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Bite force, cranial morphometrics and size

in the fishing bat Myotis vivesi (Chiroptera: Vespertilionidae)

Sandra M. Ospina-Garcés

1,2

, Efraín De Luna

L. Gerardo Herrera M.

(https://orcid.org/0000-0001-9348-5040), Joaquín Arroyo-Cabrales

& José Juan Flores-Martínez

1. Posgrado en Ciencias Biológicas, Instituto de Biología, Universidad Nacional Autónoma de México, Apartado Postal

70-153, 04510 México, Distrito Federal; ospinagarcess@gmail.com https://orcid.org/0000-0002-0950-4390

2. Instituto de Ecología, A.C. Carretera Antigua a Coatepec 351, El Haya, Xalapa, 91070, México,

ospinagarcess@gmail.com; https://orcid.org/0000-0002-0950-4390

3. Instituto de Ecología A.C. Biodiversidad y Sistemática, Xalapa, Veracruz, 91070; efrain.deluna@inecol.mx, https://

orcid.org/0000-0002-6198-3501

4. Estación de Biología de Chamela, Instituto de Biología, Universidad Nacional Autónoma de México, Apartado Postal

21, San Patricio, Jalisco, 48980, México; gherrera@ib.unam.mx

5. Laboratorio de Arqueozoología, ‘M. en C. Ticul Álvarez Solórzano’ INAH, Moneda # 16 Col. Centro, 06060 México,

Distrito Federal; arromatu5@yahoo.com.mx

6. Laboratorio de Sistemas de Información Geográfica, Departamento de Zoología. Instituto de Biología, Universidad

Nacional Autónoma de México, Circuito Exterior, Edificio Nuevo, Módulo C, Apartado Postal 70-153, 04510

México, Distrito Federal; jj@ib.unam.mx

* Correspondence

Received 06-IV-2018. Corrected 31-VII-2018. Accepted 18-IX-2018.

Abstract: Fish-eating in bats evolved independently in Myotis vivesi (Vespertillionidae) and Noctilio leporinus

(Noctilionidae). We compared cranial morphological characters and bite force between these species to test

the existence of evolutionary parallelism in piscivory. We collected cranial distances of M. vivesi, two related

insectivorous bats (M. velifer and M. keaysi), two facultatively piscivorous bats (M. daubentonii and M. capac-

cinii), and N. leporinus. We analyzed morphometric data applying multivariate methods to test for differences

among the six species. We also measured bite force in M. vivesi and evaluated if this value was well predicted

by its cranial size. Both piscivorous species were morphologically different from the facultatively piscivorous

and insectivorous species, and skull size had a significant contribution to this difference. However, we did not

find morphological and functional similarities that could be interpreted as parallelisms between M. vivesi and N.

leporinus. These two piscivorous species differed significantly in cranial measurements and in bite force. Bite

force measured for M. vivesi was well predicted by skull size. Piscivory in M. vivesi might be associated to the

existence of a vertically displaced temporal muscle and an increase in gape angle that allows a moderate bite

force to process food.

Key words: bite force; cranial morphology; Myotis; Noctilio; piscivory; size; gape angle.

The relationship between the structure of

the masticatory apparatus and diet has long

been investigated in bats (Freeman, 1981;

1984; Van Cakenberghe, Herrel, & Aguirre,

2002, Swartz, Freeman, & Stockwell, 2003;

Nogueira, Monteiro, Peracchi, & De Araújo,

2005; Dumont, Herrel, Medellin, Vargas-Con-

treras, & Santana, 2009; Nogueira, Peracchi,

Ospina-Garcés, S.M., De Luna, E.,

Herrera M., L.G., Arroyo-Cabrales, J., & Flores-Martínez,

J.J. (2018). Bite force, cranial morphometrics and size in the fishing bat Myotis vivesi

(Chiroptera: Vespertilionidae). Revista de Biología Tropical, 66(4), 1614-1628.

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Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 66(4): 1614-1628, December 2018

& Monteiro, 2009). Morphological differences

in cranial size are related to differences in diet

and have consequences in the performance of

the masticatory muscles. For example, volume,

length and position of the temporal and mas-

seter muscles vary among bat species from

different dietary groups (Dumont, 1999; Free-

man, 1981; 1984, Nogueira et al., 2005, Swartz

et al., 2003; Van Cakenberghe et al., 2002;

Dumont et al., 2009). Such morphological

variation could be related to size and hardness

of food items because these two features affect

the performance of the masticatory muscles

(Dumont, 1999; Nogueira et al., 2005; Ghazali

& Dzeverin, 2013).

Masticatory performance in bats has been

estimated analyzing bite force in relation to

body size and masticatory muscle morphology.

Body size and bite force are highly correlated,

as well as bite force and mass and length of

fibers of the masticatory muscle (Aguirre,

Herrel, Van Damme, & Matthysen, 2002;

2003; Herrel, De Smet, Aguirre, & Aerts,

2008). Cranial size, muscle masses and the

fiber length of the temporal muscle are the

best predictors of bite force: species with a

larger cranium, a larger temporal muscle mass

and shorter temporal fiber lengths bite harder

(Dumont & Herrel, 2003). In general terms,

the cranial muscular system is a good predic-

tor of bite force (Dumont & Herrel, 2003;

Dumont et al., 2009).

