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Phylogenetic and Phylogeographic
Analysis of Iberian Lynx Populations
W. E. JOHNSON,J.A.GODOY,F.PALOMARES,M.DELIBES,M.FERNANDES,E.REVILLA,
AND S. J. O’BRIEN
From the Laboratory of Genomic Diversity, National Cancer Institute-FCRDC, Frederick, MD 21702-1201 (Johnson and
O’Brien), Department of Applied Biology, Estacio
´n Biolo
´gica de Don
˜ana, CSIC, Avda. Marı
´a Luisa s/n, 41013, Sevilla,
Spain (Godoy, Palomares, Delibes, and Revilla), and Instituto da Conservac¸ a
˜o da Natureza, Rua Filipe Folque, 46, 28,
1050-114 Lisboa, Portugal (Fernandes). E. Revilla is currently at the Department of Ecological Modeling, UFZ-Center for
Environmental Research, PF 2, D-04301 Leipzig, Germany. The research was supported by DGICYT and DGES
(projects PB90-1018, PB94-0480, and PB97-1163), Consejerı
´a de Medio Ambiente de la Junta de Andalucı
´a, Instituto
Nacional para la Conservacion de la Naturaleza, and US-Spain Joint Commission for Scientific and Technological Cooperation.
We thank A. E. Pires, A. Piriz, S. Cevario, V. David, M. Menotti-Raymond, E. Eizirik, J. Martenson, J. H. Kim, A. Garfinkel,
J. Page, C. Ferris, and E. Carney for advice and support in the laboratory and J. Calzada, J. M. Fedriani, P. Ferreras, and
J. C. Rivilla for assistance in the capture of lynx. Museo Nacional de Ciencias Naturales, Don
˜ana Biological Station
scientific collection, Don
˜ana National Park, and Instituto da Conservac¸ a
˜o da Natureza of Portugal provided samples of
museum specimens. The content of this publication does not necessarily reflect the views or policies of the Department of
Health and Human Services, nor does mention of trade names, commercial products, or organizations imply
endorsement by the U.S. government.
Address correspondence to W. E. Johnson at the address above.
Abstract
The Iberian lynx (Lynx pardinus), one of the world’s most endangered cat species, is vulnerable due to habitat loss, increased
fragmentation of populations, and precipitous demographic reductions. An understanding of Iberian lynx evolutionary history
is necessary to develop rational management plans for the species. Our objectives were to assess Iberian lynx genetic diversity
at three evolutionary timescales. First we analyzed mitochondrial DNA (mtDNA) sequence variation to position the Iberian
lynx relative to other species of the genus Lynx. We then assessed the pattern of mtDNA variation of isolated populations
across the Iberian Peninsula. Finally we estimated levels of gene flow between two of the most important remaining lynx
populations (Don
˜ana National Park and the Sierra Morena Mountains) and characterized the extent of microsatellite locus
variation in these populations. Phylogenetic analyses of 1613 bp of mtDNA sequence variation supports the hypothesis that the
Iberian lynx, Eurasian lynx, and Canadian lynx diverged within a short time period around 1.53–1.68 million years ago, and that
the Iberian lynx and Eurasian lynx are sister taxa. Relative to most other felid species, genetic variation in mtDNA genes and
nuclear microsatellites were reduced in Iberian lynx, suggesting that they experienced a fairly severe demographic bottleneck.
In addition, the effects of more recent reductions in gene flow and population size are being manifested in local patterns of
molecular genetic variation. These data, combined with recent studies modeling the viability of Iberian lynx populations, should
provide greater urgency for the development and implementation of rational in situ and ex situ conservation plans.
The Iberian lynx (Lynx pardinus), the largest remaining cat
species in southwestern Europe, is one of the world’s
most endangered felids (Nowell and Jackson 1996). The
genus Lynx, including the Iberian lynx, bobcat (L. rufus),
Canadian lynx (L. canadensis), and Eurasian lynx (L. lynx)
descend from a common ancestor that diverged from
other cat species more than 6 million years ago ( Johnson
and O’Brien 1997). These four Lynx species have almost
completely disjunctive distributions and occupy different
habitats. However, they share numerous physical charac-
teristics and are distinguished primarily by their relative
sizes (Nowell and Jackson 1996) and a few cranial or
postcranial skeletal measurements (Garcia-Perea 1992).
There has been consistent support for a monophyletic
ancestry for the four species from morphological (Nowak
1999) and DNA-based analyses ( Janczewski et al. 1995;
Johnson et al. 1996; Johnson and O’Brien 1997; Pecon-
Slattery and O’Brien 1998). However, the hierarchical
relationships among lynx species and their recognition as
unique species have been debated.
