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OPEN Phylogenomics of Psammodynastes

and Buhoma (Elapoidea:
Serpentes), with the description
of a new Asian snake family
Sunandan Das 1*, Eli Greenbaum 2, Jonathan Brecko 3,4, Olivier S. G. Pauwels 3, Sara Ruane 5,
Stacy Pirro 6 & Juha Merilä 1,7
Asian mock vipers of the genus Psammodynastes and African forest snakes of the genus Buhoma
are two genera belonging to the snake superfamily Elapoidea. The phylogenetic placements of
Psammodynastes and Buhoma within Elapoidea has been extremely unstable which has resulted in
their uncertain and debated taxonomy. We used ultraconserved elements and traditional nuclear
and mitochondrial markers to infer the phylogenetic relationships of these two genera with other
elapoids. Psammodynastes, for which a reference genome has been sequenced, were found, with
strong branch support, to be a relatively early diverging split within Elapoidea that is sister to a clade
consisting of Elapidae, Micrelapidae and Lamprophiidae. Hence, we allocate Psammodynastes to its
own family, Psammodynastidae new family. However, the phylogenetic position of Buhoma could not
be resolved with a high degree of confidence. Attempts to identify the possible sources of conflict in
the rapid radiation of elapoid snakes suggest that both hybridisation/introgression during the rapid
diversification, including possible ghost introgression, as well as incomplete lineage sorting likely have
had a confounding role. The usual practice of combining mitochondrial loci with nuclear genomic data
appears to mislead phylogeny reconstructions in rapid radiation scenarios, especially in the absence of
genome scale data.

The snake superfamily Elapoidea, with over 700 species, constitutes more than one-fifth of global snake diversity.
The superfamily includes two of the major medically important venomous snake clades, namely the cosmopoli-
tan Elapidae and the Afro-Middle Eastern Atractaspidinae (Lamprophiidae), and a diverse radiation of other
snakes (namely, Cyclocoridae, Micrelapidae, and subfamilies of Lamprophiidae) spread over Africa, Madagascar,
Europe, and Asia. The phylogeny of this major group of snakes was highly debated until recently, with research-
ers producing strikingly conflicting, poorly supported topologies at deeper levels (e.g.,1–6). The recalcitrance of
the elapoid phylogeny has been attributed to an ancient, rapid r­ adiation1. Recently, Das et al.7 used over 4500
ultraconserved elements (UCE) loci and both multispecies coalescent and concatenation-based phylogenomic
methods to resolve the backbone of the elapoid phylogeny.
However, the relationships between the major (i.e., those treated at subfamily and family ranks) subclades
within Elapoidea have not been the only issue with the phylogenetic systematics of this superfamily. The phylo-
genetic positions of Buhoma, Micrelaps and Psammodynastes could not be consistently resolved with a limited
number of Sanger sequenced loci (e.g.,1,3,4). While studies a­ gree1,3,4,6,11 that these genera belong to Elapoidea, they
have not been assigned to any specific subfamily or family as their positioning within the elapoid phylogeny is
unstable across different works. Das et al.7 were able to infer the phylogenetic placement of Micrelaps with a high
degree of statistical branch support with UCE data. However, the sampling of Das et al.7 did not include Buhoma
and Psammodynastes and hence, the phylogenetic positioning of these two genera remains enigmatic. Thorough

1
Ecological Genetics Research Unit, Organismal and Evolutionary Biology Research Programme, Faculty
of Biological and Environmental Sciences, University of Helsinki, 00014 Helsinki, Finland. 2Department of
Biological Sciences, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968, USA. 3Royal
Belgian Institute of Natural Sciences, Rue Vautier 29, 1000 Brussels, Belgium. 4Royal Museum for Central Africa,
Tervuren, Belgium. 5Life Sciences Section, Negaunee Integrative Research Center, Field Museum, Chicago, IL,
USA. 6Iridian Genomes Inc., Bethesda, MD 20817, USA. 7Area of Ecology and Biodiversity, School of Biological
Sciences, Kadoorie Biological Sciences Building, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR,
China. *email: sdassnake@gmail.com; sunandan.das@helsinki.fi

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anatomical comparisons of these two genera with other major elapoid subclades have not been done either (Das
et al.7 only reported that cranial features do not support the assignment of Psammodynastes to Pseudaspidinae).
The Mock Vipers of the genus Psammodynastes have an extensive range in south and south-east Asia, includ-
ing the Indonesian ­archipelago8,9. The genus includes two species—P. pictus and P. pulverulentus. These rear-
fanged snakes have historically been assigned to the catch-all family C ­ olubridae10. The phylogenetic placement
of Psammodynastes has been contentious. Within Elapoidea, Psammodynastes has been placed—I. as sister to
­Lamprophiidae11, II. sister to Buhoma, with this cluster not being closely related to P
­ seudaspidinae1, III. basal split
to a polytomous clade consisting of Psammophiinae, Prosymninae, Pseudaspidinae and Buhoma2, IV. sister to
Buhoma, with this cluster sharing a common ancestor with ­Pseudaspidinae3,5, V. nested within ­Psammophiinae4,
and VI. sister to ­Pseudaspidinae6.
The phylogenetic position of the African forest snakes, Buhoma spp., has also been highly unstable. Points
II, III and IV on the problematic phylogenetic placements of Psammodynastes are also pertinent to Buhoma.
Apart from those, Buhoma has also been found to be—I. sister to (Psammodynastes, Lamprophiidae)11, II.
­ lapidae6, and as III. polyphyletic in the phylogeny of Figueroa et al.4, with B. procterae being sister to
sister to E
Prosymninae and B. depressiceps as sister to Elapidae.
The aim of this study is to resolve the phylogenetic relationships of the genera Psammodynastes and Buhoma
within the Elapoidea and to make appropriate taxonomic and nomenclatural decisions regarding these two
genera. We used ultraconserved elements extracted from published and newly generated reference genomes
and from a published target capture sequencing dataset to infer the phylogenetic interrelationships of Psam-
modynastes and Buhoma. For comparative purposes, we also utilized legacy nuclear and mitochondrial markers
for phylogenetic inference. To look for potential apomorphies and/or diagnostic character states, we studied and
compared micro-computed tomographic scans of crania of Psammodynastes, Buhoma and members of every
elapoid family/subfamily level clade. We also investigated the sources of conflict in different datasets.