Cranial traits related the performance

of masticatory muscles suggest parallelisms

among some bat species with the same diet. For

example, insectivorous bats have narrow and

elongated faces, and their zygomatic breadth

is about 55-70 % of the condylocanine length.

In contrast, bat species that feed on small ver-

tebrates (e.g., Noctilio leporinus, Cheiromeles

torquatus, Scotophilus gigas and Saccolaimus

peli) have wide and short faces, with a zygo-

matic breadth about 70-80 % of the condyloca-

nine length (Freeman, 1981). Cranial size and

the change to vertical orientation of the tempo-

ral muscle might allow wider gapes facilitat-

ing prey processing of vertebrate-eating bats

(Emerson & Radinsky, 1980; Van Cakenberghe

et al., 2002). Other cranial traits have been

associated with carnivory, not only in bats but

also in saber-tooth cats (Emerson & Radinsky,

1980). A laterally-flaring angular processes,

a low coronoid process, a vertically oriented

temporal fossa, and a high origin/insertion ratio

of the temporal muscle suggest wide gapes and

strong bites in carnivorous mammals (Emer-

son & Radinsky, 1980; Freeman, 1988). The

piscivorous bat N. leporinus (Linnaeus, 1758)

presents these features (Freeman, 1981; Her-

ring & Herring, 1974).

Piscivory in bats appeared in parallel in N.

leporinus (Noctilionidae) and some species in

the genus Myotis (Vespertilionidae). M. vivesi

(Menegaux, 1901) is the only truly piscivorous

species (Blood & Clark, 1998), whereas seven

other species are insectivores that occasionally

feed on fish (M. ricketti, M. daubentonii, M.

capaccinii, M. adversus, M. macrotarsus, M.

albescens and M. stalkeri; Aihartza, Almenar,

Goiti, Salsamendi, & Garin, 2008; Flan-

nery, 1995; Law & Urquhart, 2000; Siemers,

Dietz, Nill, & Schnitzler, 2001; Whitaker &

Findley, 1980). These eight species were ini-

tially grouped in the subgenus Leuconoe with

other entomophagus Myotis, due to similar

external characteristics (Findley, 1972). This

subgenus has not been supported by molecular

techniques and is treated at present not as taxon

but as an ecomorph (Ruedi & Mayer, 2001).

Typically, Leuconoe bats prey insects near the

water surface and this can possibly make easier

a transition to piscivory in large-sized Leuco-

noe. Additionally, external morphological traits

are associated with the presence of fish in their

diets including laterally compressed and large

claws to catch prey on the water surface (Free-

man, 1981; Norberg & Rayner, 1987; Lewis-

Oritt, Van Den Bussche, & Baker, 2001).

However, a phylogenetic analysis showed that

the facultatively piscivorous and piscivorous

Myotis species are not a monophyletic group

(Ruedi & Mayer, 2001; Stadelmann, Herre-

ra, Arroyo-Cabrales, Flores-Martinez, May,

Ruedi, 2004). Members of the genus Myotis

represent an excellent model to examine chang-

es in the masticatory apparatus morphology

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associated to the evolution of piscivory from

insectivorous ancestors (Lewis-Oritt et al.,

2001). Geometric morphometric analyses per-

formed recently in piscivorous Myotis species

describe morphological changes in mandibular

masticatory process position related to gape

capacity and diet, and a significant effect of

size in skull shape variation (Ospina-Garcés,

De Luna, Gerardo-Herrera, & Flores-Mar-

tínez, 2016). In the present study we explored

if there are morphological differences in the

masticatory apparatus of M. vivesi with respect

to their facultatively piscivorous (M. capaccinii

and M. daubentonii) and insectivorous rela-

tives (M. keaysi and M.velifer), and the effect

of skull size in this variation. We included

N. leporinus in the comparison to evaluate

if there are parallel trends of variation in the

masticatory apparatus of M. vivesi. In addition,

we compared masticatory performance of M.

vivesi measured as bite force in relation to skull

size with data previously reported for insec-

tivorous and facultatively piscivorous Myotis,

N. leporinus and unrelated insectivorous spe-

cies (Aguirre et al., 2002; Herrel et al., 2008).

We addressed the following questions: 1)

does cranial morphology and performance of

M. vivesi differ compared to their facultative-

ly piscivorous or insectivorous relatives and

are cranial morphotypes in M. vivesi similar

enough to be interpreted as parallelism in the

non-related piscivorous N. leporinus?, and 2)

are the morphological and functional differ-

ences explained by the increase of skull size

in both piscivorous species? We hypothesized

that cranial morphology and performance of

M. vivesi would differ with respect to insec-

tivorous and facultatively piscivorous Myotis,

but present similarities with N. leporinus. We

expected that the position and development of

the masticatory muscles would be more differ-

ent between M. vivesi and insectivorous Myotis

than with respect to the masticatory morpholo-

gy in facultatively piscivorous Myotis. We also

anticipated that bite force in M. vivesi would

be similar to that expected for its size, and that

size-corrected bite force would be similar to

that of N. leporinus.

