![Adult female of Chilabothrus angulifer bearing haplotype UD4 (Haplogroup I). Rehák I, Šťáhlavská I., Somerová B. O., Šimková O., Frynta D. 2022. A deep divergence and high diversity of mitochondrial haplotypes in an island snake: the case of Chilabothrus angulifer (Serpentes: Boidae). Acta Societatis Zoologicae Bohemicae 85: 1–22, 2021 [2022].](https://www.researchgate.net/profile/Ivan-Rehak/publication/364302546/figure/fig1/AS:11431281089068674@1665416608871/Adult-female-of-Chilabothrus-angulifer-bearing-haplotype-UD4-Haplogroup-I-Rehak-I_Q320.jpg)
![Subadult female of Chilabothrus angulifer bearing haplotype F8 (Haplogroup II). Rehák I, Šťáhlavská I., Somerová B. O., Šimková O., Frynta D. 2022. A deep divergence and high diversity of mitochondrial haplotypes in an island snake: the case of Chilabothrus angulifer (Serpentes: Boidae). Acta Societatis Zoologicae Bohemicae 85: 1–22, 2021 [2022].](https://www.researchgate.net/profile/Ivan-Rehak/publication/364302546/figure/fig2/AS:11431281089092827@1665416662584/Subadult-female-of-Chilabothrus-angulifer-bearing-haplotype-F8-Haplogroup-II-Rehak-I_Q320.jpg)
![Adult male of Chilabothrus angulifer bearing haplotype 93 (Haplogroup III). Rehák I, Šťáhlavská I., Somerová B. O., Šimková O., Frynta D. 2022. A deep divergence and high diversity of mitochondrial haplotypes in an island snake: the case of Chilabothrus angulifer (Serpentes: Boidae). Acta Societatis Zoologicae Bohemicae 85: 1–22, 2021 [2022].](https://www.researchgate.net/profile/Ivan-Rehak/publication/364302546/figure/fig3/AS:11431281089068713@1665416711387/Adult-male-of-Chilabothrus-angulifer-bearing-haplotype-93-Haplogroup-III-Rehak-I_Q320.jpg)
![Map of Cuba island with locality indication (1 – Sierra Maestra, 2 – Nicaro, 3 – Trinidad, 4 – Matanzas, 5 – Viñales). Rehák I, Šťáhlavská I., Somerová B. O., Šimková O., Frynta D. 2022. A deep divergence and high diversity of mitochondrial haplotypes in an island snake: the case of Chilabothrus angulifer (Serpentes: Boidae). Acta Societatis Zoologicae Bohemicae 85: 1–22, 2021 [2022].](https://www.researchgate.net/profile/Ivan-Rehak/publication/364302546/figure/fig4/AS:11431281089122408@1665416785691/Map-of-Cuba-island-with-locality-indication-1-Sierra-Maestra-2-Nicaro-3_Q320.jpg)
![Median-joining haplotype network computed using CYTB 1059 bp alignment containing all 96 samples of Chilabothrus angulifer, the size of the circle is proportional to the frequency of haplotypes and the mutational steps are indicated on the branches. Rehák I, Šťáhlavská I., Somerová B. O., Šimková O., Frynta D. 2022. A deep divergence and high diversity of mitochondrial haplotypes in an island snake: the case of Chilabothrus angulifer (Serpentes: Boidae). Acta Societatis Zoologicae Bohemicae 85: 1–22, 2021 [2022]. Rehák I, Šťáhlavská I., Somerová B. O., Šimková O., Frynta D. 2022. A deep divergence and high diversity of mitochondrial haplotypes in an island snake: the case of Chilabothrus angulifer (Serpentes: Boidae). Acta Societatis Zoologicae Bohemicae 85: 1–22, 2021 [2022].](https://www.researchgate.net/profile/Ivan-Rehak/publication/364302546/figure/fig5/AS:11431281089068786@1665416861358/Median-joining-haplotype-network-computed-using-CYTB-1059-bp-alignment-containing-all-96_Q320.jpg)
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1
Acta Soc. Zool. Bohem. 85: 1–22, 2021 [2022]
ISSN 2788-1369 (on-line)
ISSN 1211-376X (print)
A deep divergence and high diversity of mitochondrial haplotypes in an
island snake: the case of Chilabothrus angulifer (Serpentes: Boidae)
Ivan 1,*), Ivana 2), Barbora 2),
Olga 2) & Daniel 2,*)
1) Prague Zoo, U Trojského zámku 120/3, CZ–171 00 Praha 7 – Troja, Czech Republic
2) Ethology and Ecology Unit, Department of Zoology, Faculty of Science,
*) Corresponding authors: Ivan.Rehak@zoopraha.cz, frynta@natur.cuni.cz
Received 2 November 2021; accepted 8 July 2022
Published 16 September 2022
Abstract. For the needs of proper management, we analysed the mitochondrial haplotype structure of the European
ex situ population of Cuban boas. The results showed its extraordinary diversity. We sequenced 96 specimens and
detected 25 distinct haplotypes. Besides this haplotype diversity, the results revealed a deep divergence among
three principal haplogroups. Bayesian estimates of the divergence time (3.57 and 2.26 Mya) suggest that within
the currently only recognized species Chilabothrus angulifer
distance corresponds to or is greater than among some other – taxonomically recognized – species of the genus.
