Population genetics
implications for the conservation of the Philippine Crocodile Crocodylus
mindorensis Schmidt, 1935 (Crocodylia: Crocodylidae)
Ma. Rheyda P. Hinlo 1,
John A.G. Tabora 2, Carolyn A. Bailey 3, Steve Trewick 4,
Glenn Rebong 5, Merlijn van Weerd 6, Cayetano C. Pomares 7,Shannon E. Engberg 8, Rick A. Brenneman 9 &
Edward E. Louis, Jr.10
1 Institute for Applied
Ecology, University of Canberra, ACT, 2601, Australia
2,7 Department of Biological
Sciences, University of Southern Mindanao, Kabacan, North Cotabato, 9407
Philippines
3,8,9,10 Grewcock Center for
Conservation and Research, Omaha’s Henry Doorly Zoo and Aquarium, 3701 South 10thStreet, Omaha, NE 68107, USA
4 Phoenix Group Evolutionary
Ecology and Genetics, Massey University, Palmerston North, 4442 New Zealand.
5 Palawan Wildlife Rescue and
Conservation Center, National Road, Barangay Irawan, Puerto Princessa City,
Philippines. 6 Institute of Environmental Sciences, Leiden
University, PO Box 9518, 2300 RA Leiden, the Netherlands; Mabuwaya Foundation,
Isabela State University Garita, Cabagan 3328, Isabela, Philippines.
1 rei_vet@yahoo.com, 2 johnariestabora@yahoo.com;4 s.trewick@massey.ac.nz, 5 pwrcc.denr@gmail.com, 6 merlijnvanweerd@yahoo.com,7 cayetanop@gmail.com, 8 genetics@omahazoo.com; 9 brenne3@yahoo.com,10edlo@omahazoo.com (corresponding author)
Abstract: Limited information is available on the
Philippine Crocodile, Crocodylus mindorensis, concerning levels of
genetic diversity either relative to other crocodilian species or among
populations of the species itself. With only two known extant populations of C.
mindorensis remaining, potentially low levels of genetic diversity are a
conservation concern. Here, we evaluated 619 putative Philippine Crocodiles
using a suite of 11 microsatellite markers, and compared them to four other
crocodilian species sample sets. The two remaining populations from the island
of Luzon and the island of Mindanao, representing the extremes of the former
species’ distribution, appear to be differentiated as a result of genetic drift
rather than selection. Both extant
populations demonstrate lower genetic diversity and effective population sizes
relative to other studied crocodilian species. The 57 C. mindorensis andC. porosus, Saltwater Crocodile, hybrids identified earlier from the
Palawan Wildlife Rescue and Conservation Center were revalidated with a suite
of 20 microsatellite loci; however, the timing of the event and the prevalence
of hybridization in the species had yet to be fully determined. We defined the
hybrids as one first cross from a C. porosus female and a C.
mindorensis male and 56 C. mindorensis backcross individuals. This
hybridization event appears to be confined to the PWRCC collection.
Keywords: Crocodylus, hybrid
detection, microsatellites, Philippine crocodile, population genetics.
Abbreviations: ABI - Applied Biosystems,
Inc.; bp - base pairs; CFI - Crocodile Farming Institute; CI - confidence
interval; CSG - IUCN/SSC Crocodile Specialist Group; DENR - Department of Environment
and Natural Resources; DNA - deoxyribonucleic acid; Fis - within population
fixation index; Fst- between population fixation index; He - expected heterozygosity; Ho
- observed heterozygosity; I - Shannon Information index; IUCN -
International Union for the Conservation of Nature; LD - linkage
disequilibrium; MSA - Microsatellite Analyzer; mtDNA - mitochondrial DNA; N -
census size; N - average number of individuals genotyped per locus; Na- mean number of alleles; Ne - effective population size; Nea -
effective number of alleles; Neb - number of effective breeders; nucDNA
- nuclear DNA; PAWB - Protected Areas and Wildlife Bureau; PCA - Principal
Coordinates Analysis; PCNRT - Philippine Crocodile National Recovery Team; PCR
- polymerase chain reaction; PWRCC - Palawan Wildlife Rescue and Conservation
Center; SSC - Species Survival Commission; tI - transformed Shannon
entropy index; tHe - transformed expected heterozygosity index; tHo
- transformed observed heterozygosity index; tUHe - transformed unbiased
expected heterozygosity index; UHe - unbiased expected heterozygosity;
WGA - whole genome amplification
Filipino
Abstract: Limitado
lamang ang kaalaman na mayroon ukol sa Philippine
Crocodile (Crocodylus mindorensis), lalo na sa antas o lebel ng genetic
diversity na mayroon ito kumpara sa iba pang uri ng buwaya o kahit mismo sa
iba’t-ibang populasyon ng Philippine crocodile sa bansa. Sa kasalukuyan,
dalawang likas na populasyon na lamang ng Philippine
crocodile ang matatagpuan sa ilang, at ang potensyal ng mababang antas ng
genetic diversity na maaring matagpuan sa mga natitirang populasyon nito ay
nagdudulot ng pangamba sa kanilang pangmatagalang kabutihan. Sa artikulong ito,
aming sinuri ang 619 na Philippine Crocodile gamit ang
labing-isang microsatellite markers at inihambing ang mga ito sa apat na
pangkat na impormasyon mula sa ibang uri o species ng buwaya. Ang pagkakaibang
genetiko ng dalawang natitirang populasyon mula saisla ng Luzon at Mindanao na kumakatawan sa sukdulang distribusyon ng buwayang
ito sa Pilipinas, ay waring dulot ng genetic drift at hindi seleksyon. Aming
natuklasan na ang dalawang natitirang populasyon sa
ilang ng Philippine Crocodile ay may mas mababang genetic diversity at
effective population sizes kumpara sa ibang uri ng buwaya. Ang 57 hybrid nabuwaya na natagpuan sa isang naunang pag-aaral ay muling napatotohanan na
hybrid nga sa pag-aaral na ito gamit ang dalawampung microsatellite loci.
