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Citation: Rivas, J.A.; De La Quintana,
P.; Mancuso, M.; Pacheco, L.F.; Rivas,
G.A.; Mariotto, S.; Salazar-Valenzuela,
D.; Baihua, M.T.; Baihua, P.;
Burghardt, G.M.; et al. Disentangling
the Anacondas: Revealing a New
Green Species and Rethinking
Yellows. Diversity 2024,16, 127.
https://doi.org/10.3390/d16020127
Academic Editors: Manuel
Elias-Gutierrez, Jessica Frigerio
and Naoko Takezaki
Received: 15 January 2024
Revised: 9 February 2024
Accepted: 14 February 2024
Published: 16 February 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
diversity
Article
Disentangling the Anacondas: Revealing a New Green Species
and Rethinking Yellows
Jesús A. Rivas 1, * , Paola De La Quintana 2,3 , Marco Mancuso 4,5 , Luis F. Pacheco 6, Gilson A. Rivas 7,
Sandra Mariotto 8, David Salazar-Valenzuela 9, Marcelo Tepeña Baihua 10, Penti Baihua 10,
Gordon M. Burghardt 11 , Freek J. Vonk 12,13, Emil Hernandez 14, Juán Elías García-Pérez 15, Bryan G. Fry 5,*
and Sarah Corey-Rivas 1, *
1Biology Department, New Mexico Highlands University, 1005 Diamond Av., Las Vegas, NM 87701, USA
2Departamento de Ciências Biológicas, Universidade Estadual de Santa Cruz, Rodovia Jorge Amado, km 16,
Ilhéus 45662-900, BA, Brazil; paola.d.c.1186@gmail.com
3Red de Investigadores en Herpetología-Bolivia, La Paz 10077, Bolivia
4Amphibian Evolution Laboratory, Biology Department, Vrije Universiteit Brussel, 1050 Ixelles, Belgium;
19marcomancuso19@gmail.com
5Adaptive Biotoxicology Laboratory, School of the Environment, University of Queensland,
St. Lucia, QLD 4072, Australia
6Colección Boliviana de Fauna, Instituto de Ecología Carrera de Biología, Facultad de Ciencias Puras y
Naturales, Universidad Mayor de San Andrés, La Paz 10077, Bolivia; luispacheco11@yahoo.com
7Departamento de Biología, Facultad Experimental de Ciencias, la Universidad del Zulia,
Maracaibo 4001, Zulia, Venezuela; grivas@fec.luz.edu.ve
8Instituto Federal de Educação, Ciência e Tecnologia de Mato Grosso, Cuiabá78043-400, MT, Brazil;
sandra.mariotto@ifmt.edu.br
9Centro de Investigación de la Biodiversidad y Cambio Climático (BioCamb) e Ingeniería en Biodiversidad y
Recursos Genéticos, Facultad de Ciencias del Medio Ambiente, Universidad Indoamérica,
Quito 170103, Ecuador; davidsalazar@uti.edu.ec
10 Baihuaeri Waorani People of Bameno, Ome Yasuni, Bameno 220301, Ecuador;
guiatepena2015@gmail.com (M.T.B.); pentibaihua@hotmail.com (P.B.)
11 Departments of Psychology and Ecology & Evolutionary Biology, University of Tennessee,
Knoxville, TN 37996, USA; gburghar@utk.edu
12 Naturalis Biodiversity Center, 2333 CR Leiden, The Netherlands; freek@studiofreek.nl
13 Division of BioAnalytical Chemistry, Amsterdam Institute of Molecular and Life Sciences, Vrije Universiteit
Amsterdam, 1081 HV Amsterdam, The Netherlands
14 Laboratório de Zoologia Adriano Giorgi, Programa de Pós-Graduação em Biodiversidade e Conservação,
Campus Universitário de Altamira, Universidade Federal do Pará, Altamira 68371-155, PA, Brazil;
emilhjh@yahoo.com
15 Museo de Zoología, Programa CAM, Universidad Nacional Experimental de Los Llanos Occidentales
Ezequiel Zamora, UNELLEZ, Guanare 3350, Portuguesa, Venezuela; ecologia2unellez@gmail.com
*Correspondence: rivas@nmhu.edu (J.A.R.); bgfry@uq.edu.au (B.G.F.); sjcorey@nmhu.edu (S.C.-R.);
Tel.: +1-(505)-454-3292 (J.A.R.); Fax: +1-505-454-3103 (J.A.R.)
Abstract: Anacondas, genus Eunectes, are a group of aquatic snakes with a wide distribution in South
America. The taxonomic status of several species has been uncertain and/or controversial. Using
genetic data from four recognized anaconda species across nine countries, this study investigates
the phylogenetic relationships within the genus Eunectes. A key finding was the identification of
two distinct clades within Eunectes murinus, revealing two species as cryptic yet genetically deeply
divergent. This has led to the recognition of the Northern Green Anaconda as a separate species
(Eunectes akayima sp. nov), distinct from its southern counterpart (E. murinus), the Southern Green
Anaconda. Additionally, our data challenge the current understanding of Yellow Anaconda species
by proposing the unification of Eunectes deschauenseei and Eunectes beniensis into a single species
with Eunectes notaeus. This reclassification is based on comprehensive genetic and phylogeographic
analyses, suggesting closer relationships than previously recognized and the realization that our
understanding of their geographic ranges is insufficient to justify its use as a separation criterion. We
also present a phylogeographic hypothesis that traces the Miocene diversification of anacondas in
western South America. Beyond its academic significance, this study has vital implications for the
Diversity 2024,16, 127. https://doi.org/10.3390/d16020127 https://www.mdpi.com/journal/diversity
Diversity 2024,16, 127 2 of 28
conservation of these iconic reptile species, highlighting our lack of knowledge about the diversity of
the South American fauna and the need for revised strategies to conserve the newly identified and
reclassified species.
Keywords: cryptic diversity; Boidae; South America; Llanos; Pebas system; Orinoco basin; redundant
species
1. Introduction
South America is the most biologically diverse landmass in the world, with the high-
est diversity of species of multiple taxa compared to any other continent [
1
–
5
]. As such,
South America is a natural laboratory for studying diversity and speciation, as well as
a hotspot for conservation efforts. One problem that hinders our understanding of di-
versity in general is our ability to determine how many species there are in an area. In
addition to the inherent difficulties of thorough sampling and fieldwork across multiple
countries, understanding species diversity is complicated by the presence of both cryptic
species—species that appear morphologically identical but are genetically different (e.g.,
Astraptes spp. (Lepidoptera: Hesperiidae))—and populations that look superficially distinct
but lack the genetic divergence to infer reproductive isolation and be considered sepa-
rate species [
6
–
8
]. Despite relatively low human population densities, the economies of
most South American countries are largely dependent on extractive industries [
9
]. As a
result, habitat degradation is an increasing problem due to land fragmentation caused by
industrialized agriculture [
10
,
11
] and heavy metal pollution associated with mining activi-
ties [
12
,
13
]. These problems are exacerbated by the effect of climate change (particularly
drought), the increase in fires [
14
,
15
], and the volatile politics of the region, resulting in
drastic and frequent changes in environmental policy [16–18].
Eunectes (anacondas; Boidae) is a genus of large-bodied aquatic snakes endemic to the
east of the Andes in South America [19]. Anacondas inhabit lowland rivers and wetlands.
These snakes have the typical adaptations for an aquatic lifestyle, such as nostrils and eyes
located dorsally on the head, and displaying a dorsal coloration and pattern that blend
well with the aquatic vegetation [
20
–
22
]. Currently, four species are recognized in this
genus, with E. murinus (Linnaeus 1758) representing a sister lineage to a clade composed
of E. beniensis (Dirksen 2002), E. deschauenseei (Dunn and Conant 1936),and E. notaeus
(Cope 1862) [
23
–
25
]. The largest of these species, Eunectes murinus (or Green Anaconda),
occurs in most of the tropical regions of the continent, including the basins of the Amazon,
Esequibo, and Orinoco rivers, and several smaller watersheds [
22
,
24
]. The other three
species are smaller than E. murinus and are distributed within or adjacent to the distribution
of E. murinus. The recently described species Eunectes beniensis, or Beni Anaconda, has a
distribution restricted to the Beni region of Bolivia [
25
,
26
]. Eunectes deschauenseei, or Dark
Spotted Anaconda, is distributed in the northeast of the continent [
25
,
27
]. It is found from
the Amazon River delta in Brazil to French Guiana and possibly Suriname [
28
] (Figure 1).
Eunectes notaeus, or Yellow Anaconda, has a distribution to the south of E. murinus including
the Pantanal, Chaco, and other hyper-seasonal areas of tropical and subtropical South
America including Brazil, Bolivia, Paraguay, Argentina, and Uruguay [29].
Both E. deschauenseei and E. beniensis overlap strongly with E. murinus in their respec-
tive distributions and habitats [
30
,
31
]. The sympatry of E. murinus and E. notaeus is less
certain (Figure 1). There is no obvious biogeographic barrier separating both species. In
regions of sympatry such as the Pantanal, the extent of their syntopy is unclear; it is thought
that E. murinus may venture into the deeper rivers and ponds, while E. notaeus prefers
hyper-seasonally flooded habitats and appears to avoid the deeper river [
32
]. We know that
some of these species can interbreed [
24
] and anecdotal reports from the pet trade suggest
that their offspring may be fertile.
Diversity 2024,16, 127 3 of 28
Diversity 2024, 16, x FOR PEER REVIEW 3 of 30
Figure 1. Sampling location of samples used in this study. The green area is the known distribution
of the Green Anaconda (Eunectes murinus). The yellow area is the distribution of the Yellow Ana-
conda (E. notaeus). The orange area is the reported distribution of E. beniensis and the red area is the
distribution of E. deschauenseei.
Both E. deschauenseei and E. beniensis overlap strongly with E. murinus in their respec-
tive distributions and habitats [30,31]. The sympatry of E. murinus and E. notaeus is less
certain (Figure 1). There is no obvious biogeographic barrier separating both species. In
regions of sympatry such as the Pantanal, the extent of their syntopy is unclear; it is
thought that E. murinus may venture into the deeper rivers and ponds, while E. notaeus
prefers hyper-seasonally flooded habitats and appears to avoid the deeper river [32]. We
know that some of these species can interbreed [24] and anecdotal reports from the
pet trade suggest that their offspring may be fertile.
