The Origins and Diversification of the Exceptionally Rich Gemsnakes (Colubroidea: Lamprophiidae: Pseudoxyrhophiinae) in Madagascar

Abstract

Processes leading to spectacular diversity of both form and species on islands have been well-documented under island biogeography theory, where distance from source and island size are key factors determining immigration and extinction resistance. But far less understood are the processes governing in situ diversification on the world's mega islands, where large and isolated land masses produced morphologically distinct radiations from related taxa on continental regions. Madagascar has long been recognized as a natural laboratory due to its isolation, lack of influence from adjacent continents, and diversification of spectacular vertebrate radiations. However, only a handful of studies have examined rate shifts of in situ diversification for this island. Here, we examine rates of diversification in the Malagasy snakes of the family Pseudoxyrhophiinae (gemsnakes) to understand if rates of speciation were initially high, enhanced by diversification into distinct biomes, and associated with key dentition traits. Using a genomic sequence-capture data set for 366 samples, we determine that all previously described and newly discovered species are delimitable and therefore useful candidates for understanding diversification trajectories through time. Our analysis detected no shifts in diversification rate between clades or changes in biome or dentition type. Remarkably, we demonstrate that rates of diversification of the gemsnake radiation, which originated in Madagascar during the early Miocene, remained steady throughout the Neogene. However, we do detect a significant slowdown in diversification during the Pleistocene. We also comment on the apparent paradox where most living species originated in the Pleistocene, despite diversification rates being substantially higher during the earlier 15 myr. [Gemsnakes; in situ diversification; island biogeography; Neogene; Pseudoxyrhophiinae; speciation.].

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DOI:10.1093/sysbio/syz026
Advance Access publication April 23, 2019
The Origins and Diversification of the Exceptionally Rich Gemsnakes (Colubroidea:
Lamprophiidae: Pseudoxyrhophiinae) in Madagascar
FRANK T. BURBRINK1,∗,SARA RUANE2,ARIANNA KUHN1,3,NIRHY RABIBISOA4,BERNARD RANDRIAMAHATANTSOA4,
ACHILLE P. R ASELIMANANA5,MAMY S. M. ANDRIANARIMALALA5,JOHN E. CADLE6,ALAN R. LEMMON7,EMILY MORIARTY
LEMMON8,RONALD A. NUSSBAUM9,LEONARD N. JONES10 ,RICHARD PEARSON11 ,AND CHRISTOPHER J. RAXWORTHY1
1Department of Herpetology, The American Museum of Natural History, 79th Street at Central Park West, New York, NY 10024, USA; 2Department of
Biological Sciences, 206 Boyden Hall, Rutgers University-Newark, 195 University Ave, Newark, NJ 07102, USA; 3Department of Biology, The Graduate
School and University Center, The City University of New York, 365 Fifth Ave., New York, NY 10016, USA; 4Mention Sciences de la Vie et de
l’Environnement, Faculté des Sciences, de Technologies et de l’Environnement, Université de Mahajanga, Campus Universitaire d’Ambondrona, BP 652,
Mahajanga 401, Madagascar; 5Mention: Zoologie et Biodiversité Animale, Faculté des Sciences, Université d’Antananarivo, BP 906, Antananarivo 101,
Madagascar; 6Department of Biology, East Georgia State College, Swainsboro, GA 30401, USA; 7Department of Scientific Computing, Florida State
University, Dirac Science Library, Tallahassee, FL 32306-4102, USA; 8Department of Biological Science, Florida State University, 319 Stadium Drive,
Tallahassee, FL 32306-4295, USA; 9Division of Reptiles and Amphibians, Museum of Zoology, Research Museums Center, 3600 Varsity Drive, University
of Michigan, Ann Arbor, MI 48108, USA; 10Department of Biology, University of Washington, Seattle, WA 98195-1800, USA; and 11Centre for Biodiversity
& Environment Research, Department of Genetics, Evolution and Environment, University College London, Gower Street, London WC1E 6BT, UK
∗Correspondence to be sent to: Department of Herpetology, The American Museum of Natural History, 79th Street at Central Park West, New York,
NY 10024, USA;
E-mail: fburbrink@amnh.org.
Received 26 June 2018; reviews returned 5 April 2019; accepted 9 April 2019
Associate Editor: Nicholas Matzke
Abstract.—Processes leading to spectacular diversity of both form and species on islands have been well-documented under
island biogeography theory, where distance from source and island size are key factors determining immigration and
extinction resistance. But far less understood are the processes governing in situ diversification on the world’s mega islands,
where large and isolated land masses produced morphologically distinct radiationsfrom related taxa on continental regions.
Madagascar has long been recognized as a natural laboratory due to its isolation, lack of influence from adjacent continents,
and diversification of spectacular vertebrate radiations. However, only a handful of studies have examined rate shifts
of in situ diversification for this island. Here, we examine rates of diversification in the Malagasy snakes of the family
Pseudoxyrhophiinae (gemsnakes) to understand if rates of speciation were initially high, enhanced by diversification into
distinct biomes, and associated with key dentition traits. Using a genomic sequence-capture data set for 366 samples, we
determine that all previously described and newly discovered species are delimitable and therefore useful candidates for
understanding diversification trajectories through time. Our analysis detected no shifts in diversification rate between clades
or changes in biome or dentition type. Remarkably, we demonstrate that rates of diversification of the gemsnake radiation,
which originated in Madagascar during the early Miocene, remained steady throughout the Neogene. However, we do
detect a significant slowdown in diversification during the Pleistocene. We also comment on the apparent paradox where
most living species originated in the Pleistocene, despite diversification rates being substantially higher during the earlier
15 myr. [Gemsnakes; in situ diversification; island biogeography; Neogene; Pseudoxyrhophiinae; speciation.]
Island clades often show a pattern of rapid
diversification after colonization, which is hypothesized
to be a response for filling vacant niches (Schluter
2000;Losos and Ricklefs 2009;Presgraves and Glor
2010;Rabosky and Glor 2010). This standard pattern
of in situ island diversification has provided the basic
evidence for adaptive radiation (AR) via ecological
opportunity and has served as the intellectual template
for continental AR (Simpson 1944;Losos et al. 1998).
While studies of island biogeography have firmly
established that the essential properties of islands
such as distance to the mainland, area, and habitat
heterogeneity all influence colonization and extinction
under ecological neutrality (MacArthur and Wilson
1967), less understood are the processes of in situ
diversification as related to properties of speciation and
accumulation of diverse endemic island faunas (Losos
et al. 2009). Even though in situ island diversification
is positively associated with area, and trajectories for
diversification may vary among clades, most groups are
expected to reveal initially high speciation rates when
presented with abundant ecological opportunity upon
colonization (Paulay 1994;Lomolino and Weiser 2001;
Pinto et al. 2008;Losos and Ricklefs 2009;Pyron and
Burbrink 2014a;Igea et al. 2015).
Several taxonomic groups across large island systems
have demonstrated that speciation rates do indeed
rise rapidly after colonization. For instance, the Anolis
lizards of the West Indies (Losos et al. 2009;Rabosky
and Glor 2010) and honeycreepers and silver swords
in Hawaii (Carlquist et al. 2003;Lerner et al. 2011)
support the classic prediction of AR as applied to
island systems: rapid early speciation with associated
phenotypic adaptation. This suggests that finding
diversity dependence (DD), where rates of speciation are
initially high but trend towards an ecologically-mediated
carrying capacity, is indicative of AR over deep time
scales (Losos 2010;Yoder et al. 2010). Nevertheless, there
are a few examples that contradict these predictions.
For instance, a diverse assemblage of snakes in the West
Indies showed very little change in diversification over
time (Burbrink et al. 2012b). On the other hand, typical
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changes in the diversification trajectory often attributed
to AR may have geographic and environmental causes
not linked to saturated ecological space, such as high
speciation rates following rapid changes in habitat
that slow as the environment stabilizes (Vrba 1985;
Rosenzweig 1995;Pigot et al. 2010;Pyron and Burbrink
2012a;Quental and Marshall 2013;Moen and Morlon
2014). Moreover, AR may not be accompanied by changes
in speciation rates if the adaptive traits examined do not
directly affect speciation processes (Rabosky 2017).
One obstacle to understanding processes that produce
diverse island communities is the difficulty obtaining
reliable phylogenetic data for species-rich groups
typically associated with ecologically diverse islands.
