Exploiting genomic resources in studies of speciation and adaptive

radiation of lizards in the genus Anolis

Christopher J. Schneider
1
Biology Department, Boston University, 5 Cummington Street, Boston, MA 02215, USA
Synopsis Lizards in the genus Anolis have radiated extensively within and among islands in the Caribbean. Here,
I provide a prospectus for identifying genes underlying adaptive phenotypic traits in anoles. First I review patterns of
diversification in Anolis and the important morphological axes along which divergence occurs. Then I discuss two
features of anole diversification, the repeated, convergent evolution of ecomorphs, and phenotypic divergence among
populations within species, that provide opportunities to identify genes underlying adaptive phenotypic variation.
While small clutch size and difficulty with captive rearing currently limit the utility of quantitative trait locus analyses,
comparative analyses of gene expression, and population genomic approaches are promising.
Introduction
The diversity of organisms for which whole genomic
sequences are known offers an exciting opportunity
for evolutionary biologists and ecologists to exploit
genomic data, and associated methods, to address
long-standing questions in comparative biology,
ecology, and evolution. Not only can comparative
analyses of genomes from diverse taxa generate novel
insight into the evolution of whole genomes, but
also extending genomic information and approaches
from sequenced taxa to other related taxa offers the
opportunity to address a number of fundamental
questions in ecology and evolution.
Identifying genes underlying ecologically important
phenotypic traits is a central goal of ecological and
evolutionary genetics (Feder and Mitchell-Olds 2003;
Storz 2005; Lee and Mitchell-Olds 2006; Ellegren
and Sheldon 2008; Stinchcombe and Hoekstra 2008).
Advances in technology, theory, and applications now
allow analysis of genes underlying important pheno-
typic variation in wild populations of nonmodel
organisms. Recent examples include analyses of coat
color in beach mice (Hoekstra et al. 2006), size and
shape of the bill in Galapagos finches (Abzhanov et al.
2004, 2006), adaptive variation in wild sunflowers
(Kane and Rieseberg 2007; Sapir et al. 2007; Baack et al.
2008), and size, shape, body armor, and pigmentation
in three-spine sticklebacks (Colosimo et al. 2004;
Coyle et al. 2007; Miller et al. 2007; Albert et al. 2008;
Makinen et al. 2008).
My goal here is to assess the opportunity to
exploit the complete genomic sequence of the lizard,
Anolis carolinensis, to address ecological and evolu-
tionary questions in the adaptive radiation of the
genus Anolis. Anoles have become a model for the
study of adaptive radiation, and genomic approaches
promise novel insight into the genetic changes
underlying adaptive radiation. Difficulty with captive
breeding of anoles currently limits their utility
for genetic analyses requiring a known pedigree,
but comparative analyses of gene expression across
species, and population genomic analyses of closely
related but phenotypically varying populations
within species, provide opportunities to identify
loci involved in adaptation. Here I briefly review
Anolis as a model system in ecology and evolution,
and discuss approaches to identify the underlying
genetic basis for ecologically and evolutionarily
important phenotypic variation. I also discuss the
problem of speciation in Anolis, which is a neglected
component of their adaptive radiation, present a
model for speciation in Anolis, and discuss how
genomic information and approaches may assist in
understanding the entire breadth of speciation and
adaptive diversification of anoles.
Background on lizards in the genus
Anolis
Lizards in the genus Anolis have become a model
system for studies of adaptive radiation, community
From the symposium Reptile Genomics and Evolutionary Genetics presented at the annual meeting of the Society for Integrative and
Comparative Biology, January 26, 2008, at San Antonio, Texas.
1
E-mail: cschneid@bu.edu
520
Integrative and Comparative Biology, volume 48, number 4, pp. 520526
doi:10.1093/icb/icn082
Advance Access publication September 6, 2008
The Author 2008. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved.
For permissions please email: journals.permissions@oxfordjournals.org.

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ecology, morphology and performance, behavior,
communication, local adaptation, and biotic diversi-
fication (Losos 1994; Losos et al. 1998; Losos 2001;
Losos et al. 2003; Losos et al. 2004; Losos et al.
