibular ligament left jaw jointto mid-rostral tip of jaws right jaw joint to tip of jaws left jaw jointto the last (caudal-most) tooth on left side and right jaw joint to lasttooth on the left side Measurements of each distance were madethree times using digital calipers (Neiko 01408A) and averaged tothe nearest 001 mmThe insertion of each of the muscles on the lower jaw and

quadrate is narrow and its position for use in the model was easilylocated The origin however is broader and its position for themodel was estimated as a point in the center of the area of originThe levator mandibulae internus anterior and the levatormandibulae externus are parallel-fibered muscles whose fiberangle relative to the muscle line of action is very shallow and wastaken as zero The myofiber angles of 10 random fibers of thequadratopectoralis and levator mandibulae internus posterior wereestimated using a dissecting microscope with a camera-lucidaattachment together with a protractorMuscles were dissected free and massed (Virtual Measurements

and Control model VB-302A Santa Rosa CA USA plusmn0001 gaccuracy) The myofibers of the levator mandibulae internusposterior were teased apart to visualize their relationships to theatlanto-mandibular ligament on which they originate and insert(Fig 2BndashD) Dimensions of the atlanto-mandibular ligament weremeasured for two specimens to calculate tendon stressTheoretical isometric muscle force for each muscle was

calculated by first determining physiological cross-sectional area(PCSA) as the cosine of average fiber angle times mass divided bydensity divided by fiber length Muscle density was taken as1056 g cmminus3 Muscle force was calculated by multiplying PCSAby a value of 22 N cmminus2 isometric stress for amphibian muscle(Edman 1979)

Static model of theoretical bite forceA static model of bite force was developed that made use ofgeometric formulae to calculate muscle force vectors and lever-armlengths from straight-line distances measured on the specimenswithout the use of 3D coordinates (Fig 3) The model used thesevalues combined with theoretical isometric muscle force tocalculate the contribution of each of the three jaw muscles thatinsert directly on the mandible to bite force combined with thecontribution of the quadratopectoralis muscle via head flexion and

the resulting tension in the atlanto-mandibular ligament that insertson the mandible

The muscle force vector that contributes to biting is defined foreach of the three jaw muscles as the height of an irregulartetrahedron (ie a triangular pyramid with unequal sides) that hasthe muscle origin at its apex and its triangular base formed by themuscle insertion and the left and right jaw joints (Fig 3) Heronrsquosformula from antiquity (Metrica ca 100 BCndash100 AD) was used tocalculate the area of the base triangle from the lengths of its threesides and an extension of Heronrsquos formula was used to calculate thevolume of the tetrahedron from the lengths of its six sides(Sommerville 1939) (formulae in Workbook 1) The muscleforce vector was taken as the height of the tetrahedron calculated asthree times the volume of the tetrahedron divided by the area of itstriangular base (applying Cavalierirsquos principle) The muscle in-leverlength was taken as the height of the base triangle calculated as twiceits area divided by the length of its base (ie the distance betweenthe jaw joints) The bite out-lever length for all muscles wascalculated similarly as the height of the triangle formed from the twojaw joints at the base and the bite point at the rostral tips of the jaws(or at the last tooth) at the apex The contribution of each muscle tobiting force was calculated as the muscle force vector times the in-lever length divided by the out-lever length This quantity wasdoubled to account for both left and right muscles

The anatomical origins of the levator mandibulae internusanterior and the levator mandibulae externus were used as theorigins for these muscles in the model However for the levatormandibulae internus posterior the rostral edge of the otic-parietaldepression was used as the origin for this muscle in the model

Left jawjoint

Rightjaw joint

Muscleinsertion

Muscleorigin

U

V

w

v

Wu

L

F

Fig 3 Geometric model for calculating the jawmusclemoment producedby a jaw muscle that originates on the skull and inserts on the lower jawThe base triangle (blue with sides U V and W) is formed by the muscleinsertion and the two jaw joints the height (L) of which defines the muscle in-lever length Out-lever length is calculated similarly by using the bite point asthe apex of the triangle The height (F) of the tetrahedron (sidesU VW and uv w) defines the component of the muscle force vector (u) that liesperpendicular to the base triangle The moment produced by the muscle iscalculated as F times L See Materials and methods lsquoStatic model oftheoretical bite forcersquo for details

3590

RESEARCH ARTICLE Journal of Experimental Biology (2017) 220 3588-3597 doi101242jeb165266

Journal

ofEx

perim

entalB

iology

because the otic-parietal depression is a trochlea that redirects theforce of this muscle (Fig S1)The contribution of the quadratopectoralis muscle to biting was

calculated with analogous geometric methods using the muscleorigin on the pectoral fascia as the apex of a tetrahedron that has asits base the muscle insertion on the quadrate and the left and rightatlanto-occipital joint centers about which the skull rotatesdorsoventrally The muscle force vector was calculated as theheight of this tetrahedron and the muscle in-lever length as theheight of the base triangle as described above The out-lever lengthwas calculated as the height of the triangle formed by the twoatlanto-occipital joints and the nearest point on the left atlanto-mandibular ligament which lies just dorsal to the joint (see Fig 4)

The force that the quadratopectoralis muscle produced at theligament was calculated as the muscle force vector times the in-leverlength divided by the out-lever length this force produced anequivalent magnitude of tension in the ligament that was transmittedto the lower jaw via its path through the otic-parietal trochlea Thisforce was added to the force of the levator mandibulae internusposterior because the ligament acts as the tendon with which thismuscle inserts on the mandible This quantity was doubled toaccount for both left and right muscles

Bite force was modeled with the jaws closed and head in theresting position Manipulations of the model were used to examinethe effect of ventral head flexion on bite force Head flexion hasthree effects (see Results) that increase the contribution of thequadratopectoralis muscle to bite force (1) placing the atlanto-mandibular ligament under tension by increasing the distancebetween its origin and insertion (2) orienting the muscle so that itsentire force is applied perpendicularly to the muscle in-lever and (3)positioning the atlanto-mandibular ligaments adjacent to theatlanto-occipital joints and thereby reducing the out-leverconsiderably The latter two effects were incorporated intothe model

Measurement of in vivo bite forceVoluntary bite forcewasmeasured in fourD quadramaculatus (80ndash95 mm SVL) and three Pseudotriton ruber (65ndash75 mm SVL) allcollected in North Carolina USA Salamanders were maintained ona diet of crickets in inclined plastic shoeboxes with a fewcentimeters of water at 16ndash18degC All procedures were approvedby the University of South Florida Institutional Animal Care andUse Committee

Pseudotriton ruber is a large semiaquatic plethodontid with amore generalized cranial morphology than D quadramaculatus itwas chosen for comparison because it is of similar size and ecologyyet lacks many of the morphological features of desmognathinesthat are thought to enhance biting force such as stalked occipitalcondyles rigid quadrates robust jaws enlarged quadratopectoralismuscles and atlanto-mandibular ligaments Pseudotriton ruberdoes however possess enlarged levator mandibulae externusmuscles compared with desmognathines and thus is not entirelyunspecialized for biting

Voluntary bite force was measured with a custom-built bite meter(Herrel et al 2001) with a Kistler Type 9203 force transducerconnected to a Kistler Type 5995 charge amplifier The transducerhas a range of 0ndash50 N with a threshold force of lt1 mN making itsuitable for accurately measuring the biting forces of thesalamanders The bite meter (Fig S2) was fitted with bite platescovered in a thin layer of rubber to protect the teeth of thesalamanders and elicit strong bites (Lappin and Jones 2014) Thetransducer produced signals that were sampled at 1000 Hz usingPowerLab 1630 data acquisition hardware and LabChart software(ADInstruments Bella Vista NSW Australia) running on a DellOptiplex GX620 computer Prior to biting experiments thetransducer was calibrated with weights hung from the upperbite plate