Results
Phylogenomics
The 50, 75 and 95% complete UCE datasets consisted of 4514 (~ 5.3 M bp), 3993 (~ 4.7 M bp) and 2467
(~ 2.9 M bp) loci respectively. The concatenated Sanger loci dataset was 7439 bp long (BDNF 669 bp, C-MOS
735 bp, RAG1 1011 bp, RAG2 912 bp, 12S 979 bp, 16S 1342 bp, CYTB 1098 bp and ND4 693 bp).
In all the UCE-only wASTRAL-h MSC phylogenetic trees, Psammodynastes pulverulentus represented a
unique branch that is neither nested within nor sharing the most recent common ancestor with any of the rec-
ognised family group level taxa (Fig. 1a, Figs. S1–S3). This branch was the sister clade consisting of Elapidae,
Lamprophiidae and Micrelapidae and this bipartition received a local posterior probability (localPP) of 1, the
highest possible value.
In all the UCE-only and UCE + traditional nuclear marker concatenated data-based ML phylogenies (Fig. 1b,
2a, Figs. S4–S9), P. pulverulentus was recovered as sister to a clade comprising Elapidae, Lamprophiidae and
Micrelapidae. UFBoot branch support values, computed by resampling from within partitions and sites within
each partition to lessen the chances of getting artifactually inflated values, were 100 in all the UCE-only phylog-
enies. UFBoot values ranged from 98 to 93 in the UCE + traditional nuclear loci trees. The ExaBayes consensus
topology recovered P. pulverulentus in the same position in the tree as in the ML topologies, with moderate
posterior probability (PP) support of 0.86 (Fig. 2b, Fig. S13).
The position of the Buhoma spp., represented in our dataset only by Sanger-sequenced loci, was not resolved
with significant branch support. In all the UCE + traditional nuclear marker ML and BI phylogenies, Buhoma
genus was monophyletic (Fig. 2a, b, Figs. S7–S9). In the 50% complete UCE + traditional nuclear ML phylogeny,
the common ancestral branch of Buhoma spp. formed a polytomy with two other clades, namely (Pseudaspidinae
(Prosymninae, Lamprophiinae)) and (Atractaspidinae, Psammophiinae) (Fig. S7). In the ML and BI phylogenetic
trees estimated from respectively 75% and 95% complete UCE plus traditional nuclear markers, Buhoma genus
was sister to the Pseudaspidinae (Fig. 2a, b, Figs. S8, S9). The monophyly of Buhoma and the phylogenetic place-
ments of this genus did not receive high UFBoot or PP support in any of the phylogenies. However, the sister
taxon relationship between B. depressiceps and B. marlieri always received high UFBoot and PP branch support.
In the UCE + traditional mitochondrial and nuclear (mito-nuclear hereafter) loci ML phylogenetic trees, B.
depressiceps + B. marlieri was sister to P. pulverulentus. The P. pulverulentus plus B. depressiceps-marlieri clade was
sister to (Elapidae (Micrelapidae, Lamprophiidae)) (Figs. S10–S12). Buhoma procterae was sister to Prosymninae
in the 95% complete UCE + traditional mito-nuclear loci phylogeny. In the 50 and 75% complete UCE + tradi-
tional mito-nuclear phylogenies Buhoma was sister to Pseudaspidinae. Barring the sister taxon relationship
between B. depressiceps and B. marlieri, all the phylogenetic positions of Buhoma and Psammodynastes in the
UCE + traditional mito-nuclear phylogenies were poorly supported.
We did not recover a monophyletic Buhoma in any of the traditional loci-only phylogenies (Figs. S14–S16). In
the ML tree inferred from traditional nuclear markers, B. procterae was sister to Psammodynastes and these two
were clustered with depressiceps-marlieri. These branches received UFBoot supports of ~ 85. This subclade itself
was sister to Pseudaspidinae but with low support. In the traditional mito-nuclear phylogeny, a clade consisting
of P. pulverulentus and B depressiceps + B. marlieri was the basalmost split within Elapoidea but B. procterae was
clustered with Prosymninae in a more nested position in the tree (Fig. S16). In the mitochondrial phylogeny,
neither Psammodynastes nor B. depressiceps + B. marlieri were recovered as members of Elapoidea but B. procterae
is sister to Prosymninae within Elapoidea (Fig. S15).
Overall, the UCE-only topologies showed the same relationships between the major elapoid subclades as
­in7. The same held true for UCE + traditional nuclear and mito-nuclear loci topologies. The inferred generic
interrelationships within Elapidae, whose taxon sampling increased ­from7, is well corroborated by other studies

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Figure 1.  Weighted ASTRAL-hybrid multispecies coalescent phylogeny (a) and concatenation-based

maximum likelihood phylogeny (b) from 50% complete ultraconserved elements dataset. Branches with a
circle received > 0.95 local posterior probability or > 85% ultrafast bootstrap support. AT Atractaspidinae, CY
Cyclocoridae, EL Elapidae, LM Lamprophiinae, MC Micrelapidae, PD Psammodynastidae new family, PM
Psammophiinae, PR Prosymninae, PX Pseudoxyrhophiinae.

(e.g.,3,5). The traditional nuclear gene phylogeny inferred a monophyletic Lamprophiidae (with Psammodynastes
in it) and at the deeper level a polytomy between Micrelapidae, Cyclocoridae and Elapidae + Lamprophiidae.
Subfamilial interrelationships within Lamprophiidae in the traditional nuclear gene phylogeny differed from UCE
trees. However, the mitochondrial and mito-nuclear topologies showed marked incongruence—this includes
the placement of basal splits (assuming UCE tree to be ‘correct’), namely Cyclocoridae and Elapidae, as highly
nested within Elapoidea and a failure to recover a monophyletic Lamprophiidae.

Concordance factor, quartet support and reticulations

Site concordance factor (sCF) of the split between Psammodynastes and (Elapidae (Micrelapidae, Lamprophii-
dae)) clade was 33.95% in the UCE ML phylogeny (Fig. S17). The sCF value for other deep splits (i.e., between
clades recognised at family/subfamily rank) in this phylogeny ranged from 33.82 to 54.99%. The quartet support
for the phylogenetic position of Psammodynastes in the wASTRAL-h phylogeny was 38% (Fig. S20). The percent-
age falls within the range of the quartet support for other deep splits in the elapoid phylogeny.
In the legacy nuclear marker tree, the branch leading to Buhoma procterae and P. pulverulentus had an sCF of
45.72% (Fig. S18). The branch ancestral to B. procterae + P. pulverulentus and B. depressiceps + B. marlieri received
an sCF of 55.08% whereas the clustering of this clade to Pseudaspidinae had a 26.58% sCF. In the mitochondrial
marker phylogeny, the clustering of B. procterae with Prosymninae had 33.33% sCF whereas the clustering of B.
depressiceps and B. marlieri with the colubrid outgroup received an sCF of 31.36% (Fig. S19).
Phylogenetic networks estimated with a maximum of 2, 5 and 6 permissible reticulations were the top
three highest log-likelihood estimates in PhyloNetworks analyses, with 2 being marginally better than 5 and 6
(Fig. 3a–c, Fig. S22). None of these MSCNs had more than two inferred reticulations. A reticulation was between
Lamprophiinae (represented by Boaedon olivaceus) and Atractaspidinae (represented by Atractaspis reticulata),
with inheritance probability of the minor reticulation edge being ~ 0.25. Another reticulation event involving
either Micrelapidae or Cyclocoridae (represented by Cyclocorus), Elapidae (represented by Naja melanoleuca)
and Psammodynastes or a ghost introgression (i.e., an introgression from an extinct/unsampled t­ axon12) was
estimated but this had a minor edge inheritance probability > 0.1.