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Fossil records suggest that lynx species originated in
North America (MacFadden and Galiano 1981; Martin
1989). The bobcat appears to represent the earliest lineage to
diverge (Johnson and O’Brien 1997), but subsequent
evolutionary history is less clear. The Iberian lynx and
Eurasian lynx may be derived from an ancestral lynx species
(Lynx issiodorensis) whose remains have been found in China
(Wederlin 1981). The Canadian and Eurasian lynx have been
hypothesized to be sister taxa (Wederlin 1981). The Iberian
lynx and Eurasian lynx were both found in central Europe
during the Pleistocene (Kurten 1968; Kurten and Grandqvist
1987), but may never have had significantly overlapping
geographic ranges.
The distribution and population sizes of the Eurasian and
Iberian lynx have been reduced significantly during the last
two centuries. The Eurasian lynx was extirpated from most
of central and southern Europe during the 19th century
(Breitenmoser and Breitenmoser-Wursten 1990). Similarly,
by the beginning of the 20th century the Iberian lynx was rare
in northern Spain and by the 1960s its range was essentially
limited to isolated populations in the southwestern portion
of the peninsula (Rodriguez and Delibes 1990). By the 1990s,
it was estimated that no more than 1000 lynx were restricted
to fewer than 50 disjunct breeding areas that could be
grouped into less than 10 distinct subpopulations (Castro
and Palma 1996; Rodriguez and Delibes 1992). Only two of
these subpopulations, Sierra Morena and Montes de Toledo
(Figure 1) inhabit areas larger than 2000 km
2
, which is of
major concern since between 1960 and 1988 Iberian lynx are
presumed to have disappeared from most of the remaining
small habitat patches (from 91% of the areas smaller than
1000 km
2
). This corresponds to an 80% reduction in
occupied range (Rodriguez and Delibes 1990). However,
current geographical limits and population sizes are not well
known and continued loss of habitat and the disappearance
of lynx from previously occupied areas suggests that fewer
than 500 individuals may now remain (Beltra´n and Delibes
1994; Palomares et al. 2000; Pires and Fernandes 2003).
The best known of the Iberian lynx populations inhabits
Don
˜ana National Park, Andalusia, where field data on the
species have been collected since the 1950s and radio-
tracking studies have been conducted for more than 15 years
(e.g., Ferreras et al. 1997; Gaona et al. 1998; Palomares et al.
1991, 2001). The Don
˜ana metapopulation (1500 km
2
)of
approximately 40–50 individuals currently is distributed in at
least four distinct areas that were last completely connected
during the middle of the last century. Within the Don
˜ana
metapopulation, the population occupying the protected
parkland areas is relatively stable, while mortality in the less-
protected areas is higher, especially among dispersing
animals (70–85% mortality rate) (Ferreras et al. 1992; Gaona
et al. 1998). Because of its small numbers and isolation, the
Don
˜ana metapopulation would be susceptible to low levels
of genetic variation (Beltra´n and Delibes 1993).
The Don
˜ana metapopulation has been isolated from
other Iberian lynx populations for at least 50 years by an
expanse of more than 50 km of croplands to the north and
almost 30 km of comparatively dense human settlements to
the west (Rodriguez and Delibes 1992). The nearest lynx
populations are found in the Sierra Morena Mountains
(Figure 1), where several of the largest remaining lynx
metapopulations are suspected to still exist. This mountain
range, perhaps one of the keys to the future preservation of
the Iberian lynx, has received increasing attention by
conservationist and resource managers.
The objectives of this study were to quantify the
evolutionary relationship of the Iberian lynx with the other
Lynx species using mtDNA sequence variation, describe
patterns of lynx mtDNA variation across the Iberian
peninsula, and compare patterns of microsatellite size
variation and estimate gene flow between the important
lynx populations of Don
˜ana and Sierra Morena. An
improved understanding of patterns of Iberian lynx
molecular genetic variation and recent evolutionary history
is necessary to develop rational management plans for the
species (Vargas 2000) and provide further insights into the
biogeographical history of the Iberian peninsula.