This indicates that the Cuban boas represent in fact at least two cryptic subspecies or species. Nevertheless, after
considering the current state of the ex situ population, given the current knowledge of the phylogeography of
Cuban boas and the fact that they inhabit a single large island (and its nearby coastal islands and islets) at present,
we recommend to manage the current ex situ population as a whole.
Key words. Conservation genetics, population management, ex situ population, evolution, phylogeography, adaptive
radiation, speciation, boid snakes, Caribbean herpetofauna, Great Antilles.
INTRODUCTION
Terrestrial fauna of the Great Antilles is characterised by an extraordinary high endemism and
species diversity. It is due to unique biogeographical history of the Caribbean (cf. Hedges et al.
2019). A local Cretaceous biota was originally exterminated by Chicxulub impact at the Cretaceous-
-Tertiary boundary (Lyons et al. 2020). In spite of a strong permanent isolation by the Caribbean
Sea, this region has been repeatedly colonized from mainland. It was proposed that the immigrant
lineages dispersed through “Gaarlandia”, the putative incomplete temporary land bridge connecting
NE of the South American Continent with precursors of the Great Antilles during Oligocene and
Early Miocene periods. Currently, geological data rejected “Gaarlandia” hypothesis and molecular
references herein, but see Philippon et al. 2020). Thus colonization of the Great Antilles should be
attributed to events of overwater dispersal (Ali 2012). The earliest vertebrate fossil record is a frog
of the genus Eleutherodactylus coming from Puerto Rico Oligocene, 29 Mya (Blackburn et al.
2020). Nevertheless, the colonization events were extremely rare and thus current species richness
of the Great Antillean region is mainly a result of subsequent speciation within a few immigrant
lineages. While the diversity of native mammalian fauna of the Great Antilles has been substan-
tially reduced by recent extinctions, squamate reptiles representing another clade of terrestrial
non-volatile vertebrates have remained less affected and may serve as a model for phylogenetic
2
and population studies of Caribbean endemism. Studies performed in principal endemic clades of
squamates as anoles (Anolis sensu lato; Losos 2009 and references herein, Cádiz et al. 2018) and
iguanas of the genus Cyclura (Malone et al. 2000) clearly demonstrated that geographic isolation
among large Antillean islands typically corresponds to the deepest divergences. However, both
adaptive radiation and geographic speciation contributed to further speciation. The geographic
ranges of individual species of squamates are typically restricted to a certain part of the island as
Schettino et al. 2010, 2013). Traditionally, four main zoogeographical regions were recognized:
Western, Central, Camagüey-Maniabón and Eastern (Estrada & Ruibal 1999). Species richness
tend to be associated with higher elevations. Cladistic analysis of endemism revealed a complex
pattern with nested areas of endemism concentrated in the Western and Eastern parts of the island
(Murray & Crother 2019).
Great Antillean boas of the genus Chilabothrus
reptilian colonists of the Great Antillean region. Its relatives inhabit the South American continent
-
cording to recent molecular phylogenies, a common ancestor of anacondas of the genus Eunectes
Wagler, 1830 and rainbow boas of the genus Epicrates Wagler, 1830 represents a sister clade of
Chilabothrus. Divergence between Chilabothrus and Eunectes +
EpicratesChilabothrus
stanolseni (Vanzolini 1952) from Florida 18.5 Mya supports the view that ancestors of current
Chilabothrus reached Great Antilles as early as in lower Miocene period (Onary & Hsiou 2018).
Nevertheless, at that time boids successfully dispersed from South America also to continental
Central America as documented by a fossil record of a snake of the genus Boa Linnaeus, 1758
from Panama 19.3 Mye, i.e., prior the Great American Faunal Interchange (Head et al. 2012).
