RESEARCH ARTICLE

Morphological Variation in Wild Marmosets (Callithrix penicillataand C. geoffroyi) and Their Hybrids

Lisieux Franco Fuzessy • Ita de Oliveira Silva • Joanna Malukiewicz •

Fernanda F. Rodrigues Silva • Marcella do Carmo Ponzio • Vanner Boere •

Rebecca Rogers Ackermann

Received: 19 March 2014 / Accepted: 22 May 2014

� Springer Science+Business Media New York 2014

Abstract Evolutionary theory and observation predict

wider phenotypic variation in hybrids than parental species.

Emergent phenotypic novelty in hybrids may in turn drive

new adaptations or speciation by breaking parental phe-

notypic constraints. Primate hybridization is often docu-

mented through genetic evidence, but knowledge about the

primate hybrid phenotype remains limited due to a small

number of available studies on hybrid primate morphology.

Here, we examine pelage and morphometric variation in

two Brazilian marmoset species (Callithrix penicillata and

C. geoffroyi) and their hybrids. Hybrids were sampled in an

anthropogenic hybrid zone in the municipality of Vicosa,

Minas Gerais state, Brazil. We analyzed hybrid facial and

body pelage color variation, and compared 13

morphometric measures between hybrids and parental

species. Five different hybrid facial morphotypes were

observed, varying from intermediate to parental-like.

Hybrid facial morphotypes were biased towards C. peni-

cillata, suggesting that the pelage of this species may be

dominant to that of C. geoffroyi in this context, and indi-

cating that mate preference, and therefore gene flow/

introgression, may be biased towards C. penicillata within

the hybrid zone. Hybrid morphometric features were on

average intermediate to parental species traits, but trans-

gressive hybrids were also observed, suggesting that mor-

phometric variation for the studied traits is consistent with

Rieseberg’s complementary allele model. Finally, we

observed a decoupling of facial patterning and size/shape

in hybrids, relative to parent phenotypes, suggesting that an

important factor driving phenotypic novelty within the

Vicosa marmoset hybrid zone might be the loosening of

evolutionary constraints on phenotypic trait integration.

Keywords Hybridization � Pelage color � Transgressive

segregation � Heterosis � Phenotypic integration

Introduction

The importance of hybridization, i.e. interbreeding between

individuals from genetically differentiated lineages (typi-

cally at or above the rank of subspecies) that produces

viable offspring (Baack and Rieseberg 2007), is increas-

ingly being recognized in animal evolution. Theoretical

expectations regarding phenotypic diversity within hybrid

populations predict a complex range of phenotypes that can

include individuals that resemble either parental population

(cryptic hybrids), those that are morphologically interme-

diate to parental populations, as well as individuals that

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11692-014-9284-5) contains supplementarymaterial, which is available to authorized users.

L. F. Fuzessy

Department of Plant Biology, Universidade Federal de Minas

Gerais, Belo Horizonte, MG, Brazil

I. O. Silva � F. F. R. Silva � M. C. Ponzio

Department of Animal Biology, Universidade Federal de Vicosa,

Vicosa, MG, Brazil

J. Malukiewicz

School of Life Sciences, Arizona State University,

Tempe, AZ, USA

V. Boere

Department of Biochemistry and Molecular Biology,

Universidade Federal de Vicosa, Vicosa, MG, Brazil

R. R. Ackermann (&)

Department of Archaeology, University of Cape Town,

Rondebosch 7701, South Africa

e-mail: dr_becky@email.com

123

Evol Biol

DOI 10.1007/s11692-014-9284-5

display transgressive segregation (Ackermann 2010). This

latter phenomenon is characterized by extreme phenotypes

that fall outside of the range of parental variation (Bell and

Travis 2005; Ackermann 2010). Thus, hybrid populations

are expected to show an overall increase in phenotypic

variation in relation to pure parental populations. Although

the underlying genetic architecture responsible for this

increased phenotypic variation is incompletely understood,

it is a predictable result of allelic differences between

parental taxa, as well as genome-wide dominance and

epistatic effects (see discussion in Ackermann 2010).

Of the possible phenotypic effects of hybridization,

transgressive segregation is perhaps the most visible and

dramatic. Transgressive traits in hybrids are primarily due to

the merger of parental genomes comprised of complemen-

tary genes with additive effects (Rieseberg et al. 1999, 2003).

Transgressive segregation is common (even ubiquitous) in

hybridizing plants, but less common in animals; nonethe-

less, work by Rieseberg et al. (1999) indicated that 78 %

(45/58) of examined animal studies report transgressive

segregation, while approximately one-third of the traits

examined (200/650 animal traits) are transgressive.

Empirical studies show that transgression occurs in a wide

range of hybridizing animal taxa, including sculpins

(Czypionka et al. 2012), African cichlids (Parsons et al.

2011), land snails (Chiba 2005), bats (Larsen et al. 2010),

salamander (Dittrich-Reed and Fitzpatrick 2013), dolphins

(Amaral et al. 2014), and copepods (Pritchard et al. 2012).

This type of research supports the hypothesis that trans-

gressive segregation drives the appearance of phenotypic

novelty in hybridizing animal populations (Dittrich-Reed

and Fitzpatrick 2013), which in turn may fuel new adap-

tations and hybrid speciation (Seehausen 2004; Rieseberg

et al. 2003).

In primates, hybridization is fairly common, estimated

to occur among 7–10 % of primate species (Cortes-Ortiz

et al. 2007). While behavioral and molecular studies have

substantially informed our understanding of primate

hybridization (e.g., Nagel 1973; Phillips-Conroy and Jolly

1981; Samuels and Altmann 1986; Detwiler et al. 2005;

Bonhomme et al. 2009; Zinner et al. 2009), the number of

studies on the primate hybrid phenotype remains smaller

by comparison. Studies of morphological signatures of

primate hybridization have examined both metric traits and

non-metric traits (e.g., Kohn et al. 2001; Ackermann et al.

