Molecular Phylogenetics and Evolution 102 (2016) 265–277

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev

Review

Phylogeography of endemic Xantus’ hummingbird (Hylocharis xantusii)

shows a different history of vicariance in the Baja California Peninsula
Cristina González-Rubio a, Francisco J. García-De León a,⇑, Ricardo Rodríguez-Estrella b
a
Laboratorio de Genética para la Conservación, Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Av. IPN #195, La Paz, BCS 23096, Mexico
b
Laboratorio de Análisis Espacial, Ecología y Conservación, Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Av. IPN #195, La Paz, BCS 23096, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Studies of phylogeographic patterns provide insight into the processes driving lineage divergence in a
Received 26 August 2015 particular region. To identify the processes that caused phylogeographic breaks, it is necessary to use his-
Revised 24 May 2016 torical information and a set of appropriate molecular data to explain current patterns. To understand the
Accepted 30 May 2016
influence of geological or ecological processes on the phylogeography of the only species of hummingbird
Available online 31 May 2016
endemic to the Baja California Peninsula, Hylocharis xantusii, mitochondrial DNA sequences of three con-
catenated genes (Cyt-b, COI and ND2; 2297 bp in total) in 100 individuals were analyzed. The spatial
Keywords:
analyses of genetic variation showed phylogeographic structure consisting of a north, central and south
Hylocharis xantusii
Baja California Peninsula
regions. According to estimated divergence times, two vicariant events are supported, permanent sepa-
Phylogeographic breaks ration of the peninsula and formation of the Gulf of California at 5 mya and temporary isolation of the
Divergence times southern region at the Isthmus of La Paz at 3 mya. The temporal frame of genetic differentiation of
intraspecific haplotypes indicates that 90% of haplotypes diverged within the last 500,000 years, with a
population expansion 80,000 years ago. Only four haplotypes diverged 2.2 my and occurred in the south
(Hxan_36, 38 and 45), and north (Hxan_45 and 56) regions; only haplotype 45 is shared between south
and north populations. These regions also have the most recent haplotypes from 12,500 to 16,200 years
ago, and together with high levels of genetic diversity, we suggest two refuge areas, the Northern and
Southern regions. Our results indicate that the phylogeographic pattern first results from vicariance pro-
cesses, then is followed by historical and recent climate fluctuations that influenced conditions on the
peninsula, and it is also related to oases distribution. This study presents the first investigation of phylo-
geography of the peninsular’ endemic Xantus’ hummingbird.
Ó 2016 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
2. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
2.1. Area of study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
2.2. Sampling and laboratory procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
2.3. Phylogeographic patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
2.3.1. Estimating genetic diversity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
2.3.2. Phylogenetic analyses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
2.3.3. Analysis of molecular variance (AMOVA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
2.3.4. Spatial analysis of molecular variance (SAMOVA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
2.3.5. Isolation by distance’s test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
2.4. Historical events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
2.4.1. Estimating divergence times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
2.4.2. Historical demography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
2.4.3. Population dynamics through time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
2.5. Identifying refuge areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

⇑ Corresponding author.
E-mail address: fgarciadl@cibnor.mx (F.J. García-De León).

http://dx.doi.org/10.1016/j.ympev.2016.05.039
1055-7903/Ó 2016 Elsevier Inc. All rights reserved.
266 C. González-Rubio et al. / Molecular Phylogenetics and Evolution 102 (2016) 265–277

3. Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
3.1. Phylogeographic pattern for the Xantus’ hummingbird . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
3.1.1. Genetic diversity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
3.1.2. Phylogenetic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
3.1.3. Spatial analysis of genetic variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
3.2. Historical events: vicariance versus Pleistocene climate changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
3.2.1. Temporal framework of genetic differentiation of the Xantus’ hummingbird . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
3.2.2. Demographic history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
3.3. Identifying refuge areas of the Xantus’ hummingbird . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
4.1. Phylogenetic analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
4.1.1. Using mtDNA as a single locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
4.2. Phylogeography of the Xantus’ hummingbird . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
4.2.1. Vicariant events versus Pleistocene climatic changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
4.2.2. Historical demography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
4.3. Defining refuge areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
4.4. Conservation implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
5. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Appendix A. Supplementary material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

