Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 67-81, January-December 2022 (Published Jan. 30, 2022)

Population genetics and molecular identification of Crocodylus acutus

and C. moreletii (Crocodilia: Crocodylidae) in captive

and wildlife populations

Alejandro Villegas 1, 2; https://orcid.org/0000-0003-0319-0463

Aliet Rojas-Santoyo 3; https://orcid.org/0000-0002-5446-6389

Raúl Ulloa-Arvizu 3*; https://orcid.org/0000-0002-6181-8346

1. Facultad de Ciencias, Universidad Nacional Autónoma de México, C.P. 04510, Ciudad de México, Mexico;

alejandrovillegas@ciencias.unam.mx

2. Departamento de Ecología, Genética y Conservación de Fauna Silvestre, Ciencia y Comunidad por la Conservación,

Asociación Civil, Ciudad de México, Mexico; alejandrovillegas@ciencias.unam.mx

3. Departamento de Genética y Bioestadística, Facultad de Medicina Veterinaria y Zootecnia, Universidad

Nacional Autónoma de México, Ciudad de México, México; atl_liet@hotmail.com, ruafmvzunam@gmail.com

(*Correspondence)

Recibido 13-X-2021. Corregido 10-XI-2021. Aceptado 21-I-2022.

ABSTRACT

Introduction: There is low evidence of genetic diversity and hybridization processes within Crocodylus acutus

and C. moreletii populations.

Objetive: To evaluate genetic diversity and some phylogenetic relationships in wild and captive populations

of C. acutus and C. moreletii using the Barcode of Life Data System (COX1, cytochrome C oxidase subunit 1

gene).

Methods: 28 individuals phenotypically like C. acutus located in the state of Guerrero, Oaxaca and Quintana

Roo were sampled, as well as animals belonging to C. moreletii located in the states of Tabasco, Campeche,

and Quintana Roo. 641 base pairs of nucleotide sequence from COX1 were used to obtain the haplotype and

nucleotide diversity per population, and a phylogenetic and network analysis was performed.

Results: Evidence of hybridization was found by observing C. moreletti haplotypes in animals phenotypically

determined as C. acutus, as well as C. acutus haplotypes in animals classified as C. moreletti. Low haplotypic

diversity was observed for C. acutus (0.455 ± 0.123) and for C. moreletii (0.505 ± 0.158). A phylogenetic tree

was obtained in which the sequences of C. acutus and C. moreletii were grouped into two well-defined clades.

Organisms identified phenotypically as C. acutus but with C. moreletii genes were separated into a different

clade within the clade of C. moreletii.

Conclusions: There are reproductive individuals with haplotypes different from those of the species. This study

provides a small but significant advance in the genetic knowledge of both crocodile species and the use of

mitochondrial markers, which in this case, the COX1 gene allowed the detection of hybrid organisms in wild

and captive populations. Conservation efforts for both species of crocodiles should prevent the crossing of both

threatened species and should require the genetic identification of pure populations, to design effective conserva-

tion strategies considering the possibility of natural hybridization in areas of sympatry.

Key words: mitochondrial DNA; cytochrome C oxidase; haplotype diversity; nucleotide diversity; hybridiza-

tion; barcode of life data system.

https://doi.org/10.15517/rev.biol.trop..v70i1.46962

GENETICS

68 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 67-81, January-December 2022 (Published Jan. 30, 2022)

During the twentieth century, due to the

high demand for skin and meat in national and

international trade, Mexican crocodile species

became almost extinct because of the intensive

hunting. At the same time, ecosystems began to

transform, and crocodile habitats became frag-

mented, contaminated, and seriously limited

(Casas-Andreu, 1995; Ross, 1998; SEMAR-

NAP, 2000). In 1970, the Mexican government

declared a total and permanent ban on the

three crocodilian species distributed in Mexi-

co; the American crocodile (Crocodylus acu-

tus), Morelet’s crocodile (C. moreletii) and the

caiman (Caiman crocodilus) (Casas-Andreu,

1995), which are currently protected by the

Norma Oficial Mexicana Nom-059- SEMAR-

NAT-2010 (SEMARNAT, 2010). Recently, C.

moreletii was transferred from Appendix II to

I of the Convention on International Trade in

Endangered Species of Wild Fauna and Flora

(CITES, 2021).

The American crocodile (C. acutus), that is

distributed mainly along the Mexican coast of

the Pacific Ocean, is considered a vulnerable

species by the International Union for Conser-

vation of Nature (IUCN) Red List. On the other

hand, the Morelet’s crocodile is distributed in

the marshy areas of the coastal region of the

Caribbean and Gulf of Mexico and is consid-

ered by the IUCN as a least concern species

(IUCN, 2021).

In the Yucatan Peninsula, both species are

sympatric and eventually interbreed originat-

ing hybrids (Cedeño-Vázquez et al., 2008;

González-Trujillo et al., 2012; Hekkala et al.