2006; Bicca-Marques et al. 2008; Ackermann and Bishop

2010). In general, the empirical findings of these studies

agree with theoretical expectations for hybrid phenotypic

diversity at both the individual and population levels.

However, studies of metric morphological traits in wild

hybrid populations, exemplified by the work of Froelich

and Supriatna (1996) in macaques and Kelaita and Cortes-

Ortiz (2013) in howlers, remain underrepresented in the

general primate hybridization literature. Expanding such

work to other primate taxa enables us to further test theo-

retical expectations of the hybrid primate phenotype within

the ‘‘natural laboratory’’ of wild hybrid zones, particularly

as related to metric morphological traits. Additionally, at a

time when rates of hybridization in primates and other

animals are increasing as a result of anthropogenic activi-

ties (e.g., Allendorf et al. 2001; Detwiler et al. 2005), it is

imperative that we continue to widen our understanding of

variation in wild primate hybrid zones.

The New World genus Callithrix, made up of six species

of eastern Brazilian marmosets (C. jacchus, C. penicillata,

C. aurita, C. flaviceps, C. geoffroyi and C. kuhlii), is one

primate taxon for which reports of recent hybridization

have increased substantially (e.g., Rylands et al. 1988;

Mendes 1997a, b; Passamani et al. 1997; Ruiz-Miranda

et al. 2000; Affonso et al. 2004; Ruiz-Miranda et al. 2006;

personal observations, IOS, JM, and VB). This genus

belongs to the Callitrichidae, a unique family characterized

by rare primate characteristics such as cooperative breed-

ing, twinning, and female reproductive suppression (Digby

et al. 2007). The majority of Callithrix species reside in the

Brazilian Atlantic Forest, a biome highly devastated by

anthropogenic activity and represented by only 11 % of its

original cover (Ribeiro et al. 2009). The range of

C. penicillata also occurs outside of the Brazilian Atlantic

Forest in the Caatinga biome of NE Brazil and the Cerrado

biome of central and central-eastern Brazil (Rylands et al.

1993 and Rylands et al. 2009). The ranges of Callithrix

species are largely allopatric, with some species contact

occurring at species distribution borders, but historical

species ranges are being altered due to habitat destruction

and anthropogenic introductions of marmoset species out-

side of their historical geographical bounds (Rylands et al.

1993, 2009). C. penicillata is one species that is often

found in sympatry with several other Callithrix species due

to such range alterations (Rylands et al. 1993, 2009). While

hybridization does occur at natural contact points between

Callithrix species (e.g., Malukiewicz 2013), the above

anthropogenic factors are creating new areas of marmoset

species sympatry and in turn resulting in recent hybrid-

ization events (e.g., Alonso et al. 1987; Ruiz-Miranda et al.

2000, 2006; Affonso et al. 2004; Pereira 2010; personal

observation, ISO, JM, and VB).

Hershkovitz (1975, 1977) originally described facial

characteristics of C. penicillata 9 Callithrix spp. hybrids

from a limited number of museum specimens, and other

studies have provided similar descriptions for wild hybrid

samples (Passamani et al. 1997; Mendes 1997a, b; Ruiz-

Miranda et al. 2006; Malukiewicz 2013). However, phe-

notypic variation within wild hybrid marmoset populations

beyond these descriptions remains to be assessed. Here, we

contribute to the understanding of the diversity of the

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hybrid phenotype by examining metric and non-metric

variation within the recently diverged and widely hybridiz-

ing Callithrix marmoset genus. Specifically, we assess pel-

age and morphometric variation of pure wild populations of

C. penicillata and C. geoffroyi and a C. penicillata 9

C. geoffroyi hybrid population. Based on theoretical expec-

tations of hybrid phenotypic diversity, our main aims are not

only to test whether significant differences exist between

parental and hybrid marmoset phenotypes, but also to see

whether hybrid phenotypic variation supercedes that of

observed parental phenotypes. Thus, we specifically address

the following questions in our study: (1) Can we observe

significant differences in qualitative pelage and quantitative

morphological trait variation between pure and hybrid

marmoset populations? (2) Relative to parental species

phenotypes, are hybrid phenotypes similar, intermediate, or

outside of the observed parental range?

Materials and Methods

Marmoset Sampling

Our total sample consists of 65 individuals of C. penicillata

(n = 22), C. geoffroyi (n = 14), and their hybrids

(n = 40). Sampled individuals were aged according to

Yamamoto’s (1993) observation of dental characteristics

and genitalia growth; juveniles were considered to be

between 5 and 10 months old and all adult animals were

those above 11 months old. The C. penicillata sample was

made up of only adults: 13 males and 9 females. These

individuals were sampled within the central Brazilian cities

of Brasılia, D.F. and Goiania, Goias state, both located

within the Cerrado biome. The C. geoffroyi sample was

also comprised of only adults, and included six males and

eight females. These individuals were sampled in south-

western Brazil in close proximity, both on the campus of

the Federal University of Espırito Santo (20�1603900S and

40�1801100W) and in an Atlantic Forest fragment at Con-

vento Nossa Senhora da Penha (20�1904500S and

40�1701200W), Vila Velha, Epiritio Santo state. The hybrid

sample was composed of 19 males (15 adults and 4 juve-

niles) and 21 females (14 females and 7 juveniles) from

five wild groups (see Table 1 for group compositions). This

sample derives from a semi-deciduous submontane 75 ha

forest fragment located inside the campus of the Federal

University of Vicosa, southeastern Brazil (20�4502500S and

42�5105400W). General sampling locations of pure and

hybrid populations as well as species ranges of C. peni-

cillata and C. geoffroyi are shown in Fig. 1. Although

sampling localities vary in terms of whether they represent

more forested versus urban contexts, they are all in close

proximity to humans, and should therefore be considered

urban or near-urban environments.