2006a, 2006b). The genus Anolis (Hass et al. 1993;
Poe 1998; Frost et al. 2001) contains upwards of 400
species distributed throughout the neotropics,
Caribbean, and south-eastern United States. The
large adaptive radiation of more than 150 species in
the Caribbean has received the most attention.
Recently, a draft genome assembly of the Green
Anole (A. carolinensis) was completed (http://Broad.
mit.edu/ftp/pub/assemblies/reptiles/lizard/AnoCar
1.0). The genome of A. carolinensis is the first non-
avian reptilian genome and its phylogenetic place-
ment makes it valuable for comparative analyses of
genomic structure and evolution in both reptiles and
mammals. Importantly, the complete genome of
A. carolinensis makes Anolis one of the few genome
enabled adaptive radiations and thus provides the
opportunity to study the genetics of adaptation. The
question before us is: How can we use the genome
of A. carolinensis to further understand adaptation
and diversification in the genus Anolis?
Convergent evolution of anoles
Anoles have undergone independent adaptive radia-
tions on each of the islands in the Greater Antilles
(Cuba, Hispaniola, Puerto Rico, and Jamaica) (Losos
et al. 1998; Jackman et al. 1999). A striking pattern
of convergent evolution of morphology and behavior
is demonstrated by the independent evolution of
similar ecomorphs on each island (Losos et al.
1998). Ecomorphs are categorizations of behavior,
ecology, and morphology that reflect microhabitat
and substrate specialization. Ecomorphological diver-
gence facilitates coexistence of different species and
has resulted in complex, multispecies assemblages
on each island in the Greater Antilles. Cuba and
Hispaniola have a complicated geological history and
their anole faunas may be comprised of species
assemblages that evolved on isolated landmasses that
subsequently coalesced to form the current islands
(Hass et al. 1993; Glor et al. 2004). Thus, Cuba, with
55 species, and Hispaniola, with 40 species, contain
the most diverse anole faunas. In contrast, Puerto
Rico and Jamaica, where anole species assemblages
have largely evolved in situ, host smaller faunas
(11 and 7 species, respectively). In each case, similar
ecomorphs have evolved.
Ecomorphs differ primarily in limb length, girdle
dimensions, number of subdigital lamellae and
dimensions of the skull (Losos 1990a, 1990b;
Irschick et al. 1997). While ecomorphological
divergence has occurred convergently on each of
the Greater Antilles (Losos et al. 1998), it is not
known if convergence resulted from the same or
different changes in genes and development in each
instance. Nor is it known whether changes in
structural genes and/or regulatory regions contrib-
uted to adaptive morphological change (Hoekstra
and Coyne 2007, Wray 2007). However, the fact that
most phenotypic variation among ecomorphs occurs
on a limited number of phenotypic axes highlights
the genetic and developmental systems that we may
target for analyses.
Intraspecific variation
Anoles in both the Greater and Lesser Antilles
occupy a wide range of habitats and many species
show substantial variation in morphology associated
with habitat. The most detailed studies of geographic
variation in anoles have been on species from the
Lesser Antilles. On volcanic, topographically com-
plex, single-species islands in the Lesser Antilles
(Guadeloupe, Dominica, Martinique), species are
geographically variable, with population differentia-
tion apparently reflecting local adaptation. Most of
the species vary primarily in adult male color and
pattern, but also in size, shape and scalation, and
several subspecies have been described in each
instance (Lazell 1964, 1972). Evidence for local
adaptation derives from analyses of selection
(Malhotra and Thorpe 1991, 2000; Thorpe et al.
2005) and reduced gene flow among divergent
populations (Schneider 1996; Ogden and Thorpe
2002). Despite substantial variation in morphology
among populations within islands in the Lesser
Antilles, most populations have not diverged to the
extent that different ecomorphs have (Knox et al.
2001).
Populations of some species on the Greater
Antilles also show substantial geographic variation,
although these have been less studied. For example,
the Anolis distichus complex from Hispaniola com-
prises eight subspecies (Case 1990; Schwartz and
Henderson 1991) that differ again in size, scalation,
and, in adult males, color and pattern, especially of
the dewlap, an extensible throat fan that is important
in social communication and species recognition.