MATERIALS AND METHODS

Study system: We examined skulls and

jaws from 228 adult bat specimens of two

piscivores (Myotis vivesi: 20 females, 27 males;

Noctilio leporinus: 27 females, 24 males), two

insectivores (M. velifer: 26 females, 27 males;

M. keaysi: 31females, 19 males) and two

facultatively piscivores (M. daubentonii: 12

females, 4 males; M. capaccinii: 5 females, 6

males). Age of specimens was assessed follow-

ing the criteria proposed by Pacheco & Pat-

terson (1992). Specimens (Appendix) belong

to the National Mammal Collection (México),

the Osteological Collection of the National

Institute of Anthropology and History (Méxi-

co), the Doñana Biological Station (Spain),

and the Humboldt University Natural History

Museum (Germany).

Measurements: Inter-landmark distances

were collected using a digital caliper (Mitu-

toyo CD-6´´ Mitutoyo U.S.A.) to the nearest

0.01 mm on the right side of skull and jaw.

We measured nine dimensions on the skull and

five on the jaw (Fig. 1) from each individual:

1. Greatest skull length (GSL), 2. Maximum

zygomatic breadth (MZB), 3. Condylocanine

lenght (CCL), 4. Toothrow lenght (MTR), 5.

Maxillary breadth across M3 (M3B), 6. Tem-

poral origin (Ori T), 7. Temporal insertion (ins

T), 8. Postorbital constriction width (POC), 9.

Masseter origin (Ori M), 10. Masseter insertion

(Ins M), 11. Dentary lenght (DL), 12. Dentary

depthness under the protoconid of m2 (DD),

13. Coronoid process height (CPH), 14. Mas-

seteric fosa length (MFL), 15. Elevation of

condyle angle (ECA), 16. Coronoid process

angle (CPA).

Specific average and standard deviation

were presented in Table 1. Some of these mea-

surements are related to the development of

the masticatory muscle (Emerson & Radinsky

1980; Freeman 1984). Other measurements are

related to the size of the skull, the zygomatic

breadth, and the condyle canine length. Maxil-

lary breadth indicates the length of the skull,

and the dentary depth under the protoconid of

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m2 (DD) reflects the thickening of the dentary.

Because there are differences in skull size

among species considered, measurements were

size-adjusted with a geometric mean. Dis-

tances for each individual were divided by the

geometric mean of all 14 measurements from

the same individual (Dumont, 2004).

Two angles were estimated on digital

photographs of the right side of the mandible

of each individual with the program tpsDIG2

TABLE 1

Cranial morphological distances and angles measured in five Myotis and one Noctilio species

M. cappacinii M. daubentonii M. keaysi M. velifer M. vivesi N. leporinus

(1) GSL 15.33 (0.21) 14.80 (0.32) 13.01 (0.31) 16.33 (0.34) 21.44 (0.41) 26.72 (1.44)

(2) MZB 9.33 (0.19) 8.98 (0.31) 7.96 (0.25) 10.69 (0.26) 14.06 (0.3) 19.27 (0.73)

(3) CCL 13.45 (0.18) 12.89 (0.33) 11.51 (0.41) 14.83 (0.25) 19.57 (0.56) 23.56 (0.65)

(4) MTR 5.66 (0.08) 5.36 (0.12) 4.91 (0.22) 6.59 (0.29) 9.18 (0.24) 10.61 (0.3)

(5) M3B 6.07 (0.12) 5.79 (0.16) 5.16 (0.2) 6.88 (0.15) 8.88 (0.22) 13.02 (0.38)

(6) Ori T 8.78 (0.22) 8.51 (0.22) 7.42 (0.31) 9.16 (0.23) 11.91 (0.36) 20.5 (1.35)

(7) Ins T 5.79 (0.14) 5.42 (0.31) 4.94 (0.22) 6.09 (0.29) 6.99 (0.22) 13.91 (1.28)

(8) POC 3.79 (0.09) 4.13 (0.13) 3.31 (0.13) 3.99 (0.12) 5.56 (0.15) 7.3 (0.27)

(9) Ori M 3.83 (0.14) 3.74 (0.09) 3.4 (0.18) 4.76 (0.17) 5.91 (0.36) 7.86 (0.57)

(10) Ins M 2.23 (0.75) 2.08 (0.08) 1.81 (0.09) 2.50 (0.12) 3.03 (0.14) 4.9 (0.5)

(11) DL 11.22 (0.319) 10.63 (0.27) 9.61 (0.3) 12.92 (0.25) 17.08 (0.4) 19.42 (0.71)

(12) DD 1.13 (0.10) 1.08 (0.05) 1.04 (0.07) 1.46 (0.1) 1.74 (0.08) 3.14 (0.36)

(13) CPH 3.00 (0.13) 3.00 (0.13) 2.69 (0.16) 3.94 (0.14) 5.03 (0.17) 6.69 (0.27)

(14) MFL 2.83 (0.15) 2.73 (0.15) 2.47 (0.15) 3.51 (0.2) 4.32 (0.2) 7.04 (0.6)

(15) ECA 0.91 (0.02) 0.86 (0.05) 0.86 (0.05) 0.86 (0.05) 0.97 (0.06) 1.20 (0.05)

(16) CPA 0.99 (0.06) 1.02 (0.06) 1.06 (0.04) 1.09 (0.04) 1.15 (0.06) 1.26 (0.04)

* Values are described as mean (± SD). Abbreviations of 14 cranial measurements and two angles are ordered as in Fig. 1.