Ganoon pa man, ang panahon na nangyari ang
hybridization at kung gaano ito kalawak sa populasyon ng Philippine crocodile
ay kailangan pa ng pagsisiyasat. Sa artikulong ito, aming minumungkahi na ang
57 hybrids na natagpuan ay binubuo ng isang unang henerasyon na supling ng
lalaking C. mindorensis at babaeng C. porosus, at ang natitirang 56 na hybrid
ay mga backcross na buwaya. Ang hybridization nanatagpuan ay waring limitado lamang sa koleksyon ng Palawan Wildlife Rescue
& Conservation Centre (PWRCC).
doi: http://dx.doi.org/10.11609/JoTT.o3384.5513-33 | ZooBank: urn:lsid:zoobank.org:pub:4D6790B4-A8B5-4205-9C23-F1F6900B0904
Editor: Llewellyn D. Densmore, Texas Tech University,
Lubbock, USA. Date
of publication: 26 March 2014 (online & print)
Manuscript details: Ms # o3384 | Received 11
October 2012 | Final received 28 September 2013 | Finally accepted 15 February
2014
Citation: Hinlo, M.R.P., J.A.G. Tabora,
C.A. Bailey, S. Trewick, G. Rebong, M. van Weerd, C.C. Pomares, S.E. Engberg,
R.A. Brenneman & E.E. Louis, Jr. (2014). Population genetics implications for the
conservation of the Philippine Crocodile Crocodylus mindorensis Schmidt,
1935 (Crocodylia: Crocodylidae). Journal of Threatened Taxa 6(3):5513–5533; http://dx.doi.org/10.11609/JoTT.o3384.5513-33
Copyright: © Hinlo et al. 2014. Creative Commons Attribution 3.0 Unported
License. JoTT allows unrestricted use of this article in any medium,
reproduction and distribution by providing adequate credit to the authors and
the source of publication.
Funding: Omaha’s Henry Doorly Zoo and Aquarium, the
Crocodile Specialist Group (CSG) Student Research Fund, New Zealand Agency for
International Development postgraduate research fund, and Curt Harbsmeier, Law
Offices of Harbsmeier DeZayas, LLP.
Competing Interest: The authors declare no
competing interests.
Author Contribution: Ma.
Rheyda Hinlo was involved with the data generation and sample collection in the
Philippines and was involved in every step. John A. G. Tabora was also involved with
data generation especially the sequence data. Carolyn A. Bailey was involved with the
sample acquisition from the outgroup crocodile samples, and generated the data
on these samples. Steve Trewick,
Glenn Rebong, Merlijn van Weerd, and Cayetano Pomares, provided overall project
expertise of the Philippines and direction and academic rigour to the overall
project for the participating student authors, and participated significantly
in the final drafts of the manuscript. Shannon Engberg provided supervision and direction to the overall data
generation and of the study. Dr.
Brenneman was responsible for developing the collaborations with the Republic
of the Philippines Department of Natural Resources’ Protected Areas and
Wildlife Bureau, the Palawan Wildlife Rescue and Conservation Center, corporate
and private owners of the Crocodylus mindorensis individuals and archived
samples, and for the collection of the C. porosus samples on
Mindanao. He selected and performed
the genetic analyses of the microsatellite data, the interpretations of the
results, and wrote the majority of the manuscript. Edward Louis organized the collection of
the majority of the outgroup crocodile samples, and was primary supervisor in
the project overall design and the overall organization of the manuscript,
including the revisions.
Author Details: Ma. Rheyda P. Hinlo is a Filipino veterinarian who holds a MSc Degree in Conservation Biology from Massey University,
New Zealand, is a member of the IUCN Crocodile Specialist Group. Hinlo is the
National Project Coordinator for Protected Areas & Wildlife Bureau,
Philippines and is currently a PhD candidate in Applied Ecology at the
University of Canberra, Australia. John A. G. Tabora is an assistant
professor in the Department of Biological Sciences at the University of
Southern Mindanao. Carolyn A. Bailey is a laboratory
technician for the Conservation Genetics Department of Omaha’s Henry Doorly Zoo
and Aquarium. Steve Trewick is an evolutionist with a
special interest in speciation and the way biotas assemble. He teaches and
researches in evolutionary ecology, biogeography and systematics and is
co-leader of the Phoenix groups at Massey University evolves.massey.ac.nz. Glenn
Rebong is the Director of the Palawan Wildlife Rescue and Conservation
Center. Merlijn van Weerd is a Wildlife Biologist from the
Netherlands who has been working in the Philippines since 1999. He is connected
to the Institute of Environmental Sciences of Leiden University where he
conducts research on patterns of biodiversity distributions in the Philippines,
and on the ecology and conservation of the Philippine crocodile. In 2003 he
co-founded the Mabuwaya Foundation, of which he currently is the director.