Chilabothrus
Reynolds & Henderson 2018, Landestoy et al. 2021). Phylogenetic relationships within the genus
(1) Cuban – Chilabothrus angulifer C. inornatus (Reinhardt,
C. monensis (Zenneck, 1898) and C. granti (Stull, 1933), (3) Jamaican – C. subavus
C. ampelophis Landestoy, Reynolds et Henderson, 2021, C.
fordii (Günther, 1861), C. gracilis Fischer, 1888, and (5) Hispaniola-Bahamian one C. argentum
Reynolds, Puente-Rolón, Geneva, Aviles-Rodriguez et Herrmann, 2016, C. exsul (Netting et Goin,
C. chrysogaster (Cope, 1871), C. schwartzi (Buden, 1975), C. striatus (Fischer, 1856),
and C. strigillatus (Cope, 1862).
Molecular phylogenies placed C. angulifer as a sister taxon of either the other Chilabothrus
species with divergence time estimated to 21.7 [16.9, 26.0] Mya (Reynolds et al. 2013) or the
Puerto Rican clade with divergence time estimated to 15.3 Mya (Pyron et al. 2013, Reynolds et
al. 2015, 2016a, Landestoy et al. 2021). In any case, C. angulifer represents a phylogenetically
deep and distinct lineage of boids. Besides, its evolutionary history, there are multiple pheno-
typic characters distinguishing this species from its relatives (see also Reynolds et al. 2016a).
Chilabothrus angulifer is (1) the largest form of the genus Chilabothrus -
maternal investment per offspring (e.g., Tolson 1987, Frynta et al. 2016). A deep divergence of
C. angulifer from other Chilabothrus species and other boids as well as presence of numerous
unique morphological, physiological and behavioural characters, makes this island species a good
candidate for conservation concern.
3
Conservation genetics of both wild and ex situ populations was thoroughly studied in multiple
species of the genus Chilabothrus, especially in C. subavus (Tzika et al. 2008, 2009, Newman et
al. 2020), C. inornatusC.
monensisC. argentum (Reynolds 2016b)
and C. chrysogaster (Reynolds et al. 2011). The within-species genetic variation reported by
these studies was rather small, well-corresponding to limited population numbers and fragmented
distribution range of these endangered species (Harvey & Platenberg 2009, Newman et al. 2016,
Tucker et al. 2020). In contrast to a considerable efforts devoted to genetics of these Chilabothrus
species, C. angulifer has remained nearly neglected in this respect. There are just papers concerning
chromosomal evolution (Augstenová et al. 2019) and parthenogenesis (Seixas et al. 2020).
The Cuban boa remains the only one of the three largest Cuban reptiles for which a more de-
tailed population and habitat viability assessment (PHVA) has not yet been performed, while the
PHVA is available for the Cuban crocodile, Crocodylus rhombifer (Soberon et al. 2000) and for
the Cuban iguana, Cyclura nubila
in the Cuban National Red List assessment (González et al. 2012), as Least Concern according
to the IUCN Red List (Fong et al. 2021). However, the real conservation status of Chilabothrus
angulifer
The species is also granted international protection under the Convention on International Trade
in Endangered Species of Wild Fauna and Flora (Appendix II). An important element of species
protection is – in accordance with the modern concept of the conservation strategy (the One Plan
Approach to Conservation) – adequate perspective management of the ex situ population in human
care. The conservation breeding is listed among the recommended elements for the protection of
The effort to keep Cuban boas in human care is old (Rehák, stoodbook data). The Philadelphia
Zoo, USA, imported twenty Cuban boas already in the 19th century (from 1876 to 1893) and
survival was usually very poor and they did not reproduce (or the reproduction was unsuccessful
as in the Berlin Zoo, Germany, where the Cuban boa was also kept at the beginning of the last
century). At that time, the ex situ population consisted exclusively of specimens imported from the
wild. The birth of live young in captivity was achieved in the late 1950s (1958 in the Smithsonian
National Zoological Park, USA).
In the 1960s, 1970s and 1980s, owing to close political and economic relationships with Cuba,
Cuban boas were repeatedly imported to zoos and private holders in the former Czechoslovakia
stoodbook data), German Democratic Republic, Soviet Union, Hungary, and Poland. An inde-
pendent source of imports represented the U.S. Naval Base, Guantanamo Bay, Cuba. From the
end of the 1970s, in addition to imports from the wild, the successful captive breeding began to
ex situ Cuban boa population in human
care (e.g. Huff 1976, Nowinski 1977, Murphy et al. 1978, Vergner 1978, Tolson 1983, Bloxam
& Tonge 1981, Vergner 1989, Rehák 1992). Captive propagation of C. angulifer (although origi-
size and extremely slow life-history. Cuban boas have become common in zoos, public and also
some private collections (Marešová & Frynta 2008).