Salamanders were induced to bite the transducer by covering thebite plates with the molted exoskeletons of crickets that were part oftheir captive diet or skin secretions from a prey salamander(Desmognathus ocoee) Salamanders were placed in a retreat (acardboard paper-towel tube) and enticed to bite by placing thetransducer a few to several millimeters in front of their snouts Afterbites were obtained salamanders were rewarded with live cricketsBites were imaged in lateral view with a Canon Powershot S100

Flexed

In-lever

Out-leverBite force

C

In-lever

Quadratopectoralis muscle force

RestingB

AM ligamentRaised

1 cm

A

Out-lever

Fig 4 Cranium of D quadramaculatus in different positions relative tothe atlas showing the range of motion afforded by the stalked occipitalcondyles (A) Raised (B) resting and (C) flexed The atlanto-mandibularligament (dotted line) is placed in tension by the quadratopectoralis muscleflexing the cranium at the atlanto-occipital joint (gray dot) Tension is theproduct of quadratopectoralis muscle force (gold arrow in B) and the ratio of in-lever length (gold line) to out-lever length (green line) The out-lever lengthdecreases substantially when the cranium is flexed (C) increasing themechanical advantage of the quadratopectoralis muscle and therebyincreasing ligament tension Tension is transmitted along the atlanto-mandibular ligament over the skull to the lower jaw Bite force (arrow in C) isthe product of atlanto-mandibular ligament tension and the ratio of jaw in-lever(white line) to out-lever (black line)

3591

RESEARCH ARTICLE Journal of Experimental Biology (2017) 220 3588-3597 doi101242jeb165266

Journal

ofEx

perim

entalB

iology

Straight-line distances between morphological landmarks weremeasured on specimens with the head in the resting position and themouth closed to calculate theoretical bite force via a staticmusculoskeletal model described below A total of 27 distanceswere measured on each specimen including six distances for each ofthe four muscles that can contribute to biting For each of the threecranial jaw-levator muscles on the left side of each specimen(levator mandibulae externus levator mandibulae internus anteriorand levator mandibulae internus posterior) six distances weremeasured muscle origin to insertion left jaw joint to muscle originleft jaw joint to muscle insertion right jaw joint to muscle originright jaw joint to muscle insertion and left to right jaw joint centerFor the left quadratopectoralis muscle which can contribute tobiting by flexing the cranium ventrally six distances weremeasured muscle origin to insertion left atlanto-occipital joint tomuscle origin right atlanto-occipital joint to muscle origin leftatlanto-occipital joint to muscle insertion right atlanto-occipitaljoint to muscle insertion and left to right atlanto-occipital jointcenter Six additional distances were measured for the model leftatlanto-occipital joint center to the closest point on the left atlanto-mandibular ligament right atlanto-occipital joint center to theclosest point on the left atlanto-mandibular ligament left jaw jointto mid-rostral tip of jaws right jaw joint to tip of jaws left jaw jointto the last (caudal-most) tooth on left side and right jaw joint to lasttooth on the left side Measurements of each distance were madethree times using digital calipers (Neiko 01408A) and averaged tothe nearest 001 mmThe insertion of each of the muscles on the lower jaw and

quadrate is narrow and its position for use in the model was easilylocated The origin however is broader and its position for themodel was estimated as a point in the center of the area of originThe levator mandibulae internus anterior and the levatormandibulae externus are parallel-fibered muscles whose fiberangle relative to the muscle line of action is very shallow and wastaken as zero The myofiber angles of 10 random fibers of thequadratopectoralis and levator mandibulae internus posterior wereestimated using a dissecting microscope with a camera-lucidaattachment together with a protractorMuscles were dissected free and massed (Virtual Measurements

and Control model VB-302A Santa Rosa CA USA plusmn0001 gaccuracy) The myofibers of the levator mandibulae internusposterior were teased apart to visualize their relationships to theatlanto-mandibular ligament on which they originate and insert(Fig 2BndashD) Dimensions of the atlanto-mandibular ligament weremeasured for two specimens to calculate tendon stressTheoretical isometric muscle force for each muscle was

calculated by first determining physiological cross-sectional area(PCSA) as the cosine of average fiber angle times mass divided bydensity divided by fiber length Muscle density was taken as1056 g cmminus3 Muscle force was calculated by multiplying PCSAby a value of 22 N cmminus2 isometric stress for amphibian muscle(Edman 1979)

Static model of theoretical bite forceA static model of bite force was developed that made use ofgeometric formulae to calculate muscle force vectors and lever-armlengths from straight-line distances measured on the specimenswithout the use of 3D coordinates (Fig 3) The model used thesevalues combined with theoretical isometric muscle force tocalculate the contribution of each of the three jaw muscles thatinsert directly on the mandible to bite force combined with thecontribution of the quadratopectoralis muscle via head flexion and

the resulting tension in the atlanto-mandibular ligament that insertson the mandible

The muscle force vector that contributes to biting is defined foreach of the three jaw muscles as the height of an irregulartetrahedron (ie a triangular pyramid with unequal sides) that hasthe muscle origin at its apex and its triangular base formed by themuscle insertion and the left and right jaw joints (Fig 3) Heronrsquosformula from antiquity (Metrica ca 100 BCndash100 AD) was used tocalculate the area of the base triangle from the lengths of its threesides and an extension of Heronrsquos formula was used to calculate thevolume of the tetrahedron from the lengths of its six sides(Sommerville 1939) (formulae in Workbook 1) The muscleforce vector was taken as the height of the tetrahedron calculated asthree times the volume of the tetrahedron divided by the area of itstriangular base (applying Cavalierirsquos principle) The muscle in-leverlength was taken as the height of the base triangle calculated as twiceits area divided by the length of its base (ie the distance betweenthe jaw joints) The bite out-lever length for all muscles wascalculated similarly as the height of the triangle formed from the twojaw joints at the base and the bite point at the rostral tips of the jaws(or at the last tooth) at the apex The contribution of each muscle tobiting force was calculated as the muscle force vector times the in-lever length divided by the out-lever length This quantity wasdoubled to account for both left and right muscles

The anatomical origins of the levator mandibulae internusanterior and the levator mandibulae externus were used as theorigins for these muscles in the model However for the levatormandibulae internus posterior the rostral edge of the otic-parietaldepression was used as the origin for this muscle in the model

Left jawjoint

Rightjaw joint

Muscleinsertion

Muscleorigin

U

V

w

v

Wu

L

F

Fig 3 Geometric model for calculating the jawmusclemoment producedby a jaw muscle that originates on the skull and inserts on the lower jawThe base triangle (blue with sides U V and W) is formed by the muscleinsertion and the two jaw joints the height (L) of which defines the muscle in-lever length Out-lever length is calculated similarly by using the bite point asthe apex of the triangle The height (F) of the tetrahedron (sidesU VW and uv w) defines the component of the muscle force vector (u) that liesperpendicular to the base triangle The moment produced by the muscle iscalculated as F times L See Materials and methods lsquoStatic model oftheoretical bite forcersquo for details

3590

RESEARCH ARTICLE Journal of Experimental Biology (2017) 220 3588-3597 doi101242jeb165266