Because ecomorphological divergence is an impor-
tant component of adaptive radiation of Anolis,
populations within species that reflect the early stages
of ecomorphological divergence are of particular
interest. Anolis sagrei populations, originally derived
from Cuba, but now occupying many islands in the
Anolis genomics and adaptation 521

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Caribbean, vary in size, scalation, relative limb
length, and lamellae number (Lister 1976; Kolbe
et al. 2007). Recent studies revealed that populations
of A. sagrei occupying different habitats in the
Bahamas vary in size and relative limb length in
a direction that mirrors those among ecomorphs
(Losos et al. 1994; Calsbeek and Smith 2007;
Calsbeek et al. 2007). Also, experimental manipula-
tion of populations of A. sagrei in the Bahamas
demonstrated that evolution of size and relative limb
length can occur very rapidly and is associated with
available height and diameter of perches, as well as
with presence of predators (Losos et al. 1997, 2001,
2004, 2006b). Because of their shared genetic back-
ground, these populations may provide an excep-
tional opportunity to discover the genetic basis of
adaptive change during the early stages of ecomor-
phological divergence.
Speciation in Anolis
Anoles are highly visual animals and there is strong
sexual dimorphism in color and pattern in most
Caribbean species. The males are territorial and use
visual displays, including extension of a throat fan,
or dewlap, along with stereotypical movement
patterns, as social signals. The color of the dewlap
varies among species and is likely important in
species recognition (Losos 1985; Case 1990), as are
markings and colors on the head and body. It is
likely that divergence in color and pattern, especially
of the dewlap, is an important step in the evolution
of reproductive isolation among species.
Nearly all described subspecies of Anolis in the
Caribbean differ primarily in color and pattern of
adult males (Lazell 1964, 1972). Local adaptation
in color and pattern among populations living in
different habitats (e.g., lowland rainforest versus
montane forest versus dry forest) has led to reduced
gene flow among populations (Ogden and Thorpe
2002), suggesting that local adaptation may be
an important step in speciation. Divergence in
mate recognition systems, without ecomorpholog-
ical divergence, suggests that divergence in mate-
recognition signals, and perhaps reproductive
isolation, precedes ecomorphological divergence.
Ecomorphological divergence may then subsequently
result from ecological interactions among reproduc-
tively isolated species in sympatry. This model
contrasts with a proposed general model of adaptive
radiation (Streelman and Danley 2003) in which
habitat divergence is followed by (eco) morphologi-
cal divergence, with divergence in communication
(i.e., mate-recognition signals) occurring last.
Both sexual and ecological selections are impor-
tant in producing differences in communication
signals in anoles. Local adaptation of signal colors
to light environment may very quickly lead to
reduced gene flow among populations. Ecological
selection via predation could also influence body and
dewlap colors, with animals in areas of high
predation being less conspicuous. This prediction is
supported by a general pattern of anoles on dry
islands, where vegetation is sparse and open, tending
to be grey-brown and cryptic, whereas anoles in
rainforests and wet habitats tend to be bright green
and often contrasting with the background.
Therefore, an important step in the speciation of
anoles likely involves divergence in genes and gene
expression that control color and pattern of the
dewlap, head, and body. There are several cases in
both the Lesser and Greater Antilles where closely
related populations within species differ dramatically
in color and pattern of adult males and these
populations are good targets for population genomic
analyses to identify genes affecting variation in color
and pattern.
Identifying genes underlying ecologically
and evolutionarily important phenotypic
variation
Comparative analyses of gene expression among
ecomorphs
Convergent evolution is pervasive among anole
species. But what is the genetic basis of convergence?
A promising approach to identify genes involved in
convergent evolution of morphology is through
comparative analysis of gene expression during
developmental stages when morphological differences
among species are established. Recent studies of
development in anoles suggest that differences
among ecomorphs in the length of limbs are
established early during limb-bud development
(T. J. Sanger, unpublished data). Similarly, differ-
ences in the size of the toe pad and number of
lamellae appear to be established during a narrow
window of development when the toe pad is formed.