2015; Pacheco-Sierra et al., 2016; Ray et al.,

2004; Rodríguez et al. 2008). Due to the need

for generating genetic information on crocodile

populations, molecular markers have been used

as tools to understand the effect of various

genetic factors causing the decline of popula-

tion sizes. It is known that population frag-

mentation has an impact on the effective size

of the population and therefore on the loss of

genetic diversity and the ability of individuals

to adapt to new environmental changes (Rocha

& Gasca, 2007). Genetic analysis allows the

study of genetic diversity and gene flow among

populations, as well as to determine their

structure and measure the effect of inbreeding

crosses within a population (Amos & Balm-

ford, 2001; Rocha & Gasca, 2007). Existing

legislation and treaties governing the trade in

wildlife, such as the Convention on the Inter-

national Trade of Endangered Species (CITES)

and the United States Endangered Species Act

(ESA), are based on the recognition of distinct

population or taxonomic units.

So far, genetic studies using microsat-

ellites and mitochondrial deoxyribonucleic

acid (mtDNA) have been conducted in the

region of the caribbean and Gulf of Mexico

by Cedeño-Vázquez et al. (2008), Dever et al.

(2002), Machkour-M’Rabet et al. (2009), Ray

et al. (2004) and Rodríguez et al. (2008). While

Pacheco-Sierra et al. (2016) found evidence

that these two species have hybridization areas

and maintain genetic flow among their popula-

tions along distribution range.

A database of single gene ‘‘barcodes’’ has

been proposed to classify the complete diversi-

ty of life (Hebert et al., 2003; Ratnasingham &

Hebert, 2007). The region of the mitochondrial

cytochrome c oxidase I (COX1) gene has been

recommended as a standard for DNA barcoding

as well as for the evaluation of genetic diversity

and monitoring of legal and illegal trade of

species (Eaton et al., 2010; Hebert et al., 2003;

Ratnasingham & Hebert, 2007). Therefore, the

objective of this study was to evaluate genetic

diversity and some phylogenetic relationships

in wild and captive populations of C. acu-

tus and C. moreletii located in Southeastern

Mexico using the Barcode of Life Data System

(COX1, cytochrome C oxidase subunit 1 gene).

MATERIALS AND METHODS

Collection of samples: Species were iden-

tified according to the key characters as the

scales embedded in the base of the tail, which

correspond to fewer pronounced osteoderms

and the wide snout in C. moreletii compared to

C. acutus as Platt and Rainwater (2005) suggest.

In 2013, blood and/or tissue samples were col-

lected from five animals at the UMA-CICEA in

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 67-81, January-December 2022 (Published Jan. 30, 2022)

Tabasco and nine samples were collected from

a private farm in Campeche (Fig. 1). Blood was

drawn from the supravertebral venous sinus

and the blood samples were stored in Vacu-

tainer® tubes with 4 ml of EDTA K3; tissue

samples were preserved in ethanol. In addition,

el Colegio de la Frontera Sur provided seven

samples from Quintana Roo; and the samples

from Oaxaca (N = 12) and Guerrero (N = 12)

were provided by Serrano-Gómez (2010); The

total number of samples was 45 individuals, 28

from C. acutus and 17 samples from C. more-

letii (Fig. 1).

DNA Extraction and Sequencing: DNA

was purified from blood or scales following

the protocol from the DNeasy Blood and Tissue

kit, (QIAGEN, Austin, Texas). Using the poly-

merase chain reaction (PCR), a 641 base pair

(bp) fragment of the corresponding COX1 gene

was amplified; this fragment corresponds to

position 52-693 of the sequence of a C. acutus

(GenBank of GQ144571, (Eaton et al., 2010)).

The modified primers of Meusnier et al. (2008)

were used: forward 5’-TCCACTAATCA-

CAARGATATTGGTAC-3’ and reverse primer

5’-CCTCCGGGTGCCCGAAGAATCAG-3’.

PCR reactions was carried out in a final

volume of 20 μl containing 100 ng of total

DNA, 0.6 pmol of each primer, 0.2 mM of

dNTP’s, 1.5 mM of MgCl2, 10 mM of TrisHCl

pH 8.4, 50 mM of KCl, 10 µg/ml of gelatin; 150

µ/ml of BSA, Triton X100 0.1 % and 1 U of Taq

polymerase (BioTecMol, México).The amplifi-

cation conditions included an initial stage at 94

°C for 5 minutes followed by 30 cycles of 94

°C for 30 seconds, 56 °C for 30 seconds and 72

°C for 1 min and a final stage of 72 °C for 5

min. The amplified COX1fragments were puri-

fied with the Potassium iodide technique after

electrophoresis in 3 % agarose gel (Vogelstein

& Gillespie, 1979). The DNA fragments were

purified, and quantified. Sanger-type sequenc-

ing of both DNA strands was performed using

Fig. 1. Distribution of the haplotypes from C. acutus and C. moreletii in the sampled localities. The five haplotypes of C.

acutus are represented with the letters Ca and the eight haplotypes for C. moreletii are represented with the letters Cm. The

graphs above represent the percentage of each haplotype found in relation to the total for each species.