There have been comprehensive studies on the general natural history of the genus
Eunectes [22,33–35] including diet [36–45], diseases [46,47], habitat use and mobility
[22,30–33,44,45], allometric growth [48,49], and demography [22,50]. On the other hand,
the conservation status of anacondas throughout their range is largely unexplored, alt-
hough Eunectes species are protected from international trade by CITES’s Appendix 2 [51–
53]. Eunectes species are often persecuted by humans and used for commercial trade
[51,54–58]. All anaconda species are potentially collected locally, nationally, and interna-
tionally for medicinal and clothing purposes [51,59]. The IUCN Redlist categorizes all four
Eunectes species as “least concern”. E. murinus is listed as “least concern” due to its wide
range across eleven countries. However, population trends are unknown. E. notaeus is
listed as “least concern” overall and is estimated to have stable populations, although it is
listed as vulnerable in Argentina [60,61] and as a priority species for conservation in Uru-
guay [62]. Eunectes notaeus is locally threatened by agricultural development and
Figure 1. Sampling location of samples used in this study. The green area is the known distribution of
the Green Anaconda (Eunectes murinus). The yellow area is the distribution of the Yellow Anaconda
(E. notaeus). The orange area is the reported distribution of E. beniensis and the red area is the
distribution of E. deschauenseei.
There have been comprehensive studies on the general natural history of the
genus Eunectes [
22
,
33
–
35
] including diet [
36
–
45
], diseases [
46
,
47
], habitat use and
mobility [
22
,
30
–
33
,
44
,
45
], allometric growth [
48
,
49
], and demography [
22
,
50
]. On the other
hand, the conservation status of anacondas throughout their range is largely unexplored, al-
though Eunectes species are protected from international trade by CITES’s Appendix 2 [
51
–
53
].
Eunectes species are often persecuted by humans and used for commercial trade [
51
,
54
–
58
].
All anaconda species are potentially collected locally, nationally, and internationally for
medicinal and clothing purposes [
51
,
59
]. The IUCN Redlist categorizes all four Eunectes
species as “least concern”. E. murinus is listed as “least concern” due to its wide range
across eleven countries. However, population trends are unknown. E. notaeus is listed as
“least concern” overall and is estimated to have stable populations, although it is listed as
vulnerable in Argentina [60,61] and as a priority species for conservation in Uruguay [62].
Eunectes notaeus is locally threatened by agricultural development and hydroelectric dams.
It is also collected for the pet trade and harvested for its skin [
58
,
63
]. Eunectes beniensis is
also listed as “least concern” throughout its range [
64
]. This species has unknown popula-
tion trends and faces the same threats as other Eunectes species. Eunectes deschauenseei is the
least known species within the genus. It has a still unclear distribution range unknown
population trends, and faces habitat loss due to agricultural encroachment throughout its
known range.
The systematics of the group have been studied, but patterns of morphological and
genetic divergence within the genus are still unclear. Dirksen [
24
], in a morphological
Diversity 2024,16, 127 4 of 28
revision of the genus, found that Green Anacondas from Perúhad fewer but larger and
rounder black spots on the dorsum than specimens from Brazil. This difference was
attributed to clinal variation. The author suggested that there might be different lineages
within E. murinus associated with the different drainages of its distribution. Preliminary
data, using mitochondrial DNA on their phylogenetic relationships, showed that there may
be different clades within E. murinus, while the differences between the clades of smaller
anacondas might not be strong enough to support there being different species [
65
,
66
]. A
later study using molecular and morphological data found similar results [
23
]. However,
the phylogenetic relationships within the Eunectes complex remain unclear due to sparse
and incomplete sampling, forcing inferences across vast expanses of forest and swamps
with few representative samples. Also, because the distribution of E. murinus is so extensive,
encompassing so many aquatic habitats, ecosystems, and different major watersheds, it is
difficult to determine what biogeographic barrier may be acting to isolate different lineages
today. Furthermore, due to the dynamic paleo-history of the continent, these isolation
mechanisms may not even exist currently. A detailed knowledge of the natural history of
the anaconda is essential to formulate well-founded hypotheses about the biogeographic
barriers for each taxon, not to mention a good understanding of their divergence time in
order to understand how South America’s paleo-history may have shaped them.
In this study, we use representative samples of all Eunectes species across their dis-
tribution, including nine countries, to disentangle the phylogenetic relationships of ana-
condas. We propose new candidate species and explore the conservation implications of
our findings. We use our current knowledge of the paleo-history of South America and
the distribution of other taxa with similar, or complementary, habitats and evolutionary
histories to speculate on the speciation events that led to the diversification of this group.
2. Materials and Methods
2.1. Study Site and Sampling
We surveyed anacondas from various locations throughout the range of Eunectes
species in South America (Figure 1). While collecting demographic and ecological data, we
also collected tissue and/or blood from each specimen. In the field, we collected blood and
tissue samples from E. murinus in the Venezuelan Llanos at Hato El Cedral and Hato El
Frio (Table S1 for details of each sampled individual); the Brazilian states of Mato Grosso,
Mato Grosso do Sul, and Para; and the Bameno region of the Baihuaeri Waorani Territory
in the Ecuadorian Amazon. We collected E. beniensis and E. murinus samples in the Bolivian
Beni in the SirionóIndigenous Territory. Additional samples were donated by the Bronx
Zoo (NY), Miami Metro Zoo, National Museum of Natural History, Smithsonian Museum
of Natural History, Museu Emilio Goeldi, Muséum de Toulouse, The Naturalis Biodiversity
Center, Universidade Federal do Mato Grosso, private collectors, and Colección Boliviana
de Fauna, Bolivia (see Figure 1for locations and Table S1 for a specimen list). Blood samples
were stored in Queen’s lysis buffer [
67
], and scales were either stored in 80% ethanol or
dried at −20 ◦C.
2.2. DNA Isolation and Sequencing
Genetic work was carried out at New Mexico Highlands University in the US, Instituto
Federal do Mato Grosso, Naturalis Biodiversity Center in the Netherlands, and Universidad
Indoamérica in Ecuador. Genomic DNA was extracted from blood and scale tissue with the
Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). A standard protocol was
used for blood samples, while scales needed to initially be lysed with the kit’s ATL buffer
and proteinase K with a 12 h digest at 55
◦
C. We used the polymerase chain reaction (PCR)
to amplify portions of mitochondrial genes cytochrome b (Cytb), NADH dehydrogenase
subunit 2 (ND2), and NADH dehydrogenase subunit 4 (ND4) using both published and
newly designed primers (Table 1). We modified published primers and created new primers
using the E. notaeus mitochondrial genome [
68
] (Genbank accession AM236347.1) and the
NCBI primer-BLAST implementation of Primer 3 [
69
]. We ran 25
µ
L PCR reactions using
Diversity 2024,16, 127 5 of 28
NEB Hot Start Taq 2X Master Mix (NEB, Ipswich, Maine, USA), 50–100 ng of genomic DNA,
0.4
µ
M of each primer. Thermocycling consisted in 5 min of initial denaturation at 94
◦
C
followed by 36 cycles of a 40 s denaturation at 94
◦
C, 30 s annealing at 55
◦
C, and a 45 s
elongation at 72
◦
C. A final extension step at 72
◦
C for 7 min was specified. PCR products
were cleaned using the Monarch
®
PCR & DNA Cleanup Kit (NEB, Ipswich, MA, USA)
using the standard protocol. Cleaned PCR products were quantified with Nanodrop One
(Thermofisher, Waltham, MA, USA). Sanger sequencing was carried outin both directions
using the same PCR forward and reverse primers (Psomagen (Rockville, MD, USA).
Table 1. Summary of oligonucleotide primers and their location in the mitochondrial genome, used
for PCR and Sanger sequencing of mitochondrial genes in this study.
Gene and Primer
Primer Sequences
Mt Location and References
Cytochrome b
L14910_cytbEu 5′-GACCTGMGGTCTGAAAAACCACCG
TTG T-3′
tRNA-Glu, [
70
,
71
], modified
by this study
H16064_cytbEu 5′-CTTTGGTTTACAGAACAATGCTTTG-3′tRNA-Thr, [71], modified by
this study
ND2
En5132F 5′-AATGTCACCACGGCCTTTAC-3′tRNA-met, this study
En6262R 5′-TGCAGGCTCTACAGAAGCTAAA-3′tRNA-Trp/tRNA-Ala,
this study
Eu_ND2_6003 5′-TGGCTATTGTYGTKGCTTCT-3′ND2, this study
ND4
ND4_Eu 5′-CACCTATGACTACCAAAAGCCCAC
GTAGAAGC-3′
ND4, [72], modified by
this study
ND4_LEU 5′-CATTTCTRCYACTTGGATTTGCACCA-3′tRNA-Leu, Arévalo et al.,
1994, modified by this study
Additional samples belonging to outgroup taxa were amplified and sequenced from
specimens collected in the field, including Epicrates cenchria (Venezuela), Boa constrictor
(Venezuelan origin), Corallus ruschenbergerii (Venezuela), and Corallus hortulanus (Bolivia).
We also added published mtDNA sequences of Eunectes notaeus to the phylogenetic analysis
(Table S1).
Base calls were verified and contigs were assembled in Sequencher
®
version 5.3 (Gene
Codes Corporation, Ann Arbor, MI, USA). We used MEGA XI: Molecular Evolutionary
Genetics Analysis version 11.0.13 [
73
] to align each set of gene sequences with ClustalW
v.2.1 (default parameters). The mitochondrial gene sequences were concatenated into a
partitioned nexus file. Sequences were deposited in Genbank under accession numbers
PP273560-PP273621 (Cytb), PP334792-PP334847 (ND2), and PP334848-PP334905 (ND4)
(Table S1).
Nuclear markers CMOS, RAG1, BDNF, ODC, and NT3 were also amplified with
primers from [
74
] and TBP [
75
] and sequenced but did not provide sufficient numbers of
variable sites within the Eunectes genus to distinguish lineages and were not included in
phylogenetic analyses (Figure S2).
2.3. Phylogenetic Analysis and Genetic Divergence
Phylogenetic analyses were conducted using Bayesian inference (BI) and Maximum
Likelihood (ML) methods with the concatenated matrix of ND2, ND4, and CytB genes.