Journal

ofEx

perim

entalB

iology

because the otic-parietal depression is a trochlea that redirects theforce of this muscle (Fig S1)The contribution of the quadratopectoralis muscle to biting was

calculated with analogous geometric methods using the muscleorigin on the pectoral fascia as the apex of a tetrahedron that has asits base the muscle insertion on the quadrate and the left and rightatlanto-occipital joint centers about which the skull rotatesdorsoventrally The muscle force vector was calculated as theheight of this tetrahedron and the muscle in-lever length as theheight of the base triangle as described above The out-lever lengthwas calculated as the height of the triangle formed by the twoatlanto-occipital joints and the nearest point on the left atlanto-mandibular ligament which lies just dorsal to the joint (see Fig 4)

The force that the quadratopectoralis muscle produced at theligament was calculated as the muscle force vector times the in-leverlength divided by the out-lever length this force produced anequivalent magnitude of tension in the ligament that was transmittedto the lower jaw via its path through the otic-parietal trochlea Thisforce was added to the force of the levator mandibulae internusposterior because the ligament acts as the tendon with which thismuscle inserts on the mandible This quantity was doubled toaccount for both left and right muscles

Bite force was modeled with the jaws closed and head in theresting position Manipulations of the model were used to examinethe effect of ventral head flexion on bite force Head flexion hasthree effects (see Results) that increase the contribution of thequadratopectoralis muscle to bite force (1) placing the atlanto-mandibular ligament under tension by increasing the distancebetween its origin and insertion (2) orienting the muscle so that itsentire force is applied perpendicularly to the muscle in-lever and (3)positioning the atlanto-mandibular ligaments adjacent to theatlanto-occipital joints and thereby reducing the out-leverconsiderably The latter two effects were incorporated intothe model

Measurement of in vivo bite forceVoluntary bite forcewasmeasured in fourD quadramaculatus (80ndash95 mm SVL) and three Pseudotriton ruber (65ndash75 mm SVL) allcollected in North Carolina USA Salamanders were maintained ona diet of crickets in inclined plastic shoeboxes with a fewcentimeters of water at 16ndash18degC All procedures were approvedby the University of South Florida Institutional Animal Care andUse Committee

Pseudotriton ruber is a large semiaquatic plethodontid with amore generalized cranial morphology than D quadramaculatus itwas chosen for comparison because it is of similar size and ecologyyet lacks many of the morphological features of desmognathinesthat are thought to enhance biting force such as stalked occipitalcondyles rigid quadrates robust jaws enlarged quadratopectoralismuscles and atlanto-mandibular ligaments Pseudotriton ruberdoes however possess enlarged levator mandibulae externusmuscles compared with desmognathines and thus is not entirelyunspecialized for biting

Voluntary bite force was measured with a custom-built bite meter(Herrel et al 2001) with a Kistler Type 9203 force transducerconnected to a Kistler Type 5995 charge amplifier The transducerhas a range of 0ndash50 N with a threshold force of lt1 mN making itsuitable for accurately measuring the biting forces of thesalamanders The bite meter (Fig S2) was fitted with bite platescovered in a thin layer of rubber to protect the teeth of thesalamanders and elicit strong bites (Lappin and Jones 2014) Thetransducer produced signals that were sampled at 1000 Hz usingPowerLab 1630 data acquisition hardware and LabChart software(ADInstruments Bella Vista NSW Australia) running on a DellOptiplex GX620 computer Prior to biting experiments thetransducer was calibrated with weights hung from the upperbite plate

Salamanders were induced to bite the transducer by covering thebite plates with the molted exoskeletons of crickets that were part oftheir captive diet or skin secretions from a prey salamander(Desmognathus ocoee) Salamanders were placed in a retreat (acardboard paper-towel tube) and enticed to bite by placing thetransducer a few to several millimeters in front of their snouts Afterbites were obtained salamanders were rewarded with live cricketsBites were imaged in lateral view with a Canon Powershot S100

Flexed

In-lever

Out-leverBite force

C

In-lever

Quadratopectoralis muscle force

RestingB

AM ligamentRaised

1 cm

A

Out-lever

Fig 4 Cranium of D quadramaculatus in different positions relative tothe atlas showing the range of motion afforded by the stalked occipitalcondyles (A) Raised (B) resting and (C) flexed The atlanto-mandibularligament (dotted line) is placed in tension by the quadratopectoralis muscleflexing the cranium at the atlanto-occipital joint (gray dot) Tension is theproduct of quadratopectoralis muscle force (gold arrow in B) and the ratio of in-lever length (gold line) to out-lever length (green line) The out-lever lengthdecreases substantially when the cranium is flexed (C) increasing themechanical advantage of the quadratopectoralis muscle and therebyincreasing ligament tension Tension is transmitted along the atlanto-mandibular ligament over the skull to the lower jaw Bite force (arrow in C) isthe product of atlanto-mandibular ligament tension and the ratio of jaw in-lever(white line) to out-lever (black line)

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because the otic-parietal depression is a trochlea that redirects theforce of this muscle (Fig S1)The contribution of the quadratopectoralis muscle to biting was

calculated with analogous geometric methods using the muscleorigin on the pectoral fascia as the apex of a tetrahedron that has asits base the muscle insertion on the quadrate and the left and rightatlanto-occipital joint centers about which the skull rotatesdorsoventrally The muscle force vector was calculated as theheight of this tetrahedron and the muscle in-lever length as theheight of the base triangle as described above The out-lever lengthwas calculated as the height of the triangle formed by the twoatlanto-occipital joints and the nearest point on the left atlanto-mandibular ligament which lies just dorsal to the joint (see Fig 4)

The force that the quadratopectoralis muscle produced at theligament was calculated as the muscle force vector times the in-leverlength divided by the out-lever length this force produced anequivalent magnitude of tension in the ligament that was transmittedto the lower jaw via its path through the otic-parietal trochlea Thisforce was added to the force of the levator mandibulae internusposterior because the ligament acts as the tendon with which thismuscle inserts on the mandible This quantity was doubled toaccount for both left and right muscles

Bite force was modeled with the jaws closed and head in theresting position Manipulations of the model were used to examinethe effect of ventral head flexion on bite force Head flexion hasthree effects (see Results) that increase the contribution of thequadratopectoralis muscle to bite force (1) placing the atlanto-mandibular ligament under tension by increasing the distancebetween its origin and insertion (2) orienting the muscle so that itsentire force is applied perpendicularly to the muscle in-lever and (3)positioning the atlanto-mandibular ligaments adjacent to theatlanto-occipital joints and thereby reducing the out-leverconsiderably The latter two effects were incorporated intothe model

Measurement of in vivo bite forceVoluntary bite forcewasmeasured in fourD quadramaculatus (80ndash95 mm SVL) and three Pseudotriton ruber (65ndash75 mm SVL) allcollected in North Carolina USA Salamanders were maintained ona diet of crickets in inclined plastic shoeboxes with a fewcentimeters of water at 16ndash18degC All procedures were approvedby the University of South Florida Institutional Animal Care andUse Committee

Pseudotriton ruber is a large semiaquatic plethodontid with amore generalized cranial morphology than D quadramaculatus itwas chosen for comparison because it is of similar size and ecologyyet lacks many of the morphological features of desmognathinesthat are thought to enhance biting force such as stalked occipitalcondyles rigid quadrates robust jaws enlarged quadratopectoralismuscles and atlanto-mandibular ligaments Pseudotriton ruberdoes however possess enlarged levator mandibulae externusmuscles compared with desmognathines and thus is not entirelyunspecialized for biting