For example, even if the fundamental idea of a species-
diversity carrying capacity holds (and it may not, see
Harmon and Harrison 2015;Rabosky and Hurlbert 2015),
estimating processes and rates of diversification is often
hampered by size of the phylogeny, inadequate models
or disagreement among models, differing modes of
speciation, insufficient taxon sampling, and undetected
cryptic species (Pybus and Harvey 2000;Revell et al.
2005;Weir 2006;Rosindell et al. 2010;Pennell et al.
2012;Ruane et al. 2014). Therefore, geographically
monophyletic groups are valuable for understanding
diversification within large islands, where the standing
diversity was generated only by intra-island speciation,
and where taxon sampling is more likely to be complete.
Madagascar, at 587,041 km2with 3% of the worlds’
biodiversity, stands out among all large islands as
showing significant endemicity generated mostly by
in situ diversification (Goodman and Benstead 2003;
Vences et al. 2009). This diversity can in part be
explained by its ancient isolation from Gondwanaland
at 120+ millions of years ago (Ma) and India at
80 Ma (McLoughlin 2001;Collier et al. 2008) and
typical island biogeographic characteristics that generate
species such as area, distance from the mainland, and
sharp demarcation among distinct biomes (MacArthur
and Wilson 1967;Raxworthy et al. 2002,2015;Goodman
and Benstead 2003;Pearson and Raxworthy 2009;Vences
et al. 2009;Samonds et al. 2013;Brown et al. 2014). For
many of the diverse endemic groups on Madagascar,
colonization likely happened throughout the Cenozoic,
but dropped off rapidly after the mid-Miocene (von der
Heydt and Dijkstra 2006;Yoder and Nowak 2006;Ali and
Huber 2010;Samonds et al. 2012,2013). Some studies
have suggested that in situ diversification following
colonization for many of these vertebrate groups
remained constant through time (Crottini et al. 2012),
though research on Malagasy herpetofauna (day geckos
and chameleons) have shown early bursts of speciation
that declined through the Quaternary (Harmon et
al. 2008; Scantlebury 2013). Unfortunately, knowledge
about the diversification of groups during the Cenozoic
has relied on incomplete taxon sampling typically using
only a handful of loci, further hindered by a wide fossil
gap that excludes the critical Miocene epoch (Raxworthy
Nussbaum 1997;Burney et al. 2004;Samonds et al. 2013).
We, therefore, call attention to the diverse subfamily
of snakes, Pseudoxyrhophiinae (Colubroidea:
Lamprophiidae), which are composed of 90 described
species found in fossorial, terrestrial, aquatic, and
arboreal habitats, and occur in many of the basic
ecological niches snakes occupy throughout continental
systems (Vitt et al. 2003;Vitt and Caldwell 2009). Because
pseudoxyrhophiine snakes have no common-use name,
we herein refer to them as gemsnakes, in reference
to the locally well-known Malagasy legend of an
endemic serpent that carries a light-emitting diamond
in its stomach (Raxworthy, pers. observ.). Gemsnakes
consume a wide variety of mostly prey including
frogs, skinks, geckos, chameleons, other snakes,
birds, mammals, and occasionally invertebrates (Nagy
et al. 2003;Glaw and Vences 2007;Uetz 2009;Ruane
et al. 2015). Potentially related to dietary and habitat
preferences, gemsnakes can be divided into aglyphous
taxa without grooved rear teeth and opisthoglyphous
species, which feature enlarged, grooved rear teeth used
to hold or envenomate prey (Guibé 1958;Kardong 1980;
Mori and Mizuta 2006;D’Cruze 2008;Weinstein et al.
2011), though we note that aglyphous gemsnakes may
also envenomate prey (Razafimahatratra et al. 2015).
While dentition and diet may be correlated (Knox and
Jackson 2010), for most snakes on continental systems, it
is unclear if dentition type relates to general ecological
or prey preferences and if this trait is phylogenetically
conserved.
At least for snakes in continental communities,
diversification occurs rapidly upon colonization
(Burbrink et al. 2012a;Chen et al. 2017) and basic
ecological niches are usually filled by the colonization
and diversification of several groups across several
subfamilial taxonomic ranks (Bellini et al. 2015;
Burbrink and Myers 2015). In contrast, the majority
of Madagascar’s gemsnake diversity is suspected to
have originated via in situ diversification from a single
colonization event (Nagy et al. 2003). Importantly,
Madagascar derives most of its fauna via in situ
diversification, even when compared with other large
islands such as New Guinea and Borneo (Pyron
and Burbrink 2012a). It is unknown if processes
of diversification leading to the large radiation of
gemsnakes occurred via early bursts of speciation
with downturns as niches were filled or were constant
through time. Similarly, it is unclear how traits have
evolved to fill niches and if primary ecology predicts
key dentition types.
To address the processes of in situ diversification
that have led to the diverse snake communities in
Madagascar, we collect sequence-capture data from 366
individual gemsnakes and outgroups to first account
for extant diversity among previously named taxa and
phylogenetically deep cryptic lineages. Since under- or
over-estimating alpha taxonomy can affect downstream
estimates of diversification (Faurby et al. 2016), we
confirm species delimitations of all previously and
newly identified taxa using coalescent methods. We
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920 SYSTEMATIC BIOLOGY VOL. 68
then examine the timing of colonization and processes
of in situ species and ecological diversification, and
determine if different regions of notably contrasting
biomes were responsible for enhanced phylogenetic and
species diversity. Our conclusions provide an alternate
understanding of AR showing that constant rates (CR)
of diversification throughout the Neogene can lead to
spectacular diversification in species number and form.
METHODS AND MATERIALS
Data and Screening Pseudoxyrhophiine Samples for
High-Throughput Sequencing
Prior to high-throughput sequencing for anchored
loci used in the subsequent analyses, we Sanger-
sequenced four loci for 621 individuals (583 Malagasy
gemsnakes, plus outgroups, see Supplementary
data available on Dryad at https://datadryad.org/
resource/doi:10.5061/dryad.07h0n14): the mitochon-
drial locus cytochrome oxidase 1 (CO1) and the nuclear
loci oocyte maturation factor mos gene (CMOS),
recombination activating 2 gene (RAG2), and Nav1.4
intron 5 (NAV5) (see Supplementary data available
on Dryad). Primers and sequencing conditions follow
those used in Ruane et al. (2015). We inferred trees
using maximum likelihood in RAxML 8.0 (Stamatakis
2014) with the GTRGAMMA model of evolution and
partitioned by locus and codon position for the protein
coding genes. Tree estimates used 100 independent
tree searches and 5000 bootstrap replicates to retrieve
support values. This procedure allowed us to confirm
sample identities, determine potentially undescribed
and/or cryptic taxa, and choose individuals that would
encompass as much of the genetic diversity found
within each species and/or genus as possible for
high-throughput sequencing.
Sequence-capture data sets generating anchored
hybrid enrichment loci were collected at the
Center for Anchored Phylogenomics (www.
anchoredphylogeny.com) following Lemmon et al.
(2012) and Ruane et al. (2015; Supplementary data
available on Dryad). Including new species, we sampled
109 gemsnake taxa representing 93% of extant species
on Madagascar and outgroup samples from all major
clades of Colubroidea, resulting in a data set comprising
366 individuals and a total 732 phased sequences
(Supplementary data available on Dryad). We did
not include taxa only found on Comoros Liophidium
mayottensis, Lycodryas cococola, and Lycodryas maculatus
and were unable to sample Brygophis coulangesi,
Ithycyphus blanci, Langaha pseudoalluaudi, Liophidium
pattoni, Liophidium maintikibo, Pararhadinaea melanogaster,
Pseudoxyrhopus ankafinaensis, P. kely, and P. sokosoko.
We note that Pseudoxyrhopus ankafinaensis is probably
extinct (Raxworthy and Nussbaum 1994) and consider
Lycodryas carleti (junior synonym of L. gaimardi), and
Phisalixella iarakaensis (junior synonym of P. arctifasciata)
as invalid taxa. Few genetic differences exist between
these two species of Lycodryas; Kimura pairwise
sequence differences within L. gaimardi (∼0.043) overlap
those between L. gaimardi and L. carleti (0.027–0.047). The
one known Phisalixella iarakaensis specimen is sympatric
with P. arctifasciata, and identical in all respects except
an additional scale row on each body flank. However, as
dorsal scale row variation is common in other Phisalixella
species, we thus consider this to represent intraspecific
variation.