For both methods, we rooted the resulting trees using Boa constrictor as the outgroup.
We used ModelTest-NG [
76
] implemented in the raxmlGUI 2.0 [
77
,
78
] to determine the
best-fit model of nucleotide substitution for each gene sequence matrix using Akaike’s
Information Criterion. The BI analysis was conducted in MrBayes v3.2.7 [
79
] using the
three-gene matrix partitioned by gene and with the GTR+I+
Γ
model selected as the nearest
overparameterized model associated with the AIC-selected best-fit model (TrN+I for ND2,
Diversity 2024,16, 127 6 of 28
TPM2uf+I+G4 for ND4, and GTR+I for CytB). MrBayes rates were estimated under a GTR
model and parameter estimation was allowed to vary in each partition (ratepr = variable).
We conducted two independent runs of four-chain MCMC for 100 million generations with
25% burn-in and an automatic average standard deviation of split frequencies (ASDSF)
stop value of 0.01. Summary node and branch parameter estimates were examined to
assess convergence (PSRF converging on 1.0). The BI 50% majority-rule consensus tree is
reported with Bayesian posterior probability tree node support values. The ML analysis
was executed with the three-gene matrix partitioned by gene and run in RaxML v.8.2.12 [
77
]
using a general time-reservable (GTR) model of evolution with a gamma distribution
(GAMMA) and the thorough bootstrapping algorithm with 1000 bootstrap replicates and
100 independent searches. Genetic divergence was calculated using the three-gene matrix,
and also separately for each gene, as mean uncorrected genetic pairwise distances between
and within lineages that were identified in our phylogenetic analyses.
2.4. Divergence Time Estimation
To estimate divergence times within the genus Eunectes, we conducted multiple molec-
ular clock analyses using the software BEAST v.2.7.6 [
80
]. We implemented a GTR+I+
Γ
nucleotide substitution model for each gene partition (CytB, ND2, ND4). All analyses
were performed under the assumptions of a Birth–Death model to infer macroevolution-
ary patterns and an Optimized Relaxed Clock (ORC), which was calculated uniformly
across the three gene partitions. The complete dataset for this analysis comprised a total
of 78 sequences. The homologous sequences from Sanzinia madagascariensis, Acrantophis
dumerili (Boidae: Sanziniinae), Ungaliophis panamensis (Boidae: Ungaliophiinae), Charina
bottae, Lichanura trivirgata (Boidae: Charininae), Boa constrictor,Corallus hortulana,Corallus
ruschenbergerii, and Chilabothrus argentum (Boidae: Boinae) were added to the three-gene
matrix to allow the use of additional calibration points. Sequences from these species were
obtained from NCBI Genbank (Tables 1and S1). Five independent analyses were run for
40 million generations, and 15% burn-in values were chosen based on the output from
Tracer (v.1.7.2). Tracer was also used to assess convergence by comparing Effective Sample
Sizes (ESSs). In addition, posterior distribution plots were examined for further evidence
of convergence.
We used four different approaches to estimate divergence times. These approaches
make similar use of fossil evidence to impose ‘hard’ minimum age constraints on sev-
eral nodes in the Boidae [
81
,
82
] but differ in their consideration of Late Cretaceous land
bridges between East Gondwanan land masses to explain the divergence of Madagascan
Sanziniinae from other boids [74,83,84] (Table 2).
In our first approach, we constrained the node marking the split between Sanziniinae
and the rest of Boidae (a mostly Neotropical group) to be at least 80 Mya. The hard minimum
used for this approach takes into account the possibility of dispersal across Gondwanan
landmasses through the Gunnerus Ridge, a hypothetical land bridge that may have directly
connected Madagascar to Antarctica, by 80 Mya [
74
,
84
]. A soft maximum was applied by
setting a lognormal prior for this node, implying a <5% probability that its age exceeds
98.32 Mya. This age roughly corresponds to the Early/Late Cretaceous transition and
matches the age of Coniophis, the oldest known crown-group fossil of snakes [79].
In a second approach, we constrained the split between Sanziniinae and other Boidae to
be at least 88 Mya. This hard minimum disproves the existence of the Gunnerus Ridge [
84
]
but allows for the possibility of dispersal from western Gondwana to Madagascar via the
Indian subcontinent (which separated from Madagascar at 88 Mya [
83
]) and the Kerguelen
Plateau (a land bridge connecting Antarctica to the Indian Continent; [
84
]). The same soft
maximum of 98.32 Mya was applied to this node.
Diversity 2024,16, 127 7 of 28
Table 2. Calibration points used in this study. Maximum ages were not applied for the fossil evidence;
n.a stands for “not applied”.
Approach Calibration
Point Type Node
Prior
Implementation
in BEAST (v.2.7.6.)
Minimum Maximum
1. Divergence of
Sanziniinae from
other Boidae
assuming possible
dispersal to
Madagascar via two
Late Cretaceous land
bridges (Gunnerus
Ridge/Kerguelen
Plateau).
Paleogeographic
and fossil.
LCA of
Sanziniinae and
other boids.
Log-normal. With
hard minimum
and soft maximum
(represented by
95% percentile).
80 Million Years
Ago (Mya) based
on the latest
possible existence
of Gunnerus Ridge
[83].
98.32 Mya as soft
maximum, based
on the fossil
Coniophis, the
oldest
crown-group fossil
for Serpentes [79].
Fossil. LCA of Erycinae
and Boinae. Uniform with only
hard minimum.
58 Mya based on
Titanoboa
cerrejonensis [79]. n.a.
Fossil. LCA of Lichanura
and Charina.Uniform with only
hard minimum.
18.7 Mya based on
the fossil UNSM
125,562 [79]. n.a.
Fossil. LCA of Corallus
and its sister
lineage.
Uniform with only
hard minimum.
50.2 Mya based on
the fossil Corallus
priscus [79]. n.a.
Fossil. LCA of Eunectes
and Epicrates.Uniform with only
hard minimum.
12.375 Mya based
on Eunectes stirtoni
[79]. n.a.
Fossil. LCA of Charininae
and
Ungaliophiinae.
Uniform with only
hard minimum.
47.8 Mya based on
the fossil Rageryx
schmidi [81]. n.a.
2. Divergence of
Sanziniinae from the
rest of Boidae
assuming possible
dispersal to
Madagascar via the
Kerguelen Plateau
and the Indian
subcontinent.
Paleogeographic
and fossil.
Log-normal. With
hard minimum
and soft maximum
(represented by
95% percentile).
88 Mya based on
the latest possible
terrestrial
connection
between
Madagascar and
the Indian
subcontinent [83].
98.32 Mya as soft
maximum, based
on the fossil
Coniophis, the
oldest
crown-group fossil
for Serpentes [79].
Fossil. LCA of Erycinae
and Boinae. Uniform with only
hard minimum.
58 Mya based on
Titanoboa
cerrejonensis [79]. n.a.
Fossil. LCA of Lichanura
and Charina.Uniform with only
hard minimum.
18.7 Mya based on
the fossil UNSM
125,562 [79]. n.a.
Fossil. LCA of Corallus
and its sister
lineage.
Uniform with only
hard minimum.
50.2 Mya based on
the fossil Corallus
priscus [79]. n.a.
Fossil. LCA of Eunectes
and Epicrates.Uniform with only
hard minimum.
12.375 Mya based
on Eunectes stirtoni
[79]. n.a.
Fossil. LCA of Charininae
and
Ungaliophiinae.
Uniform with only
hard minimum.
47.8 Mya based on
the fossil Rageryx
schmidi [81]. n.a.
Diversity 2024,16, 127 8 of 28
Table 2. Cont.
Approach Calibration
Point Type Node
Prior
Implementation
in BEAST (v.2.7.6.)
Minimum Maximum
3. Divergence of
Sanziniinae from the
rest of Boidae in the
absence of Late
Cretaceous land
bridges connecting
Madagascar to
western Gondwana.
Fossil and
paleogeographic.
Log-normal. With
hard minimum
and soft maximum
(represented by
95% percentile).
120.4 Mya based
on the split
between eastern
and western
Gondwana
(Krause et al.
2020).
145 Mya based on
the oldest fossils
testifying for the
split between
snakes and
Anguimorph
lizards [79].
Fossil. LCA of Erycinae
and Boinae. Uniform with only
hard minimum.
58 Mya based on
Titanoboa
cerrejonensis [79]. n.a.
Fossil. LCA of Lichanura
and Charina.Uniform with only
hard minimum.
18.7 Mya based on
the fossil UNSM
125,562 [79]. n.a.
Fossil. LCA of Corallus
and its sister
lineage.
Uniform with only
hard minimum.
50.2 Mya based on
the fossil Corallus
priscus [79]. n.a.
Fossil. LCA of Eunectes
and Epicrates.Uniform with only
hard minimum.
12.375 Mya based
on Eunectes stirtoni
[79]. n.a.
Fossil. LCA of Charininae
and
Ungaliophiinae.
Uniform with only
hard minimum.
47.8 Mya based on
the fossil Rageryx
schmidi [81]. n.a.
4. Strictly
paleontological
calibration based
on [82].
Fossil. Pan-serpentes
crown-group age
based on Coniophis.
Log-normal. With
hard minimum
and soft maximum
(represented by
95% percentile).
98.32 Mya 113 Mya
Fossil.
LCA of Erycinae
and Boinae based
on Titanoboa
cerrejonensis.
Log-normal. With
hard minimum
and soft maximum
(represented by
95% percentile).
58 Mya 64 Mya
Fossil.
LCA of Corallus
and its sister
lineage based on
Corallus priscus.
Log-normal. With
hard minimum
and soft maximum
(represented by
95% percentile).
50.2 Mya 64 Mya
Fossil. LCA of Charininae
and
Ungaliophiinae.
Uniform with only
hard minimum. 35.2 Mya n.a.
Fossil.
LCA of Charina
and Lichanura
based on
Calamagras weigeli.
Uniform with only
hard minimum. 18.7 Mya n.a.
Fossil. LCA of Eunectes
and Epicrates.Uniform with only
hard minimum. 12.375 Mya n.a.
Diversity 2024,16, 127 9 of 28
Third, we set the split between Sanziniinae and other Boidae to be at least 120.4 Mya.