Voluntary bite force was measured with a custom-built bite meter(Herrel et al 2001) with a Kistler Type 9203 force transducerconnected to a Kistler Type 5995 charge amplifier The transducerhas a range of 0ndash50 N with a threshold force of lt1 mN making itsuitable for accurately measuring the biting forces of thesalamanders The bite meter (Fig S2) was fitted with bite platescovered in a thin layer of rubber to protect the teeth of thesalamanders and elicit strong bites (Lappin and Jones 2014) Thetransducer produced signals that were sampled at 1000 Hz usingPowerLab 1630 data acquisition hardware and LabChart software(ADInstruments Bella Vista NSW Australia) running on a DellOptiplex GX620 computer Prior to biting experiments thetransducer was calibrated with weights hung from the upperbite plate

Salamanders were induced to bite the transducer by covering thebite plates with the molted exoskeletons of crickets that were part oftheir captive diet or skin secretions from a prey salamander(Desmognathus ocoee) Salamanders were placed in a retreat (acardboard paper-towel tube) and enticed to bite by placing thetransducer a few to several millimeters in front of their snouts Afterbites were obtained salamanders were rewarded with live cricketsBites were imaged in lateral view with a Canon Powershot S100

Flexed

In-lever

Out-leverBite force

C

In-lever

Quadratopectoralis muscle force

RestingB

AM ligamentRaised

1 cm

A

Out-lever

Fig 4 Cranium of D quadramaculatus in different positions relative tothe atlas showing the range of motion afforded by the stalked occipitalcondyles (A) Raised (B) resting and (C) flexed The atlanto-mandibularligament (dotted line) is placed in tension by the quadratopectoralis muscleflexing the cranium at the atlanto-occipital joint (gray dot) Tension is theproduct of quadratopectoralis muscle force (gold arrow in B) and the ratio of in-lever length (gold line) to out-lever length (green line) The out-lever lengthdecreases substantially when the cranium is flexed (C) increasing themechanical advantage of the quadratopectoralis muscle and therebyincreasing ligament tension Tension is transmitted along the atlanto-mandibular ligament over the skull to the lower jaw Bite force (arrow in C) isthe product of atlanto-mandibular ligament tension and the ratio of jaw in-lever(white line) to out-lever (black line)

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camera recording in HD at 60 Hz to obtain the approximate positionof the jaws on the bite meter during the bite (Fig 5 Movie 1) Tovisualize the moving morphology fluoroscope images of Dquadramaculatus feeding were recorded at 1000 Hz in lateral anddorsal view with Visario Speedcam cameras (Weinberger GmbHErlangen Germany) (Fig 6 Movie 2) All in vivo experiments wereconducted at an ambient temperature of 16ndash18degC and 61ndash69relative humidityBite force data of D quadramaculatus and P ruber were log

transformed for normality and homoscedasticity and compared in Rversion 332 using an ANOVA (nlme package) that includedspecies as a fixed effect and individual as a random effect MicrosoftExcel 2016 was used to calculate effect size (Cohenrsquos d ) Means arepresented plusmnsem

RESULTSFunctional morphology of D quadramaculatus jawsManipulation of fresh specimens revealed an interaction betweenthe rotation of the mandible on the cranium and the rotation of thecranium on the atlas The jaws of D quadramaculatus closed whenthe head was flexed ventrally and opening the mouth required thecranium to be either in line with the trunk or rotated (extended)dorsally about the atlanto-occipital joint (Dunn 1926 Wake 1966Hinderstein 1971) Despite this constraint on mouth opening thecranium could be flexed ventrally to nearly 90 deg to the trunk withthe jaws in occlusion Dorsal rotation of the cranium appeared toslacken the atlanto-mandibular ligaments to allow jaw depression upto a gape angle of approximately 90 deg Flexing the craniumventrally on the atlas to bring the line of the upper jaw below theplane of the trunk placed the ligaments under tension the tautligaments pulled the jaws closed or applied force to a finger placedbetween the jaws when the cranium was further flexed ventrallyCranial flexion also moved the atlanto-mandibular ligaments closeto the atlanto-occipital joints when the cranium was fully flexedthe ligaments were in contact with the dorsal surfaces of theoccipital condyles In skinned specimens with the mouth closed andthe ligaments slackened by raising the cranium the ligaments couldbe easily displaced laterally out of the otic-parietal trochleas Whenthe atlanto-mandibular ligaments were severed interaction betweenjaw movements and head movements was eliminated confirming

earlier conclusions that these ligaments are responsible for couplinghead movements with jaw movements (Dalrymple et al 1985Schwenk and Wake 1993)

Manipulation and dissection of specimens confirmed that thethree jaw levator muscles that span the jaw joint (Fig 1) contributeto bite force directly by elevating the mandible relative to thecranium and that the quadratopectoralis muscle contributesindirectly by flexing the cranium ventrally This ventral flexion ofthe cranium is coupled with jaw elevation or the application of biteforce via the atlanto-mandibular ligaments which are placed intension when the cranium is ventroflexed Movement of the atlanto-mandibular ligaments in the otic-parietal trochlea occurs with nonoticeable friction

Morphology and estimated bite performance ofD quadramaculatusThe D quadramaculatus specimens examined for morphology hadan SVL of 935plusmn36 mm total length of 169plusmn62 mm body mass of184plusmn22 g and head width at the jaw joints of 146plusmn10 mmDistance between the jaw joint centers was 116plusmn04 mm and jawlength from a line connecting the jaw joints to the rostral-most toothwas 130plusmn05 mm (Table 1) The dorsal surface of the cranium isdevoid of musculature although the jaw levators bulge slightlylaterally just rostral to the jaw joints The enlargedquadratopectoralis muscles bulge visibly extending from just

2 cm

Fig 5 Salamanders voluntarily bit the bite plates of the force transducerbaited with exoskeletons of crickets or skin secretions ofDesmognathusocoee a prey salamander species Bite plates were covered with rubberstrips to prevent tooth damage Note the mouth is opened without headelevation Details of the bite-force transducer are shown in Fig S2

A D

B E

C F

Fig 6 Fluoroscope images of feeding in D quadramaculatus (A) Thecranium and atlas (arrow) in the resting position The cranium is raised duringtongue projection (B) and further during prey transport (C) It is lowered uponmouth closing (D) and strongly flexed relative to the atlas during biting (EF)placing the atlanto-mandibular ligaments under tension Note the curvature ofthe anterior vertebral column (EF) that changes the angle of the atlas relativeto the cranium

Table 1 Morphological measurements of Desmognathusquadramaculatus used in the biomechanical model

Variable Meanplusmnsem

Body mass (g) 184plusmn22Snoutndashvent length (mm) 935plusmn36Total length (mm) 169plusmn62Head width at jaw joints (mm) 146plusmn10Distance between jaw joint centers (mm) 116plusmn04Jaw joint to jaw tip (mm) 142plusmn05Jaw joint to last tooth (mm) 43plusmn03