Phylogeny
We generated three types of phylogenies based on the
full (732 samples), reduced (130 samples; gemsnakes and
outgroups), and core (109 samples, gemsnakes only).
We used ASTRAL II (Mirarab and Warnow 2015)to
infer species trees for all 732 phased terminals; this
method accounts for incomplete lineage sorting. We
generated support for the topology using local posterior
probabilities on quadripartitions (Sayyari and Mirarab
2016). Additionally, we concatenated data and used
maximum likelihood in IQ-TREE (Nguyen et al. 2015)
to estimate phylogeny. Here, we partitioned each locus,
assessed which among 279 models of substitution best
fit each partition using Bayesian information criterion
(BIC), and estimated phylogeny with support generated
from 1000bootstraps. We also estimated phylogeny using
maximum likelihood on the concatenated partitioned
and unpartitioned data with the GTRGAMMA model in
RAxML 8.0 (Stamatakis 2014). We compared topological
support between these estimators using Robinson–
Foulds distances (RF; Robinson and Foulds 1981)intheR
package “phangorn” (Schliep 2011) and identified nodes
with distinctly different support.
Species Delimitation
It was necessary to include deeply divergent,
unnamed species along with the currently recognized
gemsnakes to estimate diversification and run other
downstream comparative processes, and we consider 38
of these unnamed taxa as unique given that: (1) the dates
of divergence were generally greater than the Calabrian;
divergences for most snakes using phylogeographic
species delimitations have been recorded from the mid-
Pleistocene to the late Miocene (Burbrink et al. 2011;
Myers et al. 2013;Ruane et al. 2014,2016,2018;McKelvy
and Burbrink 2017), (2) the ranges of the dates of
origin of these new taxa fit within the ranges of age of
origin of previously named sister taxa within gemsnakes
(Supplementary data available on Dryad), (3) the new
taxa are geographically distinct from their sister taxa,
and (4) the new taxa have morphological diagnostic
characters (in prep).
To confirm that we can delimit all species of Malagasy
gemsnakes, including previously described and new
undescribed taxa, we used the program Bayesian
Phylogenetics and Phylogeography (BPP v4.0; Rannala
and Yang 2013). This method uses coalescent techniques
to estimate the probability that species were distinct and
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helped confirm that they were genetically isolated on
evolutionary timescales (Rannala 2015). The tree size
of core gemsnakes, which includes 109 distinct species,
prohibits delimitation using BPP as a whole. Therefore,
using a custom script in R (BPP_Maker; Supplementary
code available on Dryad), we broke up the phylogeny
into inclusive testable groups ranging from 2 to 7
terminals and all population level-samples per taxon for
a total of 32 delimitation analyses. For each analysis, we
used all 370 loci, however for Compsophis (Compsophis_5)
and Madagascarophis (Madacascarophis_1), we removed
244 and 232 loci, respectively, due to numerous missing
individuals that would have eliminated some taxa
for testing (Supplementary data available on Dryad).
We used algorithm A11 to jointly delimit species and
estimate a species tree (Rannala and Yang 2013), and
variables were fine-tuned and updated automatically
to ensure that swapping rates between Markov chain
Monte Carlo (MCMC) chains were adequate. We used
the new implementation of inverse gamma priors at
3, 0.004 and 3, 0.002 for ancestral population sizes
() and root age (), respectively. We ran these two
times for 1×10 5generations, following a burn in of
1×105generations, which were then sampled every
10 generations. We determined that all estimated
sample sizes (ESS) >200 for only using the R
package “coda” (Plummer et al. 2006). We compared
the time of speciation for all new species and pairs
of previously described taxa using a custom script
(BPP_Dater) that takes all estimates of and converts
this to time by dividing by substitutions/generation,
obtaining substitution rate and generation time from
a fitted gamma distribution for each of the 100,000
MCMC samples of (Angelis and Dos Reis 2015; see
Supplementary code available on Dryad for details).
Reduced Phylogeny
To examine processes of species, trait, and ecological
diversification in gemsnakes, we reduced the
data set from 732 phased terminals down to 130
terminal taxa (including 21 outgroups) to remove
population level sampling so that downstream
comparative methods were calculated on trees that
include only a single sample per species. The core
gemsnakes were further subsampled representing
109 monophyletic pseudoxyrhophiine species that
diversified within Madagascar and therefore represent
in situ diversification; this group does not contain
the African outgroup taxa Amplorhinus, Duberria, and
Ditypophis. Phylogenies with one terminal per taxon
(randomly sampled when >1 individual/species was
present) were generated using (1) maximum likelihood
with IQ-TREE (Nguyen et al. 2015) where each partition
was tested using ModelFinder (Chernomor et al. 2016;
Kalyaanamoorthy et al. 2017) for over 279 substitutional
models including up to six free rate gamma categories.
Support for these trees was assessed using ultrafast
nonparametric bootstrap approximation (Minh et al.
2013) over the partitioned, concatenated data sets and
over the partitioned, concatenated data set where
pseudoreplicates were created by resampling at the
full locus level, and support using the Shimodaira–
Hasegawa-like approximate likelihood ratio test
(Shimodaira and Hasegawa 1999); (2) species trees
generated using SVDQuartets (Chifman ane Kubatko
2014) and support from 1000 bootstraps; (3) ASTRAL
II (Mirarab and Warnow 2015) with support estimated
using local posterior probabilities on quadripartitions,
and (4) ASTRAL II where we did not downsample
the entire data set (n=732) but used the multiple
individuals per species feature.
Divergence Dating
We dated both the full and reduced phylogeny using
the penalized likelihood approach in TreePL (Smith and
O’Meara 2012), MCMCTree (Yang and Rannala 2006),
and BEAST2 (Bouckaert et al. 2014) and determined
that all three solutions were correlated and node error
was minimal. Implementing a full MCMC relaxed-clock
phylogenetic method was not possible given the number
of taxa and loci. Therefore, to assess error on these
date estimates, we bootstrapped the concatenated and
partitioned data set using IQ-TREE and estimated dated
trees using TreePL with fossil constraints with hard
bounds. We also used an Astral-constrained tree with
concatenated data to estimate divergence dates using
MCMCTree and Beast2. For MCMCTree, all calibrations
were made “softer” by extending probabilities of
0.025 beyond those bounds (see Supplementary data
available on Dryad for details parameterizing TreePL,
MCMCTree, and BEAST2). We used the following fossil-
based calibration locations and times, which according
to Head (2015) and Guo et al. (2012) were reliably
placed at the most recent common ancestor (mrca) with
hard bounds [mean and standard deviation (SD) in
parentheses for BEAST2] for the following groups: (1)
Crown of Colubroidea and all trees used here (mrca of
Achalinus rufescens and Compsophis albiventris) at 50.5–
72.1 Ma ( ¯
X=55.5, SD =9.0), (2) stem Elapidae (mrca
of Naja melanoleuca and Hemerophis socotrae), minimum
30.9 Ma ( ¯
X=32.9, SD =1.0), (3) stem Natricinae (mrca
of Thamnophis cyrtopsis and Helicops angulatus) 32–36
Ma ( ¯
X=29.5, SD =3.4), and (4) crown Viperidae (mrca
Crotalus molossus and Bitis gabonica) 20–23.8 Ma ( ¯
X=22.0,
SD =0.9).
Ancestral Range Estimation
Previous authors have demonstrated that Madagascar
was colonized twice by lamprophiid snakes from Africa,
an early colonization resulting in the diversification
of the core gemsnakes and a later one resulting in
two species of Mimophis (Nagy et al. 2003;Vidal et al.
2008;Ruane et al. 2018). We tested this using our new
comprehensive phylogenies that included lamprophiids
from Africa and global colubroid outgroups. Using
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the dated 130-taxon phylogeny, we estimated ancestral
areas using BioGeoBEARS (Matzke 2013,2014). We used
three base models in BioGeoBEARS (Matzke 2013): the
Dispersal–Extinction–Cladogenesis (DEC) of Ree and
Smith (2008); DIVALIKE, a likelihood implementation
of the assumptions of the parsimony DIVA program
(Ronquist 1997); and BAYAREALIKE, a likelihood
implementation of the assumptions of the BayArea
program (Landis et al. 2013). Models were run with
and without a parameter weighting jump dispersal (+J)
at cladogenesis (Matzke 2014). Terminal species were
coded as occurring in one or multiple areas that included
globally Asia, New World, and Europe, and regionally
Africa, Socotra, and Madagascar. We estimated ancestral
area and colonization times using the biogeographic
model with the lowest AICc.