Fig. 1. Cranial measurements and angles recorded in this study. Numbers designate

14 inter-landmark distances and two angles.

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(Rohlf, 2005). We used the elevation of the

coronoid process and the elevation of the con-

dyle angles to infer the direction of the masse-

ter and temporal muscles. The elevation of the

coronoid process angle (CPA) was measured

between the lines corresponding to the height

of the coronoid process and the dentary line

(Fig. 1). The elevation of the condyle angle

(ECA) was measured between the lines cor-

responding to the coronoid process height and

a line from the condyle to the coronoid process

(Fig. 1). The angles estimate the height of the

coronoid and condylar processes respect to the

dentary and therefore the orientation of the

masticatory muscles (Emerson & Radinsky,

1980). Both angle values were transformed to

radians for further statistical analyses (vari-

ables 15 and 16, Table 1).

We measured bite force using a piezo

resistive force sensor (Flexiforce A 201-100,

Tekscan, Inc., Boston), with a sensitivity range

of 0-100 pounds. This device consists of a

plate that works like a load cell in an electrical

circuit; it has a resistance that changes with the

force applied in a sensible area at the end of the

plate where the lowest electrical resistance cor-

responds to the maximum force. We obtained

the resistance of the cell with an industrial

multi-meter (Techmaster D-8500, Techmaster

de México S.A., Tijuana) with a sensibility of

400 MΩ. This instrument requires a pressure

of at least three seconds to show any recording.

The force applied is calculated by a regression

using resistance values from the application of

known weights.

Bite force was estimated from 15 adults of

M. vivesi (3 males and 12 females; body mass:

30.85 ± 4.8 g, mean ± SD). Specimens were

caught in Partida Norte Island, México (28°

53´ 30” N - 113° 02´ 25” W) and weighted

with a PESOLA precision scale (100 g maxi-

mum capacity, MICROLINE, Switzerland).

Bite force measurements were made in situ

between 09:00 am and 1:00 pm. The specimens

were placed in front of the sensor so that the

mouth occupied the first third of the sensitive

area because the plate is larger than the mouth.

This procedure was performed three times

for each individual and low-pressure readings

were discarded. We used the lowest resis-

tance measurements recorded that correspond

to the highest bite force (Freeman & Lemen,

2008). After the measurements, individuals

were released at the capture site. Procedures

followed guidelines approved by the American

Society of Mammalogists (Sikes & Gannon,

2011) and were collected under permission by

the Secretaría de Medio Ambiente y Recursos

Naturales (SEMARNAT).

Statistical analysis: Data for analyses

consisted in fourteen variables measured on

the skull and two angles (CPA: Coronoid

Process angle and ECA: Elevation of Condyle

angle) expressed as radians (Table 1). Statisti-

cal analyses were structured to compare six a

pRiori groups (six species). Data without trans-

formation and size-adjusted variables were

tested for normality within each species with

the Shapiro-Wilk test. Since differences in size

between sexes could hide differences between

species, we evaluated sexual dimorphism. We

performed a two-way analysis of variance

(ANOVA) to test differences between species

and sexes in the greatest skull length (GSL).

Post-hoc comparisons between sexes within

species were performed to identify species with

significant dimorphism.

Multivariate differences in cranial mor-

phology among six species were first examined

with a Canonical Variate Analysis (CVA) based

on variances of 14 measurements on skulls and

jaws and the two angles. These sixteen vari-

ables were transformed to base 10 logarithms.

A second CVA analysis with fifteen size-

adjusted variables, with GSL removed from

the data matrix, was performed to determine

the contribution of variables to discriminate

groups when the size effect is reduced. Size

contribution to the contrast between species

with different body mass (Freeman, 1981) was

examined by the comparison between resulting

CVA analyses and calculating the correlation

between the GLS and the scores of the first CV

from the size-adjusted CVA.

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Bite force in M. vivesi was inferred from

a linear regression between known weights of

steel nuts (N = 8), transformed in force (N), and

resistance (MΩ). We measured the resistance

of the different steel pieces added one at time

to increase the force measured in Newtons and

therefore the resistance of the sensor, and we

performed the bivariate regression between

force of each group of steel pieces and its resis-

tance (Fig. 2). Resistances of maximum bites

measured in the 15 individuals of M. vivesi

were replaced in equation of this regression

to obtain the bite force in Newtons. Bite force

mean values previously reported for 10 insec-

tivorous bat species, two facultatively piscivo-

rous Myotis and the piscivorous N. leporinus

(Aguirre et al., 2003, Freeman & Lemen, 2010;

Krüger, Clare, Greif, Siemers, Symondson, &

Sommer, 2014) were used to conduct a linear

regression analysis with the estimator of skull

size (GLS). We performed the Blomberg’s K

phylogenetic signal test on the residuals of this

linear model following Revell (2010), using the

super tree of bats (Jones, Purvis, Maclarnon,

Bininda-Emonds, & Simmons, 2002) with all

branches scaled to 1. We conducted this test

with the library Phytools (Revell, 2012) for

the statistical program R 3.3.3 (R Core Team,

2017). We examined if bite force estimated for

M. vivesi fitted the 95 % confidence interval of

the regression to determine if it corresponded

to the value predicted by its GLS. All multi-

variate analyses were performed in the program

STATISTICA 10 (StatSoft Inc, 2011) using a

significance level of α = 0.05.