70 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 67-81, January-December 2022 (Published Jan. 30, 2022)

a commercial kit following the manufactur-

er’s protocol (BigDye Terminator v3.1 Cycle

Sequencing Kit PE Applied Biosystems, Foster,

CA). The primers used for PCR were also used

for sequencing. Subsequently, the fragments

obtained were read by a matrix of 16 ABI 3100

Genetic Analyzer capillaries (Applied Bio-

systems Inc.). Forward and reverse sequences

were verified and edited in the trace editor

implemented in the MEGA X (Kumar et al.,

2018). The sequences obtained in the present

study were submitted to GenBank (accession

numbers: C. acutus: KY994087, KY994088,

KY994090, KY994093, KY994094 and C

moreletii: KY994097, KY994089, KY994091,

KY994092, KY994095, KY994096,

KY994098, KY994099).

Data analysis: A multiple alignment was

performed through a Clustal W implemented

in MEGA X (Kumar et al., 2018) using the

C. acutus sequence in GenBank as reference

(GQ144571, (Eaton et al., 2010)). To evaluate

the relationship between the sequences in this

study with the ones previously reported from

different species, seven GenBank sequences

were used (KF273840, KF273836, KF273834,

KF273841, KF273838 (Bloor et al., 2015)),

belonging to C. acutus from Colombia;

HM636894, (Man et al., 2011), belonging to C.

acutus of unreported origin; HQ585889 (Mega-

nathan et al., 2011), belonging to C. moreletti

from Belize.

The nucleotidic (π) and haplotypic (Ĥ)

diversity (Nei & Kumar, 2000) by species

was estimated using the software DnaSP 5.10

(Librado & Rozas, 2009). The neutrality test

D (Tajima, 1989) and F test (Fu & Li, 1993)

were used to measure the effect of the demo-

graphic changes of the populations on the DNA

sequences using the software DnaSP 5.10 (Lib-

rado & Rozas, 2009).

Phylogenetic analysis: The best-fitting

evolutionary model for the data and the gamma

distribution parameters were estimated using

the AICc value (Akaike Information Criterion,

corrected). Non-uniformity of evolutionary

rates among sites was modelled using a dis-

crete gamma distribution with five rate catego-

ries assuming that a certain fraction of sites is

evolutionarily invariable for which the routines

implemented in MEGA X were used (Kumar

et al., 2018).

The number of base substitutions per site

was estimated as the average of all sequence

pairs within groups using the Kimura’s two

parameter model (Kimura, 1980; Nei & Kumar,

2000). The site rate variation was modelled

using a gamma distribution (shape parameter

0.17017). The differences in the composition

bias among sequences were considered in

evolutionary comparisons (Tamura & Kumar,

2002). All positions containing gaps and miss-

ing data were eliminated. Standard errors were

computed with the bootstrap method using 1

000 replicates (Felsenstein, 1985).

Phylogenetic trees were constructed with

neighbour-joining methodology (Saitou & Nei,

1987) incorporated in the software MEGA X

(Kumar et al., 2018). Additionally, a Bayes-

ian analysis (Drummond & Rambaut, 2007)

using the BEAST 1.8 program (Drummond

et al., 2012). To choose the optimal model for

nucleotide substitution, Modeltest 3.7 (Posada

& Crandall, 1998) was used, using the value

of the Bayesian Information Criterion (BIC)

as a selection criterion. The “Yule Speciation”

model was used to estimate the evolutionary

history of the sequences. The MCMC chain ran

for 150 x 106 generations, sampling every 100

generations, using a priori the normal distribu-

tion and a 95 % probability for this range (Oaks,

2011). To evaluate the convergence values, the

effective sample size, and the “burnout” esti-

mates, the Tracer 1.7 program was used. (Ram-

baut et al., 2018). The final tree was viewed

and edited with the FigTree v.1.2.2 program.

In order to evaluate the relationship between

the sequences in this study with the four New

World Crocodylus species previously reported,

nine GenBank sequences were used, where six

belonged to C. acutus (KF273834, KF273838,

KF273836, KF273840, KF273841 (Bloor et

al., 2015); HM636894 (Man et al., 2011), one

to C. moreletii (HQ585889; (Meganathan et

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 67-81, January-December 2022 (Published Jan. 30, 2022)

al., 2011)), one to C. intermedius (JF502242,

(Meredith et al., 2011)) and one to C. rhombifer

(JF502247.1, (Meredith et al., 2011)); in order

to obtaining an adequate tree topology, we

include C. niloticus (JF502246, (Meredith et

al., 2011)), C. porosus (DQ273698, (Li et al.,

2007)) and C. johnsoni (HM488008, (Megana-

than et al., 2011)) as external groups, consider-

ing the genus Crocodylus as a monophyletic

group (Brochu, 2003).