Diversification
We determined if diversification differs throughout
the core gemsnakes, estimated with the MCMCTree
using BAMM (Rabosky 2014;Rabosky et al. 2014). We
estimated changes in diversification using BAMM with
the initial prior estimates generated using the function
setBAMMpriors over five Poisson rate priors of 0.1, 0.5,
1.0, 5, and 10 (Rabosky et al. 2014). We ran 10×106
generations for each Poisson rate prior twice to ensure
consistency, thinned by every 1000, and removed 15%
as burn in. Over these Poisson rate priors, we estimated
the number of diversification rate shifts with the highest
posterior density (HPD) in the maximum shift credibility
configuration.
Because methodologies in BAMM are controversial
given putative problems with the likelihood function
and Poisson process prior model (Moore et al. 2016;
Rabosky et al. 2017), we also used spectral densities
to identify modes or clusters that may represent
diversification shifts in this tree (Lewitus and Morlon
2016). Here, the phylogeny was converted into a graph
Laplacian and represented as a spectral density profile
of eigenvalues (). The primary and 2nd, 3rd, and 4th
moments were estimated to represent shifts, asymmetry,
and peakedness in the spectral profile, respectively. The
number of peaks in the profile represented the number
of diversification modalities across the tree. This method
was implemented using the R package “RPANDA”
(Morlon et al. 2016) using the spectR and BICompare
functions to identify distinct patterns of diversification
within the phylogeny of gemsnakes.
Diversification rates were the same over the core
gemsnakes (see Results section). We determined if
speciation or extinction rates have changed over time, or
responded to mass extinction events. Under a typical AR
model, we predicted that speciation rates should have
been highest upon colonization and decrease through
time. We tested this using rjMCMC CoMET (May et al.
2016) in the R package “TESS” (Höhna et al. 2015)
to estimate diversification parameters over time given
hyperpriors for speciation, extinction, and numbers of
mass extinction generated from the empirical data using
the TESS.analysis function with 1×105iterations. We
determined that convergence using rjMCMC occurred
for the speciation, extinction rate, and mass extinction
parameters when effective sample sizes exceeded 200.
We ran CoMET on the maximum likelihood phylogeny
to determine if Bayes factors support any mass extinction
events. With no evidence of mass extinction, we repeated
this analysis 10 times over all 1000 bootstrapped dated
trees and estimated rates of speciation and extinction
over time. We also determined if Bayes factors showed
evidence of change in speciation and extinction over
time.
To determine whether incomplete taxon sampling
from undiscovered species could explain the signal
for declining diversification rates in the Pleistocene,
we developed code to add additional artificial taxa
only in the Pleistocene without altering the height of
any existing nodes (Supplementary data available on
Dryad). This stress test was designed to determine how
many additional undiscovered speciation events would
be needed to change estimates of diversification away
from declining rates during the Pleistocene. We added
additional sets of ten taxa up to a total 109 cryptic species
(doubling the current number of taxa) replicated 100
times per set, reran TESS, and used Bayes Factors above
5.0 to account for declining or increasing rate shifts
during the Pleistocene.
To detect a signal of DD given changes in speciation
and extinction rates, we used the R package “DDD”
(Etienne et al. 2012) to test between CR and DD models.
We estimated parameters including birth (), death ,
and carrying capacity () for both DD and CR models. We
used these estimates in the function dd_LR to generate a
parametric bootstrapped (n=1000) likelihood ratio test
for DD against CR given simulations generated from
the estimated values of ,, and at 0.299 (0.162), 0
(9.718×10 −6), and 146, respectively (CR values for and
are shown in parenthesis).
In contrast, we compare results from DDD against
the coalescent-derived estimators of diversification from
Morlon et al. (2011) over the following models: (1 and 2)
saturated diversity (most similar to DD) with either time
constant or variable rates, respectively; (3) homogenous
birth death; (4a–d) expanding diversity with CR; (5)
expanding diversity with no extinction (Yule); and (6)
expanding diversity with time-varying speciation and
no extinction.
Diversity by Area
Given that Madagascar is composed of regionally
distinct habitats, we tested whether habitat type was
responsible for higher species richness and phylogenetic
diversity by first classifying all unique lineages
presented in the species tree by ecoregion. To do this,
we first obtained and confirmed locality information
using a combination of genetic samples, morphological
samples, and previously published museum records. All
genetic samples used in this study were associated with
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2019 BURBRINK ET AL.—MADAGASCAR SNAKE DIVERSIFICATION 923
localities obtained in the field (datum: WGS84, error:
Differential GPS) at 0.05 km accuracy or georeferenced
using the point-radius uncertainty method (Wieczorek
et al. 2004). Specimens that were not genetically sampled
in this study were first assigned to a particular lineage
using morphological data, then associated localities
were incorporated. Additionally, previously published
localities records (Glaw and Vences 2007;Brown et al.
2016) where lineage identity was certain were also
included. Combined, all sources provided 864 unique
locality records, ranging from 1 to 48 records per species
(Supplementary data available on Dryad).
We classified distributions of all gemsnakes into
six major terrestrial ecoregions of Madagascar, which
include arid spiny bush, central highlands, dry
deciduous forest, ericoid thicket, and evergreen
rainforest (combined with the Sambirano rainforest),
presented in Goodman and Benstead (2003) (as modified
by Yoder and Nowak (2006); see Supplementary data
available on Dryad for code and details on fitting
species to region). We then estimated species richness
and phylogenetic diversity in these regions. We used
the richness-independent measure PSV (phylogenetic
species variability) implemented in the R package
“Picante” (Helmus et al. 2007;Ives and Helmus
2010;Kembel et al. 2010). To determine if phylogenetic
diversity is either under or over dispersed, we compared
our estimates to a random shuffling of all gemsnakes,
while preserving regional species richness. We then
compared areas using PSV and species richness.
Similarly, we used the function ConDivSim in the
R package “PEZ” (Pearse et al. 2015) to simulate
mean pairwise distances in each community over
different species richness values to determine if these
communities deviated from null (n=100) community
estimates.
We also examined turnover between regions using the
measure of phylogenetic community dissimilarity (PCD)
to understand if changes between ecoregions occurred
among taxa that were phylogenetically more related
or less related in the R package “PEZ” (Pearse et al.
2015). We used PCD (where estimates lower than 1.0
indicate that dissimilar taxa between communities are
closely related) to generate hierarchical clusters in the
R package “pvclust” (Suzuki and Shimodaira 2006)over
1000 bootstraps to show which adjacent habitats have the
least phylogenetic dissimilarity.
Trait Evolution
To understand the evolution of basic ecological traits in
gemsnakes and examine how this group colonized and
diversified across Madagascar, we scored taxa as being
fossorial, terrestrial, arboreal, aquatic, or generalist. All
taxa were considered as belonging to at least one of
these basic ecological types taken from our knowledge
of these species in the field and from Glaw and Vences
(2007). Additionally, since these species show two basic
dentition types: aglyphous and opisthoglyphous, where
the latter is suggested to be associated with venom
injection (Mori and Mizuta 2006;D’Cruze 2008), we also
scored and included this dentition feature.
We estimated ancestral ecology and dentition type
using the core phylogeny with the three non-
Malagasy pseudoxyrhophiines (Duberria, Amplorhnus,
and Ditypophis). We used the fitdiscrete function in the
R package “geiger” (Harmon et al. 2008b) to compare
the fit of models with equal rates (ER), symmetric (SYM)
forwards/reverse rates, and all rates different (ARD).
We used the AICc-best model to infer ancestral state
probabilities. From this, we generated marginal ancestral
states for the best model which was determined by lowest
AICc. To compare to this maximum likelihood method,
we performed stochastic character mapping over 1000
replications in the R package “phytools” (Nielsen and
Huelsenbeck 2002;Huelsenbeck et al. 2003;Revell 2012)
given the appropriate models determined in the ace
function. These replicates were summed over all trees,
from which we estimated the number of changes from
each trait type given the proportion of time spent in
each state. We then correlated the posterior probabilities
from stochastic character mapping with the marginal
ancestral states from the maximum likelihood method.