Methods
DNA Extraction
Lynx DNA was extracted from blood and tissue samples
from 20 wild animals captured or found dead during
capturing and radio-tracking of lynx from 1985 to 2000,
and from 35 tissue and skin samples in scientific collections
from seven metapopulations throughout the range of the
Iberian lynx (Table 1). Blood samples were stored in four
volumes of lysis buffer (0.1 M Tris-HCl pH 8.0; 0.1 M Na-
EDTA; 0.01 M NaCl, 0.5% SDS) and tissue samples were
kept frozen or at room temperature in a dimethyl sulfoxide
(DMSO)-salt solution (20% DMSO, 0.25M Na-EDTA, and
NaCl to saturation, pH 8.0). From museums specimens,
approximately 1 cm
2
of skin was cut with a sterile scalpel
after superficial cleaning with 10% commercial bleach,
distilled water, and 70% ethanol. DNA extractions of
museum materials were conducted in a dedicated room
along with extraction blanks to monitor for contamination.
DNA was extracted following standard proteinase K/phenol
chloroform protocols (Sambrook et al. 1989); for museum
skin extractions, several prewashes with NTE pH 9.0 (NaCl
10 mM; Tris base 50 mM; EDTA 20 mM) were included to
remove possible enzyme inhibitors. For comparative
purposes we used DNA extracted using similar techniques
in previous studies from three European lynx (Lly 12, 15, and
16), two Canadian lynx (Lca 3 and 7), five bobcat (Lru 6, 26,
48, 68, and 73), two marbled cats (Pardofelis marmorata) (Pma 4
and 5), and one clouded leopard (Neofelis nebulosa) (Nne 80)
( Johnson and O’Brien 1997).
Mitochondrial DNA Markers
Sequence variation in portions of five mtDNA genes
(ATPase-8, 16S rRNA, 12S rRNA, NADH-5, and cyto-
chrome b) was assessed in four Iberian lynx, three European
lynx, two Canadian lynx, and five bobcat, along with two
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marbled cats and a clouded leopard as outgroups, to assess
the uniqueness of the Iberian lynx and to determine the
evolutionary relationships among Lynx species. A larger set
of Iberian lynx from across the Iberian peninsula (43
individuals from seven metapopulations; Table 1) was used
to characterize patterns of mtDNA sequence variation in
three mtDNA fragments: 191 of ATPase-8 gene, and 195
and 89 bp of two noncontiguous fragments of the hyper-
variable segment 1 of the control region (Kim et al. 2001).
DNA amplification reactions contained 67 mM Tris-HCl
pH 8.0, 16 mM (NH
4
)
2
SO
4
, 2.5 mM MgCl
2
, 0.01% Tween-
20, 0.2 mM dNTPs, 1 lM of each primer, 0.5 U of Taq
polymerase and 50–100 ng of total DNA for blood samples
or 5 ll of museum skin extracts as template. Polymerase
chain reaction (PCR) primers and primer conditions have
been published previously ( Janczewski et al. 1995; Johnson
and O’Brien 1997; Johnson et al. 1998; Palomares et al.
2002). Bovine serum albumin (BSA) was included at
a concentration of 0.1 lg/ll for amplification of blood
DNA and 0.8 lg/ll for museum and skin samples.
Amplification reactions were performed in an MJ Research
(Boston) thermocycler, model PTC-100, at an initial de-
naturation cycle of 948C for 2 min, followed by 35 cycles of
denaturation at 928C for 30 s, annealing at 55–67.58C
(depending on primers) for 30 s, extension at 728C for 30 s,
and were completed with a final extension at 728C for 5 min.
Positive and negative DNA controls were included with each
set of PCRs. Amplification products were separated by
Figure 1. Geographic distribution of the Iberian lynx populations sampled in the study modified after Rodriguez and
Delibes (1992) and Castro and Palma (1996). In Spain, the data represent estimated distributions from the 1980s and in
Portugal the data are from 1987–1996. The distribution of mtDNA haplotypes in each of the major populations (which are
labeled following Table 1) are represented in pie charts, along with the number of individuals sampled from each population.