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caudal to the jaw joints to the insertion of the forelimb and givingthe salamanders a lsquonecklessrsquo appearance (Fig S3)The levator mandibulae externus (LME) muscle originates on the

rostrolateral surfaces of the occipito-otic and squamosal and travelsventrally between the quadratomaxillary ligaments to insert on andjust caudal to the coronoid process of the prearticular Muscle fiberstravel rostroventrally and laterally nearly in parallel from origin toinsertion and are in a position to draw the mandible dorsally to closethe mouth or apply biting force as well as caudally towards thequadrate-prearticular joint The LME had a mass of 0023plusmn0003 gand was calculated to contribute a moment of 00023plusmn00003 Nm atthe jaw joint (unilaterally) and to exert a bite force of 018plusmn0023 Nat the jaw tips or 055plusmn008 N at the last tooth (Table 2) Theseforces and moments are unilateral as for the muscles discussedbelow unless otherwise notedThe levator mandibulae internus anterior (LMIA) muscle

originates on the lateral surface of the rostralmost edge of theparietal and the palatoquadrate cartilage just caudal to the eye Fibersrun ventrolaterally nearly in parallel and insert on the medialprojection of the coronoid process of the prearticular via a narrowtendon and can thus raise the mandible or apply biting force Themedial component of force that this muscle exerts on the mandiblemaybe redirected dorsally by themedial quadratomaxillary ligamentacting as a retinaculum The LMIA had amass of 0006plusmn0001 g andwas calculated to contribute a moment of 00006plusmn00003 Nm at thejaw joint (unilaterally) and to exert a bite force of 0048plusmn0023 N atthe jaw tips or 015plusmn0082 N at the last tooth (Table 2)The levator mandibulae internus posterior (LMIP) muscle lies

lateral to the LMIA It originates on the rostral surface of the atlascrest and runs rostrolaterally then rostroventrally through the otic-parietal depression and thence ventrolaterally to insert between thequadratomaxillary ligaments on the coronoid process of theprearticular via a large tendon The LMIP tendon is in fact theterminus of the robust atlanto-mandibular ligament that shares withthe LMIP its origin insertion and trajectory Muscle fibers originatefrom and insert onto the atlanto-mandibular ligament in a complexpennate arrangement (Fig 2) at an average angle of 112 deg but donot extend from origin to insertion The myofibers of the LMIP byvirtue of this arrangement can place tension on the atlanto-mandibular ligament when they contract but are limited in how farthey can be stretched when tension is placed on the ligamentduring cranial flexion The LMIP plus atlanto-mandibularligament can raise the mandible or can prevent mandibulardepression or apply biting force when the cranium is flexedventrally by the quadratopectoralis muscles As with the LMIAthe medial quadratomaxillary ligament may act as a retinaculum toredirect medial force dorsally The atlanto-mandibular ligamentwithout myofibers averaged 027plusmn006 mm in thickness 202plusmn084 mm in width and 1126plusmn012 mm in length cross-sectionalarea was thus 0489plusmn0112 mm2 The LMIP had a mass of 0009plusmn00005 g and was calculated to contribute a moment of 00013plusmn000031 Nm at the jaw joint (unilaterally) and to exert a bite forceof 0105plusmn0024 N at the jaw tips or 033plusmn008 N at the last tooth(Table 2)The quadratopectoralis (QP) muscle originates broadly on the

fascia of the pectoral-gular region and its fibers travel rostrodorsallyat an average angle of 148 deg to converge and insert on the lateralsurface of the ventral edge of the quadrate beneath the lateralquadratomaxillary ligament (Fig 1) The QP is the largest of themuscles that contribute to bite force which it accomplishes byflexing the cranium ventrally and thereby placing tension onthe atlanto-mandibular ligament The QP muscle had a mass of Ta

ble2

Morph

olog

ical

andbiom

echa

nica

lqua

ntities

from

four

mus

cles

that

contribu

teto

bitin

gin

Des

mog

nathus

quad

ramac

ulatus

Qua

dratop

ectoralis

Leva

torman

dibu

laeex

ternus

Leva

torman

dibu

laeinternus

anterio

rLe

vatorman

dibu

laeinternus

posterior

Cranium

atrest

Cranium

ventrofle

xed

Orig

inOccipito

-otic

andsq

uamos

alParietala

ndpa

latoqu

adrate

Atla

scres

tPec

toralfas

cia

Pec

toralfas

cia

Inse

rtion

Coron

oidproc

essof

prea

rticular

Coron

oidproc

essof

prea

rticular

Coron

oidproc

essof

prea

rticular

Qua

drate

Qua

drate

Joints

pann

edQua

drate-articular

(ie

jawjoint)

Qua

drate-articular

Qua

drate-articular

andatlanto-oc

cipital

Atla

nto-oc

cipital

Atla

nto-oc

cipital

Mas

s(g)

002

3plusmn000

3000

6plusmn000

1000

9plusmn000

05008

7plusmn001

3008

7plusmn001

3PCSA(cm

2)

005

5plusmn000

8002

plusmn000

8001

9plusmn000

4008

1plusmn001

008

1plusmn001

Raw

force(N

)122

plusmn017

043

plusmn017

041

plusmn007

9179

plusmn023

179

plusmn023

Fractionof

forcetran

smitted

073

plusmn005

2048

plusmn01

081

plusmn004

1084

plusmn001

610

Con

tributingforce(N

)088

plusmn010

8023

plusmn013

034

plusmn007

15plusmn

019

179

plusmn023

In-le

ver(m

m)

277

plusmn06

294

plusmn051

393

plusmn023

335

plusmn024

335

plusmn024

Out-le

ver(m

m)

1298plusmn

048

1298plusmn

048

1298plusmn

048

157

plusmn005

9081

plusmn004

3Mus

clemom

enta

bout

jawjoint(N

m)

000

23plusmn000

033

000

06plusmn000

03000

13plusmn000

031

001

1plusmn000

22002

4plusmn000

4Bite

forceco

ntrib

utionat

jawtip

s(N

)018

plusmn002

3004

8plusmn002

3010

5plusmn002

4081

plusmn015

3181

plusmn029

Bite

forceco

ntrib

utionat

last

tooth(N

)055

plusmn008

015

plusmn008

2033

plusmn008

252

plusmn054

565

plusmn107

Value

saremea

nsplusmnsemBite

forcean

dtorque

contrib

utions

areun

ilateralCon

tributionfrom

thequ

adratope

ctoralisisca

lculated

asap

pliedthroug

htheatlanto-man

dibu

larligam

ento

fthe

leva

torman

dibu

lae

internus

posteriormus

cle

PCSAp

hysiolog

ical

cros

s-se

ctiona

larea

Con

tributingforceistheco

mpo

nent

ofrawmus

cleforcethat

prod

uces

amom

enta

bout

thejawjoint

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0087plusmn0013 g and was calculated to contribute a moment of0005plusmn0001 Nm at the atlanto-occipital joint (unilaterally) and atension in the atlanto-mandibular ligament of 335plusmn063 N with thehead in the resting position (Table 2) The QP muscle is calculatedto exert via the tension in the atlanto-mandibular ligament amoment of 0011plusmn00022 Nm at the jaw joint (unilaterally) andexert a bite force of 0808plusmn0153 N at the jaw tips or 252plusmn054 N atthe last tooth With the head flexed ventrally the QP contributed amoment of 0006plusmn0001 Nm and a tension in the ligament of744plusmn106 N The QP muscle is calculated to exert via the tensionin the atlanto-mandibular ligament a moment of 0024plusmn0004 Nmat the jaw joint (unilaterally) and exert a bite force of 181plusmn029 Nat the jaw tips or 565plusmn107 N at the last toothAll muscles together contributed an estimated total moment of