RESULTS

Morphological variation: Among sixteen

variables, only CCL and the CPA were not nor-

mally distributed within the six species of bats

(W < 0.941, P < 0.05). The sixteen variables

transformed to logarithms were normally dis-

tributed (W > 0.861, P > 0.05). Size-adjusted

variables were normally distributed within the

six species of bats (W > 0.899, P > 0.05). There

were significant differences in GLS among

species (F

5, 216

= 3 111, P < 0.001), but not sig-

nificant differences between sexes (F

1, 216

= 0,

P = 0.803) or the interaction between sexes and

species (F

5, 216

= 0.5, P = 0.576). The results

of post-hoc comparisons showed significant

Fig. 2. Estimations of the bite force from the resistance measurements. Points correspond to known control weights of steel

nuts (N= 8) applied on the plate of the piezo resistive force sensor. The resistances recorded (MΩ) for Myotis vivesi were

transformed to bite force values (N) using this linear regression.

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differences between most pairs of species (P <

0.001), except for the comparison between M.

capaccinii and M. daubentonii, and with M.

velifer (P > 0.05).

The first CVA from sixteen variables

(unadjusted for size) estimated five significant

canonical vectors (P < 0.0001) for 6 a pRiori

groups. The first canonical function accounted

for 86 % of variance (Wilks’ λ < 0.0001; χ

= 2 508.21, P < 0.0001). The second canonical

variable accounted for an additional 12 % of

the variance (Wilks’ λ = 0.002; χ

= 1 340.53,

p <0.0001). The first two canonical axes

indicate differences between species groups

Fig. 3. Morphological variation of the masticatory apparatus in the fishing bat Myotis vivesi compared to insectivorous (I),

facultatively piscivorous (FP), and piscivorous (P) bat species. A. Plot of first two significant axes of the CVA analysis from

14 measurements and two angles on skulls and jaws for six species (N= 228). B. Plot of first two significant axes of the CVA

analysis from 13 size corrected measurements (GLS excluded) and two angles on skulls and jaws for six species (N= 228).

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(Fig. 3A). The contributions of variables to

the first two canonical axes are presented in

Fig. 4A. The species with insectivorous diets

(M. keaysi and M. velifer) together with the

facultatively piscivorous species (M. capac-

cinii and M daubentonii) appeared on the right-

hand side on axis 1, and the piscivorous species

N. leporinus is in the extreme left-hand side.

Along the first canonical variate, N. leporinus

had the most contrasting morphology of the six

species examined. Therefore, the comparison

of this species against all others forms the basis

of the first discriminant function. The first two

canonical variates revealed that the cranial

Fig. 4. Comparison of variable loadings from two CVA analyses of A. original and B. size corrected measurements.

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morphology of M. vivesi was very different

from the morphology of the two insectivorous

and the two facultatively psicivorous species.

Both canonical variables also reveal that the

masticatory apparatus of M. vivesi was very

different from N. leporinus. Variables that

mostly contribute to the separation of both

species were the cranial distances GLS, M3B,

POC and Ori T, on the first canonical axis. The

GLS, Ins T and DL had the highest contribution

on the second canonical axis, discriminating M.

vivesi from other Myotis species.

In the second CVA analysis (size corrected

variables and GSL excluded), the ordination of

species was different to the first CVA analysis,

with Myotis species at the same position on

CV1 and non-closely related Noctilio placed

apart (Fig. 3B). This analysis determined five

significant canonical vectors (P < 0.0001) for

6 a pRiori groups. The first canonical func-

tion accounted for 79 % of variance (Wilks’

λ = 0.00013; χ

= 1 939.26, P < 0.0001).

The second canonical variable accounted for

an additional 15 % of the variance (Wilks’

λ= 0.0081; χ

=1 041.31, P < 0.0001). The

contributions of variables to the first canonical

axes are presented in Fig. 4B. Abbreviations of

14 cranial measurements and two angles are

ordered as in Fig. 1. The values on the scale

are the correlation coefficients between each

measurement and the first two Canonical axes

from both analyses. As in the first CVA, the

first two canonical axes adequately described

differences between species groups but showed

more overlapping among Myotis species along

the first axis (Fig. 3B). This size adjusted CVA

ordination also reveals that cranial morphology

of M. vivesi differs from N. leporinus. The con-

tribution of variables was more uniformly dis-

tributed, however the DD and the MFL had the

highest contribution on the first canonical axis

(Fig. 4B). On the second canonical axis, we

observe contrasting shape differences among

M. daubentonii and M. capaccini with M. vivesi

also explained by differences in DD, MFL and

the Ori M. The scores of the size-adjusted CV1

remain highly correlated to GLS (R = 0.81, F

226

= 446.7, P < 0.001).