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Table 1. Sample identifier, sample type, population and metapopulation of origin, nucleotide residue at positions 8672 and
16,804 (domestic cat sequence, Lo´ pez et al. 1996) and mtDNA haplotype for gene segments of ATP-8, control region 1 (CR1),
and control region 2 (CR2) combined
Sample identification Tissue Population Metapopulation 8672 16,804 mtDNA haplotype Source
Barro* Blood Coto del Rey Don
˜ana C T BAuthors
Borja* Blood Coto del Rey Don
˜ana Authors
Escarlata* Blood Coto del Rey Don
˜ana Authors
Gloria* Blood Coto del Rey Don
˜ana C T BAuthors
Maki* Blood Coto del Rey Don
˜ana Authors
Nuria* Blood Coto del Rey Don
˜ana C T BAuthors
Vampi* Blood Coto del Rey Don
˜ana Authors
23122 Skin Coto del Rey Don
˜ana C T BEBD
Anibal* Blood Vera Don
˜ana Authors
Celia* Blood Vera Don
˜ana Authors
Cova* Muscle Vera Don
˜ana C T BPND
Isabel* Blood Vera Don
˜ana PND
Jabata* Tissue Vera Don
˜ana PND
Juanito* Muscle Vera Don
˜ana C T BAuthors
Navidad* Blood Vera Don
˜ana Authors
Understand* Tissue Vera Don
˜ana PND
23127 Skin Unknown Don
˜ana C T BEBD
23731 Skin Unknown Don
˜ana C T BEBD
Ibiza* Blood Sierra Morena Eastern Sierra Morena Authors
Morena* Blood Sierra Morena Eastern Sierra Morena T T APND
Pı
´riz* Blood Sierra Morena Eastern Sierra Morena T T AAuthors
Sierra* Blood Sierra Morena Eastern Sierra Morena Authors
Sofı
´a* Blood Sierra Morena Eastern Sierra Morena T T AAuthors
22650 Skin Sierra Morena Eastern Sierra Morena C T BEBD
5452 Skin Jaen Eastern Sierra Morena T T AMCNM
5463 Skin Jaen Eastern Sierra Morena T MCNM
1387 Skin Andujar Jaen Eastern Sierra Morena T T AEBD
1732 Skin Andujar Jaen Eastern Sierra Morena T EBD
19299 Skin Andujar Jaen Eastern Sierra Morena T T AEBD
23223 Skin Andujar Jaen Eastern Sierra Morena T T AEBD
c28 Skin Malcata Sierra Gata Malcata C T BICN
1376 Skin Salamanca Sierra Gata C T BEBD
c21 Skin Penamacor Sierra Gata Malcata C T BICN
c22 Skin Penamacor Sierra Gata Malcata T ICN
5456 Skin Montes de Toledo Montes de Toledo T MCNM
5458 Skin Montes de Toledo Montes de Toledo T MCNM
5446 Skin Los Yebenes To Montes de Toledo T T AMCNM
5450 Skin Los Yebenes To Montes de Toledo T T AMCNM
5451 Skin Los Yebenes To Montes de Toledo T MCNM
5462 Skin Los Yebenes To Montes de Toledo T T AMCNM
5465 Skin Los Yebenes To Montes de Toledo T MCNM
5467 Skin Los Yebenes To Montes de Toledo T T AMCNM
5470 Skin Los Yebenes To Montes de Toledo T T AMCNM
5449 Skin Toledo Montes de Toledo T T AMCNM
5454 Skin Toledo Montes de Toledo T T AMCNM
5466 Skin Toledo Montes de Toledo T MCNM
5468 Skin Toledo Montes de Toledo T MCNM
5469 Skin Toledo Montes de Toledo T MCNM
1373 Skin Huelva Western Sierra Morena T C CEBD
1377 Skin Huelva Western Sierra Morena T C CEBD
c14 Skin Alca´c¸ovas Sado Valley Algarve C T BICN
C32 Skin Alca´cer Sado Valley Algarve C T BICN
846 Skin Avila Gredos o Alto Alberche T T AEBD
23531 Skin Unknown Unknown T T AEBD
C3 Skin Unknown Portugal C T BICN
Metapopulations are also depicted on the map in Figure 1. Samples used in microsatellite analyses are marked with an asterisk (*) and samples from the
Museo de Ciencias Naturales de Madrid (MCNM), Estacion Biologica de Don
˜ana (EBD), Parque Nacional de Don
˜ana (PND), and the Instituto da
Conservac¸a
˜o da Natureza (ICN) are noted. The additional samples were collected by several of the authors during field studies.
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electrophoresis in 2–3% agarose gels in TBE buffer (89 mM
Tris base, 89 mM boric acid, 2 mM EDTA) in the presence
of 0.5 mg/L EtBr. Gels were visualized under ultraviolet
light and photographed with a digital image system (Eastman
Kodak).
Polymerase chain reaction products were cleaned by
ultrafiltration through Centricon-100 (Millipore Corp.) and
sequenced on an automated DNA sequencer (ABI 377)
using the BigDye Terminator Cycle Sequencing Kit
following the manufacturer’s instructions (Applied Biosys-
tems). Sequences were edited, assembled, and aligned using
the program Sequencher (Gene Codes Corp.) and submitted
to GenBank (accession numbers AY499248–AY499337).