0015plusmn0003 Nm at the joint unilaterally with the head in the restingposition and 0028plusmn0005 Nm with the head flexed (Table 3) TheQPmuscle contributes 73 of this moment with the head at rest and85 when the head is flexed while the LME muscle contributes15 and 8 the LMIA contributes 4 and 2 and the LMIPcontributes 9 and 5 respectively With the head in the restingposition the total moment resulted in a unilateral calculated biteforce of 114plusmn020 N at the jaw tips and 355plusmn072 N at the lasttooth With the head flexed the unilateral bite force is estimated at214plusmn034 N at the jaw tips and 668plusmn127 N at the last toothDoubling these values to account for all bilaterally paired musclesand head flexion yields a maximum theoretical applied bite force of429plusmn069 N at the jaw tips and 1337plusmn255 N at the last toothMaximum stress in the atlanto-mandibular ligament is calculated at55plusmn16 MPa with the head in the resting position and 129plusmn16 MPa with the head flexedGiven that the QP muscle contributes up to 85 of bite force and

acts indirectly via the atlanto-mandibular ligament that is absent orpoorly developed in other salamanders we calculated the requiredmuscle force and PCSA of the second greatest contributor theLME to produce the estimated maximum moment and bite forcewith the head flexed in the absence of the contribution of the QPAdding the unilateral moment of the QP of 0024 Nm to the momentof the LME of 00023 the combined moment is 00263 NmProducing this moment through the measured average in-lever of276 mm and an applied muscle force fraction of 624 (owing tothe angle of the line of action of the LME) would require an LMEmuscle force of 1527 N At a specific tension of 22 N cmminus2 theLME would need to have a PCSA of 069 cm2 or 125 times theaverage calculated PCSA of 0055 cm2 based on morphologicalmeasurements

In vivo bite performanceBoth salamander species readily bit the force transducers after aforward lunge of the entire body towards the transducer and tongueprojection in many trials The salamanders typically pressed theirsnouts against the stop on the transducer positioning the bite pointnear the mid-point of the jaws (Fig 5) The bite point therefore fellbetween the bite points that we included in the biomechanicalmodel the jaw tips and the last tooth The rigidity and immobility ofthe transducer prevented the salamanders from depressing theirheads fully during biting They presumably could flex or extendtheir anterior vertebral column dorsally to change the angle of theatlanto-occipital joint however we could not determine whetheror not they did so Thus the measured bite forces for Dquadramaculatus are likely to fall between the extreme conditionsof our biomechanical model with respect to both head angle andbite-point position

Desmognathus quadramaculatus achieved a peak bite forceof 82plusmn13 N and a bite impulse of 075plusmn012 Ns with a biteduration of 020plusmn001 s (N=21) The highest single bite fromD quadramaculatus was 143 N (Fig 7) Pseudotriton ruberachieved a peak bite force of 064plusmn020 N a bite impulse of0057plusmn0015 Ns and a bite duration of 019plusmn002 s (N=9) Thehighest single bite from P ruber was 11 N Desmognathusquadramaculatus had significantly higher peak bite force(P=00011 Cohenrsquos d=38) and bite impulse (P=00009 d=42)than P ruber but similar bite duration (P=03942 d=039 Fig S4)

Fluoroscope images of D quadramaculatus capturing a cricket(Fig 6) reveal the range of motion of the atlanto-occipital joints aswell as the head-tucking behavior after the prey has been broughtbetween the jaws by the tongue These images reveal that theanterior vertebral column is extended dorsally while the cranium isflexed ventrally These movements combine to increase the anglebetween the atlas and the cranium and thereby increase the tensionon the atlanto-mandibular ligament

DISCUSSIONFunctional morphology of D quadramaculatus jawsThe musculoskeletal cranial features of the D quadramaculatusexamined here are largely consistent with descriptions of thesefeatures in this species and other desmognathine species by previousresearchers (Dunn 1926 Wake 1966 Hinderstein 1971Dalrymple et al 1985 Schwenk and Wake 1993) Ourobservation of the solid cranium and jaws with reinforcedquadrate robust atlanto-mandibular ligaments stalked occipitalcondyles and enlarged atlas and anterior axial muscles are

Table 3 Total forces and moments from all muscles that contribute tobiting in Desmognathus quadramaculatus

Unilateral Bilateral

Cranium in resting positionTotal moment at jaw joint (Nm) 0015plusmn0003 003plusmn0006Total bite force at jaw tips (N) 114plusmn0202 228plusmn0404Total bite force at last tooth (N) 355plusmn0722 71plusmn1445Tension in A-M ligament (N) 335plusmn0632 669plusmn1263Stress in A-M ligament (MPa) 554plusmn1605 554plusmn1605

Cranium flexed ventrallyTotal moment at jaw joint (Nm) 0028plusmn0005 0056plusmn001Total bite force at jaw tips (N) 214plusmn034 429plusmn069Total bite force at last tooth (N) 668plusmn127 1337plusmn255Tension in A-M ligament (N) 744plusmn106 1489plusmn213Stress in A-M ligament (MPa) 1289plusmn155 1289plusmn155

A-M atlanto-mandibular Values are meansplusmnsem

Bite

forc

e (N

)

ndash202468

10121416

01 s

Fig 7 Representative bites of D quadramaculatus (green) measured atapproximately mid-mandible Force far exceeds that produced by the jawmuscles Bites of Pseudotriton ruber (gold) are much weaker Vertical spikeswere caused by the salamanderrsquos snout striking the stop on the bite transducer

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consistent with previous descriptions The coupled movements ofthe cranium and mandible we observed are likewise in accord withprevious descriptions However we found that the head can beflexed to nearly 90 deg relative to the trunk indicating the atlanto-mandibular ligaments do not prevent head depression Anotherdifference with earlier work lies in the description of the atlanto-mandibular ligament by Hinderstein (1971) who describes it asbeing encompassed by muscle fibers in the species he examinedincluding D quadramaculatus We found that the muscle fibers ofthe LMIP are the only myofibers attaching to the ligament and theseare restricted to the dorsal surface of the ligament and that theventral surface is smooth and devoid of myofibers This permits theatlanto-mandibular ligament to slide without noticeable friction inthe otic-parietal trochlea an important condition for thetransmission of the amplified force of the QP muscle to the bitethat is central to the unique desmognathine biting mechanismThe atlanto-mandibular ligament on each side of the skull is such

an unusual and conspicuous feature of desmognathine salamandercranial morphology that its function has long been pondered (Dunn1926 Wake 1966 Hinderstein 1971 Dalrymple et al 1985reviewed by Schwenk and Wake 1993) Our observations agreewith the findings of previous researchers that the linkage betweenthe atlas and mandible provided by this ligament limits the extent oflower jaw depression relative to the trunk (eg Wake 1966) andthereby couples cranial movements with jaw movements Howeverwe find no evidence to support the proposition that desmognathinesmust raise their heads to initially open their mouths (Hinderstein1971) finding instead that the ligament limits but does not preventmouth opening when the head is in its resting posture in linewith thetrunk (Fig 5) Further head lifting is ubiquitous during mouthopening in salamander feeding (Deban and Wake 2000 Wake andDeban 2000) and most species lack this mechanical linkage Theatlanto-mandibular ligament has also been proposed to recoilelastically to accelerate mouth closing (Hinderstein 1971Dalrymple et al 1985) however we found this conclusion to beinconsistent with our mechanical analysis and our observation thatthe ligament is far too robust to be appreciably stretched by the smalljaw depressor muscles with their narrow tendons and poorermechanical advantage The atlanto-mandibular ligament couplesmouth closing and head depression and can cause the jaws to lsquosnaprsquoshut rapidly in manipulated specimens when the snout is presseddownward but this mechanism does not require elastic recoilBased on our manipulations of specimens we found that the

robust atlanto-mandibular ligament functions as an inextensiblelink like a cable between the atlas and mandible in agreement witha previous account (Schwenk and Wake 1993) We also observedthat the position of the cranium over which the ligament passesalters the length of its path and thus the degree of mouth openingpermitted This coupling allows the cranial depressor muscle theQP to contribute to biting force when the head is flexed increasingbite performance beyond the force provided by the jaw levatormuscles (LME LMIA and LMIP)