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Figure 2.  Concatenation-based maximum likelihood (a) and Bayesian inference (b) phylogeny from 95%
complete ultraconserved elements dataset and traditional nuclear markers. Taxa with an underline are
composites (in the traditional markers), though they are labelled with species contributing the largest data
partition, i.e., UCEs, see supplementary table 1. Branches with a circle received > 0.95 posterior probability
or > 85% ultrafast bootstrap support. Abbreviations as in Fig. 1.

Figure 3.  Multispecies coalescent phylogenetic networks with the best log-likelihoods, PhyloNetworks network
with 2 (a), 5 (b) and 6 (c) permissible reticulations and PhyloNet network with 4 (d), 5 (e) and 6 (f) permissible
reticulations. Major reticulation edges are blue and minor reticulation edges are orange. Values on the edges are
the inheritance probabilities. Networks estimated with PhyloNetworks are unrooted.

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PhyloNet MSCNs, on the contrary, inferred more horizontal edges with more permissible reticulations.
MSCNs with 4, 5 and 6 permitted reticulations achieved the three best log-likelihoods, the one with 6 being
the best by a narrow margin (Fig. 3d–f, Fig. S23). These networks too inferred a reticulation event involving
Lamprophiinae or an extinct/unsampled sister and Atractaspidinae. The inheritance probability of this reticula-
tion approached 0.5. The six reticulations MSCN had one 0.27 minor edge inheritance probability reticulation
involving Elapidae and Psammodynastes. PhyloNet also inferred reticulation edges that did not originate from
either the sampled extant lineages or their common ancestral branches. Such reticulations have been interpreted
as ghost introgression, i.e., gene flow from an extinct lineage, in literature (e.g.,25). However, we refrain from
interpreting these particular horizontal edges as ghost introgression for reasons provided in the Discussion.

Timetree
The divergence of Psammodynastes from a clade consisting of Elapidae, Lamprophiidae and Micrelapidae was
dated at ~ 49 MYA (confidence interval 38.8–61.8 MYA) in the early Eocene (Fig. 4, Figs. S24, S25). The clade
comprised of Buhoma procterae, B. depressiceps + B. marlieri and Pseudaspis cana + Pythonodipsas carinatus was
dated as a polytomy at 39.6 MYA (confidence interval ~ 30 to 51.2 MYA), in the late Eocene whereas the diver-
gence of B. depressiceps and marlieri was dated to be as recent as 9.7 MYA, the late Miocene. All the other diver-
gence dates were close to those estimated ­by7 and confidence intervals also broadly overlapped.

Cranial osteology
Psammodynastes
The maxilla of Psammodynastes pulverulentus (n = 1; Table S6) is elongated, with distinct ascending, palatine
and ectopterygoid processes (Fig. 5a, c). The maxilla bears small, ungrooved teeth in the beginning, then an
ungrooved, elongated ‘fang’, then a series of small, ungrooved teeth and finally, enlarged, grooved rear-fangs. This
maxillary dentition serves to distinguish this genus from all the elapoids examined by us except Psammophis spp.
Both the maxillary and the mandibular dentition of Psammophis are almost identical to those in Psammodyn-
astes. Psammodynastes can be distinguished from Psammophis from the lack of a canthal ridge on the prefrontal
lateral lamina and a moderate-sized optic foramen (Fig. 4b) (versus very large, lacertiform optic foramen of
Psammophis).
Additionally, Psammodynastes pulverulentus possesses a very pronounced (nearly as wide as the orbital lamina
of the parietal and as high as wide), squarish postorbital crest on the anterolateral aspect of the parietal bone for
articulating the postorbital (Fig. 5b, d). This is a usual site of origin of the levator anguli oris muscle in colubroid
snakes and very likely this crest also serves that purpose. Among the examined genera, only Ophiophagus hannah
and Notechis scutatus, both belonging to Elapidae, possess a very well-developed, rounded or squarish postorbital
crest. Elapid genera Naja, Cyclocorus and Levitonius of Cyclocoridae family, Alluaudina, Compsophis, Langaha,
Leioheterodon, Lycodryas and Madagascarophis belonging to Pseudoxyrhophiinae and Buhoma can develop a
distinct, narrow or triangular, tapering process from the ventral end of the postorbital ridge; this process, how-
ever, is very different from the large, squarish crest in Psammodynastes.

Buhoma
Unlike Psammodynastes, the Buhoma (n = 5; Table S6) cranium has few distinctive features that can serve to
unequivocally distinguish this genus from other elapoids (Fig. 6a–c, Figs. S26–S29). All the examined Buhoma
spp. have a large perforation, one-third to half the diameter of the lacrimal foramen, on their prefrontal lateral
lamina. Studied elapoids, barring Naja, either lack such a foramen or possess only a very small one. However,
this character seems somewhat variable within Buhoma itself, some specimens having two small foramina on
one prefrontal lateral lamina and a large one on the other, and some Naja spp. possess a similar foramen.
Apart from the elapoid subclades with unequivocal diagnostic features (viz., dentition in elapids, micrelapids,
prosymnines, Psammodynastes and some psammophiine and atractaspidines or strong fossorial adaptation in
the snout complex bones and braincase in prosymnines and some atractaspidines), Buhoma cannot be broadly
diagnosed from any major elapoid subclade. There is no synapomorphy associating Buhoma with any recognized
family or subfamily-level subclade.
Buhoma marlieri can be distinguished from all the other congenerics in having a wide, squarish, shield-like
premaxillary ascending process, with a slightly constricted base and a slightly embayed or W-shaped upper
margin (versus a narrow, rectangle-shaped ascending process in B. depressiceps and a triangular, dorsally tapering
ascending process in B. procterae and B. vauerocegae). We did not find any cranial features clearly distinguishing
B. procterae from B. vauerocegae.