Bite force and diet: Values from the

Blomberg’s K test (K = 0.324, P = 0.938) reveal

there is no phylogenetic signal in the residuals

of the linear regression between bite force and

skull size (GSL). The original values of bite

force and GLS were highly correlated among

species with different diets (R = 0.839, F

1, 11

26.29, P < 0.001, Fig. 5). Bite force measured

in M. vivesi (10.37 ± 5.39 N) was positioned

within the 95 % confidence interval of the

regression. Size-corrected bite force of M.

vivesi (0.48 ± 0.25 N/mm GSL) was lower than

in N. leporinus (0.68 ± 0.26 N/mm GSL).

DISCUSSION

Our comparison of Myotis species sepa-

rated insectivorous, facultatively piscivorous,

and piscivorous bats based in original and size-

adjusted variables of their skull morphology.

As expected, cranial morphology in M. vivesi

diverged from its insectivorous (M. keaysi

and M. velifer) and facultatively piscivorous

relatives (M. capaccinii and M. daubentonii).

Differences in the two CVA analyses indicate

significant contribution of skull size to the

discrimination among species in the first CVA

and more uniformly distributed variable loads

in the second CVA, where similarities among

Myotis species are most evident. There were

also differences in the two CVA analyses in

the pattern of variable correlations. The con-

tributions to discrimination axes were more

dispersed among variables in the second CVA

than in the first CVA. In the analysis of vari-

ance from variables not adjusted for size, the

measurements that contributed most to the

discrimination of M. vivesi on the first two

canonical axes were the greater length of the

skull, the postorbital constriction, the maxil-

lary breadth across M3, the origin of the mas-

ticatory muscles (Ori T and Ori M), and the

elevation of the condyle angle. Most of these

traits describe the masticatory muscle position

suggesting differences among bats with differ-

ent diets in the performance of the masticatory

muscle (Nogueira et al., 2005), as in the case

of facultatively piscivorous and insectivorous

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Myotis. However, the ordination of species is

congruent with size differences with largest

species on the extreme of the first two canoni-

cal axes (N. leporinus in CV1 and M. vivesi in

CV2) and the smallest species M. keaysi in the

opposite direction.

The size adjusted CVA revealed different

trends of variation. On the first axis, differenc-

es between the non-closely related piscivorous

Noctilio and Myotis species were explained by

the contribution of mandibular distances (DD

and MFL), this could be related to the thickest

mandible in Noctilio. On the second canonical

axis, we observe contrasting shape differences

between facultative piscivorous species (M.

daubentonii and M. capaccinii) and M. vivesi,

with N. leporinus in the middle of this axis,

also explained by differences in mandibular

distances (DD, MFL) and the origin of the

masseter muscle. These trends in variation also

hint to the different values of the origin/inser-

tion ratio for the masseter muscle between M.

vivesi (1.95) and the two facultative piscivo-

rous species (< 1.79). Observed morphometric

differences in the CVA adjusted for size sup-

port the idea that masseter muscle performance

could be fundamental in diet differences inside

Myotis (Ospina-Garcés et al., 2016).

Within Myotis, the two CVA ordinations

suggest three different groups, each one includ-

ing species with more similar morphometric

distances and associated with three diets (Fig.

3). The length of the origin and insertion of

the temporal muscle (OriT and InsT) were

greater in M. vivesi than in insectivorous and

facultatively piscivorous species. These two

measurements reflect differences in the size

and development of temporal muscle fibers

(Nogueira et al., 2005). Larger temporal fibers

are related to an increase in body size and

bite force, due to a positive relationship with

muscle mass (Herrel et al., 2008; Nogueira et

al., 2005, 2009). Also, the elevation of condyle

angle in M. vivesi (52-62º) indicates a func-

tional change in the masticatory muscles posi-

tion with respect to the insectivorous species

(43-53º) and the facultatively piscivorous spe-

cies (46-53º). These differences suggest a more

Fig. 5. Relationship between bite force and skull size (GSL) in bats with piscivorous and insectivorous feeding habits.

Bite force for Myotis vivesi was well predicted by its GLS as it fell within the 95 % confidence interval (dashed lines) of

the regression.

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vertically displaced orientation of the temporal

muscle in M. vivesi than in other insectivorous

and facultatively piscivorous species of Myotis.

Cranial morphotypes in M. vivesi and

the non-related piscivorous N. leporinus were

different in size and cannot be interpreted as

parallelism. However a trend of increase size

seems to be affecting morphological descrip-

tors of masticatory muscles. In the first CVA

analysis (Fig. 3A), the measurements that

contributed most to the difference between M.

vivesi and N. leporinus were the origin (Ori T)

and the maxillary breadth across M3 (M3B).

The length and height of the temporal muscle

were smaller in M. vivesi than in N. leporinus

(Table 1), but higher than in the other Myotis.