Microsatellite Markers
Twenty-eight microsatellite loci (Fca43,71,75,80,82,90,96,
97,98,102S,117,161,132,193,232,272,369,391,424,441,
453,476,493,519,547,566,571,698) from 11 of the 19
domestic cat chromosomes were characterized in 20
presumably unrelated lynx from two populations (Vera and
Coto del Rey) in Don
˜ana and in Valquemado from Sierra
Morena (Table 1) following previously described PCR
amplification conditions (Menotti-Raymond et al. 1997,
1999). All microsatellites were dinucleotide repeats except
FCA391, FCA441, and FCA453, which had tetranucleotide
repeats. Of the 28 loci, 22 were unlinked or at least 20 cM
apart in the domestic cat and are presumed to be unlinked in
Iberian lynx (Menotti-Raymond et al. 1999; submitted).
Three pairs of loci were linked at distances of 9 cM (Fca75
and Fca96), 8 cM (Fca90 and Fca566 ), and 1 cM (Fca132 and
Fca369). The dye-labeled PCR products of the microsatellite
primer sets were pooled and diluted together based on size
range and fluorescent dye so that three to six loci could be
multiplexed and electrophoresed and subsequently analyzed
in an ABI 377 automated sequencer. Microsatellite allele
sizes were estimated by comparison with a GS350 TAMRA
(ABI) internal size standard. Data were collected and
analyzed using the ABI programs GENESCAN (version
1.2.2-1) and GENOTYPER (version 1.1). PCR product
length was used as a surrogate for actual repeat length
(Ellegren et al. 1995).
Phylogenetic and Population Analyses
Phylogenetic comparisons among lynx species were con-
ducted with sequence variation from five mtDNA gene
fragments (ATPase-8, cytochrome b, 12S rRNA, 16S rRNA,
and NADH-5). Marbled cat and clouded leopard sequences
were included for outgroup comparisons. Sequences from
each of the mtDNA gene fragments were combined into
a contig of 1613 bp after separate analysis of each gene
fragment (Huelsenbeck et al. 1996). Phylogenetic relation-
ships among the haplotypes were estimated using minimum
evolution (ME), maximum likelihood (ML), and maximum
parsimony (MP) methods using PAUP* (Swofford 2001). An
MP analysis was conducted using a heuristic search, with
random addition of taxa and tree-bisection reconnection
branch swapping. The ME approach employed a neighbor-
joining tree (Saitou and Nei 1987) constructed from Kimura
two-parameter distances, with the proportion of invari-
able sites estimated to be 0.3687 from the empirical data and
the rate for variable sites assumed to follow a gamma
distribution. After testing and comparing several models, ML
analysis was done using the HKY85 model (Hasegawa et al.
1985) with parameters estimated from the dataset. The
reliability of the nodes in each of the analyses was assessed by
100 bootstrap iterations (Hillis and Bull 1993).
Mitochondrial DNA sequence variation across Iberian
lynx was assessed in 452 bp from three mitochondrial gene
fragments (ATP-8 and two control region segments).
Measures of mtDNA sequence variation were estimated
using MEGA 2.1 (Kumar et al. 2001). The divergence date
among European lynx, Iberian lynx, and Canadian lynx was
estimated by averaging all pairwise (p) distances among
haplotypes. Feline-specific mtDNA divergence rates of
1.39% (ATPase-8), 0.97% (cytochrome b), 1.22% (NADH-
5), 0.88% (12S rRNA), and 0.97% (16S rRNA), as developed
by Lo´pez et al. (1997), were weighted based on the number
of base pairs used for each gene to obtain a composite
divergence rate of 1.04% per million years.
Estimates of microsatellite size variation, such as average
expected heterozygosity, average variance, number of unique
alleles, and average number of repeats, were derived from the
program MICROSAT (version 1.5) (Minch et al. 1995).
Deviations from Hardy-Weinberg equilibrium, following the
procedure of Guo and Thompson (1992), and estimates of
population subdivision, F
ST
, and R
ST
analogs (Michalakis
and Excoffier 1996; Slatkin 1995; Weir and Cockerham
1984) were derived using ARLEQUIN (Schneider et al.
2000).
Pairwise genetic distances among individuals using the
composite microsatellite genotypes were estimated using the
proportion of shared alleles (Dps) algorithm with a (1 – M)
correction as implemented in the program MICROSAT
(version 1.5) (Minch et al. 1995). A phylogenetic tree was
constructed from the Dps distance matrix using the
Neighbor option of the program PHYLIP (version 3.572)
(Felsenstein 1993) and was drawn using the program
TREEVIEW (version 1.5) (Page 1996).