Mabuwaya implements a community-based conservation program for the Philippine
crocodile, and in addition studies and conserves other endemic wildlife of
northern Luzon. Cayetano C. Pomaresis the Vice President for Research, Development and Extentsion at the
University of Southern Mindanao. Shannon
E. Engberg is the Conservation Genetics Research and Administration
Manager for the Conservation Genetics Department of Omaha’s Henry Doorly Zoo
and Aquarium. Rick Brenneman served as Conservation Geneticist at Omaha’s
Henry Doorly Zoo and Aquarium (2002–2013) during the time of this
study. His population genetic,
field and taxonomic studies included not only the Philippine crocodile but also
seven of the nine giraffe subspecies in Africa and 29 endangered lemur species
and two tortoise species in Madagascar. He is now a Research Associate with the Giraffe Conservation Foundation.Edward E. Louis, Jr. is the
Director of the Conservation Genetics Department of Omaha’s Henry Doorly Zoo
and Aquarium.
Acknowledgements: We would
like to thank Rainier Manalo, the late Charles Ross of Silliman University, and
Sonny Dizon of Davao Crocodile Park who contributed captive Philippine
Crocodile tissue samples that were used in this study. We thank Medel Silvosa,
Renato Cornel, Ernesto Conate, Amado Mulig, Salvador Guion, Ronnie Sumiller,
Fernando Paliza, William Tabinas, Alberto Guinto and Renato Sumiller who
assisted with the sample collection at PWRCC. We thank Willem van de Ven,
Bernard Tarun, Sammy Telan, Dominic Rodriguez and Jessie Guerrero of Mabuwaya Foundation
who assisted in procurement of samples from Isabela. Funding for tissue
collection and whole genome amplification kits were provided by grants from the
Crocodile Specialist Group (CSG) Student Research Fund, New Zealand Agency for
International Development postgraduate research fund, and Curt Harbsmeier, Law
Offices of Harbsmeier DeZayas, LLP. Assistance with the necessary prior
informed consents, gratuities, transport and CITES permits and letters of
support were provided by the Natural Resources Development Corporation,
Restituta Antolin and DENR Region II, Director Theresa Mundita Lim, Josefina de
Leon and staff of the DENR-PAWB Wildlife Management Office, along with the
United States Fish & Wildlife Service. Chris Banks and Tom Dacey of the CSG
provided contacts and information on Philippine crocodiles and funding
opportunities. We would like to thank PWRCC, Silliman University, Davao City
Crocodile Park, Calauit Game Reserve, the Valera Square Mini Zoo in Abra,
Omaha’s Henry Doorly Zoo and Aquarium, and Colette Adams and the Gladys Porter
Zoo in Brownsville, Texas, for the collection of Crocodylus mindorensissamples. We appreciate the field work by Moamar,
collecting samples in Liguasan Marsh. We thank the St. Augustine Alligator Farm
Zoological Park, St. Augustine, Florida, Peter Brazaitis and Yale Peabody
Museum of Natural History, New Haven, Connecticut for donating samples of the
other species for this study. We also like to acknowledge Lisa Kimmel for
graphic support. We appreciate the technical support of Omaha’s Henry Doorly
Zoo and Aquarium (OHDZA) Genetics Department, technicians Gary Shore and Susie
M. McGuire, along with two OHDZA docents, George Emodi and Paula Hinger, for
their expertise in DNA isolation and assistance in running countless PCR
reactions.
For figures,
images, tables -- click here
Introduction
The
application of genetics in conservation efforts has increased dramatically over
the past decades. Molecular genetic methodology has been used to address
taxonomic issues, assess genetic variability and inbreeding, track gene flow
and detect hybridization, all in an effort to conserve genetically healthy
populations and aid in the identification of ecologically significant units
(Fleischer 1998). The use of
nuclear DNA (nucDNA) and mitochondrial DNA (mtDNA) sequence data in crocodilian
research has increased our understanding of genetic variability (Flint et al.
2000; Ray et al. 2004; Russello et al. 2007), hybridization (FitzSimmons et al.
2002; Ray et al. 2004; Cedeño-Vásquez et al. 2008), differences between
individuals (Farias et al. 2004), populations (Vasconcelos et al. 2006, 2008)
and species (Li et al. 2007; Gatesy & Amato 2008; Meganathan & Dubey
2009; Meganathan et al. 2010). Microsatellites have been used to investigate population structure and
gene flow in wild populations of Morelet’s Crocodile Crocodylus moreletii Duméril
& Bibron, 1851 (Dever & Densmore 2001; Dever et al. 2002), American
Alligator Alligator mississippiensis Daudin, 1802 (Glenn et al. 1998;
Davis et al. 2002) and Black Caiman Melanosuchus nigerSpix, 1825 (de Thoisy et al. 2006). Microsatellites have also been useful in parentage analysis in Saltwater
Crocodiles C. porosus Schneider, 1801 (Isberg et al. 2004), in
determining and maintaining genetic variability in crocodiles bred for the
leather trade (Flint et al. 2000; FitzSimmons et al. 2002) and to build the
scaffolding for a genetic linkage map (Miles et al. 2009a).