This minimum denies the existence of any land bridge to Indo-Madagascar and assumes
that intercontinental dispersal across southern oceans was unlikely [
84
]. As a soft maximum,
we used 145 Mya, which roughly corresponds to the Jurassic/Cretaceous boundary and
corresponds to the oldest fossils marking the split between snakes and Anguimorph
lizards [79].
Our fourth, and final, approach dates the splits within Boidae through the aid of fossil
evidence only. Despite the limitations of dating from paleontological information alone,
especially given the poor fossil record from Central–South America [
5
,
85
–
87
], this approach
serves to test the impact of deep-time paleogeographic patterns on the resulting Boidae
timeline. Therefore, it provides an alternative perspective to the other approaches used
in this study. The timelines for the fossil calibration points were adapted from the other
methods to include soft maximum limits (based on [
79
]), which are essential for reliable
dating in the analysis.
2.5. Morphological Comparison of E. murinus between North and South
Comparisons were made with museum specimens from Venezuela (Museo de Ciencias
Naturales de Guanare: MCNG 1042, Museo de Biología Universidad Central de Venezuela:
MBUCV 1836, MBUCV 7193, MBUCV 7189), Surinam (Naturalis Biodiversity Center;
RMNH.RENA.20768), as well as Brazil (Museu Emilio Goeldi MPEG 27428), and informa-
tion from the literature on animals from Venezuela [
88
], Guyana [
89
], Peru, Bolivia, and
Brazil [
23
,
24
]. We collected the following meristic information: the number of dorsal scales
at the mid-body, number of subcaudal scales, number of ventral scales, number of ocular
scales (average of the number of scales around each eye), number of supralabial scales
(average from both sides), number of infralabial scales (average from both sides), number
of suborbitalia scales (scales simultaneously in contact with supralabials and infra-orbitals;
often called lorilabials), number of dorsal blotches from the neck to the tail (excluding the
head) in the back and sides (blotches in contact were counted as different blotches), and
number of blotches in contact with other blotches.
3. Results
3.1. Phylogenetics
3.1.1. Eunectes Overview
The three-gene matrix counted 2939 bp, charset ND4 = 1–848; charset ND2 = 849–1863;
and charset Cytb = 1864–2939, including 1015 bp of ND2, 848 bp of ND4, and 1076 bp of
cytochrome B for 71 individuals (including outgroups Boa, Corallus, Epicrates; Table S1).
The concatenated alignment had 24.5% missing data, reflecting the inclusion of published
sequence data for some but not all genes in favor of increasing phylogenetic accuracy by
increasing taxonomic coverage [
90
] (coverage by taxon and gene summarized in Table S1).
The BI consensus tree and ML tree had very similar topologies and therefore the BI tree
is presented here (Figure 2; ML tree is presented in Figure S1). Both methods confirm a
sister-clade relationship between Green Anacondas, and a clade composed of the other three
anaconda species as proposed by Dirksen [24].
3.1.2. Yellow Anaconda Phylogenetics and Taxonomy
Our analyses indicate a poorly defined phylogenetic structure in the clade composed
of E. notaeus,E. deschauenseei, and E. beniensis. Although specimens identified as E. de-
schauenseei or E. beniensis are found in well-supported clades, those identified as E. notaeus
are not, and instead represent a paraphyletic clade with E. deschauenseei and E. beniensis
(Figure 2).
Diversity 2024,16, 127 10 of 28
Diversity 2024, 16, x FOR PEER REVIEW 11 of 30
Figure 2. Bayesian consensus phylogram for Eunectes species (50% majority-rule con-
sensus tree) using the mtDNA gene sequence dataset (ND2, ND4, Cytb). Bayesian poste-
rior probability node support values > 0.95 are indicated with black circles and distal val-
ues are not shown. Refer to Table S1 for details on tip labels.
3.1.2. Yellow Anaconda Phylogenetics and Taxonomy
Our analyses indicate a poorly defined phylogenetic structure in the clade composed
of E. notaeus, E. deschauenseei, and E. beniensis. Although specimens identified as E. deschauen-
seei or E. beniensis are found in well-supported clades, those identified as E. notaeus are not,
and instead represent a paraphyletic clade with E. deschauenseei and E. beniensis (Figure 2).
Comparisons were made to relate morphology to genetic placement. One of the
snakes captured in the Bolivian Beni (B54) has markings that best fit the description of E.
Figure 2. Bayesian consensus phylogram for Eunectes species (50% majority-rule consensus tree)
using the mtDNA gene sequence dataset (ND2, ND4, Cytb). Bayesian posterior probability node
support values > 0.95 are indicated with black circles and distal values are not shown. Refer to Table
S1 for details on tip labels.
Comparisons were made to relate morphology to genetic placement. One of the
snakes captured in the Bolivian Beni (B54) has markings that best fit the description of
E. deschauenseei, as indicated by the height of the lateral flecks, which do not reach half the
height of the snake, and the dorsal blotches separated by two or three scales (Figure 3). This
specimen is recovered with other E. deschauenseei from French Guiana and Marajo Island in
the phylogenetic analysis (Figures 2and S1). In addition, other anacondas caught in Beni
(B52 and B58) also had markings that best fit the description of E. deschauenseei, but these
Diversity 2024,16, 127 11 of 28
specimens were recovered with E. beniensis in the phylogenetic analysis (Figures 2and 3).
For comparison, Figure 3also shows E. beniensis with a characteristic pattern of these
lineages (larger lateral flecks), caught in Beni, that is recovered as E. beniensis in the tree
(Figure 2). Therefore, our results challenge the validity of E. beniensis and E. deschauenseei as
distinct species from E. notaeus.
Diversity 2024, 16, x FOR PEER REVIEW 12 of 30
deschauenseei, as indicated by the height of the lateral flecks, which do not reach half the
height of the snake, and the dorsal blotches separated by two or three scales (Figure 3).
This specimen is recovered with other E. deschauenseei from French Guiana and Marajo
Island in the phylogenetic analysis (Figures 2 and S1). In addition, other anacondas caught
in Beni (B52 and B58) also had markings that best fit the description of E. deschauenseei,
but these specimens were recovered with E. beniensis in the phylogenetic analysis (Figures
2 and 3). For comparison, Figure 3 also shows E. beniensis with a characteristic paern of
these lineages (larger lateral flecks), caught in Beni, that is recovered as E. beniensis in the
tree (Figure 2). Therefore, our results challenge the validity of E. beniensis and E. deschau-
enseei as distinct species from E. notaeus.
(a) (b)
(c) (d)
Figure 3. (a) E. deschauenseei caught in Beni, Bolivia (B54). (b,c) Anacondas caught in Beni that had
markings of E. deschauenseei but were recovered as E. beniensis in the phylogenetic analysis (B52 and
B58). (d) E. beniensis recovered as E. beniensis in the phylogenetic analysis.
Consistent with a previous study [23], we recovered the Yellow Anacondas as pa-
raphyletic, with E. beniensis and E. deschauenseei nested within E. notaeus (Figure 2) and
with shallow levels of divergence between the clades (Table 3). Our sampled taxa included
one from the Bolivian Beni that was both genetically and morphologically E. deschauenseei,
despite being outside the known range of this species. In addition, two other anacondas
from the Bolivian Beni had markings that would classify them as E. deschauenseei, while
the phylogenetic analysis placed them within E. beniensis. Therefore, our results challenge
the validity of the Yellow Anaconda being split into species.
Figure 3. (a)E. deschauenseei caught in Beni, Bolivia (B54). (b,c) Anacondas caught in Beni that had
markings of E. deschauenseei but were recovered as E. beniensis in the phylogenetic analysis (B52 and
B58). (d)E. beniensis recovered as E. beniensis in the phylogenetic analysis.
Consistent with a previous study [
23
], we recovered the Yellow Anacondas as para-
phyletic, with E. beniensis and E.deschauenseei nested within E. notaeus (Figure 2) and with
shallow levels of divergence between the clades (Table 3). Our sampled taxa included
one from the Bolivian Beni that was both genetically and morphologically E. deschauenseei,
despite being outside the known range of this species. In addition, two other anacondas
from the Bolivian Beni had markings that would classify them as E. deschauenseei, while the
phylogenetic analysis placed them within E. beniensis. Therefore, our results challenge the
validity of the Yellow Anaconda being split into species.
Diversity 2024,16, 127 12 of 28
Table 3. Mean pairwise genetic distances between and within known and candidate species of
the Eunectes species complex. E. notaeus 1 refers to samples Eno6, Eno8, RGRnot1, and AM236347.
E. notaeus 2 refers to samples Eno4, Eno7, and ENU69810. Individual gene pairwise distant matrix is
presented in Table S2.
E. murinus E. akayima E. notaeus 1E. beniensis
E. deschauenseei
E. akayima 5.50%
E. notaeus 1 11.27% 10.37%
E. beniensis 10.98% 10.95% 2.25%
E. deschauenseei
10.58% 10.18% 0.67% 2.14%
E. notaeus 2 10.94% 9.91% 0.74% 2.38% 0.81%
3.1.3. Green Anaconda Phylogenetics and Taxonomy
Our analyses further identify two deeply divergent, highly supported sister clades of
the Green Anaconda. One clade is composed of specimens sampled in the northern part of
the E. murinus range; we find this clade in Ecuador, Venezuela, Trinidad, Guyana, Suriname,
and French Guiana. It can be assumed that it is also present in Colombia. The other clade
includes specimens from the southern part of South America, including Perú, Bolivia, French
Guiana, and Brazil. Specimens of both clades are found in French Guiana, suggesting that this
country may be a contact zone for these two groups (Figure 2). The northern and southern
clades have levels of divergences much higher than those for the Yellow Anaconda variants
(Figure 2, Table 3). Our morphological data show that specimens from the northern and
southern clades are indistinguishable morphologically (Table 4). Irrespective of crypsis, our
genetic data show that these two distinct lineages within E murinus form well-supported
deep clades, allowing the separation into two species based on their genetic divergence
(Tables 3and S2, Figure 2), temporal divergence (Figure 4and Table 5), and branch length
in both the Bayesian analysis and Maximum Likelihood trees (Figures 2and S1). The high
level of genetic divergence and geographic separation justifies the recognition of the northern
population as a distinct species. Therefore, we propose the scientific name Eunectes akayima
sp. Nov. (see Table 6for holotype details, and Discussion Section 4.2 for the etymology and
more in-depth considerations) and the common name Northern Green Anaconda.