1. Introduction endemic species (Grismer, 2000; Riddle et al., 2000b; Rojas-Soto

Phylogeography has proved successful in explaining how distri-
butions of natural populations have been influenced by historical
events, such as geological changes and climate fluctuations. For
example, some studies of mitochondrial DNA sequences of North
American vertebrates and plants have shown that rapid climate
change during Pleistocene glaciations, together with topographic
and ecological barriers (e.g. vicariance), were fundamental in gen-
erating phylogeographic structures within species and populations
(Hewitt, 2000; Riddle et al., 2000a; Zink et al., 2001; Lemmon et al.,
2007; Scoble and Lowe, 2010; Smith et al., 2011). Three generaliza-
tions are formulated from these works: (1) taxa were isolated into
independent lineages (species, subspecies, or phylogroups), first
because of major geological changes during the Pliocene epoch
5.3–2.6 mya (Orndorff et al., 2007), (2) climatic shifts during the
Pleistocene about 2.6–1.8 mya (Polly et al., 2009), which con-
tributed to the isolation of populations in many areas and have
led to genetic divergence and speciation, and (3) highest genetic
diversity in species or populations in areas that remained stable,
the refuges during glacial times (Hewitt, 1996, 2000).
In the Baja California Peninsula (BCP), studies of phylogeo-
graphic patterns suggest dispersal and vicariance for many taxa
(Riddle et al., 2000a,b; Zink, 2002; Zink et al., 2001, 2000; Crews
and Hedin, 2006; Devitt, 2006; Riddle and Hafner, 2006a; Leaché
and Mulcahy, 2007; Leaché et al., 2009; Munguia-Vega, 2011).
Three major events explain the phylogeographic structure in the
region: (1) Permanent separation of the peninsular and continental
populations following the formation of the Gulf of California
(5 mya; Riddle et al., 2000a,b), (2) Temporary isolation of the pop-
ulation in the southern end of the peninsula (Cape Region) from
other peninsular populations following inundation at the Isthmus
of La Paz (3 mya; Riddle et al., 2000b), and (3) a Mid-peninsular
seaway had temporarily isolated northern populations from south-
ern populations (1 mya; Riddle et al., 2000a; Zink, 2002; Zink et al.,
2001). This seaway break accounts for consistent separation of sev-
eral species (Munguia-Vega, 2011). Additionally to vicariant
events, changes in local environmental conditions (e.g. topography,
altitude) in conjunction with Pleistocene and Holocene climatic
fluctuations (2.6 mya to the present) also influenced genetic struc-
turing in peninsular populations (Leaché and Mulcahy, 2007;
Garrick et al., 2013, 2009).
The peninsula is a geologically dynamic region that includes an
array of landscapes and habitats as well as a large number of
et al., 2003). Semi-arid and arid regions have been most sensitive
to climate change, particularly during interglacial periods, when
low rainfall leads to expansion of arid vegetation, decreases in
quality and quantity of groundwater, soil degradation, salinization,
and reduction in vegetation cover (Gutierrez-Elorza, 1998). Since
the start of the Holocene (11,500 years; Gibbard and Van
Kolfschoten, 2004), several animal populations have been
restricted to their most environmental stable areas (refuges). The
southern part of the peninsula is regarded as a refuge for some
birds, as for example, the California gnatcatcher (Polioptila califor-
nica; Zink et al., 2013, 2001; Zink, 2002), and the California quail
(Callipepla californica; Zink, 2002). In this area, they show high
genetic diversity compared with northern localities of the
peninsula.
Xantus’ hummingbird Hylocharis xantusii (Lawrence, 1860)
belongs to the family Trochilidae, which has the highest degree
of specialization mainly in morphological traits, related to flight
ability, physiology along with feeding adaptations, as well as
behavior and sexual dimorphism, compared to all other families
of birds (Temeles et al., 2010; Berns and Adams, 2013). This
medium-sized hummingbird displays a white post-ocular stripe
contrasting greatly with a broad blackish auricular mask. Strong
sexual dimorphism is present: males have an orange bill and color-
ful plumage (Howell and Howell, 2000). The sister species, the
white-eared hummingbird Hylocharis leucotis (Hernández-Baños
et al., 2014), is found in southern Arizona, Nicaragua, Guatemala
and in Mexico except the peninsula (Howell and Howell, 2000).
It’s distribution does not overlap with H. xantusii’ distribution. This,
suggest that these two species diverged after the formation of the
peninsula (Zamudio-Beltrán, 2011). Little is known about the biol-