Limited
information exists concerning the Philippine Crocodile, C. mindorensis,and its comparative status with other crocodilian species. The Philippine Crocodile is a species of
special concern and has already been the focus of a breeding program for many
years (Banks 2005). A combination
of hunting for commercial exploitation, extirpation because of fear,
overfishing of prey, habitat loss and habitat fragmentation have severely
diminished the range of this species and reduced the remaining populations to
critical levels (van Weerd & van der Ploeg 2003). Fifteen years ago, the wild populations
were estimated to contain less than 100 mature individuals (Ross 1998). The most recent Crocodile Specialist
Group (CSG) status update assesses the populations of C. mindorensis in
the wild to consist of less than 250 adults (van Weerd 2010). As a result, the Philippine Crocodile is
currently listed as Critically Endangered A1c, C2a in the IUCN Red List
(Crocodile Specialist Group 1996).
Silliman
University in Dumaguete City, Philippines, in 1980, initiated the first captive
breeding of the Philippine Crocodile for conservation purposes. In 1987, the Department of Environment
and Natural Resources (DENR), in a collaboration substantially funded by the
Japanese International Cooperation Agency, established the Crocodile Farming
Institute (CFI). The CFI is now
known as the Palawan Wildlife Rescue and Conservation Center (PWRCC) in Puerto
Princessa City, Philippines, and operates under the Protected Areas and
Wildlife Bureau (PAWB). The purpose
of the facility was to conserve the two species of crocodiles found in the
Philippines, the Saltwater Crocodile and the Philippine Crocodile (Sumiller
2000; Banks 2005). Both Silliman
University and PWRCC succeeded in breeding C. mindorensis, and many of
the resulting captive-bred stock have been sent to zoos in the Philippines and
other countries via breeding loan agreements (Banks 2005). However, PWRCC temporarily discontinued
captive breeding in 2001 due to financial constraints, limited space and
ambiguities in the captive stock pedigrees (Rebong & Sumiller 2003; Banks
2005).
Philippine Crocodile reintroductions into suitable habitats have
been planned by the Philippine Crocodile National Recovery Team (PCNRT; Banks 2005). A successful in situ Philippine
Crocodile conservation program is in progress in the San Mariano municipality
in Isabela Province (van Weerd & van der Ploeg 2003; van der Ploeg et al.
2011a,b,c). The Mabuwaya Foundation began a headstart program in 2005 where
wild-born Philippine Crocodiles were captured, captive
raised (i.e., headstarted) then released after two years thus increasing
juvenile survival rates (van de Ven et al. 2009). In 2010, 50 PWRCC captive-bred
Philippine Crocodiles were released into a lake in the Divilacan municipality, geographically separated from the wild Isabela
crocodile population. This release
served as a pilot project to assess the adaptability of captive-bred Philippine
Crocodiles under wild conditions (van Weerd & General 2003; van Weerd et
al. 2010).
Recent
systematics studies identified hybrids between C. mindorensis and C.
porosus at PWRCC from the analyses of both mtDNA (D-loop and ND4) and
nucDNA (C-mos) gene sequences (Louis & Brenneman 2008; Tabora et al.
2012). These studies validated
previous concerns regarding reintroduction candidate purity, thus warranting
forensic diagnoses prior to release. Using data generated from microsatellite
loci derived from crocodilian genomes by Miles et al. (2009b,c) and this study,
we address three questions regarding the Philippine Crocodile: (1) how does the
genetic diversity in C. mindorensis compare to other crocodilian
species, (2) what are the population genetic inferences of the two populations
in the current range distribution, and (3) to what extent has hybridization
occurred between C. mindorensis and C. porosus.
Materials
and methods
Sample collection
Tissue
samples were collected from a total of 619 Philippine Crocodiles from 1999−2009. Once crocodiles were safely restrained,
scute samples were obtained by cleaning the area with 70% isopropyl alcohol and
cutting with a scalpel/razor blade. The samples were stored in 1.8ml NUNC® tubes containing a room temperature
tissue preservative (Seutin et al. 1991). The majority of the Philippine Crocodile samples were collected from the
captive population maintained at the PWRCC; the rest from Davao City Crocodile
Park on Mindanao, Calauit Game Refuge and Wildlife Sanctuary on Palawan, Valera
Square Mini Zoo in the Abra Province, Silliman University in Dumaguete City and
individuals exported to the Gladys Porter Zoo in Brownsville, TX. Tissue samples from wild C.
mindorensis were collected from the two extant populations in the
Philippines: the San Mariano region in Isabela Province on Luzon and from the
Liguasan (Ligawasan, Liguwasan) Marsh on Mindanao. These are two regions of the Philippine
archipelago where indigenous cultural traditions offered some degree of protection
to the Philippine Crocodile (van der Ploeg & van Weerd 2004; Mangansakan
2008; Pimentel et al. 2008). A
single wild sample was collected on Dalupiri Island in the province of Cagayan
north of Luzon. A list of the study areas, site descriptions and number of
crocodiles sampled from each location are described in Tabora et al.
(2012). Samples from C.
niloticus Laurenti, 1768 (n = 12), C. acutus Cuvier, 1807 (n = 11),C. siamensis Schneider, 1801 (n = 12) and C. porosus (n = 37) were
obtained from the Yale Peabody Museum of Natural History collection and from
the St. Augustine Crocodile Farm for comparison to C. mindorensis.