With the political changes at the turn of the eighties and nineties of the last century, imports
from nature came to an almost complete end. The future of the ex situ population in human care
began to depend almost exclusively on captive breeding. It become clear that zoos must focus
on the long-term perspective management of the ex situ population. At the initiative of the Pra-
gue Zoo and the Amphibian and Reptile Taxon Advisory Group of the European Association of
Zoos and Aquaria (chaired by I. Rehák), the Studbook for Cuban Boa
in a computerized form – I. Rehák, studbook keeper) and the European Endangered Species
Programme (newly the EAZA Ex situ Programme) for Cuban boa was proposed. Consequently,
the Cuban boa EEP – coordinated by Ivan Rehák/Prague Zoo – was approved by the EAZA and
launched in 1993.
The contemporary European zoo population may represent descendants of many founders
originated in multiple localities across Cuba and its neighbouring small islands. Consequently,
mitochondrial lineages from European zoos may be viewed as more or less representative po-
pulation sampling of Cuban population. Now, more than thirty years after the end of imports
from the wild (after 1989, only one import from the wild is registered in the European studbook
– Rehák, studbook data) to Europe and several generations of captive breeding, however, foun-
ding individuals already died (at present, only the last two specimens collected from the wild are
still alive – Rehak, studbook data) and records about their geographic origin are available only
in handful exceptional cases.
C. angulifer. For
this purpose, we described and analysed mitochondrial genetic variation within a population of
C. angulifer kept in European zoos and associated private holders. The sampling was performed
What can the variability estimated within ex situ population tell us about variability and historical
Fig. 1. Adult female of Chilabothrus angulifer
5
demography of wild populations? (2) Is there molecular evidence that founders of this ex situ
zoo population were variable enough to provide a good prospect for further maintenance of this
population?
MATERIAL AND METHODS
Sampling
We analysed 96 new samples of C. angulifer (Figs 1–3) a and one new sample of C. inornatus (see Table 1). The geo-
graphic origin of the sampled specimens or their maternal ancestors was certain in just few cases representing localities
The sampling, sequencing and preliminary analyses were carried out during the years 2002–2009. We decided to rely
on non-invasive sampling causing no harm and minimizing stress in sampled specimens. Thus, we selected buccal swabs
as a source of DNA. We sampled specimens kept by European zoological gardens and collaborating private breeders.
We aimed to include putative founders or their maternal descendants (daughter, granddaughter etc.). In order to avoid
multiple sampling of the same maternal lineage, we inspected pedigree data if available.
DNA Extractions and Sequencing
We sequenced two mitochondrial genes, combined the new sequences with previously published data (Campbell 1997,
Rivera et al. 2011, Reynolds et al. 2013), and supplemented them with additional sequences from GenBank (Phylogenetic
analyses).
information content in previous studies with the same taxonomic scope and availability of sequences. Genomic DNA was
extracted from 96% ethanol-preserved buccal swab with NucleoSpin Tissue kit (Macherey-Nagel) according to manu-
facturer’s protocol for buccal swab isolation. Extracted DNA was stored at –18 °C until used as a template for Polymerase
Chain Reaction (PCR, Sambrook et al. 1989). Individual markers were amplifed by PCR using the same combination
Fig. 2. Subadult female of Chilabothrus angulifer bearing haplotype F8 (Haplogroup II).
6
Fig. 3. Adult male of Chilabothrus angulifer bearing haplotype 93 (Haplogroup III).
7
Phylogenetic and demographic analyses
downloaded from GenBank used as outgroup (C. fordiiC. striatusC.
monensisC. inornatus
C. exsul C. chrysogaster C. subavus
Epicrates cenchria Epicrates maurus Eunectes murinus
Eunectes notaeus
The estimates of evolutionary divergence over sequence pairs between haplotypes of C. angulifer population were
2013). The relationship within population of C. angulifer was represented by using the Median-Joining network approach
(Bandelt et al. 1999) in the program Population Analysis with Reticulate Trees (PopART; Leigh & Bryant 2015). Two
haplotype networks were worked out – for alignment containing all 96 CYTB sequeces of C. angulifer and for alignment
Phylogenetic reconstructions were conducted using Bayesian analysis (BA), maximum likelihood (ML) and Bayesian
Table 1. List of samples collapsed to 25 haplotypes according 1059 bp long allignment of cyt b with description of three
haplogroups. The samples names were created by using unique code helping to identify the zoological garden or breeder,
where the samples were collected (AA – Aalborg Zoo, Denmark; B – Budapest Zoo, Hungary; BAR – Barcelona Zoo,
Czech Republic). If available, the geographic origin of the sampled specimen or its matriline is indicated in parentheses
(in bold); H = haplogroup, N = number of sequences
Viñales
BRF156, BRS1, BRS2, BRS3, C1, C2, F16, F17, DR115, DR117,
II MAT1 1 MAT1 (Matanzas
III TRI 1 TRI (Trinidad
Sierra Maestra
Nicaro
Nicaro
8
starting tree and run for 20,000,000 generations, with trees sampled every 1000 generations and with 25% burn-in. The
selected as the best model of nucleotide substitution by jModelTest v.2.1.6. (Darriba et al. 2012) for both partitions, the
According to Reynolds et al. (2013), we set the prior of Chilabothrus root node to 21.7 Mya (SD=1.8).