Discussion
Multispecies coalescent and concatenation-based approaches inferred highly congruent, well-supported phy-
logenies for Elapoidea and one of the focal genera, i.e., Psammodynastes, when the input dataset was UCE or
UCE + legacy nuclear loci. However, there can be discordant signals in the underlying data (viz., incongruent gene
trees) even with highly supported phylogenies. Such discordance may result from both biological and analytical
­factors13, such as incomplete lineage sorting (ILS), different types of data (viz., organellar versus nuclear genome),
hybridisation and introgression, rogue taxa etc. Site concordance factor and quartet support for the best and the
alternative topologies inferred with UCEs show the presence of a considerable amount of conflict in the data
when it comes to the backbone of the phylogeny. The sCF value was close to 33% on several branches (Figs. S17,
S20), including the split between Psammodynastes and the rest of the Elapoidea. Such sCF value is indicative
of a paucity of decisive phylogenetic signal in favour of any particular resolution of quartet topologies in these
parts of the tree and possibly also I­ LS18. These branches, including the common ancestor of Psammodynastes

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Figure 4.  RelTime time-calibrated phylogeny of Elapoidea.

and the other elapoids, were also very short in terms of both the actual time and coalescent units. The problem
of deep coalescence in rapid radiation, with a concentration of very short branches, is a well-known ­issue7,14,15.
Therefore, both low phylogenetic signal and ILS might have affected these branches. Nevertheless, this was not
a consistent pattern across the elapoid phylogeny as some very short branches (for instance, the earliest split of
Cyclocoridae) had a high sCF value and quartet support.
Gene flow can give rise to conflicts similar to those caused by ­ILS19, necessitating a test of hybridisation and
introgression prior to accepting the null hypothesis of ILS being the primary driver of discordance. Instances

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Figure 5.  Lateral (a), dorsal (b), ventral (c) and anterolateral (d) views of the cranium of Psammodynastes
pulverulentus (UMMZ:Herps 175728). Museum acronym: UMMZ University of Michigan Museum of Zoology.

Figure 6.  Lateral (a), dorsal (b) and ventral (c) views of the cranium of Buhoma depressiceps (BE-RBINS-VER-
REP-16404). Inset—anterior view of B. depressiceps (BE-RBINS-VER-REP-16404), B. marlieri (BE-RBINs-
VER-REP-8620), B. procterae (BE-RBINS-VER-REP-18541) and B. vauerocegae (BE-RBINS-VER-REP-18542),
showing differences in the premaxillary ascending process (pointed at by the yellow line). Museum acronym:
RBINS Royal Belgian Institute of Natural Sciences.

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of hybridisation and introgression are often there in the history of rapid radiations (e.g.,7,16) and may even
drive such explosive d ­ iversification17. With several reports of widespread reticulations in vertebrate phylogeny
(e.g., ), including even instances of actual hybrid s­ peciation21,22, it is now evident that reticulation events have
20

been widespread in the evolutionary history of vertebrates. The Maximum Pseudolikelihood networks estimated
with PhyloNet and PhyloNetworks differ in the former preferring hypotheses with more reticulation events. The
best networks of both methods inferred either a direct (both PhyloNet and PhyloNetworks) or ghost reticulation
(only PhyloNet) event involving Lamprophiinae and Atractaspidinae. Lamprophiinae or an extinct or unsampled
lineage sister to lamprophiines has been identified as the donor and Atractaspidinae as the recipient in most of
the higher log-likelihood networks. Such a reticulation event is not biogeographically implausible considering
both lamprophiines and atractaspidines are mostly distributed in sub-Saharan Africa. Psammodynastes was not
implicated in any genetic inflow/outflow events with an inheritance probability equalling or exceeding 0.1 in
PhyloNetworks analyses and PhyloNet reported only ghost introgression events or, with six permissible reticu-
lations, an introgression event from Elapidae (Fig. 3e–f). Phylogenetic studies usually recover Calliophis as the
earliest split within the Elapidae (e.g.,3,6), a genus with an Asian distribution like Psammodynastes. Therefore, it is
possible that the stem lineages of both elapids and Psammodynastes coexisted in the same region and experienced
some gene flow. However, there are serious caveats in these interpretations of the network analyses. Elapoidea is
a radiation dating back to the Eocene and hence, it is very likely that several early major lineages are now extinct.
Ghost introgression in the past from extinct lineages is particularly probable in such c­ ases12. The presence of such
unaccounted-for ghost introgression can significantly distort the result of many common tests of gene ­flow23,24.
MPL network methods have gained popularity owing to being computationally efficient, unlike full likelihood
or Bayesian approaches and minor reticulation edges from an interbranch to a terminal branch have routinely
been interpreted as indicative of ghost introgression (e.g.,23,25). However, recent investigations with simulation
data and reanalysis of published empirical data demonstrated that pseudolikelihood approaches are not capable
of discerning ghost introgression from other plausible scenarios and the direction of gene flow estimated with
such methods may not be reliable ­either24. Therefore, we caution against regarding the reticulation edges from
ancestor to terminal branches in our networks as evidence of gene flow from unsampled, or more likely, extinct
lineages. We further stress that care should be exercised with the donor-recipient directions in estimated reticula-
tion events in the present study. We also refrain from interpreting some inheritance probabilities approaching 0.5
as suggestive of hybrid origin of a lineage because continuous gene flow can also result in such a ­scenario26. Phy-
loNetworks and PhyloNet disagree over these scenarios and phylogenetic networks in general are not designed
to discern true hybridisation from other types of reticulation e­ vents27. A set of conservative conclusions would
be that – 1. reticulation events probably occurred in the Afro-Malagasy radiation (Lamprophiidae), the stem
lineages (or their extinct sister taxa) of Lamprophiinae and Atractaspidinae being the more likely contenders,
2. some relatively less probable reticulation events may involve early stem elapids, cyclocorids, Psammodynastes
and possibly lineages that are now extinct.
Given that reticulation edges were present in phylogenetic networks, including the ones having the best log-
likelihood, the null hypothesis that ILS solely explains all the conflicts in the backbone of the elapoid Tree of
Life cannot be accepted. Also, not all the deep branches have been consistently implicated in reticulation events.
Thus, ILS cannot be rejected as the principal factor (probably in conjunction with low phylogenetic signal) in
producing discordant signal on those bipartitions.
Site concordance factors frequently went down below 33% for deep splits in traditional mito-nuclear loci
phylogenies. Branch supports were also low in these phylogenies. The mitochondrial topology was especially
dissimilar from those estimated with UCEs. Concatenation of mitochondrial loci with traditional nuclear mark-
ers and UCEs lowered the ultrafast bootstrap support throughout the backbone of the phylogeny and in the
case of Sanger nuclear markers alone, changed the topology as well. Evolutionary geneticists have long treated
mitochondrial genomic sequences separately from nuclear data, and this has resulted in identification of inter-
esting evolutionary phenomena such as directional gene flow mediated by one sex (e.g.,28), possible despecia-
tion/reverse speciation (e.g.,29) etc. In contrast, it has been the usual practice in phylogenetics to concatenate
mitochondrial and nuclear sequences, even when the taxonomic group under investigation is an old radiation
(e.g.,1,4,6) for which mitochondrial genes with fast substitution rates may not be a suitable phylogenetic marker.
The obscuring of the already low phylogenetic signal on the short, deep edges by the mutational saturation on
the long descendant edges is indeed an issue with resolving ancient, rapid r­ adiations14. Mitochondrial genes
can and probably do exacerbate this problem. Therefore, it seems advisable to treat mitochondrial and nuclear
markers separately when studying old, rapid radiations.
Genus Buhoma is an interesting case; this genus was not monophyletic in the phylogeny ­of4 and in our
UCE + traditional mito-nuclear marker phylogenies and the phylogenies computed only with traditional mark-
ers. These phylogenies also recovered some of the controversial placements of Buhoma spp. inferred in past
studies, such as Buhoma as sister to Psammodynastes (as ­in3) or Prosymna (as i­ n4), even though our taxon and
gene sampling both were higher for this genus than those in previous studies. What can possibly explain these
conflicts? The common ancestral branch of the three Buhoma spp. is short in the combined UCE-Sanger nuclear
loci phylogenies. The timetree has a polytomy of B. procterae, B. depressiceps + B. marlieri and Pseudaspis + Pytho-
nodipsas but the branches leading to B. procterae and B. depressiceps + B. marlieri are long. It therefore appears
that Buhoma itself could be a case of ‘ancient, rapid radiation’. Rapid diversification soon after the origin of the
stem Buhoma might have resulted in the accumulation of little molecular and morphological synapomorphies
that could help cluster them in a cladistic analysis. Millions of years of evolution on the long descent lineages then
possibly led to mutational saturation which caused further obfuscation of any meaningful phylogenetic signal.
Lack of decisive support for monophyly in small scale molecular data, combined with a potentially misleading
‘signal’ (phylogenetic noise) causes some or all the Buhoma spp. to behave as rogue taxa. The association of
Buhoma with Psammodynastes is also possibly caused by the same factor. Homoplasy most probably resulted in