DNA extraction
Genomic
DNA from the great majority of the tissue samples was extracted and amplified
using a whole genome amplification kit (WGA; Illustra TempliPhi®, GE Healthcare,
Piscataway, NJ). The WGA yielded an
average of 500ng of DNA per µL and all products were diluted to 50ng/µL. DNA from the remaining C. mindorensistissue samples were extracted using a standard
phenol/chloroform/isoamyl alcohol extraction method as described in Sambrook et
al. (1989).
Microsatellite amplification
A
subset of the sampled species was screened with an initial 31 microsatellite
loci (Miles et al. 2009b,c) discovered in the C. porosus genome. A locus was eliminated from the comparative
study if it failed to amplify in any one species or was monomorphic in at least
two species. Microsatellite loci
4HDZ27, 4HDZ35 and 4HDZ391 were discovered in the C. mindorensis genome
following the general protocol of Moraga-Amador et al. (2001) at Omaha’s Henry
Doorly Zoo and Aquarium’s Center for Conservation and Research (Table 1).
PCR
amplifications were performed in MBA Satellite 0.2G thermal cyclers (Thermo
Fisher Scientific, Inc., Waltham, MA) in final reaction volumes of 25µL and
containing 20−50 ng of DNA template. Final amplification conditions
consisted of 12.5 pmol unlabeled reverse primer, 12.5 pmol fluorescently
labeled forward primer, 1.5 mM MgCl2, 200 µM each dNTP, and 0.5
units of Taq DNA polymerase (Promega; Madison, WI). One of two PCR
thermal cycling methods were used depending on the microsatellite locus
amplified. Stratified touchdown programs were used for three loci: TD55 for
CpP4116 and TD65 for CpP302 and CpP2516 as described in Miles et al. (2009b). Standard PCR profile parameters for all
other markers used in this study were: 34 cycles of 950C for 30s, a
primer-specific annealing temperature for 45s, and 720C for 45s, and
a final extension step of 720C for 10 min. Optimum annealing temperatures were
determined as follows: 58°C for CpP305, CpP801 and CpP4004; 600C for
CpP1708, CpP3008 and 4HDZ391; 620C for 4HDZ35; and 640C
for 4HDZ27. PCR products were
visualized to verify amplification on 2% agarose gels stained with ethidium
bromide. For the comparison between C. mindorensis and C. porosusand hybridization analysis CpP305, CpP1708, CpP2516, CpP3008, CpP4004 and
CpP4116 were amplified with the above standard conditions. An additional 12 loci were found to be
informative for these analyses. The
stratified touchdown programs TD55 for CpP3313 and CpP4301 and TD65 for CpP4311
were used as described in Miles et al. (2009b). The following loci were amplified with
standard PCR as described above at the following annealing temperatures: 560C
for CpP208 and CpP1610; 580C for CpP80 and CpP3601; 600C
for CpP405, CpP1002 and CpP3220; and 620C for CpP203 and
CpP610. Allele sizes were
determined by separation of the PCR products via POP 4 capillary buffer
electrophoresed on ABI 3100/ABI 3130xl Genetic Analyzers (Applied
Biosystems, Inc., Foster City, CA). Fragment length genotypes were assigned by GeneScan
using GeneScan™ 500XL ROX™ size standard in the GeneMapper software version 4.0.
Data analysis
MICRO-CHECKER
(Van Oosterhaut et al. 2004) and Microsatellite Analyzer (MSA; Dieringer &
Schlötterer 2003) were used to analyze the data set for genotyping and
typographical errors. Null allele
frequencies were estimated using CERVUS 2.0 (Marshall et al. 1998; Slate et al.
2000). Excessive frequencies of
null alleles can bias the data interpretation by either overestimating
homozygosity or underestimating heterozygosity (Callen et al. 1993; Hoffman
& Amos 2005). Loci with high
null allele frequency estimates (nf>0.2) were removed from further
analysis (Chapuis & Estoup 2007). The population genetic parameters: observed (Ho), expected (He),
and unbiased expected heterozygosity (UHe), mean number of alleles (Na),
effective number of alleles (Ne), Shannon Information index (I;
Shannon 1948), and the within population fixation index (Fis) were estimated using
GenAlEx 6.41 (Peakall & Smouse 2006). The Shannon entropy index was transformed by Diversity of Order 1 =
exponential of I (Jost 2009). Heterozygosity estimates were transformed by Diversity of Order 2 = 1/
(1-He) (Jost 2008). The
between population fixation index (Fst)
with significance was estimated with FSTAT 4.3 (Goudet 1995, 2001). For intraspecific diversity study, we
neglected the captive populations because (1) the collections do not represent
true populations; (2) the sample sizes for most were too small; and (3) hybrids
had been previously discovered in PWRCC and thus we expect that C. porosusalleles would be present in the population inflating estimates reflecting intraspecific
genetic diversity.