Results and Discussion
Phylogenetic Analyses
Analysis of the evolutionary relationships among the four
Lynx species from 1613 bp of sequence from five mtDNA
genes (ATPase-8, 16S rRNA, 12S rRNA, NADH-5, and
cytochrome b) confirmed the taxonomic status of the Iberian
lynx as a unique species with a relatively long evolutionary
history (Beltra´n et al. 1996). Among the four Lynx species,
there were 158 variable sites, of which 141 were parsimo-
niously informative (Figure 2). There were two Iberian lynx
haplotypes from four individuals, two haplotypes from three
European lynx, two haplotypes from two Canadian lynx, and
three haplotypes from five bobcats, along with two marbled
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cat haplotypes (Figures 2 and 3). Among the Lynx species,
using the marbled cat and clouded leopard as outgroup
species, there was strong support (96–100% bootstrap
support from MP, ME, and ML phylogenetic analyses) that
the bobcat was the most basal lineage, or the first to diverge
(Figure 3). The relative relationships among the Iberian lynx,
Eurasian lynx, and Canadian lynx were less well defined, with
bootstrap support varying depending upon the method of
analysis. However, each analysis suggested that the Iberian
lynx and Eurasian lynx were sister taxa (58% MP, 50% ME,
and 60% ML bootstrap support) that together shared
a common ancestor with the Canadian lynx. This finding
differs from the findings of Beltra´n et al. (1996) that the
Iberian lynx and the Canadian lynx were sister taxa. The
pairwise genetic distances among these three species were
low, ranging from 53 to 58 bp (of 1639 bp), or 3.2–3.5%.
Figure 2. Variable sites among Iberian lynx (Lyp), Eurasian lynx (Lly), Canadian lynx (Lca), Bobcat (Lru), and Marbled cat
(Pma) for 12S, 16S, Atp8, NADH-5, and CytB mtDNA gene fragments. Sample codes refer to the individual Iberian lynx (Lyp),
European lynx (Lly), Canadian lynx (Lca), bobcat (Lru), and marbled cat (Pma) depicted in the phylogenetic tree of Figure 3.
Position numbers correspond to the complete domestic cat mtDNA sequence (Lo´ pez et al. 1996).
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These results confirm that the Iberian lynx is a unique species
and that the Eurasian lynx, Canadian lynx, and Iberian lynx
all speciated or diverged into monophyletic lineages around
the same time. Assuming an mtDNA divergence rate of
1.04% per million years, based on a feline gene-specific
mtDNA mutation rate (Culver et al. 2000; Johnson and
O’Brien 1997), the rapid divergence among the three lynx
species occurred around 1.53–1.68 million years ago.
Mitochondrial DNA Diversity
Mitochondrial DNA sequence variation across 46 Iberian
lynx from throughout most of their distribution on the
Iberian Peninsula (from seven metapopulations) was
assessed in 452 bp from three mitochondrial gene fragments
(positions 8657 to 8818, as numbered in the complete Felis
catus mtDNA sequence of Lo´ pez et al. [1996] and two
control region segments). Because many of these samples
were hides from museums, not all individuals amplified for
each of the three gene segments. MtDNA diversity among
Iberian lynx was low (Figure 2 and Table 2). Among the 46
Iberian lynx sequenced, there were two variable sites in 452
bp from three mtDNA fragments that defined three
haplotypes (A, B, and C). At position number 8672
(nucleotide numbers from the reference domestic cat
sequence; Lo´pez et al. 1996) in the ATP-8 gene, there was
either a T (haplotypes A and C) or a C (haplotype B). At
position number 16,804 of the control region, two
individuals from the Sado-Algarve metapopulation had a C
(haplotype C), compared with T for the other lynx
(haplotypes A and B). All eight Iberian lynx from the
southernmost metapopulation of the Don
˜ana National Park
area had haplotype B, as did one lynx from eastern Sierra
Morena, two lynx from Sado-Algarve, Portugal, and four
lynx from Sierra Gata Malcata, Portugal. Haplotype A was
found in the easternmost metapopulations of eastern Sierra
Morena and Montes de Toledo. Haplotype C was restricted
to the two samples from the western Sierra Morena
metapopulation (Figure 1).