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clustering of one or two Buhoma spp. with Psammodynastes. In the absence of genome-wide data, this resulted
in a more nested position for Psammodynastes in the phylogeny, i.e., within Pseudaspidinae (as i­ n3 and our tra-
ditional marker trees). When combined with UCE data, the position of Psammodynastes was correctly inferred
but this had the effect of pulling some Buhoma spp. down the tree when the mitochondrial partition was added.
Based on these considerations and biogeography, we regard the association of Buhoma with Psammodynastes as
an artefact of inadequate data and homoplasy. One theoretical expectation of rogue taxa is that by being able to
get placed almost anywhere in the phylogeny, such taxa lower the overall branch support throughout the t­ ree5.
The marked lowering of branch support in the UCE + Sanger sequenced mito-nuclear loci tree may be a combined
effect of both highly conflicting signals from the mitochondrial genes and the rogue behaviour of Buhoma. Some
phylogenetic problems are resolvable only with either whole or reduced representation genome-scale d ­ ata30 and
Buhoma seems to be one such problem. However, there is support, albeit weak, in the nuclear genes to unite
Buhoma with Pseudaspidinae and therefore, we provisionally regard Buhoma as a member of Pseudaspidinae,
as was done by Pyron et al.3. It is noteworthy that the divergence between B. depressiceps and B. marlieri is older
than that between several other species pairs in our timetree (Fig. S24). This and the osteological difference in
the premaxilla support the decision of Chippaux and ­Jackson31 to treat B. marlieri as a separate species.
The phylogenetic position of the mock vipers, Psammodynastes, has been well-resolved and consistent across
methods when using target capture data in the present study. There are also distinct apomorphic character states
diagnosing this lineage from other elapoid subclades. Psammodynastes is the sister taxon to the common ancestral
branch of Elapidae, Micrelapidae and Lamprophiidae and is not nested within any family group taxon. Therefore,
the only Linnean rank appropriate for it would be a family. There are no available suprageneric nomina to which
this genus was exclusively assigned in the past. Hence, we describe Psammodynastidae new family, belonging to
the Elapoidea superfamily, to accommodate Psammodynastes:
Psammodynastidae new family
ZooBank LSID: urn:lsid:zoobank.org:pub:1F332821-51B7-4E47-9FBD-84E79E3269EB
Type genus: Psammodynastes Günther, 1858
Type species: Psammodynastes pulverulentus (Boie, 1827)
Etymology: The generic name is a combination of ancient Greek word psammos (ψάμμος, meaning sand) and
dynastes (δυνάστης, meaning ruler). We derive the family name by adding -idae to the stem of the generic suffix.
Contents: Psammodynastes pulverulentus (Boie, 1827), P. pictus Günther, 1858
Diagnosis and definition: Maxilla of Psammodynastes bears 2 ungrooved, small teeth at the anterior end, fol-
lowed by 1–2 enlarged, ungrooved teeth (occasionally with several shallow, non-venom carrying ­grooves32), then
5–9 small, ungrooved teeth and finally, 2 enlarged, grooved rear fangs (present s­ tudy10,33). The mandible bears
enlarged teeth at the anterior end (present ­study33). The Psammodynastes cranium has a very well-developed
postorbital crest from the parietal. Vertebrae of Psammodynastes bear hypapophyses t­ hroughout10. The combina-
tion of—I. maxillary dentition, II. pronounced postorbital crest, III. prefrontal bone devoid of a canthal ridge and
IV. moderate-sized optic foramen (i.e., frontal orbital laminae contact the crest on the parasphenoid cultriform
process for at least two-thirds of the length of the latter) distinguishes Psammodynastidae new family from all
other elapoid families and subfamilies.
In external appearance, the head is distinct from the neck and has a canthal ridge (not to be confused with the
canthal ridge on the prefrontal bone in Psammophiinae) anterodorsal to a large eye with a vertical pupil. There
are 8–9 supralabials, of which the 3rd to 5th enter the orbit, 8–9 infralabials, and the temporal formula is 2 + 2
or 2 + ­38,33. Dorsal scales are smooth, devoid of apical pits and there are usually 17 (rarely 19) dorsal scale rows
at ­midbody8,10,33. There are 146–175 ventrals, a single anal plate and 44–88 paired subcaudal s­ cales8.
Distribution: South Asia (North-eastern India, Nepal), southern China, Southeast Asia (Myanmar, Thailand,
Malaysia, Indonesian archipelago, Cambodia, Laos, Vietnam, Philippines) and Taiwan.