Network analysis: Phylogenetic methods

may not lead to the desired resolution at the

intraspecific level due to the lower genetic

diversity; complementary network approach-

es might be valuable alternatives for study-

ing phylogenetic structures and haplogroups

at the population level; therefore, the pro-

gram PopART (Leigh & Bryant, 2015) was

used to reconstruct median-joining network

(Bandelt et al., 1999).

RESULTS

The final sequences obtained began at

position 52 of the COX1 gene and corre-

sponded to the 5 370 position of the complete

mitogenome of C. acutus (HM636894). A total

of 28 sequences of 641 bp for C. acutus COX1

gene were obtained with 24 variable sites, 22

of them were informative and two were single

mutations (singletons). For C. moreletii, 17

sequences were obtained with 24 variable

sites, where 20 were informative sites and 4

were singletons. The polymorphic sites of the

obtained sequences are shown in Table 1.

Five haplotypes for C. acutus (CaI-CaV)

and eight for C. moreletii (CmI-CmVIII) were

identified in crocodile populations studied in

Mexico. CaI haplotype was the most frequent

and was found in both the Pacific and the

Caribbean. The CaII haplotype was the second

most frequent haplotype and was found in an

TABLE 1

Polymorphic sites at the Cytochrome Oxidase subunit I gene in Crocodylus acutus (Ca) and C. moreletii (Cm) haplotypes

1112223344444455555666666666

8891662464802667805789011226677

7812258015283254618967028156828

GQ144571 A G A C C C A C T A G A A A G C T G A C T G A G C T C A C A T

Ca I ...............................

Ca II ..............................A

Ca III ..T............................

Ca IV .............................G.

Ca V .........................C....A

Cac08 ...............T...............

Cac03 ........C..G......G....A.......

Cac01 ...........G......G..A.A.......

Cac05 ........C.........G....A.......

Cm I . . . . T T G T . C . . G G A . C A G T C A C A T C T G . . A

Cm II . . . . T T G T . C . . G G A . C A G T C A C A T C T G G . A

Cm III . A . . T T G T . C . . G G A . C A G T C A C A T C T G . . A

Cm IV G . . . T T G T . C . . G G A . C A G T C A C A T C T G . . A

Cm V . . . G T T G T . C . . G G A . C A G T C A C A T C T G . . A

Cm VI . . . . . T G T . C . . G G A . C A G T C A C A T C T G . . A

Cm VII . . . . . T G T . C A . G G A . C A G T C A C A T C T G . . A

Cm VIII . . . . T T G T . C . . G G A . C A G T . A . A . C T G . . A

The upper numbers correspond to the position (pb) of the sites. The dots indicate equal nucleotides. Reference sequence of

C. acutus GenBank GQ144571. Cac are haplotypes of C. acutus reported by Bloor et al. (2015).

72 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 67-81, January-December 2022 (Published Jan. 30, 2022)

organism in the Ventanilla lagoon (Oax.) in

the Pacific, but an individual from the farm in

Tabasco and another individual in Río Hondo

(Quintana Roo), who were phenotypically clas-

sified as C. moreletii, they presented this hap-

lotype. While the CaIII haplotype was found in

the Chacahua lagoon (Oax), and the CaIV and

CaV haplotypes appear in Fortuna (Gro).

For C. moreletii, the most frequent hap-

lotype was CmI (Fig. 1), and it is is equal to

a sequence of an animal sampled in Belize

(HQ585889, (Meganathan et al., 2011)). Also

with this haplotype, a phenotypically identi-

fied organism was found as C. acutus, which

was sampled in Boca Paila, Quintana Roo. The

CmVI haplotype was presented in Quintana

Roo and Chacahua Lagoon (Oax) and CmVII

and CmVIII haplotypes were from organisms

identified as C. acutus, two individuals from

Chacahua, Oaxaca.

Table 2 shows the results of the haplotype

and nucleotide diversity of the two species.

A first analysis was made including all the

sequences; but since hybridization has an influ-

ence on the estimates, a second analysis was

carried out, discarding the sequences of organ-

isms with a phenotype different from their gen-

otype. For C. acutus, the haplotype diversity

was 0.455 (± 0.123) and nucleotide diversity

was 0.0009 (± 0.0003); while for C. moreletii

the haplotype diversity was 0.505 (± 0.158) and

the nucleotide diversity was 0.0009 (± 0.0003).