Effective
population sizes were estimated with the linkage disequilibrium (LD) method
using LDNe 1.31 (Waples & Do 2008) that corrects for small sample sizes
bias (Waples 2006), an advantage over NeEstimator (Peel et al. 2004). The LD method is grounded on the
principal that the loss of genetic variation is intensified by an increase in
linkage disequilibrium. Testing
allelic associations among multiple loci allows inbreeding estimation in the
effective population size. Waples
& Do (2008) determined that estimates of effective population size may
become slightly less accurate but more precise as alleles with lower allele
frequencies are included in the estimation. LDNe estimates effective population
sizes excluding allele frequencies below the critical values of 0.05, 0.02, and
0.01 to assess the effects of rare alleles in the data. The ratio of the effective population
size to the census size (Ne/N) can be used to predict inbreeding and
genetic variation loss in wildlife populations (Frankham 1995).
Since
it is possible that the two extant C. mindorensis populations, being
from the northern and southern extremes of the distribution, might exhibit
detectable selection, we tested for selection using both Lositran (Beaumont
& Nichols 1996; Antao et al. 2008) and BayeScan 2.0 (Foll & Gaggiotti
2008). Lositran implements an FSToutlier method to identify loci likely under selection whereas BayeScan employs
a maximum likelihood posterior probability. Relevance of the BayeScan posterior
probabilities were interpreted with Jeffreys’ scale of
evidence (Jeffreys 1961). Considering that the extant populations are small, all within-population
dyads were tested for relatedness (Queller & Goodnight 1989) using SPAGeDi
(Hardy & Vekemans 2002) and compared to a simulation of 10,000 individuals
of known pedigree relationships (Queller & Goodnight 1989).
Crocodylus porosus x C. mindorensis hybridization was identified in Tabora et
al. (2012) where 57 captive crocodiles expected to be C. mindorensis by
breeding records had inherited mtDNA haplotypes and nucDNA C-mosdiagnostic sites found in C. porosus. We examined the microsatellite loci
screened for the species diversity comparison to identify markers that would be
informative in comparing the two species of crocodiles found in the
Philippines. Eight additional loci
found to be monomorphic in C. mindorensis and polymorphic in C.
porosus for diagnostic alleles not present in the genotype data of C.
mindorensis populations and collections exclusive of PWRCC (CpP2516,
CpP208, CpP405, CpP610, CpP1002, CpP3601, CpP4301, and CpP4311) were included
to test for evidence of hybridization. We generated multilocus data on 619 C.
mindorensis from both wild populations and the captive collections comprising
a great majority of the freshwater crocodiles in the Philippines and 37 C.
porosus from samples collected in Republic of Palau (RP) by Russello et al.
(2007).
Population
structure was inferred using STRUCTURE v2.1 (Pritchard et al. 2000; Falush et
al. 2003) to determine the differentiation between the northern and southern C.
mindorensis populations and to test for potential hybridization in the
populations with C. porosus. The program uses a Bayesian clustering based method to determine whether
the two extant populations could be identified by genetic clustering and to
determine if populations harboring allelic structure demonstrated genetic
admixture of the parental species clusters. STRUCTURE attempts to identify
population subsets that maximize Hardy Weinberg expectations and minimize LD
from multilocus genotypes (Pritchard et al. 2000). We chose the ancestry model, correlated
allele frequencies, different FST values assumed for each
subpopulation, a uniform prior for alpha (max: 10, SD for updating: 0.025),
constant lambda value of 1, prior FST mean (0.01) and
standard deviation (0.05). We set
the range to consider 1−11 genetic clusters as Evanno et al. (2005)
suggests estimating over a range of at least three clusters more than sampling
locations. The burnin period was
set at 105 repetitions followed by 106 MCMC repetitions
for 20 iterations of the Gibbs sampler for each K value. Occasionally STRUCTURE overestimates the
optimal K value; hence, Evanno et al. (2005) developed an ad hoctest statistic ΔK to evaluate the output files in addition to
approximating the asymptote of the posterior probability curve. At K-max,
we applied a conservative threshold of q≥0.05 to the membership
coefficient (q-value) of the cluster attributed to the introgressing
species to identify hybrids (Hapke et al. 2011).
In
addition, we used the Principal Coordinates Analysis (PCoA) in GenAlEx v6.41 to
detect shifts in multilocus genotype groupings that might indicate individual
affinity drifting away from expected parental groups. We charted the first two
axes of inertia using genetic distance as the criteria with the covariance
standardized method of calculation.
Results
Eleven
informative microsatellite loci amplified and were used to generate the data
set from the two wild-sampled C. mindorensis populations and the samples
of C. acutus, C. niloticus, C. porosus and C. siamensis. The average number of alleles ranged
from 3.7 in the C. mindorensis samples from the population of Liguasan
Marsh to six in C. niloticus. The number of effective alleles ranged
from 2.159 in the C. mindorensis of Isabela to 3.847 in C. niloticus. The observed heterozygosity ranged from
0.408 in samples from the Isabela population to 0.630 in C. porosus and
expected heterozygosity ranged from 0.423 in the Isabela population to 0.663 inC. niloticus (Table 2). Regardless of the estimate or index, the two extant C. mindorensis populations ranked lowest in genetic diversity compared
to the sample collections of C. acutus, C. niloticus, C. porosusand C. siamensis. F-statistics measuring within population
fixation or inbreeding (Fis)
ranged from -0.149 to 0.160 but were not significant. Population fixation
indices (Fst)
and their significances are presented in Table 3.