ogy, ecology, and distribution patterns of Xantus’ hummingbird,
but it is restricted to the central and southern parts of the penin-
sula with discontinuous distribution; it has two breeding seasons
each year, usually two chicks per nest, and is found from sea level
to about 1800 m (Howell and Howell, 2000).
Three features endorse this hummingbird as a model to under-
stand the influence of geological and ecological dynamics in penin-
sular populations of birds, mammals, and reptiles. First, it is the
only neotropical hummingbird species endemic to the peninsula
(Arriaga et al., 1990); second, this species inhabits the Sierra La
Laguna region (Arriaga et al., 1990), which is the most outstanding
region in terms of the number of endemic taxa, and also the only
area that contains pine-oak forests within the south of the
 C. González-Rubio et al. / Molecular Phylogenetics and Evolution 102 (2016) 265–277
peninsula (Rodríguez-Estrella et al., 2005, 1999). Third, outside of
the Sierra La Laguna, it is mostly restricted to oases, refuges that
contain relict mesic habitats from the Pleistocene (Maya et al.,
1997; Valero et al., 2014). The oases, dispersed throughout the
peninsula, are highly valuable for the unique biotas that they
support, containing rich food sources for many small animals
(Arriaga et al., 1997; Rodríguez-Estrella et al., 2005, 1999,
1997). Understanding the phylogeographic pattern of the Xantus’
hummingbird and its relation to historical processes, is particularly
important in evolutionary and adaptive terms, since it is the only
neotropical endemic species that has adapted to an arid peninsula
compared to other endemic species that have Nearctic origin
which presently only inhabit the pine-oak and tropical deciduous
forests in southern Baja California peninsula and have failed to
extend beyond this habitat (Rodríguez-Estrella, 1987).
In this study, we analyzed Xantus’ hummingbird phylogeo-
graphic structure, specifically the intraspecific evolutionary history
in light of current biogeography and historical climatic conditions.
We analyzed fragments of three mitochondrial DNA concatenated
genes of individuals covering its entire geographic distribution to
test whether: (1) there is a phylogeographic pattern of intraspecific
lineages, (2) phylogeographic patterns are related to specific his-
torical events, either a vicariant event or Pleistocene climate
changes, and (3) there are refuge areas representing Pleistocene
climatic conditions.
2. Materials and methods
2.1. Area of study
The Baja California Peninsula contains a wide array of habitats.
Vegetation types include xerophytic scrub (including the arroyos’
vegetation), pine-oak forests (only in the Sierra La Laguna, which
is above 1400 m), tropical deciduous forest (only in the Sierra La
Laguna, 300–800 m), coastal vegetation, mesic vegetation of oases
(including palms and reed grass), and similar vegetation of adja-
cent off-coast islands in the Pacific and the Gulf of California. Mean
annual precipitation ranges from 100 mm in the Vizcaino Desert to
more than 765 mm in the Sierra La Laguna (Rodríguez-Estrella
et al., 2005). An important feature of the arid and semi-arid regions
of the peninsula is the highly seasonal nature of rainfall, summer
tropical storms bring considerable flash floods, mostly in two-
thirds southern portion of the peninsula. In more arid areas, tem-
peratures can rise to 50 °C, while they do not exceed 30 °C in
the humid Sierra La Laguna and other oases. Oases are separated
by at least 10 km, with some that are more than 50 km from the
nearest neighbor, their size varies from 0.05 to 2.7 km2, and only
48% of them have surface water. Oases contain the highest bird
species richness, diversity and abundances from all the peninsula
habitats (Arriaga and Rodríguez-Estrella, 1997; Rodríguez-Estrella
et al., 1997). H. xantusii, is a species related to oases regions, mainly
those with surface water and vegetation of arroyos. It is found
more abundant in the oases of greater size, particularly towards
the southern region of the peninsula. In this region, the species is
very common, especially after rains in the pine-oak forests of Sierra
La Laguna (Rodríguez-Estrella, 1997, 1987; Arriaga et al., 1990). It
is found less abundant in the northern part of the species’ distribu-
tion, where oases are more distant from each other (Rodríguez-
Estrella, 1997).
2.2. Sampling and laboratory procedures
Six related hummingbirds’ species were used in this work:
Hylocharis xantusii, H. leucotis, Cynanthus latirostris, Amazilia versi-