Capture and Anesthesia

Marmosets were captured with a multiple-entrance trap and

anesthetized using ketamine hydrochloride (Vetaset, Fort

Dodge, Kansas, USA) at 10 mg/kg of body weight and

with xylazine hydrochloride (Anasedan, Divisao Vetbrands

Saude Animal, Sao Paulo, Brazil) at 0.5 mg/kg of body,

and weighed immediately after trappings. To avoid

stressing captured groups, no infants and pregnant females

were ever captured. After collection of biological data (see

below), the marmosets were kept in a warm and darkened

room until they recovered from the anesthesia. Afterwards,

the animals were released at the same location where they

were captured. All used protocols and procedures were

reviewed and approved by the Federal University of Vicosa

Ethics Committee in Animal Use (number: 89/2011) and

the Brazilian Institute of Environment and Natural

Resources/Brazilian Environmental Ministry (SISBIO/

ICMBio, protocol number: 28632-1).

Pelage Measurements and Analysis

We used all individuals captured within the Vicosa hybrid

zone to analyze variation in pelage color and patterns in

this population because juveniles had similarly developed

coat color to adults (personal observation, LFF and IOS).

The C. penicillata facial phenotype is defined by buff to

dark brown cheeks, dark temples, the forehead possessing a

well defined white to yellowish blaze (or ‘‘star’’), blackish

periauricular tufts, dark brown or reddish brown to buff

neck and throat area (Hershkovitz 1977). Other C. peni-

cillata chromogenetic fields (distinctively colored areas of

the coat, Hershkovitz 1968, 1977) included dark brown to

buff or black upper surfaces of the hands and feet and black

and grey striated hairs of the back with an orange under-

coat. C. geoffroyi has fuller black ear-tufts than C. peni-

cillata, and is also striking in its facial phenotype through

possession of a fully white facial mask (Hershkovitz 1977).

The throat of C. geoffroyi is also white, the upper surfaces

of the hands and feet are black or dark brown, and back

coloration is similar to that of C. penicillata (Hershkovitz

1977). Both C. penicillata and C. geoffroyi possess a stri-

ated tail (Hershkovitz 1977; Vivo 1991). Hybrid identifi-

cation was based on phenotypic facial traits of C.

penicillata 9 C. geoffroyi hybrids as originally described

and illustrated in Hershkovitz (1975, 1977): the presence of

a conspicuous white spot on the forehead reminiscent of C.

penicillata mixed with a white or light-grey blaze on the

forehead and face reminiscent of C. geoffroyi.

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Hybrid pelage patterns and color were analyzed across

various chromogenetic fields of the face and body (see

Fig. 2) originally identified by above coat color descriptions

by Hershkovitz (1977) and personal observation by LFF and

IOS. Two researchers (LFF and IOS) independently

inspected all chromogenetic fields across all sampled

specimens and visually assigned color to each field. The

presence of any striations within each chromogenetic field

was also noted during pelage inspections. There was a 95 %

level of concordance between inter-observer pelage color

and pattern classifications, and in cases of observer dis-

agreement individuals were rechecked and an inter-obser-

ver agreement was reached.

Morphometric Measurements and Analysis

Morphometric measurements and analyses were carried

out only in fully adult animals. Captured individuals

were measured with a tape measure and calipers (King.

Tools) and weighed while under anesthesia. The primates

were measured according to the methods described by

Nagorsen and Peterson (1980). Metric data are repre-

sented by one measure of body weight (WEIGHT) taken

in grams, and 12 linear distances (in centimeters) as

follows: tail length (TAIL); body length (BODY); ear

length—left (EARL) and right (EARR); intercranial-lat-

eral distance (ILD); fronto-occipital distance (FOD);

wrist-longer claw—left (WLCL) and right (WLCR);

femur length—left (FEMURL) and right (FEMURR);

calcaneus-longer claw—left (CLCL) and right (CLCR).

Raw metric data are presented in Table S1. The trait

mean values, standard deviations, and sample sizes for

C. penicillata, C. geoffroyi and their hybrids are shown

in Table 2. A multivariate analysis of variance (MA-

NOVA) including all 13 traits indicates that these vari-

ables do not differ significantly among the sexes

(p = 0.66; five individuals deleted due to missing data).

Similarly, univariate analysis of variance (ANOVA)

indicates that none of the traits are significantly different

between males and females at the p \ 0.05 level of

significance. Examination of stem-and-leaf plots also

indicates no deviations from normality. Therefore traits

were left uncorrected, and we do not expect there to be

any confounding effects from sexual dimorphism.