It is expected that differences in cranial morphology
and other characters likewise will be established
during specific stages of morphogenesis. Compara-
tive analysis of gene expression from specific tissues
at developmental stages when interspecific differences
are established provides a good opportunity to
identify genes responsible for adaptive phenotypic
evolution.
But, how best to compare gene expression among
species that may have diverged many millions of
522 C. J. Schneider

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years ago? Until recently, gene expression was
analyzed primarily using DNA microarrays. While
the technical and analytical aspects of microarray
gene expression analyses have advanced significantly
in recent years, it has become clear that there are
limitations to DNA microarrays in cross-species
comparisons (Gilad et al. 2005). Given nearly
160,000 expressed sequence tags (ESTs) that have
been produced from diverse tissues of A. carolinensis,
DNA microarrays could be designed that contain
nearly all expressed genes from that species.
However, comparative analysis of gene expression
using microarrays designed for A. carolinensis may be
limited to comparisons of closely related species.
Given the age of divergence within Anolis (50
million years) an approach that allows comparative
analyses of gene expression among more distantly
related species is desirable.
A recently developed approach, known as RNA
sequencing or cDNA censusing, uses massively
parallel sequencing technology to directly sequence
mRNA or cDNA from target tissues (Torres et al.
2008; Wold and Myers 2008). The sequence reads are
then compared against a reference genome, tran-
scripts identified to gene, and their frequency tallied.
This approach does not require sequencing of entire
cDNAs, as short sequence reads of 2530 bases are
sufficient to place most reads on a reference genome.
However, longer reads are desirable since they
provide additional information on splice variants
and nucleotide polymorphisms within genes. The
results of RNA sequencing or cDNA censusing are
spectra of EST frequencies which can be compared
directly among species to discern differences in gene
expression. This approach is promising as it accesses
the entire transcriptome, but it is not without its
own set of problems. Presently, short reads of 2540
bases that are produced by some sequencing
technologies may result in 1520% of reads being
difficult to assign to a single region in the reference
genome due to multiple copies of short sequences in
the genome or poor matches of sequence to reference
(Wilhelm et al. 2008; Wold and Myers 2008). The
latter becomes more of a problem as species
examined become more evolutionarily distant from
the species from which the reference sequence was
derived. Again, longer sequence reads may provide
an advantage in placing sequences on a reference
genome. In a best case scenario, Torres and others
(2008) were able to map 97% of ESTs from
Drosophila melanogaster, with read lengths of 200
base pairs, to the D. melanogaster genome, and 90%
of the sequences were unambiguously mapped to
annotated genes. RNA sequencing is the most
promising approach for comparative analyses of
gene expression among species of Anolis.
Population genomics of intraspecific phenotypic
variation
Because population differentiation is the first step
in adaptive radiation, it is important to understand
the genetic basis of adaptation at this level. Several
recent reviews discuss approaches to identify
loci underlying phenotypic variation within and
among populations (Feder and Mitchell-Olds 2003;
Beaumont and Balding 2004; Slate 2005; Storz 2005;
Ellegren and Sheldon 2008; Stinchcombe and
Hoekstra 2008). There are three general approaches.
All rely on genome-wide surveys of variation using
genetic markers (e.g., microsatellites, SNPs, or
AFLPs) to identify regions under selection. The
first, and most general, approach has come to be
known as Population Genomics (Black et al. 2001;
Goldstein and Weale 2001; Jorde et al. 2001). The
term population genomics refers to analyses of
variation at a large number of marker loci to identify
those linked to regions under selection, while
controlling for demographic events that affect levels
of genetic variation across the entire genome. The
simplest application of population genomic scans is
to use the variance in allele frequency at marker loci
to calculate a measure of divergence or variation
between two populations and identify marker loci
that have higher or lower values than would be
expected by chance (Luikart et al. 2003; Schlotterer
2003; Beaumont and Balding 2004; Storz 2005;
Stinchcombe and Hoekstra 2008). These outlier
marker loci may be linked to regions under selection
and thus have a pattern of population differentiation
that differs from neutral genetic variation across the
genome. It is unlikely that the marker itself is the
target of selection, but if these markers are mapped,
or can be localized in a published genome, then
candidate loci in regions to which they are linked can
be identified. Variation upstream, and downstream
of the marker(s) can then be examined directly to
identify genes and regulatory regions that may be, or
have recently been, targets of selection (see e.g.,
Sutter et al. 2007). If markers are developed from
sequence reads of the target species, those reads may
be mapped to the A. carolinensis genome and,
assuming shared synteny, genes, or other functional
features in the region would be candidate loci.