Table 4. Morphological and meristic comparison of different species of Green Anaconda. The
specimen listed as Linnaeus’s is the combination of type series 319 described in Systema Naturae [
91
]
and Gronovious [
92
]. The other morphological info reported is from NRM-9 from Stockholm Museum.
In addition, Roze [
88
] reports 242–262 ventral, 63–73 subcaudal, and 57–64 dorsal scales at mid-body
for Venezuela. Gorzula and Pilgrim [
89
] scale count is within these ranges for Guyana. Data from
E. akayima from this study come from Venezuela (n= 3). Other data from the literature come from
Dirksen [
24
], reported by Tarkhnishvili et al. [
23
]. Suborbitalia are also called lorilabials in other
references. Here, we followed Dirksen (2002) for consistency.
E. akayima
This Study
E. akayima
Dirksen (2002)
E. murinus
Dirksen (2002)
Linnaeus 1758
(NRM-9)
Ventral 247–249 243–259 254
Subcaudal 67–68 61–69 61–78 65–69
Dorsal scales, mid-body 65–65 59–66 58–74
Dorsal blotches 80–90 104–116 81–148 113
Spots in contact 10–20
Supralabials 14–15 16–17 14–18 damaged
Infralabials 16–20 20–21 18–25 16
Infraoculars 2–3 3
In contact with eye 8–9 6–8 5–10 damaged
Loreal 4–5 3–9 3–9 damaged
Supraocular 1–1 1
Diversity 2024,16, 127 13 of 28
Diversity 2024, 16, x FOR PEER REVIEW 14 of 30
Figure 4. Calibrated species tree depicting inferred lineage splits, assuming the scenario of one land
bridge. Node bars on the tree represent the 95% highest posterior density (HPD95%) divergence
interval of each node. Legend at the top shows the split of the E. akayima and E. murinus under the
three other scenarios that we tested for.
Intriguingly, within both E. akayima and E. murinus, there are well-supported sub-
clades with divergences at or above the level of the structures within the Yellow Anaconda
clade. For the Southern Green Anaconda clade, further geographic structure is evident:
one subclade is restricted to the east, around the Xingu river basin (eastern Brazil, in the
states of Para, Mato Grosso, and Mato Grosso do Sul), and the other subclade extends
from Peru and Bolivia to French Guiana, probably including the main channel of the Am-
azon River.
Table 4. Morphological and meristic comparison of different species of Green Anaconda. The spec-
imen listed as Linnaeus’s is the combination of type series 319 described in Systema Naturae [91] and
Gronovious [92]. The other morphological info reported is from NRM-9 from Stockholm Museum.
In addition, Roze [88] reports 242–262 ventral, 63–73 subcaudal, and 57–64 dorsal scales at mid-body
for Venezuela. Gorzula and Pilgrim [89] scale count is within these ranges for Guyana. Data from E.
akayima from this study come from Venezuela (n = 3). Other data from the literature come from
Dirksen [24], reported by Tarkhnishvili et al. [23]. Suborbitalia are also called lorilabials in other
references. Here, we followed Dirksen (2002) for consistency.
E. akayima
This Study
E. akayima
Dirksen (2002)
E. murinus
Dirksen (2002)
Linnaeus 1758
(NRM-9)
Ventral 247–249 243–259 254
Subcaudal 67–68 61–69 61–78 65–69
Dorsal scales
,
mid-body 65–65 59–66 58–74
Dorsal blotches 80–90 104–116 81–148 113
Figure 4. Calibrated species tree depicting inferred lineage splits, assuming the scenario of one land
bridge. Node bars on the tree represent the 95% highest posterior density (HPD95%) divergence
interval of each node. Legend at the top shows the split of the E. akayima and E. murinus under the
three other scenarios that we tested for.
Table 5. Calculated median ages for the split of the different lineages under different evolutionary
scenarios. HPD95% confidence intervals are listed in parentheses.
Scenario Epicrates -Eunectes
Node
Basal Eunectes Genus
Node
LCA of E. akayima
and E. murinus
LCA of Yellow
Anaconda Node
Two land bridges
(Gunnerus
Ridge/Kerguelen Plateau)
37.68
(51.28–24.01)
Mya
20.81
(31.93–11.18)
Mya
8.70
(15.06–3.95)
Mya
2.85
(5.21–1.31)
Mya
One land bridge
(Kerguelen Plateau only)
38.62
(52.47–25.10)
Mya
21.54
(33.15–11.68)
Mya
9.08
(15.57–4.44)
Mya
2.96
(5.45–1.38)
Mya
No land bridges 46.30
(66.58–28.40)
Mya
26.30
(40.97–14.67)
Mya
11.30
(19.46–5.48)
Mya
3.87
(7.14–1.75)
Mya
Fossil calibration only [82]35.34
(45.41–23.98)
Mya
19.88
(29.61–11.24)
Mya
8.59
(14.43–4.08)
Mya
2.87
(5.28–1.32)
Mya
Intriguingly, within both E. akayima and E. murinus, there are well-supported subclades
with divergences at or above the level of the structures within the Yellow Anaconda clade.
For the Southern Green Anaconda clade, further geographic structure is evident: one
subclade is restricted to the east, around the Xingu river basin (eastern Brazil, in the states
Diversity 2024,16, 127 14 of 28
of Para, Mato Grosso, and Mato Grosso do Sul), and the other subclade extends from Peru
and Bolivia to French Guiana, probably including the main channel of the Amazon River.
Table 6. Morphological comparison of the holotype and paratype of E. akayima, and the lectotype of
E. murinus.
Eunectes akayima
(MCNG 1042)
Holotype 1
Eunectes akayima
(RMNH.RENA.20768)
Paratype 2
Eunectes murinus
(MPEG 27428)
Lectotype 3
Ventral 241 252 254
Subcaudal 53 45 66
Dorsal scales, anterior body
50 45 50
Dorsal scales, mid-body 60 61 58
Dorsal, posterior 41 38 42
Dorsal spots 94 96 94
Spots in contact 19 17 18
Supralabials 14/15 16/16 16/16
Infralabials damaged 22 left side, right side
is damaged 22/22
Infraoculars damaged 2 2/2
In contact with eye 7/8 7 7/7
Suborbitalia damaged 4 5/5
Supraocular 1/1 1 1/1
1
Holotype is in UNELLEZ Museo de Ciencias Naturales, Venezuela.
2
Paratype is in the Naturalis Biodiversity
Center, the Netherlands. 3Lectotype is in Museu Emilio Goeldi, Brazil.
3.2. Divergence Time Estimation
The results of our multiple molecular clock analyses showed that the analysis based
on paleontological information alone yielded slightly more recent splits than the other
setups. On the other hand, the approach excluding the possibility of dispersal across
Cretaceous land bridges gave older divergence times. Despite these minor differences,
the ranges obtained from the different analyses were found to overlap to a large extent
(Figure 4, Table 5). Our estimate for the divergence of Eunectes from its sister lineage Epicrates
is approximately 46–35 Mya (95% HPD: 66.58–28.40 to 45.41–23.98; Paleocene/Eocene),
depending on the approach.
4. Discussion
Our phylogenetic analyses reveal two major clades within what we currently recognize
as E. murinus, one distributed in the northern part of South America and another one
distributed toward the central and southern parts of the continent. The genetic distances
inferred here, as well as the molecular clock analyses, suggest that these clades are divergent
enough to justify their separation into two distinct species (E. akayima and E. murinus). In
contrast, our analyses reveal much lower genetic divergence among three smaller-bodied
species and fail to recover the monophyly of E. notaeus. These results, together with the
lack of reliable diagnostic morphological characters, cast doubt on E. notaeus,E. beniensis,
and E. deschauenseei as separate species.
4.1. Taxonomic Implications for Yellow Anacondas
Tarkhnishvili et al. [
23
] found similarly small genetic distances, and paraphyly, within
the Yellow Anaconda clade and concluded that the involved species had not reached
lineage sorting, with both E. beniensis and E.deschauenseei appearing as sister taxa but both
nested within E. notaeus. Despite their genetic proximity and paraphyletic nesting position
Diversity 2024,16, 127 15 of 28
within E. notaeus, Tarkhnishvili et al. [
23
] suggested that they should be kept as separate
species due to the physical geographic distance separating their known distributions. Our
data show a similar genetic proximity between these groups and the same paraphyletic
pattern within E. notaeus. However, we identified one individual who is morphologically
and genetically similar to E. deschauenseei in the Bolivian Beni, more than 1700 Km from
its known distribution. This finding calls into question the currently known geographic
distributions of these species (Figures 1and 5), and even whether they are different species
at all. At least two anacondas from the Bolivian Beni have markings that classify them as
E. deschauenseei, while the phylogenetic analysis places them as clustered with E. beniensis
samples. Taken together, this calls into question both the morphological differences found
by Dirksen and Böhme [25] as well as the differences in their geographic distribution.
A crucial aspect considering E. beniensis and E. deschauenseei as valid species or even
subspecies is that in biological taxonomy, a clade is a group of organisms that includes
a common ancestor and all its descendants. If we were to recognize either of these as
subspecies of E. notaeus, this would make E. notaeus a paraphyletic species, which is not
desirable in modern taxonomy. Thus, despite the fact that E. beniensis and E. deschauenseei
each form monophyletic groups, they do so by rendering E. notaeus paraphyletic (Figure 2
in this study, which is consistent with previous work [
23
]). Therefore, by adhering to
phylogenetic principles, we do not recognize them as either species or subspecies. It may
well be that there are other subspecies within E. notaeus that our dataset is insufficient to
detect but with the data available, we prefer to be conservative. We propose that E. beniensis
and E. deschauenseei be grouped together within E. notaeus until more detailed studies using
nuclear DNA and more complete geographic sampling can determine their relationships,
if any.