We also asked if dentition was correlated with
primary ecology over the evolutionary history of the
gemsnakes, here coded into binomial traits as arboreal
(0) against all other states (1). We used this coding as
we noted that aglyphous and opisthoglyphous dentition
appeared to be associated with arboreal and terrestrial
ecology. To assess this correlation, we used the threshold
model (Felsenstein 2005,2012) in “phytools” to examine
correlated character change among dentition (aglyphous
and opistoglyphous) and habitat (arboreal and non-
arboreal). Tests were run for 5×106generations with
the first 25% of samples discarded as burn-in (ESS
>200) and thinned by 1000. The mean and 95% HPD
of r(correlation) were calculated from the remaining
samples.
Finally, to understand if traits influence rates of
diversification across the gemsnakes, we used the
semiparametric correction to estimate binary, trait-
driven diversification using the traitDependentBAMM
function of Rabosky and Huang (2016)intheRpackage
“BAMMTools”. Using the function set for 1000 replicates
and the Mann–Whitney Ustatistical test, we determined
if dentition type or basic habit preference (arboreal/non-
arboreal) influenced rates of diversification over the
dated phylogeny of the gemsnakes. We also modeled
trait influence on diversification using binary state
(BiSSE) and hidden state (HiSSE) speciation and
extinction methods (Maddison et al. 2007;Beaulieu
and O’Meara 2016). Whereas BiSSE only examines
whether a discrete character influences the rate of lineage
diversification, HiSSE additionally accounts for hidden
states (unsampled states) that could affect diversification
rates for any of the observed states. We estimated AIC for
null BiSSE and HiSSE models, alternative models where
there was a binary state effect of traits on diversification,
and equal and unequal hidden and observed state
transitions and their effects on diversification.
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924 SYSTEMATIC BIOLOGY VOL. 68
RESULTS
Data and Phylogenies
We successfully sequenced 607 individuals for COI
(674 bp), 571 individuals for CMOS (558 bp), 499
individuals for RAG2 (645 bp), and 436 individuals
for NAV5 (599 bp). Maximum likelihood inference
using RAxML with mtDNA only and with the entire
concatenated data set resulted in largely congruent
phylogenies with minor differences regarding some
outgroup placement (Supplementary Fig. S1 available
on Dryad).
We sequenced 371 anchored loci for 366 samples
of gemsnakes and outgroups (359 guided from the
Sanger tree; Supplementary data available on Dryad),
and estimated phylogenies for 732 phased terminals. For
the 732 terminals (all phased samples), our average locus
length for the 371 loci was 1575 bp (SD 34, range: 532–
2481) with a mean number of parsimony informative
sites of 190 (SD 135.615, range: 0–534). Mean number
of missing individuals per locus was 2.75% (SD = 4.1%,
Range 0.13%–24.3%) when compared with the full data
set of all individuals.
The ASTRAL II species tree generated high support for
most nodes (Supplementary Fig. S2 available on Dryad)
and the topology and support was consistent with IQ-
TREE and the RAxML partitioned and unpartitioned
bootstrap trees. Normalized RF distances ranged from
0.01 (RAxML vs. IQ-TREE) to 0.22 (RAxML partitioned
vs. ASTRAL II; Supplementary Material available on
Dryad).
All gemsnake genera were monophyletic and generic
relationships among them were similar to Ruane
et al. (2015) showing two major clades of generally
aglyphous and opisthoglyphous snakes, see exceptions
below (Fig. 1; Supplementary data available on Dryad).
Outgroup colubroid relationships were typical of recent
multilocus and NGS studies (Pyron et al. 2013,2014;
Streicher and Wiens 2016) showing xenodermatids
as sister to the remaining colubroids, followed
by pectinate relationships for pareatids, viperids,
homolopsids, colubrids, elapids, and lamprophiids.
Here, the Malagasy Mimophis wassistertothe
psammophiine African beaked snakes (Rhamphiophis).
Among Pseudoxyrhophiinae, the Malagasy gemsnake
radiation was sister to a clade composed of East African
genera Amplorhinus and Duberria. Sister to all other
pseudoxyrhophiines was the Socotran island endemic
Ditypophis (Fig. 2). Divergence dates with the full tree
generally agree with those generated with the reduced
phylogeny (see below).
Species Delimitation
To examine diversification of species and traits in the
gemsnakes, we reduced this phylogeny to account for
only terminal species that also included undescribed
candidate species. Here, using a combination of deep-
time divergence between sister taxa and geographic
distribution, we suggest that there are 38 unrecognized
species distributed across all genera of gemsnakes (with
exception to Alluaudina) and we included them in the
reduced tree (Fig. 2, Supplementary data available on
Dryad).
We confirmed these 38 species delimitations using
BPP. ESS were all >300 (mean ESS across all delimitation
=19,296) for all 32 separate runs of BPP. All previously
described taxa and newly identified species were
delimited at a probability of 100%. In contrast to the
gene/tree-based estimates of divergence times, for these
coalescent estimates the average speciation time for
undescribed taxa was 7.85×105years (range 0.50–2.97
×106) and for pairs of previously described taxa was
1.65 ×106years (range 1.64–9.36 ×10 6). For the newly
identified species, 96.1% of these speciation events
(drawn from the MCMC distribution) occurred during
the Pleistocene and 3.7% during the Neogene. For
the previously described species, we found that 87%
of speciation events occurred during the Pleistocene
and 13% during the Neogene (Fig. 1). We note that
for several taxa only a few individuals were sampled
due to their rarity, and thus this may compromise
species delimitations if greater sampling and isolation-
migration models revealed high levels of migration
(Zhang et al. 2011;Burbrink and Guiher 2015). However,
we are confident that including these proposed taxa
with such ancient divergences are useful for downstream
approaches to understanding diversification.
Reduced Trees
We inferred the phylogeny for a reduced set of
terminals, where each species was represented by a
single individual. Over all loci, 19 different substitution
models fit these data using Modelfit, with the highest
number of loci (n=82) fit to the HKY model, whereas
the most parameter-rich model, GTR, was best fit to
only nine loci. All models included some form of
rate heterogeneity with the FreeRate model and three
categories fit to 74% of loci. Across all loci, the number
of parameters to model substitution rates ranged from 3
to 10 parameters, with a median of 7 parameters.
Normalized RF distances among trees generated using
partitioned bootstrapped were lowest between RAxML
and IQ-TREE concatenated and locus bootstraps (0), and
greatest between SVDQuartets and ASTRAL II with or
without using multiple individuals per species (0.06).
All trees supported monophyly of all genera, similar
species relationships, and similar relationships among
genera (Figs. 1and 2), which agree in part with
Ruane et al. (2015). Nearly all nodes among methods
were well supported. We found that the relationships
among Langaha, Micropisthodon, and Ithycyphus differed
among all methods, where SVDQuartets indicated a
sister relationship between Ithycyphus and Langaha at
54% support, ASTRAL II showed Micropisthodon and
Ithycyphus as sister taxa (98–100% support), and RAxML
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2019 BURBRINK ET AL.—MADAGASCAR SNAKE DIVERSIFICATION 925
FIGURE 1. Circular phylogeny of Pseudoxyrhophiinae of all 732 phased samples from 371 loci generated in ASTRAL II (see Supplementary
Fig. S2 available on Dryad for a detailed view of this phylogeny) with photographs showing representative species from all included Malagasy
genera. Inset: Distribution of pairwise divergence dates for 38 newly discovered species pairs (dark blue) against previously described species
pairs (light pink) using BPP.
and all IQ-TREE showed Langaha and Micropisthodon
as sister taxa (93% support for RAxML, 84.7%, 82%,
and 87% for SH, bootstrapped, and locus bootstrapped
IQ Tree support, respectively). Among all 371 loci,
276 provided definitive sister relationships between
these genera, where support was similar showing
Micropisthodon and Ithycyphus at 38.7% loci, Langaha and
Micropisthodon at 32.6%, and Ithycyphus and Langaha at
28.6%.