No significant differences were found

between the values of nucleotide diversity

among both species (H = 3.30, P = 0.770). Fur-

thermore, no differences were found between

nucleotide diversity between C. acutus and

C. moreletii populations. The same statistical

value was obtained for both tests (H = 2, P =

0.368). With the values of haplotype diversity,

no significant differences were found between

the C. acutus populations (H = 2.7, P = 0.440),

nor between C. moreletii populations (H = 2, P

= 0.368) and neither among the total of the two

TABLE 2

Genetic diversity estimated in Crocodylus acutus and C. moreletii by locality

Species site n S h Ĥ ± SE π ± SE

C. acutus

Ventanilla 4 1 2 0.667 ± 0.2040 0.0010 ± 0.0003

Chacahua* 8 23 5 0.786 ± 0.1510 0.0177 ± 0.0039

Chacahua** 5 1 2 0.400 ± 0.2370 0.0006 ± 0.0003

La Fortuna 7330.667 ± 0.1600 0.0016 ± 0.0006

El Medano 5 0- 1 0 0

Q Roo* 4 21 3 0.833 ± 0.2220 0.0221 ± 0.0064

Q Roo ** 2 0 1 0 0

Total* 28 24 9 0.632 ± 0.1030 0.0103 ± 0.0029

Total** 23 4 5 0.455 ± 0.1230 0.0009 ± 0.0003

C. moreletii

L. Ilusiones 5120.400 ± 0.2370 0.0006 ± 0.0003

Buenavista* 3 20 2 0.667 ± 0.3140 0.0213 ± 0.0098

Buenavista** 1 - - - -

Campeche 6 2 3 0.600 ± 0.2150 0.0011 ± 0.0004

Q. Roo* 3 21 3 1.000 ± 0.2720 0.0223 ± 0.0090

Q. Roo** 2221.000 ± 0.5000 0.0016 ± 0.0007

Total* 17 24 6 0.675 ± 0.1170 0.0111 ± 0.0038

Total** 14 4 5 0.505 ± 0.1580 0.0008 ± 0.0003

*Localities with atypical haplotypes. **Analysis without atypical haplotypes, n: Number of sampled individuals, S:

Polymorphic sites, h: Number of haplotypes, Ĥ: Haplotype diversity, π: Nucleotide diversity, SE: standard error. Q. Roo:

Quintana Roo.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 67-81, January-December 2022 (Published Jan. 30, 2022)

species (H = 1, P = 0.317). For C. acutus, the

neutrality tests showed negative values for D

and F, but not different from zero (D = -1.2951,

P > 0.05, F = -1.1139, P > 0.05) suggesting

that the analyzed sequences belonging to the

COX1 gene for this species are not subject

to selection. On the other hand, both indices

were negative and significant for C. moreletii

(D = -1.7975, P < 0.05, F = -2.4488, P < 0.05);

this may be indicative that populations of this

species are expanding or emerging from a

recent bottleneck.

A phylogenetic analysis was carried out

that includes 13 haplotypes of the partial

COX1 fragment found in Mexico plus 10 hap-

lotypes retrieved from the data bank. Fig. 2A

shows the C. acutus and C. moreletii sequences

in two well-defined clades. The Colombian

C. acutus sequences obtained from GenBank

(KF273834, KF273838, KF273836) together

Fig. 2. A. The phylogenetic tree was constructed using the neighbor union method with 1 000 bootstrap replicates. Bootstrap

values are expressed as a percentage. B. The phylogenetic tree was reconstructed using Bayesian analysis. The values are

the posterior propabilities of the topology. COX1 sequences and the Kimura 2P mutation model were used and C. niloticus

(JF502246), C. porosus (DQ273698) and C. johnsoni (HM488008) were used as external groups and C. intermedius

(JF502242) and C. rhombifer (JF502247.1) were also included. Accession numbers for the Colombian haplotypes of C.

acutus are presented.

74 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 67-81, January-December 2022 (Published Jan. 30, 2022)

with HM636894 seem to form a subgroup

within the C. acutus clade apart from the

sequences found in Mexico, while the sequence

of a Colombian C. acutus (KF273840, Cac07)

is equal to the CaI and the reference sequence

GQ14457; also, the sequence KF273841

(Cac08) showed that Colombian specimens are

close to the Guerrero and Oaxaca populations.

For its part, the CmI haplotype found in croco-

diles of Tabasco, Campeche and Quintana Roo

is equal to a sequence of an animal sampled in

Belize (HQ585889, (Meganathan et al., 2011)).

Fig. 2B was reconstructed using Bayesian

analysis and the sequence of C. intermedius

(HM636895) is within a specific clade of C.

acutus like the neighbor phylogenetic tree. This

analysis shows too the C. acutus and C. more-

letii clades similar to Fig. 2A.

The median junction network (MJ) shows

the two haplogroups formed by C. moreletii

and C. acutus (Fig. 3), the most frequent hap-

lotypes were CmI and CaI, respectively. The

Colombian Cac07 haplotype and CaI haplo-

type are the same, and from this haplotype

the Mexican haplotypes and the Colombian

Cac05 and Cac08 haplotypes are derived. The

connection between C. acutus and C. moreletii

haplogroups appears in two lines that join the

CmVIII haplotype. On the other hand, C. inter-

medius derives from CaI presenting four muta-

tions, while C. rhombifer derives from CmVI

with 20 mutations difference.