Twenty
loci were found to be informative for intraspecific evaluation and to compare C.mindorensis with C. porosus. Analysis of the estimated effective population sizes of the Isabela and
Liguasan Marsh populations showed that those populations have much lower
effective population sizes than the population of C. porosus from
Republic of Palau using the more precise 0.01 rare allele threshold (Table
4). The SPAGeDi dyad analysis
revealed overall relatedness within the Isabela Philippine Crocodile population
to be slightly more than what might be expected from matings of unrelated
individuals (Fig. 1A). This trend
was not detected, though, in the Liguasan Marsh population (Fig. 1B). The population of Saltwater Crocodiles
showed little relatedness differing from the simulation of unrelated
individuals (Fig. 2).
Both
Lositran and BayeScan identified two outlier loci as potentially linked to
genes that might be under some degree of selection. However, the two approaches agreed on
only one locus (CpP801). Lositran found CpP801 to be a significant FSToutlier whereas BayeScan found it “barely worth mentioning” using the Jeffreys’
scale of evidence (data not shown). The sequences flanking the CpP801 repeat motif were submitted to the
BLASTn algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_SPEC=WGS&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch)
to search for potential candidate genes that might be under selection. Minimal sequence fragments ranging 25−50
bp in length were found in other species but no long sequence homologies and
none of the queries returned candidates common to both flanking regions. Two short sequences were found in
multiple species although corresponding to different genes. They were also found on multiple
chromosomes in a single species indicating that these two sequences were both
conserved and duplicated in the genome.
From
the STRUCTURE analysis, K=3 was found to be the optimal number of
clusters represented in the data by Evanno et al.’s (2005) ΔK (Fig.
3). These clusters represent the
Isabela C. mindorensis population, the Liguasan Marsh C. mindorensispopulation and the Republic of Palau C. porosus population. At K-max, a total of 59 putative C.
mindorensis individuals had q-values above the noise threshold of
0.05 in the cluster represented by C. porosus (Fig. 4, see also Appendix
1). The PCoA suggested the same C.
mindorensis individuals as previously identified with affinity to the C.
porosus sample set (Fig. 5). The PCoA also identified individuals in the Isabela population that
appear to group with the southern populations; a
phenomenon which cannot be verified with records or observations. The PWRCC bred crocodiles reintroduced
in Isabela were not included as Isabela members in this study.
DISCUSSION and CONCLUSIONS
Previous
studies have estimated genetic diversity in crocodilian species but making
direct comparisons was difficult since the same marker systems were not applied
across each study. Here, we used
the same microsatellite loci to compare the genetic diversity of C.
mindorensis to C. acutus, C. niloticus, C. porosus andC. siamensis. The heterozygosity estimates from our data for C.
acutus, C. niloticus, C. porosus and C. siamensis fall
within the ranges of estimates previously reported for captive purebred C. siamensis,Ho = 0.42±0.17 (FitzSimmons et al. 2002), farmed C. porosus, Ho
= 0.59 (Isberg et al. 2004) and in wild populations of C. niloticus, He
= 0.27–0.61 (Hekkala et al. 2010) and Ho = 0.51 (Bishop et al.
2009), C. moreletti, Ho = 0.49 (Dever et al. 2002) and Melanosuchus niger, Ho = 0.47–0.70 (de Thoisy et al. 2006). We found
that genetic diversity measures for C. mindorensis were lower compared
to C. acutus, C. niloticus, C. porosus and C. siamensis,
whether using traditional FST and heterozygosity measures or
by transforming such measures into diversity indices.
The
LDNe analysis of the effective population sizes allows the interpretation at
three levels dictated by thresholds for rare alleles in the data. Considering the lowest accepted
frequency for rare alleles to be 0.01, the estimates of effective breeders were
4.8 (95% CI: 3.5−7.3) in Isabela, 7.9 (95% CI: 3.0−20.2) in
Liguasan Marsh and 22.6 (95% CI: 18.8−27.6) in the collection of C.
porosus from RP. In 2008, the minimum census of the Isabela population was
86 individual crocodiles comprised of 10 adults, 41 sub-adults/juveniles and 35
hatchlings with six nests in four distinct localities (van Weerd 2010 and van
Weerd unpublished data). The
Philippine Crocodile population in Liguasan Marsh remains poorly known but was estimated in 2008 to include at least 258
individuals in all age classes (Pomares et al. 2008). This estimate is based on interviews
with the local inhabitants of the marsh, which in all likelihood contain
multiple sightings of individual animals. The ratios of effective breeders to the estimated population sizes were
determined to be 0.06 in Isabela and 0.03 in Liguasan Marsh. These estimates hover about the 0.05
ratio threshold which Frankham (1995) considers quite
low, and is, when compared to recent studies in Steelhead Trout (Oncorhynchus
mykiss, Araki et al. 2007) and the European Common Frog (Rana temporaria,
Schmeller & Merila 2007), 0.10–0.40 and 0.23–1.67,
respectively. We did find evidence
for increasing relatedness in the small isolated Isabela population. This
estimate would be expected as hatchlings were sampled from the nests. We did not find excessive Fis values, but could expect
those to rise in future generations if mating among related individuals becomes
commonplace due to the small effective population sizes.