One possibility is that E. beniensis and E. deschauenseei are forest-dwelling ecotypes of
E. notaeus, rather than different species. The darker coloration and similarities that they
share with E. murinus could be homoplasy as a result of adaptation to the same habitat. It
has been proposed that the preference of E. notaeus for open habitats keeps its distribution
separate from that of E. murinus [
23
]. However, while it is true that some studies show that
E. notaeus and E. beniensis may prefer open habitats, the same studies also show that both
species can be found in closed-canopy forests [
30
,
32
], so closed-canopy forests would not
be a barrier for their distribution. Furthermore, we have found evidence that E. notaeus
does occur in closed-canopy forest (see below). Alternatively, Dirksen and Böhme [
93
]
proposed that E. beniensis was a hybrid between E. notaeus and E. murinus but later revised
this interpretation and described it as separate species [
24
,
25
]. This possibility could be
re-examined. A more complete study, including nuclear patterns of lineage sorting and
introgression, is needed to answer this question.
Either scenario, hybridization or a forest ecotype of E. notaeus, would imply the
existence of a population of E. notaeus living in Beni (Bolivia) and northeastern Brazil, or
throughout the Amazon basin, for which there is no evidence. Unfortunately, because
the distribution of each species is taken for granted, collected specimens are generally
not systematically keyed. Instead, the criterion used to identify a species is where it is
found [
25
]. For example, a recent paper on snakes from Rondonia (Brazil) includes an image
of a Yellow Anaconda (E. notaeus) that is misidentified as a Green Anaconda (Figure 5) [
94
].
Also, a recent paper on Beni Anacondas also includes a picture of a specimen with small
lateral flecks that best fits the description of E. deschauenseei [
26
]. We also made this mistake
when working in Beni. We assumed that all anacondas that were not E. murinus were
E. beniensis without properly keying all specimens. Therefore, the presence of a low-density
population of E. notaeus in the Amazon basin that escaped detection cannot be ruled out.
Diversity 2024,16, 127 16 of 28
Diversity 2024, 16, x FOR PEER REVIEW 17 of 30
has been proposed that the preference of E. notaeus for open habitats keeps its distribution
separate from that of E. murinus [23]. However, while it is true that some studies show
that E. notaeus and E. beniensis may prefer open habitats, the same studies also show that
both species can be found in closed-canopy forests [30,32], so closed-canopy forests would
not be a barrier for their distribution. Furthermore, we have found evidence that E. notaeus
does occur in closed-canopy forest (see below). Alternatively, Dirksen and Böhme [93]
proposed that E. beniensis was a hybrid between E. notaeus and E. murinus but later revised
this interpretation and described it as separate species [24,25]. This possibility could be re-
examined. A more complete study, including nuclear paerns of lineage sorting and in-
trogression, is needed to answer this question.
Figure 5. Distribution of E. akayima and E. murinus samples in this study. The light-yellow dot rep-
resents E. notaeus of the Beni ecotype (formerly E. beniensis). The orange dots represent E. notaeus of
the Dark Spoed Anaconda ecotypes (formerly E. deschauenseei). Notice the substantial distance be-
tween the mouth of the Amazon where the Dark Spoed Anaconda general distribution is and one
of our samples found in the Bolivian Beni. The yellow triangle shows the location of a recent Yellow
Anaconda reported in Rondonia, Brazil [94]. The Casiquiare river is presented in dark blue, con-
necting the Orinoco river (turquoise) with the Rio Negro (turquoise), which is a tributary of the
Amazon. The Vaupes arch indicates where waters from the north and the south were divided in
geological time.
Either scenario, hybridization or a forest ecotype of E. notaeus, would imply the ex-
istence of a population of E. notaeus living in Beni (Bolivia) and northeastern Brazil, or
throughout the Amazon basin, for which there is no evidence. Unfortunately, because the
distribution of each species is taken for granted, collected specimens are generally not
systematically keyed. Instead, the criterion used to identify a species is where it is found
[25]. For example, a recent paper on snakes from Rondonia (Brazil) includes an image of
a Yellow Anaconda (E. notaeus) that is misidentified as a Green Anaconda (Figure 5) [94].
Also, a recent paper on Beni Anacondas also includes a picture of a specimen with small
lateral flecks that best fits the description of E. deschauenseei [26]. We also made this mis-
take when working in Beni. We assumed that all anacondas that were not E. murinus were
E. beniensis without properly keying all specimens. Therefore, the presence of a low-
Figure 5. Distribution of E. akayima and E. murinus samples in this study. The light-yellow dot
represents E. notaeus of the Beni ecotype (formerly E. beniensis). The orange dots represent E. notaeus
of the Dark Spotted Anaconda ecotypes (formerly E. deschauenseei). Notice the substantial distance
between the mouth of the Amazon where the Dark Spotted Anaconda general distribution is and
one of our samples found in the Bolivian Beni. The yellow triangle shows the location of a recent
Yellow Anaconda reported in Rondonia, Brazil [
94
]. The Casiquiare river is presented in dark blue,
connecting the Orinoco river (turquoise) with the Rio Negro (turquoise), which is a tributary of the
Amazon. The Vaupes arch indicates where waters from the north and the south were divided in
geological time.
4.2. A New Species of Green Anaconda
Our data show that two distinct lineages within the former E. murinus form well-
supported deep clades, allowing the separation of two species based on their genetic
divergence, time divergence, and branch length in both the Bayesian analysis and Maximum
Likelihood trees: E. akayima sp. nov. and E. murinus. Although we are aware that our data
come only from mitochondrial DNA, the divergence of these clades is substantial. Male
and female anacondas have comparable dispersal patterns, showing strong philopatry in
both sexes [
22
]; therefore, it is unlikely that the structure found in mDNA is the result
of differential dispersal of males and females within the same species. We believe that
the lack of support from nuclear genes for the separation of these clades is due to the
low rate of variation at these loci, rather than a lack of separation between taxa. We also
examined TATA-binding protein (TBP) and intron data, which also failed to distinguish the
northern and southern clades. It separates E. murinus from E. notaeus with an extremely
short branch length and a difference of one pair of bases. If two clades that separated at
24 Mya (Table 5, Figure 4) show such a small difference (Figure S2), it stands to reason
that this marker would not be able to detect a split that occurred at 10 Mya. Thus, the lack
of nuclear support is more likely to be related to inappropriate markers than to a lack of
difference. The mitochondrial support for the separation of these two clades is superior to
that found in other vertebrates in the recent literature [95,96].
In addition to the strong mitochondrial DNA support, there is a well-established
pattern of the presence of sister species with northern and southern distributions on the
continent. These include lizards of the genus Tupinambis, with T. cryptus occupying a distri-
bution similar to the northern species and other species in the south [
97
]; Dracaena, with
Diversity 2024,16, 127 17 of 28
D. guianensis in the north and D. paraguayensis in the south [
98
,
99
]; matamata turtles with
Chelus orinocensis in the north and C. fimbriata in the south [
100
]; Red-headed Amazon River
turtles (Podocnemis erythrocephala) [
101
]; the arboreal boas Corallus with C. ruschenbergerii in
the north and others to the south [
102
,
103
]; boas of the genus Epicrates with E. maurus in
the north and the other lineages in the south [
104
]. While there may not be a clear barrier
separating the northern clades from the southern species today, these patterns likely speak
to paleogeographic events that produced this split at the continental scale in a variety of
taxa (see below). Thus, the separation of E. akayima sp. nov. from E. murinus is not unique
and is likely part of this continent-wide biogeographic pattern.
The first challenge in describing this new species of Green Anaconda is to determine
which is the new species. In his 1758 Systema Naturae, Linnaeus gave only “America” as
the place of origin [
91
]. The Adolphi Friderici Museum has a specimen labelled NRM9,
identified as Boa murina, which could be the specimen described by Linnaeus. The record
of this specimen is unclear and there is no provenance for it, but it appears to be a specimen
in Linnaeus’ collection and its scale number matches that of #319 in Linnaeus’ Systema
Naturae. Attempts to obtain tissue samples from this specimen were unsuccessful, which
is to be expected given the low probability of obtaining usable DNA from such an old,
formalin-fixed specimen. It is likely that the specimen described by Linnaeus was from
Suriname, as much of the trade to Europe came from this area (E. Åhlander pers. comm.).
However, our data show that French Guiana is probably a contact zone where both species
can be found, and Suriname may also be a contact zone. Therefore, even if we knew for
sure that specimen #319 in Linnaeus’ collection was from Suriname, we would still not
know which species it was, because both species are truly cryptic, and there is no way to
tell from morphological data which species the type belongs to, as far as anyone can tell.
When Linnaeus described the Boa murina, he provided reference to other specimens from
Seba [
105
] and Gronovious [
92
], who in turn cited two specimens by Seba and added his
description of a third specimen. The plate 29 specimen cited by Linnaeus has no source in
Seba’s catalogue. Gronovius refers to this specimen in one of his entries, entry 44, and also
to specimen 1 from plate 23 of Seba’s catalogue. This snake, whose drawing resembles that
of E. deschauenseei, has a source, Guianensis, which probably refers to present-day French
Guiana. As there is no scale number on either of Seba’s specimens, it is uncertain whether
Linnaeus ever examined the specimens himself, or whether he simply based his inclusion
of these specimens on the drawings in Seba’s catalogue. Seba’s second collection was sold
at auction after his death [
106
] and is probably lost. Gronovious also provides a description
of a specimen of his own and alludes to specimen A in plate 606 of Physica Sacra [
107
]. This
specimen is doubtlessly an anaconda that was in the collection of Johann Heinrich Linck,
Leipzig. However, there is no type specimen of anacondas in this collection today (Bauer,
per com).
Since the provenance of specimen NRM9 is unknown, and given that both Green
Anaconda species are truly cryptic, there is no way to determine which clade the syntypes
belong to other than genotyping, which is not possible with such an old specimen. We
propose to name E. murinus as the southern species because of its larger distribution and
for historical reasons. We believe that naming the new species as the one with the smallest
distribution will contribute to the stability of the nomenclature code, as it will result in less
geographical change. In addition, although E. akayima sp. nov. is found in French Guiana
and Suriname, it is a species of the Orinoco Basin, which was not explored by European
naturalists until later. The lectotype for E. murinus is a specimen from the Xingu River in
Para, Brazil, labelled MPEG 27,428 in the Museu Paraense Emílio Goeldi. It was found in
Altamira, State of Para, Brazil (3
◦
9
′
16
′′
S, 52
◦
14
′
11
′′
W) in October 2011 by Emil Hernández.