DISCUSSION

Mitochondrial DNA is by far the most

widely used population genetic marker in ani-

mals. The reasons for its use depend on its

high level of variability, maternal (clonal)

inheritance and mtDNA diversity that can be

correlated with demographic effects (varia-

tions in population size between species or

populations), which makes it reliable in the

context of biological conservation (Harrison,

1989; Nabholz et al., 2008; Roman & Palumbi,

2003). In the phylogenetic tree obtained during

this study using a partial sequence of the COX1

gene, the relationships between the New World

crocodiles: C. acutus (American crocodile), C.

moreletii (Morelet’s crocodile), C. intermedius

(Orinoco crocodile) and C. rhombifer (Cuban

crocodile) are consistent with other studies

using the mitogenome sequence or partial

ND6-tRNAglu-cytb, COX1-Cytb (Meganathan

et al., 2011; Meredith et al., 2011).

Fig. 3. Median-joining network with the haplotypes obtained in this study and those obtained from C. acutus from Colombia.

The size of the circle is proportional to the frequency of the haplotype. Marks (|) indicate mutations between two haplotypes.

The Colombian Cac07 haplotype and CaI haplotype are the same.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 67-81, January-December 2022 (Published Jan. 30, 2022)

The CaI haplotype in this COX1 region,

is equal to the colombian haplotype KF273840

and to GQ144571 and, according to Eaton et

al. (2010), tissue from an organism collected

in Oaxaca, Mexico (possibly in the Chacahua

Lagoon) was used. This haplotype occurs on

the coasts of the Pacific and in the Mexi-

can Caribbean; another Colombian haplotype

(KF273841, Cac08) also appears in the Mexican

clade; in addition, the haplotypes Cac01, Cac3

and Cac05 that correspond to the sequences

KF273834, KF273836, KF273838 reported for

Colombia by Bloor et al. (2015) form another

branch in the clade of C. acutus, as well as the

sequence HM636894 reported by Man et al.

(2011), which does not indicate the locality of

sampling, form another branch in the clade of

C. acutus and it is very likely that the crocodile

that was sampled comes from Colombia. This

supports what was mentioned by Bloor et al.

(2015), who consider the possibility of two

lineages: The North American haplogroup,

where CaI is located and the Central American

haplotypes (C. acutus) and Southern american

haplogroup. The North American haplogroup,

where CaI is located and the Central American

haplotypes (C. acutus) and Southern american

haplogroup. Unfortunately we were unable to

include C. acutus sequences from United State

of America, Central America and the Caribbean

reported in other studies (Milián-García et al.,

2011; Milián-García et al., 2015; Milián-García

et al., 2018) since they used another region of

the COX1 gene sequence. Recently, studies

were published that used fragments of control

mtDNA sequences and microsatellites (Milián-

García et al., 2020) and SNPs (Rossi et al.,

2020), where there is a differentiation between

the populations of the Caribbean (Mexico and

Belize) and the populations of South America

that coincide with those observed in this study

using only information from the COX1 gene.

On the other hand, it was observed that

the values of genetic diversity are like those

obtained by Cedeño-Vázquez et al. (2008),

Glenn et al. (2002), Ray et al. (2004) and

Rodríguez (2007) using the control region

of the mitochondria as well as what was

observed in the COX1 sequence studied by

Milián-García et al. (2018); these values have

been characterized as low by these authors.

The haplotype diversity was also like the one

reported by Ray et al. (2004); these authors

found values of 0.251 for C. moreletii, whereas

Rodríguez (2007) found values of 0.518 for

C. acutus. Milián-García et al. (2018) found,

in C. acutus from captives and wild in Cuba,

four haplotypes of the COX1 fragment with a

haplotype diversity per population that varies

from 0.286-0.558.

The low genetic diversity found with mito-

chondrial markers may be due to the low muta-

tion rate that they present for being conserved

genes or be interpreted as consequence of

bottleneck when populations remain reduced

in a demographic context. Vasconcelos et al.

(2008), suggest that severe declines in croco-

diles can be masked in mitochondrial DNA

information, first, because previous popula-

tion expansions may have left signature on

mitochondrial DNA; second, signal cannot

be detected on mitochondrial because of long

generation periods in crocodiles, when com-

pared to 100 years of continuous exploitation;

and third, because crocodile counts can still be

large, even when they represent only a fraction

of ancient abundance. In this sense, population

recovery may still be an artifact. Our neutrality

test values did not detect significant deviations

indicative of non-neutrality; also, we did not

find any positive D values that could indicate

a positive selection, balancing or a reduction

of the population size (Perfectti et al., 2009).