The Iberian lynx displayed among the lowest levels of
mtDNA diversity that have been documented for a felid
species, as is apparent from a comparison of the sequence
variation across the roughly 880 bp from the mtDNA gene
fragments of NADH-5, 16S, and ATP-8 among several cat
species (Table 2). This overall pattern suggests that Iberian
lynx is descended from a recent founder effect or
a population bottleneck. In addition, the disjunct distribution
of the haplotypes (Figure 1) suggests that recent isolation of
populations and reduced population sizes may have led to
haplotype fixation. All three haplotypes were found between
Table 2. Measures of mtDNA sequence variation in the same combined fragments of NADH-5 (positions 12,647 to 12,946
from the complete Felis catus mtDNA sequence of Lo´ pez et al. 1996), 16S (positions 2904 to 3285), and ATP-8 (positions 8657 to
8818) (a total of about 880 bp) among eight felid species
Species Sample size
Number of
variable sites
Number of
haplotypes p3100 Reference
Puma (Puma concolor) 286 15 13 0.32 Culver et al. (2000)
Andean mountain cat (Oreailurus jacobita) 9 6 9 0.45 Johnson et al. (1998)
Tigrina (Leopardus tigrinus) 32 44 11 0.97 Johnson et al. (1999)
Pampas cat (Lynchailurus colocolo) 22 44 14 1.12 Johnson et al. (1999)
Geoffroy’s cat (Oncifelis geoffroyi) 38 48 32 1.25 Johnson et al. (1999)
Kodkod (Oncifelis guigna) 6 7 3 0.25 Johnson et al. (1999)
Iberian lynx (Lynx pardinus) 20 3 3 0.05 This study
Figure 3. Phylogenetic relationships among Lynx species
and outgroup Felid species from 1639 bp of sequence from six
combined mtDNA gene fragments. Depicted is a maximum
likelihood phlylogenetic tree constructed with the HKY85
model using empirical nucleotide frequencies, a transition/
transversion ratio of 12.6, an assumed proportion of invariable
sites of 0.295, and a shape parameter (a) of 0.206. Above the
branches are bootstrap values (100 iterations) for maximum
parsimony/minimum evolution/maximum likelihood analyses
and below the branches are the number of base substitutions/
number of homoplasies from the maximum parsimony
analyses. Maximum parsimony trees were obtained via
a tree-bisection reconnection algorithm with starting trees
obtained by stepwise addition. Minimum evolution trees were
depicted using the neighbor-joining algorithm using Kimura
two-parameter distances.
25
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the eastern and western Sierra Morena metapopulations, in
south-central Spain, in the middle of the historic distribution
of lynx.
Microsatellite Diversity
The amount of microsatellite allele variation among Iberian
lynx from the two Don
˜ana populations was very similar
(Table 3). Average observed heterozygosity was 26.7% in
Coto del Rey and 29.4% in Vera and the average range in
allele sizes in both populations was 1.57. Estimates of
microsatellite size variation were slightly higher in Sierra
Morena than in Don
˜ana (Table 3), including a larger
percentage of polymorphic microsatellite loci (75% versus
71.4%). Overall, this amount of microsatellite variation is less
than or comparable to that seen in felids such as cheetahs
and North American pumas (Table 4), which experienced
demographic bottlenecks around the time of the Pleistocene
ice ages (Culver et al. 2000).
The populations of Coto del Rey and Vera each had three
unique alleles that were not observed in the other. In
contrast, the metapopulation of Don
˜ana had 19 unique
alleles not observed in Sierra Morena, and Sierra Morena had
26 that were not seen in Don
˜ana (Table 3). These differences
were reflected in the analyses of population structure. F
ST
values among all three populations were significant (P,.05),
but were highest between Sierra Morena and the two Don
˜ana
populations (0.378 with Coto del Rey and 0.227 with Vera;
0.132 between Coto del Rey and Vera). R
ST
values were only
significant between Sierra Morena and Coto de Rey (R
ST
¼
0.576). However, several loci were significantly out of Hardy-
Weinberg equilibrium in the Don
˜ana metapopulation and in
the Vera population when analyzed separately. In each case
there was a deficiency of heterozygotes that may reflect
inbreeding, disproportionate reproductive success of some
individuals, or some degree of allele dropout.
The pattern of differentiation among populations can be
visualized in the dendrograms resulting from the phyloge-
netic analyses of individual composite genotypes (Figure 4).
The six lynx from Sierra Morena are separated from Don
˜ana
individuals with high bootstrap support (93%). In compar-
ison, lynx from the two Don
˜ana populations were
intermixed, although animals from the same population
tended to be most closely linked with another individual
from the same population. During the last 15 years there has
been only one documented instance of a lynx that moved
from Vera to Coto del Rey and another three cases of lynx
moving from Coto del Rey to Vera (Ferreras 2001). Of these,
only the lynx that emigrated to Coto del Rey established
a territory and successfully bred.