Age and Colonization
Estimating timed trees using penalized TreePL,
MCMCTree, and BEAST2 produced dates that were
correlated 0.96–0.99 (P<2.2×106) with mean absolute
differences between shared node dates among methods
ranging from 0.33 to 0.92 my. While all dated trees
were highly correlated, we note that trees and dates
were not burned in after two separate long runs
of BEAST2 due primarily to the size of the data
set and slow parameter estimation. The BEAST2 tree
was not used in any downstream calculations. The
root height of this tree representing the origin of
Colubroidea occurring earlier than the Cretaceous-
Tertiary boundary was consistent with recent studies,
though error around this estimate is high for most
studies (Burbrink and Pyron 2008,2010;Burbrink
and Crother 2011;Pyron and Burbrink 2014b;Hsiang
et al. 2015). Moreover, the origins of colubroid groups,
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926 SYSTEMATIC BIOLOGY VOL. 68
FIGURE 2. Dated phylogeny of all sampled species of Pseudoxyrhophiinae using ASTRALII and TreePL. Estimates of speciation and extinction
rates over time using CoMET with probabilities of those rates changing indicated by Bayes factors over 1000 bootstrapped phylogenies.
which included Pareidae, Viperidae, Homolopsidae,
Elapidae, Lamprophiidae, and Colubridae, occurred
throughout the Paleogene which was consistent with
recent studies and the fossil record (Fig. 2;Burbrink
and Pyron 2008; Pyron and Burbrink 2012; Head 2015;
Hsiang et al. 2015).
Using the dated MCMCTree tree, we estimated
the ancestral area of origin for the core gemsnakes.
BioGeoBEARS shows strongest support for the DEC+J
model (AICc = 3.71–54.95; AICc weight = 0.86),
where estimating the jump (“J” parameter) is consistent
with a single over-ocean island colonization. Ancestral
area estimates were the same with or without the
“J” parameter. Madagascar was colonized twice by
lamprophiids, first in the early-to-mid Miocene resulting
in the core gemsnake radiation (Supplementary data
available on Dryad) and second in the mid-to-late
Miocene, resulting in the two species within the genus
Mimophis. This agrees with previous studies attempting
to date and number these colonization events, relying
on mainly mtDNA data and reduced sampling of
gemsnake taxa (Nagy et al. 2003;Vidal et al. 2008;Kelly
et al. 2009). However, our result conflicts with other
studies that found support for paraphyletic gemsnakes
in Madagascar. These previous results supported more
complex multiple-dispersal scenarios between Africa,
Socotra, and Madagascar (Lawson et al. 2005;Pyron et al.
2011,2013), but relied on a few independent markers with
very incomplete taxon sampling.
Diversification
We tested whether diversification rates changed over
the phylogeny of core gemsnakes using both BAMM
and spectral distances. For all Poisson shift priors (0.1,
0.5, 1.0, 5.0), we obtained ESS >707 from BAMM and
similar results for either the PLTree or MCMCTree.
The posterior distribution for 0 shifts was always the
highest, from 0.98 (BF>16), 0.93 (BF>7), 0.90 (BF>5), and
0.84 (BF>2.8), respectively for each Poisson shift prior.
Similarly, spectral distances estimated in RPANDA only
identified a single mode across the phylogeny from the
eigengap metric for either tree (here asymmetry = 10.70,
peakedness = 477.86, principal eigenvalue =1.20).
Given that rates did not change within this tree,
we estimated time-dependent speciation and extinction
rates from the bootstrapped distribution of dated trees
using CoMET producing similar results for all dated
trees. There was no evidence for mass extinction in these
snakes (BF <1.0). Rates of extinction, which are typically
difficult to estimate using phylogeny alone (Quental and
Marshall 2009;Rabosky 2010), were estimated to be low
(close to 0) and constant through time (Fig. 2). Speciation
rates remained constant above 0.1–0.3/myr throughout
the Neogene, possibly declining by the Pliocene and
certainly dropping sharply during the Pleistocene (Fig. 2,
BF = 7.0–7.5). Our bootstrap likelihood ratio test for DD
and CR using the program DDD for the MCMCTree
showed support for rates of diversification declining
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2019 BURBRINK ET AL.—MADAGASCAR SNAKE DIVERSIFICATION 927
50 60 70 80 90 100
Bayes Factors speciation shifts
0 20406080100
0.6 0.7 0.80.9 1.0
Number of additional terminal taxa
Fraction of down shifts in the PL
Down Shift Mixed
Down &
Up Shifts
FIGURE 3. Testing the effect of unsampled taxa in the Pleistocene
on predicted downshifts in diversification. The upper graph shows
the percentage of Bayes factors (>5.0) from 100 simulations using
the program CoMET that detect a decrease in Pleistocene speciation
rates given the addition of terminal taxa imputed onto the gemsnake
phylogeny occurring only in the Pleistocene. The lower graph shows
the fraction of down shifts (regardless of strength of Bayes factors) in
diversification during the Pleistocene detected out of 100 simulations
per number of additional taxa added to the phylogeny of gemsnakes.
Note that upward shifts in diversification in the Pleistocene become
more common with the addition of more than 40 taxa.
against a carrying capacity [Likelihood ratio (LR) =
3.845; P<0.0059] with the power of this test at 0.81.
This measure estimated speciation rates, extinction rates,
and carrying capacities at 0.29/myr, 0.0/myr, and 146,
respectively. We contrasted these results against the
coalescent models from Morlon et al. (2011), which
supported the saturated species diversity model with
time-varying rates (Model 1=−219.110) slightly over
the declining speciation rate model (Model 6=−220.27;
Supplementary data available on Dryad).
Simulations to stress–test models of diversification
where we added cryptic taxa diverging within the
Pleistocene to the core gemsnakes phylogeny revealed
that Bayes Factors (>5.0) using CoMET supported
reduced rates of diversification in the Quaternary up
to the addition of 30 species in more than 90% of
simulations (Fig. 3). Above 50 imputed species, the
fraction of simulations showing reduction in speciation
during the Pleistocene decreased rapidly, leading to both
increases and decreases in diversification during this
period.
Area Diversity
Species occurred in a median of 2 ecoregions (SD
=0.98) and 36% of all taxa were endemic to a single
ecoregion. Endemicity was correlated with habitat size
(=0.96, P=0.009; species endemicity in parentheses):
dry deciduous (13), evergreen rainforest (12), central
highlands (8), arid spiny bush (3), and montane ericoid
thicket (2). Species richness was lowest in the smallest
ecoregion, montane ericoid thicket (n=7), and highest
in the evergreen rainforest (n=73). PSV was similar
among habitats and randomly dispersed across most
habitats (Fig. 4). Only the dry deciduous forest showed
phylogenetic underdispersion (reduced variability of
phylogenetic diversity due to the absence of some
humid adapted lineages; P=0.015; Fig. 4) yet still
has an appreciably high species diversity (n=56).
Paradoxically, habitat endemicity as a proportion of
taxa was lowest in the expansive central highlands
(10.6%) and largest in the region of lowest diversity,
montane ericoid thicket (28.5%; see also Raxworthy
and Nussbaum 1996). Both species richness and PSV
increased marginally with larger biomes covering larger
areas (Fig. 5). Additionally, variance in mean pairwise
differences (MPD) by community increased with greater
richness, which was expected when larger communities
contain both a higher proportion of species and
phylogenetic diversity. These communities by biome
fell within the 95% confidence interval (CI) of null
community MPDs given richness (Fig. 5). Finally,
phylogenetic turnover as measured by PCD was lowest
between communities sharing similar precipitation
profiles, where PCD was lower than 1.0 between dry
deciduous forests and arid spiny bush, and similarly
between the central highlands/montane ericoid thickets
and evergreen rainforests (Fig. 5).
Trait Origins and Diversification
Maximum likelihood and stochastic character
methods of ancestral state reconstructions for
all Pseudoxyrhophiinae (with non-Malagasy
outgroups) were highly correlated for both habitat
and dentition state reconstructions (Pearson’s
r=0.999,P=2.2×10−16 ). The equal rates character model
of evolution for habitat type was always preferred
(AIC weights =0.99). On average, 13.05 changes from
arboreal to generalist (n=3.25) and then terrestrial to
fossorial (2.88) were observed during the evolution of
the gemsnakes. For dentition, the all rates different model
of character evolution was preferred (AICweigths =0.73;
Supplementary data available on Dryad). On average,
4.5 changes all from opisthoglyphous to aglyphous
were observed with no reversals. The ancestral states
for the gemsnakes upon colonization of Madagascar
were opisthoglyphous and terrestrial (Fig. 6). We used
the threshold model to determine if dentition type and
arboreal vs. non-arboreal (generalist, aquatic, fossorial,
terrestrial) ecologies were correlated. With an ESS
=437, we found that dentition was correlated with
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928 SYSTEMATIC BIOLOGY VOL. 68
FIGURE 4. Phylogenetic species variability (PSV) for Pseudoxyrhophiinae in the five biomes of Madagascar, with null distributions showing
randomly sampled taxa preserving richness relative to the actual community (vertical line). Values in the upper right-hand corner of each box
are from top to bottom: species richness, P-value of observed PSV, and percentage number of ecoregion endemic species/richness.
basic ecology at a median correlation coefficient of 0.44
(Fig. 6; 95% CI 0.28–0.58).