To test for significant quantitative differences among

the three taxa (C. penicillata, C. geoffroyi, hybrids), a

MANOVA was performed on all 13 variables. Following

this, each of the 13 measurements was analyzed indi-

vidually using ANOVA to test for differences between

the three groups. For each of the 13 traits, the means of

the purebred groups were then compared to one another

and to the means of each hybrid sample using t tests

(two-tailed) to assess the significance of observed dif-

ferences; separate variance tests for significance were

employed due to differences in sample size (Zimmerman

and Zumbo 2009). Bonferroni adjusted probabilities for

multiple tests are also calculated. A principal compo-

nents analysis (PCA) was also performed on the data in

order to visualize differences among the purebreds and

hybrids. PCA reduces the dimensionality of a data set,

Table 1 Vicosa hybrid group

size and compositionGroup Male Female Group

Juveniles Adults Male Total Juveniles Adults Female Total Total

Belvedere 1 2 5 7 2 4 6 13

Belvedere 2 2 1 3 1 3 4 7

Casa 32 0 3 3 1 4 5 8

Casa 41 0 2 2 1 3 4 6

Casa 50 1 3 4 2 3 5 9

Mean 3.80 4.80 8.60

Fig. 1 Figure shows general sampling locations of hybrid and non-

hybrid marmoset populations. Latitude is represented by the x-axis

and longitude is represented on the y-axis

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producing a smaller number of uncorrelated variables

that nonetheless retain all of the original size and shape

information. The PCA was based on a correlation matrix

due to the differences in variable nature and scale (e.g.,

mass vs linear distances). We chose not to center data

across the purebred and hybrid groups (i.e. use a pooled

within-group correlation matrix), as it is unclear whether

correlation matrices should be expected to differ between

the purebred species and their hybrids. Moreover, we are

interested in illuminating both within and between group

differences.

Results

Pelage Variation

There were no observed intersexual differences in coat

color among the hybrids, and indeed little variation in

chromogenetic fields between hybrid individuals (outside

of the facial region, as discussed below). The upper and

lower portions of the back showed a striated pattern

composed of orange, dark grey, and light grey similar to

what can be observed in the upper and lower back

Fig. 2 Chromogenetic fields

observed in marmoset

individuals across the (a) body

and (b) face. The vertex

represents the part of the head

anterior to the forehead and the

menton region is analogous to

the chin region in humans

Table 2 Sample sizes (n), means, standard deviations (SD), ANOVA probabilities (p), and univariate probabilities of intergroup differences in

morphometric measurements among all three groups and among pairs of taxa

Variable C. penicillata (P) C. geoffroyi (G) Hybrids (H) ANOVA t test pairwise comparison p values

n Mean SD n Mean SD n Mean SD p PG PH GH

WEIGHT 22 327.27 47.12 14 410.86 30.00 29 337.72 33.19 0.00 0.00* 0.38 0.00*

TAIL 21 28.77 1.15 14 33.24 1.65 28 31.05 3.23 0.00 0.00* 0.00* 0.01

BODY 22 18.13 0.90 14 18.90 1.19 29 18.81 1.97 0.13 0.05 0.11 0.85

CLCR 21 5.63 0.31 14 6.08 0.21 28 5.90 0.31 0.00 0.00* 0.00* 0.03

CLCL 22 5.59 0.24 14 6.08 0.23 28 5.93 0.31 0.00 0.00* 0.00* 0.07

FEMURR 22 6.06 0.40 14 6.90 0.20 29 6.76 0.35 0.00 0.00* 0.00* 0.31

FEMURL 22 6.05 0.33 14 6.90 0.21 29 6.84 0.27 0.00 0.00* 0.00* 0.42

EARR 22 2.35 0.16 14 2.55 0.12 29 2.57 0.26 0.00 0.00* 0.00* 0.70

EARL 22 2.29 0.16 14 2.63 0.25 29 2.55 0.27 0.00 0.00* 0.00* 0.25

ILD 22 3.19 0.18 14 3.36 0.23 29 3.16 0.26 0.04 0.02 0.64 0.01

FOD 22 5.97 0.41 14 6.79 0.47 29 6.22 0.56 0.00 0.00* 0.08 0.00*

WLCR 21 3.54 0.34 14 3.86 0.26 28 3.82 0.25 0.01 0.00* 0.00* 0.66

WLCL 22 3.51 0.30 14 3.81 0.24 28 3.83 0.33 0.01 0.00* 0.00* 0.85

P represents C. penicillata, G represents C. geoffroyi, and H represents hybrids. Pairwise comparisons are presented by these letter abbreviations.

Pairwise comparisons that remain significant (at p B 0.05) using a Bonferroni adjusted probability are indicated with an asterisk. Measurements

are in centimeters

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pelage of the parental species. The proximal portions of

the arms and legs were grey with orange basal hair,

while the distal portions were grey. The tail of all

studied individuals displayed a ringed pattern consisting

of black, dark grey, and light grey (see Fig. 3), matching

published descriptions described above for C. geoffroyi

and C. penicillata.

All C. penicillata and C. geoffroyi hybrids possessed a

conspicuous white spot on the forehead (a C. penicillata

characteristic) and different degrees of light grey and white

on the sides of the face, menton region and front half of

vertex. Although hybrids faces were polymorphic with

respect to color patterning, and the range of variation was

continuous, nonetheless they could be generally divided

into five morphotypes (Fig. 4) as follows: (A) side face and

menton region with light color, evident white spot on

forehead, front and back half of the vertex greyish, dark

grey neck, and black tuft; (B) side face and menton region

with light color, evident white spot on forehead, front half

of the vertex with light color, back half of the vertex

greyish, dark grey neck, and black tuft; (C) side face and

menton region with light color, evident white spot on

forehead, front half of the vertex with light color, back half

of the vertex greyish, black neck, and black tuft; (D) all

face with light color with evident white spot on forehead,

dark grey neck, and black tuft; (E) all face with light color

with evident white spot on forehead, black neck, and black

tuft. Pattern A is more like C. penicillata individuals,

whereas patterns D and E are more like C. geoffroyi indi-

viduals. Patterns B and C represent hybrids possessing

intermediate features between the parental species. The

percentage of hybrid individuals that fell into each mor-

photype is as follows: A 35.5 %, B 16.13 %, C 12.9 %, D

19.35 %, E 16.13 %.