The population genomics approach does not
require knowledge of the location of markers, or
even of the existence of phenotypic variation among
populations (e.g., Bonin et al. 2006), but if such
Anolis genomics and adaptation 523

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information exists, then statistical associations
between phenotypes and genetic markers can be
detected using linkage disequilibrium (LD) mapp-
ing or quantitative trait locus (QTL) analyses
(Stinchcombe and Hoekstra 2008). QTL analysis
requires the measurement of phenotype and marker
genotype in a controlled breeding design, but can
be applied to natural populations in cases in which
pedigrees can be determined unambiguously (Slate
2005). QTL analyses are most appropriate to organ-
isms that can be readily reared in captivity and that
have large numbers of offspring and, therefore,
anoles may not be good subjects for QTL analyses.
LD mapping, on the other hand, may be applicable
to natural populations. LD mapping in natural
populations will require many more marker loci
than QTL analysis (hundreds or thousands instead of
dozens) because the breeding design of QTL analysis
results in large linkage groups. Natural populations
are likely to have smaller linkage groups due to a
long history of recombination but, with sufficient
marker density, small linkage groups may provide
higher resolution for identifying regions under
selection since any marker consistently associated
with phenotype is likely to be closely linked to
candidate loci. As with any association study, LD
mapping requires controlling for population struc-
ture and careful attention must be paid to the impact
of population structure on statistics for association
(Freedman et al. 2004; Kimmel et al. 2007).
Which of these approaches is applicable to
populations of anoles? Because of difficulty in
husbandry and small clutch size, QTL analyses may
not be feasible for Anolis in the near term. However,
we may exploit population genomic and LD studies
in closely related, but phenotypically varying, popu-
lations to identify regions of the genome that
underlie important variation in body size, limb
length, color and pattern, and other characteristics.
Therefore, the most fruitful approaches are likely to
involve population genome surveys using microsa-
tellite or SNP markers to identify markers associated
with phenotypes of interest. Marker loci can then
be mapped by comparing the sequences from
which they were derived to the genome sequence
of A. carolinensis. Closely related populations of
Anolis, in which most neutral genetic variation is still
segregating in both populations (i.e., those in which
genetic drift has not fixed populations for many
loci), but which are fixed or nearly so for phenotypic
characters potentially under selection, offer the best
opportunity for identifying marker loci associated
with phenotypic variants. Populations of A. sagrei in
the Bahamas, and several polytypic species in the
Lesser Antilles, are good targets.
Summary and conclusions
Despite difficulties with captive breeding of Anolis,
comparative analyses of gene expression across
species and population genomic surveys and LD
mapping in natural and experimental populations,
offers great opportunity to identify and study genes
underlying ecologically and evolutionarily important
variation. The completion, and future annotation, of
the A. carolinensis genome will facilitate the study of
genes involved in adaptation, speciation, and adap-
tive radiation by providing a framework for identify-
ing candidate genes and gene regions underlying
adaptive phenotypic evolution. The radiation of
Anolis in the Caribbean is a textbook example of
adaptive radiation and the integrated historical and
experimental approaches applied to date have
resulted in a deep and rich understanding of the
causes of diversification in anoles (Losos 2007).
Information on the genetic basis of adaptation and
diversification in this group will complete the picture
and provide insight into the genetic basis and
architecture of adaptation and adaptive radiation.
Acknowledgments
Thanks to Daniel Janes, Christopher Organ, and
Nicole Valenzuela for organizing the Reptile geno-
mics symposium. Thanks to Travis Glenn, Nicholas
Crawford, Jonathan Losos, and Hopi Hoekstra for
insightful discussions, and thanks to three anon-
ymous reviewers for constructive criticism on the
manuscript. Special thanks to Thomas Sanger for
sharing his work in progress on embryological
development in Anolis.
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