Since most of the distribution of E. murinus is in Brazil, we consider it appropriate that the
lectotype is in that country, even though the original specimen may have been collected in
Suriname. We also designate specimen MCNG 1042 from UNELLEZ Museo de Ciencias
Naturales, Venezuela, as the holotype for E. akayima sp. nov. This specimen was collected
by Jesús Rivas in March 1993 at Hato El Cedral, Apure Estate, Venezuela (7
◦
25
′
0.4
′′
N,
Diversity 2024,16, 127 18 of 28
69
◦
19
′
51
′′
W). The diagnostic features of this species, which are morphologically cryptic,
required DNA sequencing. We also designate a paratype for E. akayima sp. nov. specimen
RMNH.RENA.20768 deposited in the Naturalis Biodiversity Center in the Netherlands and
MBUCV 7189 located at the Museo the Biología de la Universidad Central de Venezuela.
Information for other type specimens can be found in Table S3.
Etymology
We propose the common name, Northern Green Anaconda, for Eunectes akayima sp.
nov. Before the arrival of the Spaniards, northern Venezuela was occupied by various
Indigenous nations, among which the Caribs were an important group. Several Carib
nations remain including the Kariña, Panare, Yekuana, Pemones, and Akawaio. The
word for anaconda in various Cariban languages is a variant of akayima/okoyimo/okoimo, in
which akayi/okoyi/okoi means “snake” and the suffix -ima/-imo means “large”. The suffix
-ima/-imo
does not necessarily mean ‘large’ in a physical sense. Rather, it is used to denote
the kind of largeness that indicates a different category of being. The literal translation of
akayima is “The Great Snake” (S. Gildea pers. Communication [
52
]). The species name akay-
ima is pronounced as follows:
e
k
e
yim
e
in standard dictionary pronunciation font;
˘
uk
˘
uy
¯
em
˘
u
using the phonics; and uh-kuh-yee-muh using the Plotkin method for English-like writing
to capture Cariban language pronunciations [
108
]. The word akayima is also used to refer to
the rainbow, probably associated with a feathered serpent in their belief system that came
out after rains to dry its feathers [
109
]. We, therefore, acknowledge the culture of these
Indigenous people who share their territories with this species by adopting their word
for anaconda as the specific epithet for this new species. We propose the common name
for E. murinus as Southern Green Anaconda, to promote taxonomic stability for the most
widely distributed species and avoid confusion. Table 6provides a comparison between
the E. akayima sp. nov. holotype, one of its paratypes, and the E. murinus lectotype.
Previous work had identified other candidate species and subspecies of the anaconda
in the Orinoco basin with somewhat similar distribution to E. akayima [
110
]. However, all of
these differences have been found to be inconsistent [
24
,
27
,
111
]; therefore, these synonyms
are all invalid. In addition, the word “akayima” has been indigenously used to designate this
species for at least hundreds (and perhaps even thousands) of years before the use of any of
the other synonyms. It was certainly in use in 1758 when the Code started counting names
as valid; so, akayima is clearly the senior synonym. This is, admittedly, an unorthodox
position regarding the International Code of Zoological Nomenclature [
112
], which prefers
the names that have been published in Western science as “valid”. However, it is well due
time that Western science starts recognizing the ancestral knowledge and cultural legacy
of non-Westernized society. If we respect and honor the culture of these original nations,
accepting akayima as the senior synonym is unavoidable.
4.3. Paleogeographic Events Triggered the Origin of Large-Bodied Aquatic Snakes
Our estimate for the divergence of Eunectes from its sister lineage Epicrates is approx.
46–35 Mya (Paleocene/Eocene) depending on the approach (Table 5). Previous studies
have claimed this divergence to be more recent [
74
,
113
], but this estimate has been subject
to great variability [
103
]. The differences we found are likely the result of the impossibility
to apply soft maximum limits to approximately half of our fossil-based calibration points in
our analysis. Therefore, those were only treated as hard minima. While the problem of the
underestimation of divergence dates due to fossil calibration featuring hard maxima may
be a common issue in molecular clocks, this might be particularly impactful for regions
with notoriously poor fossil records, like South America. During much of the Cenozoic
large extensions of the Amazon basin were flooded forest and flooded habitats covered by
black water systems with a very low pH [
5
,
85
,
86
]. This low pH would have dissolved the
calcium phosphate from the bones in a short time, making fossilization substantially less
likely than it normally is.
Diversity 2024,16, 127 19 of 28
A Paleocene/Eocene date for the origin of Eunectes as an aquatic lineage seems plau-
sible because it matches relevant paleogeographic events and similar origins of other
South American aquatic taxa. As the Nazca plate subsided under South America, circa
90 Mya [
114
], it would have made the mouth of the Proto-Orinoco/Amazon shallower, pre-
venting it from draining all its volume and flooding extensive parts of the continent. This
was a process of general flooding of the western part of the continent at geological speed.
It likely started with the river backing up and permanently flooding its flood plains [
5
,
87
].
The proportion of flooded forest, Varzea and Igapo, increased over time, gaining surface over
Terra Firme forest, with Varzea forest expanding from west to east into the continent [
115
].
Because of the slow nature of this process, it would have allowed for natural selection to
develop aquatic lineages as the aquatic habitat became more abundant and aquatic niches
became available [87].
Previous studies have estimated the appearance of Chelus, an aquatic turtle specializing
in small forest creeks around 70 Mya [116]. Around 60 Mya, South American side-necked
turtles (Podocnemididae) diversified into new lineages [
117
]. At approximately the same
time, alligatorids split into two lineages [
118
]: a larger one, Caiman, that prefers rivers
and lagoons, and Paleosuchus, a smaller forest specialist living in small creeks inside the
forest [
119
]. Around 40 Mya, a lineage of very large caimans, Melanosuchus, also split
from the same lineages [
118
]. In addition, approximately 40–35 Mya, Teiidae produced
two aquatic linages, Crocodilurus and Dracaena, from terrestrial ancestors [
120
]. Lastly,
approximately 49 Mya, we see the appearance of a strictly arboreal lineage of boids: Coral-
lus [
103
,
113
]. Specialization to living on the trees could be an evolutionary response to a
flooded understory that was unavailable. Taken together, this scenario speaks of a gener-
alized increase in habitats for aquatic lineages throughout the continent, and throughout
the Cenozoic, that supports the notion that Eunectes split from Epicrates earlier than other
studies have estimated.
We hypothesize that Eunectes diverged from Epicrates due to the increased occurrence
of flooded forest resulting from the initial damming of the Proto-Orinoco/Amazon. This
process would have opened extensive habitats for aquatic snakes to exploit. The evolution
of the dorsal eye and nostril placement and cryptic coloration would have been beneficito
hunt terrestrial prey in the flooded forest. Surprisingly, despite Eunectes being an aquatic
lineage, and the abundance of variety of fish in South America, fish are not an important
part of the diet of any extant lineages of anacondas. While E. notaeus has been reported
scavenging on fish dead in droughts [
40
], the importance of fish in anacondas’ diets is
negligible [
33
,
39
,
40
,
121
]. The shallow water of the flooded forest likely became quickly
dysoxic or anoxic with the warmth of the area [
122
,
123
], not allowing for fish as reliable
prey. Thus, as anacondas adapted to their new aquatic habitats, they continued preying on
terrestrial prey in the flooded forest, rather than undergoing a dietary shift to fish [52].
It is likely that the last common ancestor of all Eunectes species was not much larger
than a regular Epicrates. The large size of today’s Eunectes probably evolved later, as
living in water released them from the constraints of gravity and the aquatic vegetation
allowed them to hide from their prey despite their large size. The split between the
large- and the small-bodied clades occurred an estimated 26 (95% HPD: 40.97–14.67)-20
(95% HPD: 29.61–12.24) Mya (Oligocene), a time when the western Amazon was covered
by a mega wetland [
124
,
125
]. At this point, the Andes had completely blocked the passage
of the Proto-Orinoco/Amazon and the river was diverted to the north, forming the Pebas
system [
5
,
87
,
114
,
115
,
126
]. While the permanent waters of the Pebas system may have
allowed the evolution of large-bodied aquatic specialists, the specific topology of the
adjacent area might be the reason for the evolutionary preservation of smaller-bodied
relatives. Due to the extremely flat relief of the area (1.5 cm/km; [
127
]), a small change in
the water level would have resulted in a substantial displacement of the water edge [
5
].
The Eunectes populations living at the edges of the hyper-seasonal Pebas system would,
therefore, need to travel long distances on dry land to track the receding waters in every
dry season. The need to move across dry land might have constrained their growth, thus
Diversity 2024,16, 127 20 of 28
maintaining a lineage of small-bodied anacondas (E. notaeus). Since Pebas drained toward
the north, there would have been a constant volume of water in this direction, causing only
large-bodied anacondas (E. akayima) to be found toward the north of the Pebas system. This
would explain today’s lack of small-bodied Eunectes to the north of Pebas, even today with
part of the area possessing developed hyper-seasonal Savannahs. These are no older than
ten thousand years [128,129].
4.4. Miocene Divergence of Northern and Southern Green Anacondas
Our molecular clock analyses indicate that E. akayima and E. murinus diverged in the
Miocene at the same time that other South American taxa were undergoing similar-aged
north–south divergences (Table 5, Figure 4). The vicariant event splitting these lineages
might have been associated with the uplift of the Vaupés arch, an elevation that connected
the Andes with the Guyana shield on the southern end (Figure 5). The rise of this arch
separated the north from the south, in what is now the Venezuelan and Colombian Llanos.
This was the result of the continent-wide readjustment of the landscape that resulted
in tilting the continent to the east and the separation of the Proto-Orinoco and Proto-
Amazon River into their current descendants [
114
,
126
]. The rise of this arch occurred
almost synchronously to the split of these clades. So, this was likely the vicariant event
that separated these two species. However, the current distribution of E. akayima is far
south of the Vaupés arch, all the way to the Yasuni National Park, in the Ecuadorian
Amazon, as sedimentation has changed the current topology of the region [
127
]. Looking
at the big picture, combined, the presence of the Pebas system as a barrier for dispersal of
shallow water organisms [
130
] as well as the Vaupés arch splitting the watersheds likely
explain the separation between the north and south of much of the aquatic fauna in South
America including not only anacondas but also caimans [131,132], matamata turtles [100],
stingrays [127], and lizards [97,133].