Morphometric Traits

Quantitative differences among the purebred taxa and their

hybrids are significant (MANOVA; n = 60; 5 cases

deleted due to missing data; p \ 0.001). Univariate

ANOVA tests (Table 2) indicate significant differences

among the three groups at p \ 0.05 for all but one of the

traits (BODY). Pairwise taxa comparisons conducted with

the t test (Table 2) indicate that the purebred C. penicillata

and C. geoffroyi marmosets are significantly different from

each other for all of the traits; all but two differences

remain significant with a Bonferroni correction. Callithrix

geoffroyi is on average larger than C. penicillata for all

measured metric traits. The hybrids are not significantly

larger than both parental species for any traits. The hybrids

are significantly larger than C. penicillata for nine traits

(TAIL, CLCR, CLCL, FEMURR, FEMURL, EARR,

EARL, WLCR, WLCL); they are not significantly smaller

for any traits. Conversely, the hybrids are significantly

smaller than C. geoffroyi for five traits (WEIGHT, TAIL,

CLCR, ILD, FOD; only WEIGHT and FOD remain sig-

nificant with Bonferroni correction), and are not signifi-

cantly larger for any traits. These results indicate that while

the hybrids are, on average, intermediate to the two

parental taxa, and are similar to both in body length, they

are not strictly intermediate. Instead they show a mosaic

pattern, being more like C. penicillata in traits that measure

body mass and head size, as well as foot length, while

being more like C. geoffroyi in femur and hand length, and

in ear size.

Component loadings for the PCA are presented in

Table 3. Component scores for the individuals are plotted in

Fig. 5 for the first four PCs (representing 72.97 % of the total

variation). These are labeled in different ways to illuminate

differences between taxa (C. geoffroyi, C. penicillata, and

hybrid; Fig. 5a–c), between the different sexes of each taxon

(Fig. 5d–f), and between purebred and hybrid facial color

patterns (Fig. 5g–i). The first component (PC1) has positive

loadings across all variables, indicating that this is an overall

size component, with the relatively larger C. geoffroyi at the

right side of the axis, and the relatively smaller C. penicillata

at the left side. The other components combine positive and

negative values indicating that they portray aspects of shape.

Fig. 3 Body pattern observed on all captured individuals: upper back

and lower back showing a striated pattern colored orange, dark grey

and light grey; arms and legs with orange and grey color in proximal

portion and grey color in distal portion; ringed tail colored black,

dark grey and light grey

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Considering taxon differences first (Fig. 5a–c), results of

the PCA support what was seen in the quantitative tests,

and suggest that on average the hybrids occupy an

intermediate shape space between the two parent species.

This is particularly apparent on PC1. However, these

results also indicate that for the most part the variation seen

Fig. 4 Five different facial

patterns observed across all

captured individuals with

observed percentages of each

pattern

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in the hybrids does not overlap with that of the parental

species (i.e. they are transgressive), and that the overall

magnitude of variation seen in the hybrids exceeds that of

the parental taxa. In particular, there are a few hybrid

individuals who are well outside of the shape-space of the

parental taxa. When considering the sex of the individuals

(Fig. 5d–f), the hybrid individual most extreme in the PC1

versus PC2 plot (bottom right of Fig. 5d) is a female. The

two hybrids most extreme in the PC2 versus PC3 plot

(bottom and top left of Fig. 5e) and in the PC3 versus PC4

plot (furthest left and right) are also female.

By labeling the positions of the individuals on the PCA

plot according to facial pelage categories (Fig. 5g–i) it is

possible to associate size/shape differences with external

appearance. Note that two individuals have morphometric

data but no recorded facial color/pattern data; these are

labeled as unknown (U). Overall, the PCA plots show

individuals possessing facial phenotype A (as well as one

individual with unknown coat color/pattern), whose facial

patterns are similar to that of C. penicillata, as the most

transgressive group in terms of body size/shape. Addi-

tionally, a few individuals with the E facial pattern, which

resembles that of C. geoffroyi, are at or outside the range of

variation of either parental species. Interestingly, all of the

hybrids, with the exception of group C, associate more

closely with C. geoffroyi in terms of body size (Fig. 5g;

PC1) than they do with C. penicillata, despite spanning the

range from C. penicillata-like facial patterning (morpho-

type A) to C. geoffroyi-like facial patterning (morphotype

E). Only morphotype C, the most intermediate of the

hybrid facial patterning groups, falls firmly within the

C. penicillata size range.

Discussion

Origin of the Vicosa Hybrid Zone

The origin of the Vicosa marmoset hybrid zone is unique

compared to that of most other wild primate hybrid zones

that have been described either morphologically or genet-

ically. The hybrid zone is found on a former sugar and

coffee plantation that was abandoned 90 years ago and that

reestablished itself as a secondary growth forest (IOS,

personal observation). While the highly endangered C.

aurita is thought to be the native marmoset species in this

area of Brazil (Rylands et al. 1993, 2009), the species is

currently not found in Vicosa, possibly due to high levels

of anthropogenic disturbance within the Atlantic Forest

biome and resultant declines in native marmoset popula-

tions (Rylands et al. 1993, 2009). Instead, current mar-

moset populations within Vicosa are descended from non-

native species introduced into the area during 1970s as pets

(IOS, personal observation). Other Vicosa marmoset

groups not sampled in this study also show morphological

evidence of C. jacchus ancestry.