Evolutionary Implications
Our estimation, based on mtDNA sequence variation and
a felid-specific divergence rate for these genes, that L.
pardinus diverged as an unique species 1.53–1.68 million years
ago is compatible with paleontological evidence that the
Lynx species inhabiting Europe during the late Pliocene and
early Pleistocene was probably a common ancestor (fre-
quently called L. issiodorensis) of three current Lynx species, L.
lynx, L. pardinus, and L. canadensis (Kurten 1968; Werderlin
1981). The earliest paleontological evidence of L. pardinus has
been found in France from around several hundred thousand
years ago, in the Middle Pleistocene and later (Kurten and
Table 4. Measures of microsatellite size variation at 12 loci among populations of four felid species
Population Sample size Loci typed
Observed
heterozygosity
Mean microsatellite
variance
Number of
alleles/locus Reference
Asian lion 10 12 16.7 0.314 1.67 Driscoll et al. (2002)
Lion, Crater 10 12 37.8 3.223 2.92 Driscoll et al. (2002)
Lion, Serengeti 10 12 48.2 3.732 3.50 Driscoll et al. (2002)
Cheetah, East Africa 10 11 53.0 5.067 4.09 Driscoll et al. (2002)
Cheetah, West Africa 10 11 51.0 3.935 4.00 Driscoll et al. (2002)
Puma, Big Cypress Swamp 10 11 26.0 3.908 2.09 Driscoll et al. (2002)
Puma, Idaho 10 11 61.7 11.651 3.91 Driscoll et al. (2002)
Puma, South America 10 11 81.3 31.667 7.82 Driscoll et al. (2002)
Iberian lynx, Don
˜ana 10 12 25.4 0.484 1.75 This study
Iberian lynx, Sierra Morena 5 11 38.0 1.540 2.27 This study
Table 3. Measures of variation in 28 microsatellites in three Iberian lynx populations
Sample size
Percent
polymorphic
Percent observed
heterozygosity
Microsatellite
variance
Average allele
size range
Mean number
alleles per locus Unique alleles
Sierra Morena 5 75.0 36.4 1.655 2.57 2.30 26
Don
˜ana 15 71.4 29.4 0.825 1.68 1.88 19a
Coto del Rey 7 71.4 26.7 0.773 1.57 1.86 1b,3c
Vera 8 67.9 29.4 0.825 1.57 1.89 1b,3c
Total 20 85.7 32.9 1.240 2.12 2.05
a
Relative to Sierra Morena.
b
Relative to Sierra Morena and Vera population (or Coto del Rey).
c
Between Coto del Rey and Vera.
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Journal of Heredity 2004:95(1)
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Granqvist 1987; Werderlin 1981). These lynx, often referred
to as cave lynx (L. p. speleus), were larger than current
L. pardinus.
Ecological and biogeographical data suggest that the
Iberian lynx was restricted to a glacial refugium in southern
Iberia during one or more of the frequent ice periods, as
occurred with numerous other species (Bennet et al. 1991;
Hewitt 1996), such as the grasshopper Chorthippus parallelus
(Cooper et al. 1995) and some shrews of the Sorex araneus
group (Taberlet et al. 1994). The relatively low levels of
mtDNA sequence and microsatellite size variation relative to
other cat species in evidence today may have resulted from at
least one demographic bottleneck during this time period.
Conservation Implications
There currently are no recognized subspecies of Iberian lynx.
Most of the conservation programs that have been envisaged
for the Iberian lynx have been based on metapopulations.
These molecular genetic results suggest that there is modest
genetic differentiation among microsatellites between Do-
n
˜ana versus Sierra Morena metapopulations. However, the
differences are slight and inapparent with mtDNA. These
results suggest that the overriding genetic concern of Iberian
lynx populations may be their small effective population
sizes at risk for extinction and further genetic reduction. If
current trends continue, it is very likely that much more
active management of some populations will be necessary in
order to maintain sufficient population sizes and existing
levels of genetic variation (Johnson et al. 2001).
We recommend that any future movement of animals in
the wild or the establishment of captive programs be
accompanied by more thorough analysis of the population
sizes and levels of microsatellite variation in the other lynx
metapopulations in addition to those of Don
˜ana and Sierra
Morena. This will necessitate an increased emphasis on
coordinated conservation action plans, both in situ and ex
situ, among the numerous jurisdictions encompassing lynx
distributions in Portugal and Spain (Vargas 2000).
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Received February 24, 2003
Accepted August 28, 2003
Corresponding Editor: Rob DeSalle
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