Materials and methods

Genome sequencing, processing of target capture and whole genome sequence data
The ultraconserved elements (UCE) of 47 taxa, including 44 elapoids and three of the four outgroups, are from
Das et al.7. These samples were specifically enriched and sequenced for U ­ CEs7.
UCEs of 11 more elapoids, including one of the focal taxa of this paper—Psammodynastes pulverulentus,
and ten elapids, and one additional outgroup taxon belonging to Homalopsidae, Myanophis thanlyinensis, were
harvested in silico from whole genome sequences. The reference genome sequencing of P. pulverulentus and
assembly were performed at Iridian Genomes (Bethesda, USA). Sequencing of reference genome, described in
this section, and Sanger sequencing and CT scans, reported under other sections, utilised only preserved museum
specimens and no live animals were collected/experimented upon/euthanised for our study. Genomic DNA from
a P. pulverulentus (FMNH 273629) from the collection at the Field Museum of Natural History (Chicago, USA)
was extracted with the Qiagen DNAEasy kit following the standard instructions. Library preparation was done
using Illumina TruSeq kit with standard adapters and following the standard protocol. Sequencing at 105X cov-
erage was carried out on llumina X-Ten platform. Raw reads were preassembled to scaffold (103,289 scaffolds)
level with SPAdes version 3.15.434 and Zanfona (https://​github.​com/​zanfo​na734/​zanfo​na) was used for genome
finishing. The assembly has been made available on the NCBI Genome database (https://​www.​ncbi.​nlm.​nih.​gov/​
datas​ets/​genome/​GCA_​02580​2295.1/). Rest of the genome assemblies were obtained from the NCBI Genome
database (NCBI Taxonomy since July, 2023). Reference assembly name, link and associated publications, if any,
for all the reference genomes, including that of P. pulverulentus, are listed in the Supplementary Information.
Reference genome sequences for all the elapoids and a homalopsid available in the NCBI Genome (NCBI
Taxonomy) database were downloaded. The assembly of Hydrophis hardwickii has assembly contamination and
hence, was not incorporated in the downstream analyses. The phyluce pipeline, version 1.7.2,35 was used to

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match the Tetrapods-UCE-5Kv1 bait set (https://​www.​ultra​conse​r ved.​org/) to the genome sequences and create
a database using the phyluce_probe_run_multiple_lastzs_sqlite function. Using the lastz database generated in
that step, loci matching the UCE probe were extracted with the phyluce_probe_slice_sequence_from_genomes
function, retaining 400 bp of downstream and upstream flanks. The outputs were symlinked to the contigs gener-
ated from the dataset of Das et al.7. To generate the necessary probe match database and configuration files for the
downstream processing, the probe matching step was repeated with phyluce_assembly_match_contigs_to_probes
command with the same Tetrapods-UCE-5Kv1 probe set. Finally, the phyluce_assembly_get_match_counts and
the phyluce_assembly_get_fastas_from_match_counts functions were used to extract the UCEs for all the 59
taxa. ­MAFFT36 was used, in conjunction with edge trimming, to align each individual UCEs locus dataset on
phyluce. Preparation of the final UCE multiple sequence alignments for phylogenomic analyses was carried out
in the phyluce pipeline and includes three steps, namely, removal of UCE locus name from the taxon nomen,
preparing datasets of three different levels of completion (viz., 50, 75 and 95% complete datasets) and generating
concatenated datasets for combined data analyses.

Sanger sequencing and preparation of Sanger sequence data

For this study, we have sequenced mitochondrial cytochrome b (CYTB), NADH dehydrogenase subunit 4 (ND4)
and 16S ribosomal RNA gene (16S) and nuclear brain-derived neurotrophic factor (BDNF), oocyte maturation
factor mos (C-MOS) and recombination activating gene 1 (RAG1) of Buhoma depressiceps (UTEP 22599) and B.
marlieri (UTEP 22598) specimen in the collection of the University of Texas at El Paso (El Paso, USA). The PCR
and sequencing primers for BDNF were as i­n37. The rest of the primers were f­ rom38. The laboratory protocols,
including those for genomic DNA extraction, PCR amplification and Sanger sequencing f­ ollow38.
The raw Sanger sequencing reads were assembled using the Pearl programme of the Tracy ­toolkit39 hosted at
GEAR-GENOMICS (https://​www.​gear-​genom​ics.​com/). The assembly was visually checked in Pearl and base
calls were rectified, based on thorough inspection of the chromatogram, if needed. The ND4 and CYTB sequences
of B. marlieri were of low quality and hence, those were discarded from subsequent analyses. Open Reading
Frames (ORF) of the protein-coding sequences were detected with NCBI ORF Finder (https://​www.​ncbi.​nlm.​
nih.g​ ov/o
​ rffin
​ der/), with setting the ORF start codon as any sense codon. Afterwards, we performed a megablast
on NCBI BLAST with default parameters to confirm the identity of the sequences we generated.
We searched for and obtained sequences of mitochondrial CYTB, ND4, 16S and 12S (12S ribosomal RNA
gene) and nuclear BDNF, C-MOS, RAG1 and RAG2 (recombination activating gene 2) for elapoids and outgroups
for which we have UCE data from NCBI GenBank. While most of the Sanger sequences in our dataset belong
to the same species as in the UCE dataset, in a few instances we have included the sequence of a closely related
congener if the sequence of a gene for a particular taxon is unavailable. This was done only if the taxon is the sole
representative of its genus in our dataset and the congeneric status of the replacement is taxonomically uncon-
troversial. GenBank accession numbers of the sequences, including the ones sequenced by us for this study, and
any other associated information are provided in the Supplementary Information (Table S1).
We do not have tissue sample of Buhoma procterae and therefore used CYTB, ND4, 12S, 16S, C-MOS and
RAG2 sequences f­ rom11. We also obtained 12S and RAG2 sequences of B. depressiceps generated in the same study.
The B. depressiceps specimen used in that work originated from Gabon and hence, is unambiguously referable
to B. depressiceps and not B. marlieri.
For taxa with a reference genome, we harvested nuclear genes from the genome assembly itself to alleviate
the need for creating composites. To do this, we BLAST-ed conspecific/congeneric/confamilial sequences of the
four nuclear loci separately against a genome with megablast and default parameters. The matched region was
extracted and megablast-ed against the GenBank database, irrespective of whether the genome was annotated
or not, to ensure that we have got the exact gene we were looking for. Nuclear loci sequences harvested in this
way were added to our Sanger loci dataset. For the query sequence accession number, see Supplementary Infor-
mation (Table S2–S5).
Sequences were aligned with ­MUSCLE40, implemented on MEGA 1­ 141, with default parameters. Alignments
were scrutinised visually. Overhangs of a few very long sequences at the ends that were missing in other sequences
were trimmed. External gaps were coded as missing data with SequenceMatrix 1.942. SequenceMatrix 1.9 was
used to concatenate the Sanger loci. We have prepared the following concatenated datasets—(1) four nuclear
loci, (2) four mitochondrial loci, (3) all eight Sanger loci, (4) four nuclear loci plus 50, 75 and 95% complete, con-
catenated UCE datasets, and (5) all eight Sanger loci plus 50, 75 and 95% complete, concatenated UCE datasets,
using SequenceMatrix 1.9. Prior to combining Sanger nuclear and UCE loci, we ran local blastn (blast-2.10.0 +)
of unaligned multifasta query of Sanger nuclear loci, with e-values 1­ e−50 and 0.05, against a database (prepared
with makeblastdb [blast-2.10.0 +]) of unaligned multifasta of UCEs to ensure that UCEs flanks did not overlap
any of the four nuclear genes. No match was detected in the local blastn searches.