Polymorphisms within C. angulifer population were worked out by the statistic software DnaSP v6 6.12.03 (Rozas
estimate population dynamics through time we have constructed a model in BEAUti, we have run Markov chain Monte
Carlo simulations with 30 million iterations and 10 million burn-ins using the GTR model and molecular clock with set-
v1.7.1 and displayed them as Bayesian skyline plot drawing the effective population sizes over time.
RESULTS
We sequenced cyt b in 96 specimens of Chilabothrus angulifer
(Table 1). While 13 of them were detected exclusively in a single specimen, the remaining ones
Median-Joining Network (MJN) revealed a presence of three clearly distinct main groups,
further referred to as Haplogroup I, Haplogroup II, and Haplogroup III (Fig. 5). Maximum unco-
rrected p-distances among CYTB haplotypes belonging to different groups were 0.0220 (mean
Corresponding values for within group comparisons were 0.0121 (0.0076), 0.0083 (0.0033) and
0.0293 (0.0101), for groups I, II and III, respectively (Table 2).
Bayesian spline-plot of this dataset revealed a long-term stability of effective population size
during the last three millions of years followed by a recent decline (Fig. 6). Population parameters
estimated separately for each haplogroup, as well as for pooled ones, are provided (Table 3). These
parameters are congruent with relative stability of the population numbers in the past. The only
exception represents Haplogroup II exhibiting negative values of parameters indicating recent
Table 2. Estimates of evolutionary divergence over sequence pairs between haplotypes of Chilabothrus angulifer popula-
tion. The mean number, minimum, and maximum of base substitutions per site overall sequence pairs within each group
is shown. Analyses were conducted using the maximum composite likelihood model implemented in MEGA6 (Tamura
et al. 2013); alignment 1059 bp of CYTB; H = haplogroup
H I II III
min max average min max average min max average
I 0.000628 0.012102 0.007595 – – – – – –
9
resembled that of CYTB one (Fig. 5).
In order to recover phylogenetic relationships among C. angulifer haplotypes, we run Baye-
sian analysis. The analysed alignment contained both examined mitochondrial genes for all 25
C. angulifer haplotypes and outgroups. The results corroborated that the groups II and III form
well-supported monophyletic clades (posterior probability = 1). In contrast to this, the group I
splits into two lineages. One of them has a sister relationship to a clade including both remaining
groups (II+III), while the other represents a basal most offshoot of C. angulifer tree (Fig. 8).
C.
angulifer haplogroup, other Chilabothrus species and outgroups (Eunectes and Epicrates). We
employed Bayesian and Maximum Likelihood approaches. The results of both computation me-
Finally, we run BEAST and constructed time-calibrated tree to estimate timing of divergence
among C. angulifer haplogroups (Fig. 10). In contrast to the previous analyses, it placed group
Fig. 5. Median-joining haplotype network computed using CYTB 1059 bp alignment containing all 96 samples of
Chilabothrus angulifer, the size of the circle is proportional to the frequency of haplotypes and the mutational steps are
indicated on the branches.
10
III as a sister of groups I+II. The last common ancestor of all C. angulifer haplogroups was esti-
DISCUSSION
Haplotype diversity
We sampled 96 individuals and detected presence of 25 mitochondrial haplotypes in examined
captive population of C. angulifer. Although, we tried to avoid sampling of close maternal relati-
ves, we repeatedly found multiple occurrence of the same haplotype coming from the same insti-
tution (cf. Table 1). It is likely, that we sampled multiple maternal descendants of the same foun-
der in some cases. Thus, we even underestimated haplotype diversity among the founders of the
Table 3. Demographic characteristics for the Chilabothrus angulifer based on the 1059 bp mitochondrial CYTB alignment.