Neither dentition or habitat type had any effect on
speciation rates (P=0.995 for both); species-specific rates
were similar between opisthoglyphous and aglyphous
taxa (0.134, 0.133) and between arboreal and terrestrial
taxa (0.133, 0.134). Using HiSSE, we found greatest
support for the BiSSE null model (min AICc BiSSE
Null and HiSSE null =3.56) suggesting no connection
between traits and diversification.
DISCUSSION
Diversification
The Malagasy gemsnakes show a pattern of constant
diversification rates throughout the Neogene with a
decline in speciation during the Plio-Pleistocene (Fig. 2).
This suggests that constant speciation throughout the
Neogene accounts for most of the diversification in this
group. Speciation, however, declined in the last 2 myr of
gemsnake history, likely due to diversity-dependence.
Furthermore, there is no support for mass extinction on
Madagascar in the gemsnakes. Upon colonization from
Africa in the early Miocene, divergence into most of the
basic ecologies occurred early, and within those groups,
diversification was constant over time.
These constant rates (CR) of diversification upon
colonization might seem at odds with a long-standing
phylogenetic view of AR and exploration of adaptive
zones (Mitter et al. 1988;Schluter 2000;Losos 2010),
particularly in comparison to some other diverse reptile
groups in Madagascar (Harmon et al. 2008a;Scantlebury
2013). Diversity of both species and form occurred
at CR over the first 15–17 myr after colonization,
though we do not know about comparative speciation
rates of the related lamprophiid snakes on Africa. The
process of AR can generate a diverse faunal group
on Madagascar yet still be divorced from changing
rates of species diversification through time (Givnish
2015;Stroud and Losos 2016;Rabosky 2017). Similar
to Madagascar, evidence from both molecular and
fossil studies suggest that colubroid snakes colonized
North America and Europe in the late Oligocene/early
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2019 BURBRINK ET AL.—MADAGASCAR SNAKE DIVERSIFICATION 929
FIGURE 5. Estimating the relationships between richness, PSV, and biome. The five biomes are shown on the map to the right. Top graph shows
heatmaps based on phylogenetic community differences, where values lower than one on the PCD tree (left tree) indicate turnover between
biomes shows low dissimilarity (i.e., dissimilar species are closely related). The tree on the top of the heatmap shows support for these groupings
based on approximate unbiased (AU) and 1000 bootstraps (BP) probabilities. The middle graph shows the relationships between richness and
mean pairwise distance for each biome relative to a random sampling of individuals from the total pool relative to local species richness (dark
to light representing 95% CI and white represents the median trend). Bottom graph shows the relationship between area and richness and PSV.
Miocene, and that diversification was highest in the
Miocene and declined during the Pleistocene (Holman
2000;Burbrink et al. 2012a;Chen et al. 2017). While
competition from other snakes could prevent an early
burst of diversification, there is no evidence that
other groups of colubroids ever colonized Madagascar;
currently only extant blindsnakes (11 species) and boas
(4 species), and extinct Late Cretaceous Madtsoiidae
and Nigerophiidae snakes ever occurred in Madagascar
(Glaw and Vences 2007;Laduke et al. 2010). We also did
not find that Madagascar was subject to major pulses of
extinction during the Neogene and speciation proceeded
without major external disruption during this period.
Apparently for the gemsnakes, the constant rate of
speciation for 15–17 myr occurred before niches become
saturated and speciation rates were reduced during the
Pleistocene.
Difficulty estimating extinction rates from molecular
phylogenies is well known, especially in the absence of
a fossil record (Quental and Marshall 2009;Rabosky
2010). It is unclear how these estimates affect tests of
diversification over time or interpretation of changing
speciation rates, though we point out that these trends
seen on Madagascar are similar to other large but
similarly young snake groups (Burbrink et al. 2012a).
Unfortunately, whereas the fossil record also supports
a pattern of sustained and high diversification of snakes
throughout the Miocene in Europe and North America
(Holman 2000;Szyndlar 2009), a Miocene fossil record
of terrestrial vertebrates is unknown for Madagascar
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930 SYSTEMATIC BIOLOGY VOL. 68
FIGURE 6. Stochastic character mapping showing ancestral dentition and ecological reconstructions for Pseudoxyrhophiinae with the
distribution of correlation between dentition and habitat (arboreal v. others) over the phylogeny using the Bayesian Threshold model.
(Goodman and Benstead 2003;Samonds 2009). Marshall
(2017) stressed that extinction rates may be just below
speciation rates even in expanding groups, and failing
to account for this may hinder reliable estimates of
diversification over time using molecular phylogeny.
Without credible extinction estimates, it is possible that
initial diversification rates were extremely high in the
gemsnakes for a short period of time upon colonization,
reached saturated diversity rapidly, and then finally
was reduced with enhanced extinction rates during
climate change in the Pleistocene. This last phase might
appear as a slowdown in speciation rates without proper
estimates of extinction.
The evolution of traits allowing taxa to occupy novel
niches in unique ways or, in this case, colonize empty
ecological niches, may have little to do with the rate at
which speciation occurs. Moreover, when considering
the traits that allow taxa to colonize unique niches, it is
uncertain that these same traits necessarily reduce gene
flow among populations necessary for rapid speciation
to occur (Servedio et al. 2011). Instead, we show that taxa
evolve into distinct niches early in their history despite
CR of speciation. Excluding Australia (Grundler and
Rabosky 2014), this is unique among snakes, where at
least in continental systems, taxa from different families
assemble to fill all local niches (Burbrink and Myers
2015).
Gemsnakes occurring in the basic arboreal, terrestrial,
general, fossorial, and aquatic habitats are also mostly
found across the five distinct ecoregions of Madagascar,
though the aquatic taxon does not occur in the montane
ecoregion. Given species richness for each ecoregion,
phylogenetic diversity is no different from a random
draw of PSV (phylogenetic species diversity) across these
regions, excluding the dry deciduous biome (Figs. 4
and 5). The largest biomes, evergreen forest, central
highlands, and dry deciduous, contain the greatest
diversity. However, at least within all extant biomes,
no single habitat is generating most of the phylogenetic
diversity. Therefore, all biomes across the island are
likely responsible for gross in situ diversity of the
gemsnakes, though realistically it is unclear if these
biomes persisted continuously through the Neogene or
were even responsible for the diversification of their local
assemblages. Finally, among these biomes, phylogenetic
dissimilarity is lowest between the two adjacent dry
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2019 BURBRINK ET AL.—MADAGASCAR SNAKE DIVERSIFICATION 931
habitats and also among the three mesic habitats (Fig. 5),
suggesting that colonization between similar biomes is
related to having similar adaptive traits (e.g., as a result
of having conserved ecological niches) given the reduced
phylogenetic dissimilarity.
Pleistocene Divergence
While rates of speciation may have been highest
throughout the Miocene, most of the standing extant
diversity was likely generated during the Pleistocene
(Fig. 1). Using coalescent species delimitation methods,
we have shown strong support that 109 terminal
species in our phylogeny are useful for understanding
diversification; we assessed strong support for the
existence of 38 undescribed species and date the origins
of all taxa. We show for the first time a strong role
for the Pleistocene generating extant diversity of a
major faunal component in Madagascar. This role of
Pleistocene speciation has been debated for other areas
of the world (Avise et al. 1998;Klicka and Zink 1999;
Knowles 2001;Weir and Schluter 2004;Carstens and
Knowles 2007;Moyle et al. 2009;Ruane et al. 2014;
Oliveira et al. 2015;Rull 2015), though it is likely that
Pleistocene shifts in habitat due to global climate change
may have generated many new species via isolation
and divergence. Mirroring Last Glacial Maxima habitat
changes in the Northern Hemisphere (Waltari et al.
2007;Hollingsworth and Near 2009;Burbrink et al.