Phylogenomic analyses
We inferred quartet-based multispecies coalescent species trees from gene trees of individual UCE loci, maxi-
mum likelihood (ML) phylogenies from concatenated UCE only dataset, concatenated UCE + traditional marker
dataset and combined traditional marker dataset and Bayesian Inference (BI) phylogeny from concatenated
UCE + traditional marker dataset.
For the multispecies coalescent (MSC) phylogenetic tree estimation, we used weighted ASTRAL hybrid
(wASTRAL-h) ­method43. Algorithms of weighted ASTRAL class weigh quartets based on branch support, branch
length or both without requiring any arbitrarily specified threshold. This has been demonstrated to result in
phylogenies more accurate than those produced by ASTRAL-III ­algorithm43. Among the three different weight-
ing approaches, the one that weighs based on both the branch length and support (i.e., wASTRAL-h) performs

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the best. ML gene trees were estimated for 50, 75 and 95% complete UCE loci set with IQ-TREE 2.2.0.544 with
1000 ultrafast ­bootstrap45 replicates per gene tree. Model selection for each locus was performed with the default
­ModelFinder46 option. MSC species tree was then inferred from the gene trees set with wASTRAL-h with default
settings.
We used IQ-TREE 2.2.0.5 to infer ML species tree from the UCE-only concatenated matrices of 50, 75 and
95% completeness. The default model selection and optimum partitioning scheme finding algorithm of IQ-TREE,
i.e., ModelFinder plus partition merging (MPF + MERGE option), proved to be computationally infeasible on
our cluster. Hence, we chose the TESTMERGE option, which functions identically to ­PartitionFinder47 and is
less computationally taxing. TESTMERGE differs from ModelFinder in that it does not consider the FreeR-
ate heterogeneity ­model46 implemented in the latter. Additionally, we used the relaxed hierarchical clustering
­method48 to consider the top 10% most similar partition (individual UCE locus) pairs (by setting -rcluster to 10)
which are then merged until no better partition merging schemes can be estimated for further computational
efficiency. Branch support was assessed with 1000 ultrafast bootstrap (UFBoot) replicates. To reduce the possibil-
ity of wrong splits receiving high UFBoot s­ upport49, we resampled from within the partitions and sites within a
partition by setting the –sampling to GENESITE.
We inferred ML phylogenies with UFBoot branch support from UCE + all eight Sanger loci and UCE + only
the Sanger nuclear loci combined datasets with the same software and parameter settings as with the UCE-only
ML analyses.
ML phylogenies were also inferred from concatenated Sanger-sequenced mito-nuclear, mitochondrial and
nuclear loci datasets separately with IQ-TREE. Small sizes of these datasets allowed a full MFP + MERGE model
selection and greedy search for the best partitioning scheme. For protein-coding genes, partitions were inputted
as individual codon positions. UFBoot support, with within-partition and sites-within-partition resampling
enabled, was computed for these phylogenies in the same manner as the UCE and UCE + traditional marker
ML analyses.
Finally, we also computed a BI phylogeny from the 95% complete UCE and traditional nuclear loci dataset
with ExaBayes 1.5.150 to test if BI corroborates the result from the ML analyses of UCE and traditional marker
combined data. We ran the MPI (message passing interface) version ExaBayes 1.5.1, i.e., exabayes, on unpar-
titioned UCE and nuclear loci matrix with 200 MPI processes. Two independent runs, each with four coupled
chains (one cold, three heated), were run as parallel processes. Number of swap attempts per generation was
set to 2. Sampling frequency was set to 500 generations. Diagnostics for convergence were checked every 5000
generations. Other parameters were left at default settings. Though the number of generations was set to 100 M
prior to the commencement of the analysis, two consecutive runs (a second one from the checkpoint of the
timed-out first run on the cluster) could reach only 2.7 M generations. However, the average standard deviation
of the split frequencies (ASDSF), the default divergence metric of ExaBayes, fell to 3.88% which is lower than
the default 5% convergence diagnostic value, thus indicating an acceptable level of convergence. A majority
rule consensus topology was estimated from the posterior distribution of trees after discarding the first 25% as
burn-in with the consense utility of ExaBayes.
All the phylogenomic analyses were run on the Puhti supercomputing cluster of the Finnish CSC-IT Centre
for Science Ltd. Phylogeny visualisation was done with iTOL 6.7.651.