Sequences: number of individuals sequenced (Ns), number of segregating sites (S), number of haplotypes (H), haplotype
clade Ns S H h F* F&L D* Fu’s Fs Tajima’s D
Fig. 6. The Bayesian skyline plot (BSP) of sequence variability in CYTB 1059 bp long for the Chilabothrus angulifer
population, visualizing the effective population sizes over time.
11
examined captive population. This clearly supports the view that haplotype diversity in the sou-
rce natural populations of this endangered snake was extremely high.
Divergence among haplogroups
The deepest divergence among principal haplogroups of C. angulifer, we report here (3.57 Mya,
magnitude as those previously reported between clearly distinct species of the genus Chilaboth-
rus belonging to the same major clade of this genus (for these clades see under Introduction).
ampelophis-fordii, exsul-schwarzi-argentum-striatus-strigillatus
and monensis-granti clades are possibly even more recent (cf. Landestoy et al. 2021). Of course,
it can be assumed that in widely distributed Cuban boas inhabiting a relatively large island (and
its nearby coastal archipelagos) with a complicated geological history, the genetic structure and
phylogeography will be much more complicated, making the interpretation of detected diver-
Chilabo-
thrus angulifer, the size of the circle is proportional to the frequency of haplotypes and the mutational steps are indicated
on the branches.
12
small islands or islets. Current advanced genomic studies in continental rattlesnakes of the genus
Crotalus showed complex evolutionary history of these snakes. Episodes of temporal allopatry
accompanied by genetic drift and divergent selection were repeatedly followed by secondary
2017, 2018, 2019).
Sequence divergence of mitochondrial genes among mainland species of boas tend to be higher
than among the haplogroups of Chilabothrus angulifer, e.g., Epicrates (Passos & Fernandes 2008,
Rivera et al. 2011), Boa (Hynková et al. 2009), Corallus (Colston et al. 2013), Eryx (Eskandarza-
deh et al. 2020a, b), Acrantophis (Vences & Glaw 2003), and Candoia (Austin 2000). There are,
however, multiple exceptions, e.g., sequence divergence between the South American Epicrates
cenchria and its sister species E. maurus with predominantly Central American range (Rivera et
al. 2011) is very close to that between Chilabothrus angulifer haplogroups III and I+II. Similarly,
geographically localized haplogroups within Boa imperator exhibiting distinct parapatric geogra-
Card et al. 2016). Island populations of B. imperator are challenging. The discordance between
very small sequence divergence and parallel change in morphological and developmental traits
reminds us that magnitude of the adaptive evolution is not necessarily proportional to expired time
(Boback 2006, Boback & Siefferman 2010, Green 2010, Bushar et al. 2015, Card et al. 2019). In
2016), we can found multiple examples demonstrating above discussed phenomena (Rawlings
et al. 2008, Esquerré et al. 2020).
Hybridization between snakes belonging to different mitochondrial clades
Sequence divergence in mitochondrial genes can be used as a predictor of ability to hybridize
of C. angulifer is comparable with that among distinct species of boids and pythons (see abo-
ve), it is still much smaller than that between the most distant species of squamates that are still
expressed as a posterior probability (posterior probabilities >0.95 are shown)
13
C. angulifer, we already proved fertility of hybrids
between individuals belonging to different haplogroups (unpublished data).
Why are the haplogroups so divergent within a single island
We report here a surprisingly deep divergence among mitochondrial haplogroups of C. angulifer.
This requires persistence of maternal lineages within this species for a long period of last few
millions of years. According to the phylogeographic and coalescent theory (cf. Avise 2000), this
may be explained either by (1) an extremely large and stable population size, or by (2) spatial
subdivision of the species into multiple isolated populations, each locally maintaining a certain
haplogroup.
that prior colonization of the island by humans, C. angulifer belonged to principal top terrestrial
predators of Cuba. Compared to “warm blooded” predators, the ratio between biomass of pre-
dators and their prey is much higher in the case of “cold-blooded” predators like snakes. Thus,
we cannot exclude that C. angulifer was present in high densities and distributed throughout the
territory of this large island. Although recent records of C. angulifer are absent in some zones
continuous. Although, C. angulifer was repeatedly reported from several Pleistocene localities (cf.
Syromyatnikova et al. 2021 and reference herein), we may only speculate about real population
size of C. angulifer in the Pleistocene or Pre-Colombian periods. Our Bayesian-spline estimating
demography for a quasi-unstructured population revealed that except very recent decline the
population was stable during the last three millions of years.
Although, slow life history of C. angulifer may also contribute to extension of coalescence time
Fig. 9. Bayesian MCMC consensus tree from the concatenated and partitioned 2-gene dataset. Nodes with posterior
probabilities >0.95 are shown, while numbers indicate posterior probabilities at nodes with lower support are without
number marking. Refer to Table 1 for more information on tip labels.