It can be inferred that current levels of genotypic and

phenotypic variation in the Vicosa marmosets derive solely

or largely from the founder populations originally intro-

duced into the area, with limited current gene flow from

parental species. This is true at least in the case of

descendants of C. jacchus and C. geoffroyi; ranges of the

parental species are located beyond the limits of Vicosa

(Rylands et al. 1993, 2009), and are therefore geographi-

cally isolated from the Vicosa hybrid zone. The forest

fragment housing the Vicosa hybrid zone does come into

Table 3 Principal component analysis component loadings and the proportion of variance explained by each component

PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 PC9 PC10 PC11 PC12 PC13

WEIGHT 0.55 -0.23 -0.42 0.44 0.02 0.02 -0.49 -0.10 -0.14 -0.07 -0.05 -0.05 0.03

TAIL 0.69 -0.11 -0.10 -0.15 -0.27 -0.48 -0.02 0.40 -0.03 -0.11 0.00 0.03 0.02

BODY 0.27 0.52 -0.31 0.26 -0.59 0.28 0.13 0.03 0.18 0.03 -0.10 0.00 0.00

CLCR 0.70 0.40 0.02 0.27 -0.04 -0.10 0.29 -0.07 -0.40 0.10 0.08 -0.02 -0.01

CLCL 0.82 0.13 0.14 0.00 0.19 -0.24 0.18 -0.26 0.10 -0.21 -0.19 0.03 -0.05

FEMURR 0.74 0.05 -0.43 -0.34 0.17 0.26 -0.04 0.09 0.00 0.02 0.05 -0.05 -0.18

FEMURL 0.76 0.01 -0.31 -0.31 0.29 0.30 0.14 0.05 -0.02 -0.03 -0.02 0.04 0.18

EARR 0.59 -0.44 0.45 -0.28 -0.22 0.12 -0.11 -0.01 -0.13 0.21 -0.20 0.01 -0.01

EARL 0.59 -0.51 0.27 -0.11 -0.39 0.22 0.02 -0.19 0.01 -0.18 0.20 0.01 0.01

ILD 0.15 -0.62 0.21 0.55 0.21 0.26 0.24 0.26 0.04 -0.05 -0.04 -0.04 -0.03

FOD 0.51 -0.57 -0.39 0.12 0.02 -0.31 0.14 -0.15 0.22 0.24 0.07 0.02 0.01

WLCR 0.71 0.39 0.35 0.23 0.16 0.07 -0.21 0.07 0.12 0.07 0.09 0.26 -0.02

WLCL 0.72 0.40 0.42 0.02 0.11 -0.06 -0.12 0.03 0.18 0.06 0.06 -0.27 0.05

% var EXP 39.52 15.21 10.35 7.89 6.62 5.97 4.21 2.99 2.62 1.68 1.21 1.15 0.58

Evol Biol

123

contact with other forest fragments, but presence or

absence of marmoset groups in these outer fragments has

not yet been determined. However, one of us (IOS) has

identified C. penicillata groups as close as 39 and 24 km to

the Vicosa hybrid zone, and Vicosa itself is located on the

cusp of the geographical range of this species, making the

flow of C. penicillata genes into the Vicosa hybrid zone a

remote possibility. Based on this information, the hybrid

individuals studied here most likely represent highly

admixed multigenerational hybrids that may eventually

form a hybrid swarm.

Pelage Variation Within the Vicosa Hybrid Zone

All hybrids in our sample were similar to parental species

in chromogenetic fields of the arms, legs, back, and tail.

Such similarities between hybrid and parental individuals

suggest that underlining genetic differences between

Fig. 5 Bivariate plots of scores for the first four principal compo-

nents/factors labelled and colored to indicate species affiliation only

(a–c), species affiliation plus sex (d–f), and facial color patterns (g–i).The legends for plots a–f indicate species affiliation and sex as

follows: G = C. geoffroyi, P = C. penicillata, H = hybrids,

F = female, M = male. The legends for the three facial color pattern

plots (g–i) indicate color patterning as follows: A–E = hybrid

patterns described in the text, G = a parental C. geoffroyi-like

pattern, P = a parental C. penicillata-like pattern, U = an unknown

(not recorded) pattern

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123

parental species are minor for these pelage traits; as a result

there is no expectation for hybrids to be highly variable.

Coimbra-Filho et al. (2006) do point out that Callithrix

species vary in shades of the undercoat in hairs of the back,

flanks and outer thighs (between yellow, brown, and red

hues). For example, C. kuhlii has an intensely reddish

brown undercoat that seems to be dominant to C. geoffroyi,

as the C. kuhlii phenotype is often transmitted to C. ku-

hlii 9 C. geoffroyi hybrids (Coimbra-Filho et al. 2006).

However, we observed no such dominance effects of coat

coloration in our studied C. geoffroyi 9 C. penicillata

hybrids, suggesting that patterns of dominance in coat color

may be species specific.

In contrast, we identified five distinct C. penicilla-

ta 9 C. geoffroyi facial morphotypes in our hybrids,

ranging from phenotypes that closely resembled parental

species to those that were intermediate between parental

phenotypes. These results support expectations for higher

phenotypic variation among hybrids relative to parental

populations. The largest percentage of hybrid individuals

possessed a C. penicillata-like facial morphotype for both

males and females separately, and as a whole. Given the

high percentage of hybrids possessing a C. penicillata

facial morphotype, the C. penicillata facial phenotype may

be dominant to that of C. geoffroyi. The notable presence of

the C. penicillata forehead star across our entire range of

hybrid facial morphotypes, even those morphotypes that

more closely resembled C. geoffroyi, also supports the idea

of dominance in facial features of the former species over

the latter.