We think that a possible hypothesis in this

regard is that crocodiles have remained largely

unchanged throughout their evolutionary his-

tory, causing a low mutation rate in conserved

genes as it is mentioned by Field (1988) and

Li and Graur (2000). In this regard, Laird et

al. (1969), suggested that the rate of nucleotide

substitution may be negatively correlated with

generational time, that is, species with short

generational periods have high substitution

rates, this is supported by Kimura (1983).

Particularly in poikilotherm organ-

isms, Mooers and Harvey (1994) found low

76 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 67-81, January-December 2022 (Published Jan. 30, 2022)

nucleotide substitution rates in mitochondrial

DNA compared to homeoterm organisms of

the same size, indicating that the metabolic

rate influences the rate of molecular evolution

(Allen et al., 2006; Martin & Palumbi, 1993),

which could also respond to the low rate of

nucleotide substitution found in the croco-

diles of this study. Ray et al. (2004) using the

mitochondrial DNA control region calculated

a nucleotide substitution rate of 1.66 x 10-8,

which is 3.6 times lower than the standard rate

of 5.9 x 10-8 of mitochondrial genes for homi-

nids calculated by Brown et al. (1982). Recent-

ly the mutation rate in the complete genome of

crocodiles was estimated at 7.9 x 10-9 (Green

et al., 2014) which is very low compared to the

rate of nuclear genetic mutation in humans that

are 1-10 x 10-5.

The variation in this gene is lower com-

pared to nuclear markers, such as microsatel-

lites; an example of this is the study of Dever

et al. (2002), who found heterozygosity values

of 0.49 in C. moreletii populations from Belize.

Therefore, the highly polymorphic pattern of

nuclear markers can detect higher polymor-

phism in crocodile populations. Flint et al.

(2000) and Lawson et al. (1989) mentioned

that patterns of low genetic variation in mito-

chondrial genes found in crocodile populations

have been attempted to explain because of

directional selection, which favors a particu-

lar phenotype in response to long periods of

environmental stability due to its adaptation to

relatively unchanging aquatic environments.

The analysis of the obtained sequences

for both species evidenced individuals pheno-

typically characterized as C. acutus with C.

moreletii haplotypes and viceversa. Specimens

from the UMA in Tabasco, Quintana Roo and

Oaxaca showed a different phenotype. Regard-

ing the captive individuals from Buenavista,

Tabasco, that had haplotypes of another spe-

cies, at the time of collection, they were part

of a breeding female group from a leather and

meat marketing crocodile farm in Campeche. It

is well known that these females have had off-

spring previously; therefore, this supports the

analyzes of Rodríguez et al. (2008) in which

they show that the hybrid individuals grouped

in intermediate positions in their phylogenetic

reconstructions are backcrosses towards one or

the other species, so interspecific hybrid indi-

viduals are reproductively viable.

Of the crocodiles that were sampled in

Quintana Roo, an individual phenotypically

identified as C. moreletii corresponded to the

CaII haplotype (characteristic of C. acutus)

and two phenotypically C. acutus individu-

als had haplotype of C. moreletii had CmI y

CmVI haplotype (characteristic of C. more-

letii); this is very likely since they could cor-

respond to hybrid organisms of C. acutus x C.

moreletii. Hybridization within the Crocodylus

genus has been reported in several species,

mainly in captive organisms (FitzSimmons et

al., 2002; Milián-García, et al., 2015; Milián-

García, et al., 2018; Rodríguez et al., 2011;

Tabora et al., 2012; Weaver et al., 2008). The

presence of hybrids in Belize is the result

of crosses between C. moreletii males and

C. acutus females, which have a fertile off-

spring, as reported by Hekkala (2004) and

Ray et al. (2004). However, in this study, we

found that the interbreed between these spe-

cies has been bidirectional, as also reported

by Cedeño-Vázquez et al. (2008); since C.

acutus haplotypes were grouped into organ-

isms phenotypically determined as C. moreletii

and viceversa.

The Pacific coast of Mexico is the ances-

tral habitat of C. acutus. It is known that in the

last century (1970 approximately) in the lagoon

of Chacahua (Oaxaca), a farm of C. morelleti

was established started its operations with a

batch of 40 animals of C. moreletii from the

Atlanta Zoo, but by the scourge of a hurricane

the crocodiles escaped towards the lagoon

and eventually crossed with the C. acutus

(Muñíz et al., 1997).

The results obtained in this study showed

that there are reproductive individuals with

different haplotypes to those of the species. It

is important to take this into account for future

reintroductions, since having the genetically

identified individuals is paramount for the con-

servation of these species. The translocation of

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 70: 67-81, January-December 2022 (Published Jan. 30, 2022)

specimens into non-original distribution zones

has also resulted in hybridization processes,

such as in the case of the Chacahua Lagoons

(Cedeño-Vázquez et al., 2008; Muñíz et al.,

1997). Another case reported by Sánchez-

Vilchis (2007) corresponded to the Alcazahue

lagoon in Colima, where C. moreletii indi-

viduals were introduced in 1985 to perform

intensive breeding. Currently, it is believed

that C. moreletii has displaced C. acutus in this

locality, even some C. acutus individuals with

atypical nuchal osteoderms patterns have been

observed, reason why it is believed that hybrid-

ization took place in this area; however, until

now the individuals of these populations have

not been studied with nuclear markers. There-

fore, it is important to determine the actual con-

servation status of both species and the effect

of anthropogenic hybridization on populations

before developing exploitation strategies.