-
ported in other Cuban reptiles (see below for rock-iguanas).
The second hypothesis might be supported by the fact that Cuba consists from multiple con-
tinental fragments, originally representing separate islands. During the Lower Middle Miocene
Eastern islands that have been joined only recently. The Western island was the most distant
from the others. Formation of Cuba in its current form has been completed in Pliocene, roughly
C. angulifer
haplogroups (3.57 Mya) is not old enough to be compatible with the scenario suggesting a se-
condary contact of haplogroups initially evolved in isolation on precursor islands. Nevertheless,
ruled out entirely.
Putative subdivision of C. angulifer
Pliocene is more consistent with our estimates of divergence time among three principal haplo-
groups. We have no evidence about permanent geographic barrier dividing the island during the
during the interglacial maxima. This process temporally separated the area into three or even more
pieces. On the contrary, the Isla de la Juventud would merge with mainland Cuba when the sea
level would drop by about 18 m, while during the last glacial maximum (ca. 20,000 years ago)
Fig. 10. Time-calibrated species tree for the clade Chilabothrus, Epicrates and Eunectes. Nodes are labelled with letters
and 95% HPD intervals are shown. Estimated divergence times in million years, [95% HPD] are following: A – 20.0,
all posterior probabilities are 1.00.
15
the sea level was about 125 m lower than it is today (Fairbanks 1989, Tolson & Henderson 1993,
Poore et al. 2000, Steadman & Franklin 2017).
A strong male-biased dispersal was reported in many snake species (e.g., Rivera et al. 2006,
geographically limited distribution of haplotypes. Therefore, an isolation by distance should be
also considered, besides a true geographic barrier. A study performed in continental coral snakes
recently demonstrated that spatial sorting of their mitochondrial haplotypes can be attributed to
-
gence (Streicher et al. 2016).
In general, all scenarios involving the second hypothesis predict presence of a clear geographic
distribution pattern of the haplogroups. Below we discuss evidence supporting this prediction.
Geographic distribution of the haplogroups
We sampled captive population, decades after last imports from wild. Thus, we rely on only
anecdotic records about the precise geographic origin of examined haplotypes.
Western part of Cuba is the only clearly localized sample of the Haplogroup I comprising 7 ha-
plotypes (23 individuals). The most common origin of imported specimens of C. angulifer were
probably provinces surrounding the capital Havana. Multiple founders possessing haplotypes
-
dividuals), probably come from this part of the island, nevertheless we can be sure about it in just
only four are of known geographic origin. All of them come from Central (Trinidad) and Eastern
Cuba (Nicaro and Sierra Maestra). Another record of Haplogroup III from Eastern part of Cuba
was reported from Guantánamo by Reynolds et al. (2013). This may suggest, that Haplogroup III
is distributed predominantly in the Central-Eastern parts of the Island, while Haplogroups I and
II in the Western areas (see below for a comparison with Cuban rock iguanas).
Although, it is likely that C. angulifer haplogroups follow the above suggested geographic
is needed to solve this problem.
A comparison with phylogeography of other species
Cuban rock-iguana (Cyclura nubila) belongs to the most charismatic species of squamates in-
habiting Cuba. Because its large body size and similar history on the Great Antilles, this species
provides a reliable comparison with Chilabothrus angulifer. Similarly, as in C. angulifer, there
is a deep divergence among mitochondrial haplotypes of this iguana (Frynta et al. 2010). Recent-
ly, Shaney et al. (2020) sampled multiple populations of Cyclura nubila and demonstrated that
C. nubila is characterized by an exclusive group of
closely related haplotypes. Moreover, the haplotypes coming from the western and eastern parts
of Cuba form mutually clearly distinct clades exhibiting parapatric distribution. Each of these
clades further splits into several local haplogroups. The split between these principal clades is
major mitochondrial clades (I+II versus III) of Chilabothrus angulifer
underlying process.
16
A deep split between Western and Eastern clades of CYTB (5.2%) was also reported in endemic
rodents of the genus Capromys. Nevertheless, as a result of more rapid substitution rate in rodents,
a divergence time between these clades was estimated just to 1.1 My (Upham & Borroto-Páez
2017). Interestingly enough, geographic distribution of these Capromys clades follows almost
precisely the pattern reported in Cyclura nubila (Shaney et al. 2020).