These two variables had a size component and

indicate the development of the masseter mus-

cle in these piscivorous species being gradual

to the insectivorous Myotis to piscivoros Myo-

tis and Noctilio. Our results from the second

CVA analysis with size-corrected variables and

GLS excluded still suggest that most differ-

ences in cranial morphology between M. vivesi

and N. leporinus are due to their differences in

cranial proportions (Fig. 3B). In this analysis,

the strong correlation between skull size (GLS)

and the scores of the first CV indicate that size

effect was reduced but not completely eliminat-

ed, as, it has been found for diverse methods to

correct size effects on traditional morphometric

variables (Adams, Rohlf, & Slice, 2004).

Differences between the two piscivorous

species can be further explained probably in

function of phylogenetic distance, because

they belong to unrelated families with differ-

ent geographic origins (Jones et al., 2002), and

this can be explored using additional charac-

ters and other methodological approaches to

study shape and including lineages represent-

ing size variation between these two genera

and families.

Skull size (GLS) and bite force were sig-

nificantly related in bat species with different

diets as previously reported (Aguirre et al.,

2002). Bite force measured for M. vivesi fitted

well the predictions for its size. Size-corrected

bite force in M. vivesi was lower than that

measured in N. leporinus, suggesting differenc-

es in the performance of the masticatory appa-

ratus of these piscivores. Size-corrected bite

force in M. vivesi (0.48 N/mm GSL) was higher

than in insectivorous and facultatively piscivo-

rous Myotis (0.16-0.2 N/mm GSL; Aguirre et

al., 2003), which suggests that piscivory in this

genus required stronger bites. However, size-

corrected bite force appears to increase with

size in bats (Fig. 5), and its value in M. vivesi

is similar to that of unrelated insectivorous bats

of similar size, such as N. albiventris (0.55 N/

mm GSL; Aguirre et al., 2003) and Molossus

rufus (0.41 N/mm GSL; Aguirre et al. 2003).

Therefore, the importance of bite force for the

adoption of piscivorous habits in Myotis bats is

unclear and warrants further tests that include

congeners similar to M. vivesi size. However,

the revealed differences in skull size, shape,

and bite force among facultative piscivorous

and piscivorous Myotis species can possibly be

adaptations for prey on vaRious fish species.

Expected morphological similarities in

gape due to parallelism in piscivorous species

are not evident in M. vivesi and N. leporinus. A

difference in the elevation condyle angle seems

to have functional implications in the piscivo-

rous diet. A high gape angle is possible when

the position of the temporal muscle allows a

higher moment around the temporo-mandibu-

lar joint (TMJ), as it has been shown in mam-

mals with hard diets (Herring & Herring, 1974;

Reduker, 1983; Santana, Dumont, & Davis,

2010). This pattern is documented in N. lepori-

nus, which displays a vertical temporal muscle

and a high gape angle related to the capture of

prey on water surface (Freeman, 1988). Myotis

vivesi had a lower elevation of the condyle

angle than N. leporinus (64 to 74º). This angle

indicates the orientation of the temporal muscle

(Emerson & Radinsky, 1980; Freeman, 1988;

Van Cakenberghe et al., 2002; Dumont et al.,

2009), and determines the action line of the

force (Nogueira et al., 2005; Radinsky, 1982).

A vertical orientation of the temporal muscle

of N. leporinus is associated with a greater

gape and lower effort required to process food

(Freeman, 1988). The angles measured for M.

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vivesi were intermediate between N. leporinus

and the group of insectivorous and faculta-

tively piscivorous Myotis species. However,

bite force might have a synergistic effect with

the gape angle in M. vivesi. The orientation of

the temporal muscle in M. vivesi is close to

being vertical, which may explain its capacity

to generate just enough bite force in function of

its skull size to process fish but at a lower gape

angle than N. leporinus.

Our morphometric analyses did not find

cranial morphological similarities as expected

between the piscivorous M. vivesi and N. lepo-

rinus. The two piscivorous species differed in

size and particularly in the angles and propor-

tions of the masticatory apparatus; this suggests

that there is no parallelism associated to fish-

eating in the measured variables. Bite force in

M. vivesi is explained by a moderate increase of

skull size. M. vivesi had a more robust cranial

morphology than its facultatively piscivorous

and insectivorous congeners, but not as large

as in N. leporinus. The increase in cranial size,

the existence of a vertically displaced temporal

muscle, and a greater gape angle that allows

a moderate bite force, appear to be associated

with the appearance of piscivory in M. vivesi.

Ethical statement: authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we

followed all pertinent ethical and legal proce-

dures and requirements. A signed document has

been filed in the journal archives.

ACKNOWLEDGMENTS

This study was funded by a research grant

from Dirección General de Asuntos del Per-

sonal Académico (DGAPA, UNAM), and sab-

batical grants (CONACyT #146770, DGAPA

#1404) to LGHM (#IN201108). A graduate

student fellowship by the Consejo Nacional

de Ciencia y Tecnología (CONACyT, México)

supported SMOG. Transportation to Partida

Norte island was generously provided by Sec-

retaría de Marina-Armada de México. The

Prescott College Kino Bay Center provided

invaluable logistic support during fieldwork.