Novel biting mechanismOur mechanical analysis reveals a novel mechanism of forceamplification in which the force contribution of the atlanto-mandibular ligament to biting is greater than the force producedby the QP muscle in flexing the cranium ndash a biting mechanism thatmay be unique to desmognathine salamanders The high tensionproduced in the atlanto-mandibular ligament is generated by thehigh mechanical advantage of the QP muscle resulting fromthe long in-lever of the QP muscle defined as the distance of the

quadrate to the atlanto-occipital joint and the very short out-leverdefined as the distance from the atlanto-occipital joint to the atlanto-mandibular ligament (Fig 4) With the head fully flexed the QPforce is better aligned to produce a moment about the atlanto-occipital joint using the full length of the in-lever from the QPinsertion to the atlanto-occipital joint a length that is increased bythe presence of stalked occipital condyles Perhaps moreimportantly the distance from the atlanto-mandibular ligament tothe atlanto-occipital joint drops by half when the head is fullyflexed by virtue of the stalked occipital condyles As a result of thisincrease in mechanical advantage the QP muscle contributes anestimated 74 N to the tension of the atlanto-mandibular ligamentwhen the cranium is fully flexed despite exerting an estimatedforce of only 18 N at its insertion on the quadrate (Tables 2 and 3Fig 4) ndash a force amplification of over four times

The novel biting mechanism of desmognathines may providebenefits in jaw mechanics beyond simply increasing peak bitingforce A dual mechanism of mouth closing may expand the range ofhead and jaw angles over which high bite force can be applied Theintrinsic jaw levator muscles (LME LMIA and LMIP) may be usedto close the jaws with relatively low force and high velocity at arange of head angles such as when prey are first engulfed whilethe QP may be incapable of producing rapid mouth closing becauseof its high mechanical advantage but instead may be recruitedsubsequently to apply high biting forces Such an expandedfunctional range of force application might be demonstratedwith additional biomechanical modeling and in vivo bitingexperiments

The biting mechanism described here is an emergent propertyof many of the distinguishing morphological features ofdesmognathine salamanders The robust skull and jaws are able towithstand elevated biting forces compared with other salamandersThe enlarged QP muscles that give desmognathines their distinctivelsquonecklessrsquo appearance account for 70 of biting-muscle mass butexert 85 of biting force by virtue of the robust atlanto-mandibularligament that originates on the enlarged atlas The stalked occipitalcondyles are critical they enable a wide range of head movementsincluding the characteristic lsquohead tuckrsquo that desmognathines exhibitwhen biting (Dalrymple et al 1985) During the head tuck thecondyles improve mechanical advantage of the QP muscles andamplify its force contribution by simultaneously increasing the in-lever length and decreasing the out-lever length

Static model of theoretical bite forceWe derived a novel geometric method to compute muscleforce components (ie vectors) and lever-arm lengths inD quadramaculatus that makes use of straight-line distancesmeasured using calipers on dissected specimens without the needfor 3D coordinates of morphological features The vectors and leverarms produced were combined with estimates of muscle force (egvia PCSA) to calculate muscle moments The same geometricmethods were then used to convert muscle moments to force outputat the end of a lever such as a jaw tip (see Workbook 1)

Our geometric method enabled us to quantify the contribution ofeach of the jaw muscles and the unusual jaw mechanism describedabove to bite force in D quadramaculatus We conclude from theseanalyses that the unusual mechanism of force amplification andtransmission via the atlanto-mandibular ligament significantlyincreases biting performance in desmognathine salamandersbeyond what is possible with intrinsic jaw levator muscles Inaddition flexion of the head is important for fully engaging thismechanism and achieving the highest bite forces

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In vivo bite performanceBite force calculated from the model a maximum of 134 N at thelast tooth with the head flexed is comparable to the maximummeasured in vivo 143 N indicating that the model captures theessential elements of the biting mechanism We do not comparepredicted with in vivo bite force statistically because we cannot becertain of the exact bite position along the jaw in each bite (Lappinand Jones 2014) or the degree of flexion of the cranium relative tothe atlas in the absence of fluoroscope images for each in vivo biteOur measured biting performance of D quadramaculatus is

impressive for a salamander and significantly exceeds what thecomparably sized P ruber exhibited (143 versus 11 N maximumsingle bite Fig 7 Fig S4) However we conclude that themeasured biting force of D quadramaculatus falls below what theyare capable of for three reasons First the bite transducer is rigid andtherefore prevented full head flexion which our model indicates isimportant for generating maximum biting force with the jaws inocclusion Second we measured the force of rapid feeding bites(Movie 1) rather than static-pressure bites used in defense in combator to crush prey (Brodie 1978 Schwenk and Wake 1993) Thirdthe value of isometric muscle stress that we used in the model(22 N cmminus2) is conservative because it is derived from frog-limbmyofibers operating at 29degC myofibers whose temperaturecoefficient of isometric force was calculated at 124 (Edman1979) We estimate that maximum in vivo bite forces may be 136times greater at 18degC (the room temperature of our bitingexperiments) at which temperature the isometric muscle force isextrapolated to be 30 N cmminus2 Estimated peak bite force forD quadramaculatus at 18degC is therefore approximately 18 N atthe last tooth

Significance and comparison with other systemsDesmognathine salamanders of all species possess the same unusualmorphological features of the jaw mechanism described here andelsewhere (Dunn 1926Wake 1966 Schwenk andWake 1993) andare therefore expected to display similarly elevated bitingperformance to what we observed in D quadramaculatus Whymight desmognathines produce high biting forces Desmognathineslike most salamanders are opportunistic feeders that will eat mostprey that can fit in the mouth (Deban and Wake 2000 Wake andDeban 2000) including arthropod prey such as insects and in thecase of D quadramaculatus crayfish that must be subdued by apowerful bite The ability to inflict crushing bites on prey likelyexpands the dietary range of desmognathines beyond that of othercomparably sized salamanders That some prey may inflict injurymay explain the presence in D quadramaculatus of unusualsubocular mineralizations (Fig 2A) that are perfectly positioned toprotect the eyes fromdamage by prey that are inside the buccal cavityDesmognathines are unusual among salamanders in biting readilywhen cornered or handled (Baldauf 1947 Dalrymple et al 1985Bakkegard 2005) notably D quadramaculatus was found to usebiting to repel the attacks of shrews (Brodie 1978) We haveobserved D ocoee defend against attacks by a larger plethodontidsalamander speciesGyrinophilus porphyriticus by tenacious bitingand have observed scars on G porphyriticus of the appropriate sizeand shape to be the result of D ocoee or small D monticola bitesFinally the diminutive D aeneus and D wrighti are unusual amongplethodontids in using prolonged biting during courtship(Promislow 1987) biting that is forceful enough that the malebreaks the skin of the female and is able to lift her from the substrateIn addition to enhancing biting force the distinctive skull and

jaw morphology and mechanism of desmognathines have been

proposed to improve the ability of these salamanders to burrow andto wedge themselves beneath stones behaviors that they exhibit(Dunn 1926 Wake 1966 Worthington and Wake 1972Bakkegard 2005) The tapered skull and head that lacks dorsallyprotruding jaw levator muscles would require less force to penetratea substrate or crevice than a skull with greater diameter while itsheavy ossification can withstand the forces involved The atlanto-mandibular ligaments would indeed hold the mouth closed toprevent the entry of debris when the head is flexed