It is noteworthy that the hybrids differ from the parent

species in facial patterning, but not body patterning; this is

undoubtedly a reflection of the divergence in the former

(but not the latter) of the parent species themselves. The

auricular ornaments, patterns, and colorations that charac-

terize marmoset facial morphotypes are among the most

complex facial patterns of New World primates, and may

serve an adaptive function of intraspecific communication

(Santana et al. 2012). Cavalcanti and Langguth (2008)

presented behavioral evidence that head coloration in

marmosets may also be important in recognition of con-

specifics, but whether facial traits play a role in mate rec-

ognition for marmosets is still unclear. We are unable to

conclude from our dataset whether hybridization alters the

communicative role of marmoset facial patterns. However,

the tendency for Vicosa hybrids to display C. penicillata-

like facial patterns suggests that within this population

individuals with such features may be preferred as mates to

individuals possessing a C. geoffroyi-like facial phenotype,

resulting in uni-directional (or directionally biased) gene

flow favoring backcrossing into C. penicillata-like animals.

It is also possible that the isolation of the Vicosa hybrid

zone from native parental populations may limit mate

choice for marmosets within the zone, a condition which

may change any adaptive role that marmoset facial patterns

play in mate recognition. It is also noteworthy that

chemosensory communication is well developed in Calli-

thrix species (Epple et al. 1993), although how chemo-

sensory signals play into conspecific identification has not

been well established. Ultimately, further studies are nee-

ded of marmoset visual and chemical communications to

understand their role in conspecific and mate recognition,

and to extrapolate such findings to hybrids.

Morphometric Variation Within the Vicosa Hybrid

Zone

Analysis of morphological trait variation between parental

C. penicillata and C. geoffroyi shows that the parental forms

are distinct from one another. Hybrids of the two species

showed a mosaic of parental traits in being closer to C.

penicillata for weight and head size but closer to C. geoffroyi

for femur and hand length. Despite this mosaic, hybrids were

on average intermediate between the parental taxa for the

majority of morphological traits. Most notably, we found

evidence of greater morphological variation among indi-

viduals of mixed ancestry than in individuals of pure

ancestry, with a number of hybrids falling outside of the

parental range of shape variation. This general pattern was

upheld when our sample data were examined in terms of

species, sex, and facial coloration phenotypes (discussed

further below). In short, these results also support the

expectation that hybrid populations are more phenotypically

variable than their parental populations (Ackermann 2010).

Heterosis, dysgenesis, and trangressive segregation are

among the main mechanisms used to explain the existence

of phenotypic variation within hybrid populations that

deviates away from that of parental taxa. Different under-

lining genetic architectures are suggested for these three

mechanisms, which have different evolutionary implica-

tions. Taxa whose crosses display dysgenesis, or hybrid

dysfunction, are thought to possess distinct coadapted gene

complexes that break down during hybridization (Falconer

1981; Templeton 1987). Dysgenesis is predicted in the

hybrids between taxa that are more distantly related and

adaptively distinct. One possible morphometric indicator of

dysgenesis in hybrids is trait values below parental trait

averages (sensu Kohn et al. 2001; Ackermann et al. 2006).

Here, hybrid trait averages were not smaller than both

parental trait averages, suggesting that dysgenesis is not

present in our hybrid marmoset sample, and therefore that

the gene complexes of the parental species are similar.

Heterosis, or hybrid vigor, is observed in genetically

isolated taxa with differences in gene frequencies or

dominance deviations and is maximal when different

alleles are fixed between taxa (Kohn et al. 2001). Previous

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123

primate studies showing evidence for heterosis in primate

hybrids were based on early generation hybrid samples

with known pedigrees (e.g., Cheverud et al. 1993; Kohn

et al. 2001; Ackermann et al. 2006), which allowed for

distinction among different kinds of hybrids (e.g., F1 vs

backcross). Heterosis is expected to be strongest in early

generation hybrids that are genetically intermediate

between parental populations (i.e. F1s), due to restoration

of heterozygosity after mating of differentiated lineages.

Thus, heterosis may become diluted in multigenerational

hybrids where genetic contributions from each parental

species may be become biased due to the effects of genetic

drift, selection, and environment. As our sample is most

likely comprised of wild multigenerational hybrids with

unknown pedigrees, we did not test directly for heterosis.

However, trait values within our hybrid population on

average tended to be intermediate between parental taxa

values. These results suggest similar allele frequencies or

dominance deviations between the parental taxa. Testing

for heterosis in a sample of known F1 C. penicillata and C.

geoffroyi hybrids would provide stronger evidence for such

genetic similarities in the two parental species than is

possible with our wild multigenerational hybrid sample.

As the occurrence of heterosis or dysgenesis implies a

certain level of genetic distinctness of parental taxa, it is

not surprising we did not observe either in our sample

given the young age of the Callithrix genus as a whole.

Based on nuclear data, Callithrix separated from its sister

clade of Mico and Cebuella 2.5 million years ago (Perel-

man et al. 2011), and the young age of Callithrix species

probably has not yet allowed for the evolution of either

distinct coadapted gene complexes or significant differ-

ences in allele frequencies between these species. For

example, C. penicillata diverged from its species C. jac-

chus less than a million years ago (Perelman et al. 2011),

and these two species showed overlapping allele size fre-

quencies and no diagnostic loci between the two species

(loci with non-overlapping alleles between the two species)

in a panel of 42 nuclear microsatellite loci (Malukiewicz

2013). Further, mitochondrial phylogenies produced for

Callithrix species often show evidence of incomplete

lineage sorting and unresolved species relationships (e.g.,

Tagliaro et al. 1997; Malukiewicz 2013). In summary,

these genetic and morphometric data suggest that there is

minimal divergence between Callithrix species.