2016;Gehara et al. 2017), environments throughout
Madagascar were likely much cooler and drier with
glacial vegetation found at lower elevations (Goodman
and Benstead 2003;Burney et al. 2004;Raxworthy
et al. 2008a;Goodman and Jungers 2014). Although
anthropogenic effects are often highlighted as primary
contributors to habitat change (e.g., Goodman and
Patterson 1997;Burney et al. 2003,Salmona et al.
2017), evidence now shows forest fragmentation and
modern grass communities in Madagascar predate
colonization by humans (Vorontsova et al. 2016).
Pleistocene fragmentation and shifting vegetation and
habitats may possibly have elevated extinction risks for
some species (Raxworthy et al. 2008b) and reduced rates
of speciation in the gemsnakes.
It is possible that there may be many undetected
Pleistocene speciation events given that young cryptic
reptile species may be difficult to detect and diagnose
and yet appear to be widespread in Madagascar
(Raxworthy et al. 2007;Pearson and Raxworthy 2009;
Florio et al. 2012;Florio and Raxworthy 2016;Ruane et al.
2018), which in turn would cast doubt that Pleistocene
speciation rates declined. However, our method to stress
test these conclusions show, remarkably, the results
are immune even with the addition of as many as 30
unsampled taxa (Fig. 3). It is unlikely that this many
new snake taxa are still remaining to be discovered
given the care taken to survey across the full range of
taxon distributions to identify possible cryptic species of
gemsnakes, including regions predicted to have disjunct
populations and unrecognized endemism (Raxworthy
et al. 2003). Coalescent delimitation and dating was
used to include all candidate taxa not yet described and
therefore as much undetected diversity as possible was
included in our estimates. Ongoing studies should help
clarify population structure for poorly sampled taxa here
and assess processes of speciation to provide a more
granular view of diversification rates and changes in
demography during the Pleistocene.
Decreases in Pleistocene diversification rates in
Madagascar contradict predictions that multiple
Pleistocene shifts in habitat (associated with glacial and
interglacial periods) accelerate speciation as a result
of enhanced isolation and divergence in allopatry. As
an alternative explanation, this speciation decrease
may have been due to interspecific competition as
niches became saturated (Burbrink and Pyron 2010;
Rabosky 2013). Our models show support for this
DD relationship. In a semi-closed system over a
smaller area with limited immigration like Madagascar,
diversity-dependent dynamics are more likely to
prevail. Harmon and Harrison (2015) suggested that
there is little evidence for bounded diversity given
evidence from biological invasions, the fossil record,
experimental manipulation, and weak estimates from
phylogenetic studies (also see Marshall and Quental
2016). Alternatively, changes in speciation rates from
the Neogene to the Pleistocene may not be related
to carrying capacities if: (1) rates of speciation were
directly associated with environmental instability and
habitats changed more rapidly in the Neogene than
the Pleistocene, (2) rates were time dependent and
reduced as groups failed to diversify with changing
environments, or (3) extinction rates were suitably high
(Vrba 1985; Rosenzweig 1995;Pigot et al. 2010;Pyron
and Burbrink 2012a;Quental and Marshall 2013;Moen
and Morlon 2014).
This paradox, where standing diversity was most
likely generated in the Pleistocene yet may also have
been a period of declining diversification rates, has been
addressed in other contexts (Zink and Slowinski 1995). In
summary, if we consider the life span of a species, from
origin to extinction, as lasting on average 2 myr (Marshall
2017), then the probability of discovering extant species
pairs originating in the Pleistocene is not remarkable
given that the probability of finding divergence dates
older than this declines with age as extinction probability
becomes higher. This begs the question,what unique role
does the Pleistocene play in speciation other than being
suitably young for divergence and survival through the
Holocene?
Traits History
Upon colonization of Madagascar from Africa in
the Miocene via oceanic dispersal, diversification into
the main ecological types were generated early with
terrestrial and opisthoglyphous conditions likely being
the original state at colonization (Fig. 6). Arboreal
ecology and the correlated opisthoglyphous dentition
originated soon after colonization. Given that rates
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of diversification did not change within clades of
gemsnakes, it is not surprising that changes in basic
ecology or dentition had no influence on speciation
rates. This demonstrates that while these traits expanded
ecological diversity in gemsnakes, rates of speciation and
accumulation of diversity in Madagascar do not follow
common patterns of trait-driven diversification seen in
many other groups of taxa here and elsewhere (Agrawal
et al. 2009;FitzJohn et al. 2009;FitzJohn 2010;Pyron and
Burbrink 2012b).
Globally, rear-fanged snakes capable of envenomation
are represented in a variety of niches, including fully
terrestrial, aquatic, semi-fossorial, and arboreal habitats
(Weinstein et al. 2011;Peichoto et al. 2012). However, in
Madagascar, we show that opisthoglyphous dentition
is correlated with arboreality, which may serve to hold
or puncture prey in these habitats (Knox and Jackson
2010) where full constriction may be difficult (Harrington
et al. 2018). Envenomating arboreal prey, such as lizards
or frogs, may be required for successful capture, but
we note that some aglyphous taxa have enlarged rear
teeth, yet with no groove, but may still envenomate
prey (Razafimahatratra et al. 2015). Further work on
mandibular and tooth morphology here, which includes
hinged teeth, gaps in tooth rows, enlarged anterior teeth,
and edentulous jaws (Savitzky 1983;Cadle 1996,1999,
2003,2014), integrated with prey capture, envenomation,
and production or constitution of venom should be
examined for a more nuanced view of gemsnake trait
evolution.
Taxonomy
Phylogenetic relationships among gemsnakes and the
taxonomy of this group are now stable; results are
similar to those in Ruane et al. (2015) showing the basic
taxonomic split into opisthoglyphous and aglyphous
clades (Figs. 2and 6; Supplementary data available on
Dryad). As opposed to expectation and other empirical
studies (Edwards 2009;Lambert et al. 2015;Mirarab
et al. 2016), our species-tree and concatenated phylogeny
estimates were similar, showing the same intergeneric
relationships and monophyly of all genera with high
support, with the exception of the relationships among
Ithycyphus, Langaha, and Micropisthodon. While current
techniques cannot handle data sets of this size, we note
that future research should consider the likelihood that
the gemsnake phylogeny may not be best represented
as purely bifurcating, given current research on these
methods (Mallet et al. 2016;Solís-Lemus and Ané
2016) and estimates from other snakes (Burbrink
and Gehara 2018). While the basic relationships are
accurately captured in our phylogeny, instances of
ancient reticulation may provide additional insight into
diversification of endemic species on Madagascar.
SUPPLEMENTARY MATERIAL
Data available from the Dryad Digital Repository:
https://doi.org/10.5061/dryad.07h0n14.
FUNDING
This research was supported by the National Science
Foundation (DEB 1257926 to F.T.B.; DEB 1257610,
0641023 to C.J.R. and R.P., DEB 0423286, 9984496 to
C.J.R., and DEB 9625873, 9322600, BSR 9024505 to
R.A.N. and C.J.R.), the American Museum of Natural
History Gerstner Scholars Program (Gerstner Family
Foundation), and the Richard Gilder Graduate School.
Field research in Madagascar was made possible due to
the assistance of the Ministère de l’Environnement, de
l’Ecologie et des Forêts; Madagascar National Parks; the
Faculté des Sciences (Mention: Zoologie et Biodiversité
Animale), Université d’Antananarivo; and the Faculté
des Sciences, de Technologies et de l’Environnement
(Mention Sciences de la Vie et de l’Environnement),
Université de Mahajanga.
ACKNOWLEDGMENTS
We thank the following for tissue loans for this
work: Port Elizabeth Museum (W. Conradie), Natural
History Museum of Zimbabwe (D. Broadley), J. Reissig,
Museum of Vertebrate Zoology (C. Spencer), Field
Museum of Natural History (A. Resetar), E. Razzetti, R.
Vasconcelos, S. Carranza, R. Gandola, E. Courtois, the
California Academy of Sciences (J. Vindum), American
Museum of Natural History Ambrose Monell Cryo
Collection (J. Feinstein), University of Michigan Museum
of Zoology (G. Schneider), and R. Lawson. We thank
Henry Ferguson-Gow for help digitizing the ecoregion
map.
The authors thank Michelle Kortyna, Sean Holland,
Alyssa Bigelow, and Kirby Birch at the Center for
Anchored Phylogenomics for assistance in generating
and processing the phylogenomic data. We also thank
Liam Revell for assistance with code development.
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