Analyses of conflict in data and topology

To better identify the potential causal factors that complicate the inference of the rapid radiation of elapoids,
including the focal taxa, we investigated site concordance factor, quartet support and tested for the presence of
any deep reticulation events.
We have computed the site concordance factor (sCF)18 for the 50% complete UCE wASTRAL-h and IQ-
TREE species trees, traditional nuclear loci phylogeny and the mitochondrial marker-based phylogeny with
IQ-TREE 2.2.0.5. ASTRAL 5.7.852 was used to compute the quartet support for branches in 50% complete UCE
wASTRAL-h phylogeny.
We prepared a 100% complete UCE dataset consisting of a colubrid outgroup (Spalerosophis diadema), one
of the focal genera, namely Psammodynastes, and one representative taxon from every other elapoid families/
subfamilies. This 11-taxon dataset was prepared with the phyluce pipeline. A set of unrooted ML gene trees was
estimated with IQ-TREE 2.2.0.5 with the same parameters as with the gene trees estimated for wASTRAL-h
analyses in the previous section. Another set of ML gene trees, rooted by specifying the outgroup with -o com-
mand, was also inferred in the same manner. The unrooted gene trees were used as input to P ­ hyloNetworks53 in
Julia 1.9.1 to estimate the quartet concordance factors (quartetCF). The quartetCFs were used to infer Maximum
Pseudolikelihood (MPL) multispecies coalescent networks (MSCN) with the snaq! a­ lgorithm54 of PhyloNetworks.
Firstly, one MSCN with zero reticulations (hence, essentially a tree) was inferred with the quartetCFs from the
unrooted gene trees and a wASTRAL-h phylogeny computed from the same set of gene trees. For the subsequent
MSCN estimation, with 1 to 6 reticulations (hmax), we used the gene trees set and the first MSCN (hmax = 0)
as input. The best number of hybridisations was determined by plotting the log-likelihoods from the aforemen-
tioned runs against the hmax following the recommendation in the online manual. A second set of MPL-based
MSCNs, with 0–6 reticulations, was estimated from the rooted gene trees with PhyloNet 3.8.255. Unlike the snaq!
of PhyloNetworks, the MPL algorithm of PhyloNet uses rooted triplets induced by the gene trees to compute the
­MSCN56. This method produced five MSCN per analysis. The best number of reticulations was determined by
plotting the best log-likelihood from each analysis against the number of reticulations f­ ollowing57.
All the aforementioned analyses were conducted on a local machine. All the MSCNs were visualised with
the PhyloPlots package on Julia 1.9.1.

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Timetree analysis
We estimated a time-calibrated phylogeny with the 95% UCE plus traditional nuclear loci dataset using the
RelTime-ML58,59 as implemented on MEGA ­1141. Log-normal calibration d ­ ensities60 used were the same as
­in7. We set GTR + G + I as the substitution model for the analysis. The resulting Timetree was visualised with
FigTree 1.4.461.

Micro‑CT scan and comparison of anatomical data

We have μ -CT scanned the heads of Buhoma depressiceps, B. marlieri, B. procterae and B. vauerocegae in the
holdings of the Royal Belgian Institute of Natural Sciences (Brussels, Belgium) and the Royal Museum for Cen-
tral Africa (Tervuren, Belgium). These include the holotype of B. marlieri (RMCA-VER-REP 18091). The μ-CT
scan of the cranium of Psammodynastes pulverulentus was obtained from MorphoSource. We also scanned four
elapids, five lamprophiines, four psammophiines, one pseudaspidine and two prosymnines for comparison.
We also examined additional μ-CT scans studied b ­ y7 to broaden the scope of the comparison. The μ-CT scans
generated in the present study have been uploaded to MorphoSource and are listed in the Supplementary Infor-
mation (Table S6).
Several specimens were scanned at the μCT facility of the Royal Belgian Institute of Natural Sciences. Depend-
ing on the size of the specimen, scans were performed using: a) an EasyTom 150 (RX Solutions, Chavanod,
France) with an aluminum filter at 10–30 W, 110 kV, 5.5–12.5frames/s, 1440 projections per rotation and 6–26 μm
isotropic voxelsize; b) an XRE UniTom (Tescan XRE, Ghent, Belgium) at 10–22 W, 75 kV, 150–400 ms frame
rate, 1800 projections per rotation, and 6 to 11 μm isotropic voxelsize. Segmentation of the scans was done using
Dragonfly software, Version 4.1 for Windows (Object Research Systems (ORS) Inc., Montreal, Canada, 2020,
https://​www.​theob​jects.​com/​drago​nfly/​index.​html). Cleaning of the resulting 3D models was done using GOM
Inspect (https://​www.​gom.​com/). Terminology used for cranial bones and muscle attachment sites f­ ollows62,63.

Data availability
Ultraconserved elements, traditional nuclear and mitochondrial marker sequence datasets and the best partition-
ing schemes for the concatenation-based analysis of each dataset are available at https://​figsh​are.​com/​accou​nt/​
home#/p ​ rojec​ ts/1​ 73346. Micro-CT scans, Sanger sequences and the reference genome sequence generated for this
study are uploaded to MorphoSource, NCBI Nucleotide and Genome databases respectively and the relevant ark,
accession number and assembly names and links are listed and/or provided in the Supplementary Information.

Received: 23 July 2023; Accepted: 19 April 2024

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Acknowledgements
We thank the support from the Kone Foundation (fellowship grant to SD) and Helsinki Institute for Life Sci-
ences (HiLife; grant to JM). Funding for the reference genome was provided by Iridian Genomes, grant IRGEN_
RG_2021-1345 Genomic Studies of Eukaryotic Taxa. We further want to acknowledge CSC-IT Center for Science,
Finland, for access to computational resources. Fieldwork by EG in Democratic Republic of the Congo was
funded by the Percy Sladen Memorial Fund, an IUCN/SSC Amphibian Specialist Group Seed Grant, K. Reed,
M.D., research funds from the Department of Biology at Villanova University, two National Geographic Research
and Exploration Grants (nos. 8556-08 and WW-R018-17), UTEP, and the US National Science Foundation (DEB-
1145459). EG thanks his field companions Chifundera Kusamba, Wandege M. Muninga, and Mwenebatu M.
Aristote; the Centre de Recherche en Sciences Naturelles provided project support and permits. Ana Betancourt
of the Border Biomedical Research Center (BBRC) Genomics Analysis Core Facility is acknowledged for ser-
vices and facilities provided. This work was supported by Grant 5U54MD007592 from the National Institute on
Minority Health and Health Disparities (NIMHD), a component of the US National Institutes of Health (NIH).
We are grateful to the University of Helsinki library for open access support. We thank three anonymous referees
for rigorous, constructive reviews which helped us in improving the manuscript.

Author contributions
Conceptualisation, SD; methodology, SD; data, SD, EG, JB, OSGP, SP, SR; analyses, SD; writing original draft,
SD; resources, SD, EG, JM, SP; review and editing of draft, all authors.

Competing interests
The authors declare no competing interests.

Additional information
Supplementary Information The online version contains supplementary material available at https://​doi.​org/​
10.​1038/​s41598-​024-​60215-2.
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