With
only two extant populations of C. mindorensis known to remain today, it
is imperative to evaluate the similarity or differences between the two. Biogeographic differences might exist
since the Isabela population exists in the northern extreme of the distribution
whereas the Liguasan Marsh population is found in the southern extreme. One might expect that if the populations
were highly differentiated, molecular testing could detect a genetic selection
signature associated with some of the neutral markers. We did find positive results using two
testing methods, but for only one of the 11 loci. We searched the repeat motif
flanking sequences against sequences stored in the BLASTn database, but we did
not identify a potential candidate gene. In fact, in both flanking regions,
small fragments (25−50 bp) were highly conserved among species and
duplicated within genomes. With one
method identifying this locus as a significant FST outlier
and the other as marginal, we suggest that this locus is not under selection
but a false positive in both tests. False positives can be the result of
hierarchical structure perhaps created from the pooling of samples from four
distinct breeding areas in the San Mariano area of the Isabela region
(Excoffier et al. 2009). Likewise, the data set or the number of remaining
Philippine Crocodiles in the wild may simply be too small to detect selection
(Hohenlohe et al. 2010). Regardless, we cannot suggest that evidence was found
to support selection that might be differentiating the populations. If the two populations differed greatly,
then the populations might require separate management. However, the populations differ only
slightly, which we assume may simply be caused by genetic drift thus mixing may
reestablish or maximize genetic diversity supporting positive genetic health of
the species.
Tabora
et al. (2012) identified a total of 57 putative hybrids in that study. From the
STRUCTURE analysis of the same set of samples, we identified 59 individuals
with genotypic proportions exceeding a background noise level (q>0.05)
in the cluster generated by the C. porosus samples (Appendix 1). The PCoA analysis also identified the
same individuals to be closer to the C. porosus grouping than C.
mindorensis below the nominal q-value threshold. Only two individuals approached the q= 0.50 genotypic proportions expected of an F1 individual (PWc005, q =
0.512; PWb097, q = 0.409). The former, PWc005, possesses both a C.
porosus D-loop haplotype and the C. porosus C-mos diagnostic
characters. We consider this individual to be an F1 from a C. mindorensismale and a C. porosus female. The latter, PWb097, possesses the C.
porosus D-loop haplotype yet is homozygous for the C. mindorensis C-mosdiagnostic sites. We consider this individual to be a C. mindorensisbackcross falling in the upper tail of the backcross q-distribution. Two individuals from Abra (K7895 and
K7897) exceeded the conservative 0.05 q-threshold for background noise
though did not possess C. porosus D-loop or C-mos markers. We accept these to be C. mindorensiswith slightly higher background noise than the conservative threshold we
imposed in our criteria. The
remaining 55 fell in a q-distribution around 0.25 (avg q = 0.253±0.067) which approximates the proportion of introgressed genes
expected to be retained in the first backcross generation. Thus, we suggest one first generation
hybrid cross and 56 backcross individuals only in the PWRCC-sampled group.
The morphological identification of
hybrids, and particularly among the hybrids in this study, proves to be
problematic. Hybrid detection through morphological characteristics is not
always effective because hybrids can express mosaics of phenotypes (Campton
1987) due to incomplete penetrance or partial dominance of the diagnostic
character. Hybrids in the PWRCC
population were undetected since all express the post occipital scutes
indicative of C. mindorensis (Image 1A). This suggests a single gene effect where
the allele conferring the diagnostic scutes expressed in C. mindorensisis dominant over the allele fixed in C. porosus that suppresses the
expression of that phenotype (Image 1B). Had F1 inter se mating occurred, one would expect that one fourth
of the offspring should have inherited both C. porosus C-mosalleles and one fourth should express the absence of post occipital scutes.
Neither scenario was detected in the data. Considering the multilocus allele frequency distributions, there is no
indication that F1 inter se mating has occurred since the average of theq-distribution of an F2 generation would be higher (closer to
0.50). Backcrossing to C.
mindorensis would ensure at least one C. mindorensis allele at all loci which is exactly what the data shows. This comprehensive genetic testing
identifies hybrids in the collection that can be separated out of the gene pool
before a hybrid swarm is created that could have a detrimental effect on the
conservation management of the species (Allendorf et al. 2001). The removal of suspected hybrids could
protect the genetic integrity of the species, especially if used as
reintroduction candidates or to augment the genetic diversity of the wild
populations (Rhymer & Simberloff 1996).
The
two distantly isolated extant populations of C. mindorensis, Isabela and
Liguasan Marsh, present several concerns for long-term conservation
management. Both show less genetic
diversity than what has been detected in other crocodilian species in this and
previous studies. Both populations have
low effective population sizes and low effective population size to census
ratios. The recent systematics
study (Tabora et al. 2012) did not indicate branch lengths that would suggest
more than population level differentiation. There is no indication of selection
being a differentiating factor but the distance and isolation would be expected
to drive genetic drift. Slightly
elevated relatedness estimates suggest that future generations within both
populations could face unavoidable mating of related individuals and the
potential consequences of inbreeding. Genetic augmentation should be considered to offset these potential
problems, whether by reintroduction from captive populations or by
translocation between the populations. The most difficult constraint for successful conservation is securing
the necessary funding to engage and monitor the programs. Whether genetic mixing between the two
extant populations, augmentation from captive collections, or reintroduction of
headstarted or captive born candidates is decided upon, funding will be crucial
to monitor the success of the effort and protect remaining habitats for the
future of the species.
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