However, while morphometric traits studied in our

sample of C. penicillata 9 C. geoffroyi hybrids were on

average intermediate between those of the parental species,

with no obvious evidence for heterosis or dysgenesis,

nonetheless, we did observe individual transgressive

hybrids. This is a manifestation of the general increase in

variation seen in the hybrids, and suggests that within the

Vicosa hybrid zone morphometric variation for the studied

traits can best be explained by the complementary allele

model proposed by Rieseberg et al. (1999, 2003). In other

words, morphometric traits in parental species are likely to

be influenced by additive alleles of opposing effects, with

non-opposing alleles recombining in the transgressive

hybrids. As a result, transgressive hybrids show extreme

phenotypes relative to parental species in both negative and

positive directions.

Discordance in Facial and Morphometric Features

Among Vicosa Hybrid Marmosets

One intriguing pattern apparent in our results is discor-

dance between parental facial and morphometric traits, as

well as differences in overall levels of variation, among

hybrid phenotypes in the Vicosa zone. For example, the

individuals with the most C. penicillata-like facial mor-

photype (pattern A) were on average more C. geoffroyi-like

in body size, but were also highly variable relative to the

other hybrid facial groups, with a number of individuals

who were strongly transgressive in body shape relative to

the purebred species. Similarly, the hybrid group with the

intermediate facial pattern C is the only hybrid group that

is C. penicillata-sized and shaped, while the facially C.

geoffroyi-like hybrids are fully intermediate between the

two parental species in size. It has been suggested and

evidenced that hybridization may release evolutionary

constraints on phenotypic integration, affecting not only

variation in traits but also the relationship between traits

(e.g., head shape in African cichlids, Parsons et al. 2011;

modularity in mouse mandible, Renaud et al. 2012). These

cichlid and mouse data show that release from evolutionary

constraints of phenotypic traits can occur in the F2 gen-

eration and then continue in later hybrid generations. Given

the mixture of parental traits seen in our hybrid sample, it is

plausible that a similar process has also occurred here. If

non-native marmosets were originally introduced to this

area in the 1970s, and assuming approximately 1.5 years

per marmoset generation (Tardif et al. 2003), we would

expect that marmosets currently living in the area are

members of approximately the 25th–30th generation of

marmoset hybrids within Vicosa. From that time, different

evolutionary, heritable, and environmental factors could

have shaped the hybrid phenotypes in the Vicosa zone,

releasing evolutionary constraints and leading to the

emergence of new phenotypic combinations. The produc-

tion of new forms, or new combinations of traits, is one

important result of hybridization (Arnold 2006, 2008). We

can also speculate that the apparent dominance of C.

penicillata facial features over C. geoffroyi facial features

in hybrids, despite an overall tendency for hybrids to be

more C. geoffroyi-like in body size/shape, suggests that C.

penicillata communication might have played a prominent

Evol Biol

123

role in decoupling these phenotypic traits. However, whe-

ther these new combinations are associated with adaptive

success of the overall phenotypes or simply due to genetic

drift on genes acting on these traits remains to be tested.

Further research is needed to better understand Callithrix

trait integration and relaxation of trait associations under

hybridization.

Conclusion

The results presented here have shown that hybrids of C.

penicillata and C. geoffroyi differ from parental taxa, as

well as from each other, in terms of body size/shape and

facial patterning. We also provided some evidence for the

breakdown of association between size/shape and color

patterning in these hybrids. However, without additional

genetic and behavioral data it is difficult to take these

results further. One of the pitfalls of using only morpho-

logical data in the identification of hybrids is the lack of

information on hybrid status (i.e. F1 vs backcross), and

inability to identify cryptic hybrids—individuals that phe-

notypically resemble parent taxa but are of mixed ancestry.

The latter leads to underestimation of actual levels of

hybridization (or numbers of hybrids) within a population

(Ackermann 2010; Kelaita and Cortes-Ortiz 2013). Cryptic

hybridization has been observed at C. jacchus 9 C. peni-

cillata hybrid zones where discordance exists between

marmosets possessing the phenotype of one parental spe-

cies and the mitochondrial D-loop haplotype of another

species (Malukiewicz 2013). Such discordance is also

widely observed throughout baboon hybrid zones (Zinner

et al. 2009). Thus it is likely that levels of hybridization

may currently be underestimated at Vicosa.

Management of hybrid populations is a controversial

topic (Allendorf et al. 2001; Detwiler et al. 2005), and

anthropogenic activities will likely continue to increase

incidence and prevalence of animal hybridization. How-

ever, hybridization offers many different evolutionary

outcomes and wild hybrid populations offer immense

opportunities to examine hallmark issues in the evolution

of primates, including examining the forces that drive

speciation, helping to define taxonomic boundaries, and

understanding the influence of genotype on phenotype.

Thus future work on marmoset hybrids in Vicosa will

include genetic and genomic profiling of hybridization and

admixture levels within the hybrid zone, as well as

examination of hybrid fitness and adaptations. This

research will help to determine the hybrid status of sampled

individuals, and provide more nuanced information on the

correlation between genetic, phenotypic, and behavioral

data. As we have already shown great phenotypic diversity

in the morphometric traits of wild marmoset hybrids, these

unique primate populations will surely continue to prove

themselves as novel natural laboratories for answering

questions regarding primate evolution.

Acknowledgments The authors would like to thank CAPES for

providing a graduate scholarship to L.F.F. A Fulbright Fellowship and

National Science Foundation Doctoral Dissertation Grant #1061508

to J.M. also provided additional funding for this work. We thank the

Brasilia Zoo and Brasilia Botanical Garden for access to their

premises.

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