Phylogenetic relationships among the haplogroups I, II and III
The topologies recovered by MrBayes and maximum likelihood proved that haplogroups II
and III are monophyletic and mutually exclusive. These methods, however, failed to support
monophyly of Haplogroup I and placed lineages belonging to this group as sister clades of the
remaining haplotypes belonging to groups II and III (Figs 8 and 9). In contrast to this topology,
a time-calibrated tree produced by BEAST reveal basal split between Haplogroup III and the clade
including mutually sister clades formed by haplogroups I and II. We prefer the topology of the
time-calibrated tree (Fig. 10) because of following reasons: (1) The divergence between C. angu-
lifer and its closest relatives of the the Puerto Rican clade (15.3 Mya) is much longer compared
to the deepest split between C. angulifer
accord with the haplotype networks, (3) In the analyses placing haplogroup III with II, the branch
leading from their common ancestor to the Haplogroup III is long.
Conservation genetics of European ex situ population
On one hand, we reported the deep splits among principal mitochondrial haplogroups and thus
we have to expect that natural populations of Chilabothrus angulifer, similarly like those of the
Cuban rock-iguanas (Cyclura nubila), are divided into multiple conservation units according to
coalescence of their genes (cf. Shaney et al. 2020). After several generations of captive breeding
(the generation time for C. angulifer is about 11.0 years, the oldest captive born Cuban boa, who
contributed to the current European population with offsprings, is a female born in 1973 – Rehák,
studbook data) and interbreeding among C. angulifer originated from multiple populations, pure-
(depending on the criteria and applied species concept). would require derivation of new ex situ
populations from the wild (as pointed out already by Rehák 2006, 2008).
On the other hand, zoo populations of the reptiles are typically extremely small (Marešová
& Frynta 2008, Frynta et al. 2010) and thus suffer from inbreeding rather than outbreeding de-
of founders and their genetic variability at the beginning of this millennium were large enough
to create a viable ex situ population of this endangered snake species. Nevertheless, an initial
required to prevent loss of genetic variation and viability of the population.
Recent population of Cuban boas in zoos (as well as in private collections) consists mostly
of captive born animals originated of founders imported from wild – especially in the 1970s and
1980s (Rehák, studbook data). The species is currently mainly found in European institutions, with
just a few others in Asia and North America, so the management at the European level (European
ex situ population) is a convenient option. The current population is descended from at least of
of specimens have unknown pedigree. For the same reason, a more detailed population assess-
ment, including demographic and genetic analysis, cannot be performed, population projections
17
cannot be accurately created and thus important sources for the well based establishing of the
Long-term Management Plan for Cuban boas in human care are missing. Currently, 233 living
Cuban boas (68 males, 80 females and 85 of undetermined sex – held in 63 cooperating insti-
tutions) are registered in the European studbook for Cuban boas (Rehák, studbook data), which
random demographic and catastrophic events for the long-term) and at the same time a number
reproductive problems in connection with crossbreeding.
At the same time, we consider that although the divergence among some populations of Cuban
boas is greater than that of some other, taxonomically recognized, species (see above), it is important
to note that most of these species are isolated island species “doomed” to evolve independently,
whereas in the case of Cuban boas, the individual evolutionary lines previously formed during
Cuba’s complex geological history, currently inhabit a single island (with the surrounding nearby
coastal archipelagos), and it can be assumed that their future evolution is likely to be associated
with the unavoidable hybridization.
In conclusion, we therefore recommend the management of the existing ex situ population of
Cuban boas in human care as a whole, as a single unit. At least until a more accurate picture of
the phylogeography of Cuban boas, the geographical distribution of their individual evolutionary
lines, their natural hybridization and the possible existence of hybrid zones is available.
Limitations of the study
Our study is based on genetic samples from captive animals. Thus, we relied solely on maternally
inherited mitochondrial genes. As the examined individuals were mostly descendants of indivi-
duals coming from different regions of Cuba, the utility of biparentally inherited nuclear genes
was greatly limited. Moreover, sex chromosomes of C. angulifer are not clearly differentiated
(Augstenová et al. 2019), which prevented us to employ Y-chomosome. Therefore, further genetic
examination of wild populations of C. angulifer including genomic approach is urgently needed
to complete the picture.
A c k n o w l e d g e m e n t s
We thank all zoos and breeders who provided DNA samples and/or allowed us to collect buccal swabs from their Cuban
boas. Special thanks belong to Zuzana Starostová (Charles University, Prague) who performed a considerable part of
help with BEAST and Petr Velenský (Prague Zoo) for consultations on the Cuban boa husbandry. This project was sup-
. IR and DF designed, conceived and supervised the research; IŠ performed laboratory work, curation
IR, DF and OŠ collected the samples; DF and IR funding acquisition; DF, IR and BOS wrote the paper.
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