Management decisions should be based

on the strongest possible evidence to allow a

reliable estimate of the genetic processes that

are occurring in crocodile populations. This

study provides a small but significant advance

in the genetic knowledge of both crocodile

species and the use of mitochondrial markers,

which in this case, the COX1 gene allowed the

detection of hybrid organisms in wild and cap-

tive populations. Conservation efforts for both

crocodile species should prevent interbreeding

of both threatened species and would require

the genetic identification of pure populations,

to design effective conservation strategies con-

sidering the possibility of natural hybridization

in sympatry areas.

Ethical statement: the authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

The authors are grateful to Erzy, Eri-

ane and Ileana of the UMA-CICEA from

the Autonomous Juarez University of Tabasco

and Tixchel Vázquez Flores and Juan Carlos

Cremiux from the Coco Maya Farm, for the

facilities and support received during the sam-

pling. The authors also thank Carmen Pozo

de la Tijera and Enrique Escobedo Cabrera

from the Museum of El Colegio de la Fron-

tera Sur (ECOSUR) for the samples’ dona-

tion belonging to the collection. ARS thanks

CONACYT for the received scholarship No.

264646. We thanks to Programa de Apoyo a

Proyectos de Investigación e Innovación Tec-

nológica (PAPIIT IN222017) UNAM. Finally,

the authors are grateful to Samuel Said Serrano

Gómez for providing the samples for this study

and to the staff of the Genetics Laboratory of

the Genetics and Biostatistics Department,

especially to Amanda Gayosso and J. Pablo

Pintor for their assistance.

RESUMEN

Genética de poblaciones e identificación molecular

de Crocodylus acutus y C. moreletii

(Crocodilia: Crocodylidae) en poblaciones

de cautiverio y de vida libre

Introducción: Existe poca evidencia de la diversidad

genética y los procesos de hibridación dentro de las pobla-

ciones de Crocodylus acutus y C. moreletii.

Objetivo: Evaluar la diversidad genética y algunas rela-

ciones filogenéticas en poblaciones silvestres y cautivas de

C. acutus y C. moreletii utilizando el Sistema de Código

de Barras de la vida (COX1, subunidad I del gen del cito-

cromo C oxidasa).

Métodos: se muestrearon 28 individuos fenotípicamente

similares a C. acutus ubicados en los estados de Guerrero,

Oaxaca y Quintana Roo, así como animales pertenecientes

a C. moreletii ubicados en los estados de Tabasco, Campe-

che y Quintana Roo. Se utilizaron 641 pares de bases de

la secuencia de nucleótidos de la subunidad I del gen del

citocromo C oxidasa para obtener el haplotipo y la diver-

sidad de nucleótidos por población, y se realizó un análisis

filogenético y de redes.

Resultados: Se encontró evidencia de hibridación al obser-

var haplotipos de C. moreletti en animales determinados

fenotípicamente como C. acutus, así como haplotipos de

C. acutus en animales clasificados como C. moreletti. Se

78 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 70: 67-81, January-December 2022 (Published Jan. 30, 2022)

observó una baja diversidad haplotípica para C. acutus

(0.455 ± 0.123) y para C. moreletii (0.505 ± 0.158). Se

obtuvo un árbol filogenético en el que las secuencias

propias de C. acutus y C. moreletii se agruparon en dos

grandes y bien definidos clados. Los organismos identifi-

cados fenotípicamente como C. acutus pero con genes de

C. moreletii se separaron en un clado diferente dentro del

clado de C. moreletii.

Conclusiones: Existen individuos reproductores con

haplotipos diferentes a los de la especie. Este estudio apor-

ta un pequeño pero significativo avance en el conocimiento

genético tanto de las especies de cocodrilos como del uso

de marcadores mitocondriales, que, en este caso, el gen

COX1 permitió la detección de organismos híbridos en

poblaciones silvestres y cautivas. Los esfuerzos de conser-

vación para ambas especies de cocodrilos deben evitar el

cruce de ambas especies amenazadas y deben requerir la

identificación genética de poblaciones puras, para diseñar

estrategias de conservación efectivas considerando la posi-

bilidad de hibridación natural en áreas de simpatría.

Palabras clave: ADN mitocondrial; citocromo C oxidasa;

diversidad haplotípica; diversidad de nucleótidos; hibrida-

ción; sistema de código de barras de la vida.

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