The desmognathine system represents a means of generating highbite forces while eliminating dorsally protruding jaw levatormuscles that would increase head diameter and the consequentcost of burrowing (Larsen and Beneski 1988 Schwenk and Wake1993) Other salamanders that achieve high biting performance doso with enlarged jaw levator muscles in the case of Aneides(Stebbins 1985 Staub 1993) or by a combination of large levatormuscles and large body size as in Amphiuma and Cryptobranchus(Elwood and Cundall 1994 Erdman and Cundall 1984) Ourbiomechanical analysis reveals that for D quadramaculatus toproduce the predicted maximum biting force in the absence of theQP muscle and its force-amplification mechanism that we describethe LME would need to be 12 times larger in cross-sectional areawhich would greatly increase the diameter of the head Anothergroup of burrowing amphibians the limbless caecilians haveevolved an unusual bite-force enhancing mechanism in whicha post-cranial muscle the interhyoideus posterior pullsventrocaudally on the retroarticular process of the mandible Thecaecilian jaw mechanism is thought to have evolved in response to asimilar selective pressure to reduce head cross-sectional area andthereby reduce the cost of constructing burrows (Bemis et al 1983Kleinteich et al 2008 Nussbaum 1983 Summers and Wake2005) Interestingly the interhyoideus posterior muscle ofcaecilians is a homologue of the QP of desmognathines (Bauer1992) An alternative strategy of increasing bite force in the face ofspace constraints is seen in crevice-dwelling chuckwalla lizards andinvolves widening rather than deepening the head (both dimensionsare increased in many lizards) males have four times the bite forceof females and wider but not deeper heads (Lappin et al 2006)

The highest in vivo biting forces of D quadramaculatusmeasured here (143 N maximum) fall within the range of thosemeasured in lizards of similar size Anoliswere measured to producesim11 N at 10 cm SVL (Herrel and OrsquoReilly 2006) which Dquadramaculatus can exceed Xenosaurid lizards produced up to204 N at 11 cm SVL (Herrel et al 2001) which exceeds ourmeasured biting force in D quadramaculatus but is close to theforce predicted by the model (18 N) operating with full head flexionat a comparable temperature If D quadramaculatus couldwithstand the higher temperatures that many lizards preferthey might well outperform lizards That the bite force ofD quadramaculatus is comparable to that of lizards especiallywhen compared with the maximum performance we measured inP ruber of 11 N is a testament to the effectiveness of the forceamplification mechanism The importance of biting in the naturalhistory of desmognathine salamanders ndash in feeding defense andcourtship ndash suggests that selection for elevated biting performanceplayed a role in the evolution of their unique morphological featuresand highly effective jaw mechanism

AcknowledgementsWe thank members of the Deban lab for assistance collecting salamanders JamesOrsquoReilly for use of the bite-force transducer and William Ryerson for recording themovie of Desmognathus quadramaculatus capturing a mealworm Fluoroscopeimages were obtained in collaboration with Nadja Schilling at the Institute of

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Systematic Zoology and Evolutionary Biology Friedrich-Schiller-University in JenaGermany Charlotte Stinson Jeffrey Olberding Mary Kate OrsquoDonnell and ChristianBrown provided helpful comments on the manuscript

Competing interestsThe authors declare no competing or financial interests

Author contributionsConceptualization SMD JCR Methodology SMD JCR Formal analysisSMD JCR Investigation SMD Writing - original draft SMD Writing - reviewamp editing SMD JCR Project administration SMD Funding acquisitionSMD

FundingThis research was supported by National Science Foundation grant IOS 1350929 toSMD

Supplementary informationSupplementary information available online athttpjebbiologistsorglookupdoi101242jeb165266supplemental

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Baldauf R J (1947) Desmognathus f fuscus eating eggs of its own speciesCopeia 1947 66

Bauer W J (1992) A contribution to the morphology of the m interhyoideusposterior (VII) of urodele Amphibia Zool Jahrb Anat 122 129-139

BemisW E Schwenk K andWakeM H (1983) Morphology and function of thefeeding apparatus in Dermophis mexicanus (Amphibia Gymnophiona)Zool J Linn Soc 77 75-96

Brodie E D Jr (1978) Biting and vocalization as antipredator mechanisms interrestrial salamanders Copeia 1978 127-129

Dalrymple G H Juterbock J E and La Valley A L (1985) Function of theatlanto-mandibular ligaments of desmognathine salamanders Copeia 1985254-257

Deban S M and Wake D B (2000) Aquatic feeding in salamanders In Feedingform Function and Evolution in Tetrapod Vertebrates (ed K Schwenk) pp95-116 San Diego CA Academic Press

Dunn E R (1926) The Salamanders of the Family Plethodontidae NorthamptonMA Smith College

Edman K A (1979) The velocity of unloaded shortening and its relation tosarcomere length and isometric force in vertebrate muscle fibres J Physiol 291143-159

Elwood J R L and Cundall D (1994) Morphology and behavior of the feedingapparatus inCryptobranchus alleganiensis (Amphibia Caudata) J Morphol 22047-70

Erdman S and Cundall D (1984) The feeding apparatus of the salamanderAmphiuma tridactylum morphology and behavior J Morphol 181 175-204

Herrel A and OrsquoReilly J C (2006) Ontogenetic scaling of bite force in lizards andturtles Physiol Biochem Zool 79 31-42

Herrel A De Grauw E and Lemos Espinal J A (2001) Head shape and biteperformance in xenosaurid lizards J Exp Zool 290 101-107

Hinderstein B (1971) The desmognathine jaw mechanism (Amphibia CaudataPlethodontidae) Herpetologica 27 467-476

Kleinteich T Haas A and Summers A P (2008) Caecilian jaw-closingmechanics integrating two muscle systems J R Soc Interface 5 1491-1504

Lappin A K and Jones M E H (2014) Reliable quantification of bite-forceperformance requires use of appropriate biting substrate and standardization ofbite out-lever J Exp Biol 217 4303-4312

Lappin A K Hamilton P S and Sullivan B K (2006) Bite-force performanceand head shape in a sexually dimorphic crevice-dwelling lizard the commonchuckwalla [Sauromalus ater (=obesus)] Biol J Linn Soc 88 215-222

Larsen J H Jr and Beneski J T Jr (1988) Quantitative-analysis of feedingkinematics in dusky salamanders (Desmognathus) Can J Zool 66 1309-1317

Nussbaum R A (1983) The evolution of a unique dual jaw-closing mechanism incaecilians (Amphibia Gymnophiona) and its bearing on caecilian ancestryJ Zool 199 545-554

Promislow D E L (1987) Courtship behavior of a plethodontid salamanderDesmognathus aeneus J Herpetol 21 298-306

Schwenk K and Wake D B (1993) Prey processing in Leurognathusmarmoratus and the evolution of form and function in desmognathinesalamanders (Plethodontidae) Biol J Linn Soc 49 141-162

Soler E I (1950) On the status of the family Desmognathidae (AmphibiaCaudata) Univ Kansas Sci Bull 33 459-480

Sommerville D M L Y (1939) Analytical Geometry of Three DimensionsCambridge Cambridge University Press

Staub N L (1993) Intraspecific agonistic behavior of the salamander Aneidesflavipunctatus (Amphibia Plethodontidae) with comparisons to other plethodontidspecies Herpetologica 271-282

Stebbins R C (1985) A Field Guide toWestern Reptiles and Amphibians BostonMA Houghton Mifflin

Summers A P and Wake M H (2005) The retro-articular process streptostylyand the caecilian jaw closing system Zoology 108 307

Wake D B (1966) Comparative osteology and evolution of the lunglesssalamanders family Plethodontidae Mem South Calif Acad Sci 4 1-111

Wake D B and Deban S M (2000) Terrestrial feeding in salamanders InFeeding form Function and Evolution in Tetrapod Vertebrates (ed K Schwenk)pp 95-116 San Diego CA Academic Press

Worthington R D and Wake D B (1972) Patterns of regional variation in thevertebral column of terrestrial salamanders J Morphol 137 257-277

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