Samples were collected with permission from

the Secretaría de Medio Ambiente y Recursos

Naturales and CONANP (México).

RESUMEN

Fuerza de mordida, morfología craneal y tamaño

del murciélago pescador Myotis vivesi (Chiroptera: Ves-

pertilionidae). La alimentación por peces en murciélagos

evolucionó independientemente en Myotis vivesi (Vesper-

tilionidae) y Noctilio leporinus (Phyllostomidae). En este

estudio se compararon características craneales morfológi-

cas y fuerza de mordida entre estas especies, para probar la

existencia de paralelismo evolucionario en piscivoría. Se

recolectaron distancias craneales en M. vivesi, dos parien-

tes insectívoros (M. velifer y M. keaysi), dos murciélagos

piscívoros facultativos (M. daubentonii y M. capaccinii), y

N. leporinus. Se analizaron datos morfométricos aplicando

múltiples métodos para probar las diferencias entre las seis

especies. Se midió la fuerza de mordida en M. vivesi y se

evalúo si puede ser predicha por el tamaño del cráneo. Las

especies piscívoras fueron morfológicamente diferentes

de las facultativamente piscívoras y las insectívoras, el

tamaño del cráneo tuvo una contribución significativa en

esta diferencia. Sin embargo, no encontramos semejanzas

morfológicas y funcionales que puedan ser interpretadas

como paralelismos entre M. vivesi y N. leporinus. Estas dos

especies piscívoras difieren significativamente en medidas

craneales y fuerza de mordida. La fuerza de mordida en

M. vivesi fue efectivamente predicha por el tamaño de

cráneo. La piscivoría en M. vivesi puede estar asociada

con la existencia de un músculo temporal verticalmente

desplazado y el incremento en el ángulo de apertura man-

dibular que permite moderar la fuerza de mordida para

procesar el alimento.

Palabras clave: fuerza de mordida; morfología cra-

neal; Myotis; Noctilio; piscivoría; tamaño; ángulo de

apertura mandibular.

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APPENDIX

Specimens examined. Specimen catalog numbers are listed according

to country and locality or province

Museum acronyms are as follows: CNM= National Mammal Collection, Universidad Nacional

Autónoma de México (México). EBD= the Doñana Biological Station, Sevilla (Spain). INAH=

Osteological Collection, Instituto Nacional de Antropología e Historia (México). IPN= Mammal

collection, Instituto Politécnico Nacional, México. ZMB= Humboldt University Natural History

Museum, Berlin (Germany).

M. capaccinii- EBD: Taforalt, Morocco: 15528, 15533, 15594, 15615. ZMB: 67025, 67023,

67022, 85321, 15002, 85322, 4049.

M. daubentonii- ZMB: Braunschweig, Germany: 577, 44053, 44050, 67563, 59320, 55170,

55511, 55168. Rostock, Germany: 70805, 70811, 92492, 85854, 85862, 92495, 92494, 93583.

M. keaysi- CNM: Tabasco, México: 6804, 7723, 7724, 7727, 7876, 7877, 7878. Yucatán,

México: 16345, 18510, 18951, 19278, 19279, 19283, 20268, 20938, 20939, 20940, 20942, 20944,

20946, 20947, 20948, 20949, 22726, 24472, 28872, 28868, 28869, 32853, 32854, 32855, 32857,

32858, 34756, 36660, 43536. INAH: 5382, 5206 IPN: 18685, 18697, 18696, 18694, 18869, 18695,

18693, 18691, 18688, 18692, 24194, 18686.

M. velifer- CNM: Morelos, México: 5157, 5158, 5161, 5159, 5167, 7156, 7902, 7903, 8241,

9069, 9450, 9761. Coahuila, México: 13820 13567.Baja California, México: 15571, 15572.

Puebla, México: 16867,18605, 18607, 18609, 18610. Durango, México: 19684, 19685, 19687,

19750, 19996, 19999, 20000, 20001, 28855, 28856, 28857, 28858, 28859, 28860, 28861. INAH:

5700, 5701, 5702. IPN: 7414, 7415, 7417, 2151, 2155, 2157, 2156, 2159, 2160, 2161, 2162,

2164, 2165, 2168.

M. vivesi- CNM: Isla Partida Norte, México: 2193, 7716, 15658, 15660, 15661, 15662,

15663, 15664, 15659, 16803, 16805, 16806, 16807, 23909, 23910, 23912, 23913, 23914, 23915,

30327, 39327, 45675, 45676, 45677, 45679, 45680, 45681, 45682, 41683, 45684, 45685, 45686,

45689, 45690, 45691, 45693, 45696, 45697. IPN: 38764, 38765, 5168, 5183, 5184, 38766, 38767,

38768, 38769.

N. leporinus- CNM: Tonalá, Chiapas, México: 5921, 6208, 6209, 6210, 6211, 6212, 6213,

6214, 6215, 6216, 6217, 6218, 6219, 6220, 6221, 6222, 6490, 6623, 6931. Rio popoyuta, Micho-

acán, México: 17133, 17135, 18118, 19117, 19116, 19119, 19120. Guerrero, México: 34400,

34401, 23701, 27886, 34402, 34403.