Eunectes murinus is composed of an eastern clade associated with the Xingu river,
which diverged approximately 3.5-2.53 Mya from the one found in the west, associated
with the Beni drainage. It is possible that rivers Tapajos and Madeiras might also have
independent lineages since they have similar topologies. The similarities between anacon-
das in the western Amazon and those in French Guiana speak of the Amazon River as a
waterway connecting these areas.
Unfortunately, before drawing more precise conclusions about the distribution ranges
of both species of Green Anaconda and potential interaction zones, a more comprehensive
sampling of the intervening areas will be needed. One problem hindering our understand-
ing of these distribution patterns is the difficulty of identifying what would constitute
a biogeographic barrier for anacondas. At first glance, the whole basin seems like fair
ground for anaconda dispersal. It seems like they should be able to disperse more broadly
into their landscape given today’s homogeneity of many habitats. Indeed, there does not
seem to be any barrier preventing E. akayima from moving into the southern part of the
continent or preventing E. murinus from dispersing north. The Casiquiare river flows from
the Orinoco to the Rio Negro, which in turn flows into the Amazon (Figure 5) [
114
,
127
].
Consequently, E. akayima could disperse down to the northern bank of the Amazon. At
its broadest section, and perhaps starting at Manaus, the Amazon is a formidable water
body that anacondas might not be inclined to swim across, since they much prefer shallow
water bodies with aquatic coverage [
22
], but the presence of E. murinus in French Guiana
and the ecotype of E. notaeus known as the Dark Spotted Anaconda north and south of
the lower Amazon suggests that the river is not a definite barrier for anaconda dispersal.
Our Peruvian samples come from the Iquitos region. The Amazon in this region is narrow
enough not to constitute a barrier against anaconda dispersal. In addition, E. akayima is
also found in Yasuni, Ecuador, where the Napo River flows into the Amazon from eastern
Ecuador, so there would be a clear path for this species to colonize the rest of the Amazon
basin. Clearly, more sampling is needed to determine possible contact zones between E.
akayima and E. murinus.
Diversity 2024,16, 127 21 of 28
4.5. The Arrival of the Pleistocene
It is intriguing that circa 3 Mya, all lineages underwent further splits. We see this in
the E. akayima splitting in Venezuela with the watersheds to the south of the mouth of the
Orinoco. We find the same split between the Peru/Bolivia clade and the eastern one in
E. murinus. It is noteworthy to mention that at this same time, E. notaeus was splitting into
the three lineages we find today. While these splits are not deep enough to grant separation
into multiple species, they do tell us about the dynamics in the continent. They coincide
with the start of the Pleistocene at its glaciations. As the South American continent was
rearranging its drainage, the Amazon and the Orinoco had their current location. There
were still extensive wetlands and marshes covering a good part of the continent [
134
] where
likely anacondas thrived. During the glaciations, the icecaps sequestered a lot of the global
water, producing the expansion of forest [
135
] as the marshes were receding. This would
have led to the separation of aquatic habitats, likely resulting in the synchronous split
among these aquatic lineages.
4.6. Conservation Assessments
The proposed division of distinctive Green Anaconda lineages into two species may
prompt further conservation assessment in their respective ranges and countries. This
study stresses the lack of knowledge on the distribution of species representing large top
predators. As top predators, anacondas are especially vulnerable to habitat degradation:
not only do they suffer from the damage to the habitat, they are also heavily impacted by
the damage to their prey base [
136
,
137
]. In addition, conflict with humans also threatens
predators, since they may accidentally prey upon livestock, as well as being generally
perceived as a danger to humans [
22
,
51
,
52
,
138
]. The reason behind E. murinus being
considered of least concern is its large distribution [
52
,
53
]. Evidence pointing toward two
species in its original range may change that conclusion. Our study highlights our lack
of knowledge on the distribution of different populations and their possible connectivity;
we do not know which populations may be under stress due to inbreeding. As habitat
fragmentation and other forms of habitat degradation continue, the conservation status of
these top predators may change.
Further surveys should aim to decipher the status of the different lineages in E. no-
taeus. The newly documented distribution of E. notaeus makes us wonder about its true
conservation status. The forest-dwelling ecotype seems to have very small densities and
would represent a very vulnerable lineage. Alternatively, E. notaeus may be more common
in the Amazon basin but misidentified in the field, demonstrating the need to further clarify
morphological distinctions of these subspecies.
Introgression among Eunectes species is undocumented, but possible. Future studies
using nuclear markers may help to identify the geographic areas and extent of potential
introgression and comment on the potential conservation concerns of hybridization zone
expansion with climate change and threats of outbreeding depression for any of our
proposed species or subspecies groups.
The deforestation of the Amazon basin due to agricultural expansion has resulted in
an estimated 20 to 31% habitat loss [
11
,
15
], which might impact up to 40% of its forests by
2050 [
15
]. Anaconda species are likely to be threatened by deforestation processes driven
by agriculture, forest fires, climate change, and drought [
52
]. These phenomena affect their
prey base and habitat in general. This emphasizes the need to further delineate Eunectes
species ranges and population trends.
5. Conclusions
This study provides the most extensive sampling of anacondas to date and raises new
questions about the distinctive lineages, geological history, and conservation status of the
Eunectes group. Historical, geographic, and landscape-scale events may have shaped the
current distribution and composition of the species. Looking at the ecology of present-day
anacondas, it would seem that the entire Amazon/Orinoco basin would be an area of
Diversity 2024,16, 127 22 of 28
free dispersal for anacondas. However, the presence of a new cryptic species in the north
and the E. murinus in the south tells us that we still know very little about the gene flow
dynamics of a large vertebrate in the world’s most diverse terrestrial ecosystem. The idea
that there could be a population of E. notaeus living throughout the Amazon basin that
has managed to evade detection thanks to a coloration that superficially resembles that
of E. murinus is puzzling, and speaks loudly to the need for thorough sampling to better
document the diversity we still have.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/d16020127/s1: File S1: Sequences analyzed in this study. Figure
S1: Phylogeny constructed from mitochondrial three-gene concatenated partitioned Maximum
Likelihood analysis; Figure S2: Maximum Likelihood using TBP showing lack of resolution between
the clades; Table S1: Samples analyzed in this study; Table S2: Mean pairwise genetic distances using
individual genes; Table S3: Location of the types designated in this study for this study.
Author Contributions: Conceptualization, J.A.R. and S.C.-R.; methodology, S.C.-R. and B.G.F.;
software, S.C.-R. and M.M.; validation, B.G.F. and F.J.V.; formal analysis, S.C.-R., D.S.-V. and S.M.;
investigation, J.A.R. and S.C.-R.; resources, M.T.B. and P.B. (capture of E. akayima anacondas in
Baihuaeri Waorani Territory), F.J.V., L.F.P., P.D.L.Q. and E.H.; data curation, S.C.-R.; writing—original
draft preparation, J.A.R. and S.C.-R.; writing—review and editing, J.A.R., P.D.L.Q., M.M., L.F.P.,
G.A.R., S.M., D.S.-V., M.T.B., P.B., G.M.B., F.J.V., E.H., J.E.G.-P., B.G.F. and S.C.-R.; visualization, J.A.R.,
S.C.-R., M.M., M.T.B. and P.B.; supervision, J.A.R. and B.G.F.; funding acquisition, J.A.R., S.C.-R.,
B.G.F. and F.J.V. All authors have read and agreed to the published version of the manuscript.
Funding: Partial Sequencing charges were under-written by National Geographic, NM Inbre, NMHU
FRC, and Disney. BGF was funded as a National Geographic Explorer and the Waorani expedition
was documented by National Geographic for their upcoming series Pole to Pole.
Institutional Review Board Statement: Animal handling was carried out and tissues were obtained
under IACUC protocol at New Mexico Highlands University, Approval 2012/7-12-2012, and Univer-
sity of Queensland, Animal Ethics Approval 15 March 2021/AE000075. Registration of E. akayima in
zoobank: A58A262E-2E07-48D3-B712-209CCDFFD038. Permit for working on Indigenous land in
Ecuador: MAATE-DBI-CM-2022-0259.
Data Availability Statement: All Genbank accession numbers of gene sequences are available in the
Supplementary Materials.
Acknowledgments: We thank The Wildlife Conservation Society, The National Geographic Society,
the Doue de le Fountain Zoological Park, Miami Metro Zoo, Bronx Zoo, American Museum of Natural
History, and private collectors. We are very grateful to the following four people from the Naturalis
Biodiversity Center for their great help in sequencing the DNA of two (museum) Northern Green
Anaconda specimens (RMNH.RENA.20768 and RMNH.RENA.29769): Elza Duijm, Esther Dondorp,
Stacey Dubbeldam, and Dick Groenenberg. We would also like to express our gratitude toward the
Naturalis Biodiversity Center for swiftly providing us with the necessary permission to perform the
invasive sampling in these two anaconda specimens. We also thank COVEGAN, Estación Biologica
Hato El Frío, for logistic assistance and for permission to perform this study on their property. We
thank New Mexico Highlands University FRC and NM INBRE for partial funding. We also thank
the governments of Bolivia, Brazil, Ecuador, France, Perú, Venezuela, Suriname, and Trinidad and
Tobago and the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES)
for research permits. We also thank Erik Åhlander and Claudia Samuelsson for information on NRM9
from the museum Adolphi Friderici. We thank the staff of the Museo de Biología of the Universidad
Central de Venezuela (MBUCV) for providing access to their collection of amphibians and reptiles,
especially Professors Mercedes Salazar and Hedelvy J. Guada. We are in debt to Anthony Fouquet for
early comments on the ms and providing tissue samples. We thank Christine Strussmann and Amalia
Espinoza for help and collaboration during fieldwork in Brazil and Ecuador. We also thank Kim
Roelants for early comments on the ms and assistance in the phylogenetic dating analysis. We thank
Andi Wolfe for help, encouragement, and assistance in the beginning of the molecular study. We thank
E. Lavilla for advice and help navigating taxonomic questions. In addition, we thank Spike Gildea for
help with Carib language and its pronunciation. Finally, we thank Will Smith for his help in collecting
the E. akayima samples from the Bameno region of the Baihuaeri Waorani Territory in Ecuador.
Diversity 2024,16, 127 23 of 28
Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
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