color, Calypte costae, and Archilochus colubris (McGuire et al.,
267
2014, 2007; Hernández-Baños et al., 2014). Species selection was
based on the following criteria: H. leucotis and H. xantusii are sister
species; C. latirostris occurs with H. leucotis; Calypte costae occurs
with H. xantusii; Amazilia versicolor is related to Hylocharis species
and C. latirostris; and A. colubris is related to C. costae. The genomes
of the last two species were used as reference to delineate testing
sequences. We downloaded complete genomes from the GenBank
database (A. versicolor accession number KF624601, and A. colubris
no. EF532935).
Samples were obtained from hummingbirds in the field (except
A. versicolor and A. colubris). We collected samples of Xantus’ hum-
mingbird along its entire geographic distribution; the locations and
sample numbers per site are shown in Fig. 1 and Table 1 respec-
tively. We have included two mainland locations for H. leucotis
and C. latirostris (Fig. 1). Samples of C. costae were obtained within
the peninsula’s locations. Birds were captured with mist nets, then
blood was collected from the tarsus, and birds were released. No
injuries were noted during blood sampling; standard procedures
were used for managing the hummingbirds.
Genomic DNA from the blood was obtained using the rapid salt
extraction protocol (Aljanabi and Martínez, 1997). We analyzed
three fragments of mtDNA in the six related hummingbirds’ spe-
cies: (1) nicotinamide adenine dinucleotide dehydrogenase sub-
unit 2 (ND2), which were amplified with H6313 and L5219
primers (Sorenson et al., 1999); (2) cytochrome c oxidase subunit
1 (COI) amplified with primers H8191 and L7165 (De Filippis and
Moore, 2000); and (3) cytochrome b (Cyt-b) amplified with primers
H16065 and L14841 (Kocher et al., 1989). For the hummingbird
field samples, all sequences were deposited in GenBank (http://
www.ncbi.nlm.nih.gov/).
All PCR reactions were performed in 14 lL total volume, con-
taining about 40 ng of DNA, 1 PCR buffer (20 mM Tris-HCl,
50 mM KCl at pH 8.4), 0.2 mM of each dNTP, 0.4 lM of each primer,
2.0 lM MgCl2, and 0.5 unit Taq DNA polymerase (Invitrogen, Carls-
bad, CA). The temperature profiles included initial denaturation at
94 °C for 5 min, 30 cycles at 94 °C for 1 min each, 30 s at 55 °C
annealing temperature, and a final extension at 72 °C for 5 min.
Sequences were reported by DNA sequencing services (Genewiz,
Plainfield, NJ; http://www.genewiz.com/; Macrogen (Seoul, South
Korea; http://www.macrogen.com/eng/). Sequences were edited
with ChromasPro 1.7.6 and aligned with MUSCLE in MEGA 6
(Tamura et al., 2013), and revised manually, using UltraEdit Profes-
sional 14.20.1.
2.3. Phylogeographic patterns
In order to determine if there is a phylogeographic structure for
the Xantus’ hummingbird, we characterized the genetic diversity
by conducting three approaches: a phylogenetic analysis, molecu-
lar variance and spatial distribution of molecular variance analysis,
and Isolation by distance’ test.
2.3.1. Estimating genetic diversity
The characterization of genetic diversity at the intraspecific
level was estimated from nucleotide and haplotype diversity, the
number of segregating sites, and the number of haplotypes. These
analyses were performed for each group (see below), using sepa-
rated mtDNA genes (Cyt-b, COI, ND2), and concatenated sequences,
all this was performed using DnaSP 5 software (Librado and Rozas,
2009).
2.3.2. Phylogenetic analyses
First, we used maximum likelihood in GARLI 2.0 (Zwickl, 2011)
to recover phylogenetic relationships as well as to obtain a non-
parametric bootstrap support value for the nodes. Individual solu-
tions of trees were selected after 10,000,000 generations and when
268 C. González-Rubio et al. / Molecular Phylogenetics and Evolution 102 (2016) 265–277

Fig. 1. Map of the Baja California Peninsula showing Xantus’ hummingbird (Hylocharis xantusii) localities sampled in the present study (labeled 1–15). The number of
individuals per locality is shown in Table 1. Three major vicariant events are showed in dashed lines (Gulf of California, Isthmus of La Paz, and Mid-peninsular seaway; Riddle
et al., 2000a,b; Zink et al., 2001; Zink, 2002). Mainland localities (16 and 17) belongs to samples of Hylocharis leucotis and Cynanthus latirostris used in this work.

the total improvement in likelihood score was 0.05. We also per-

formed 1000 bootstrap replicates, each included two independent
Table 1 runs. Second, phylogenetic reconstruction and posterior probabil-
Sampling sites used in this work. Species sample sizes are indicated for the endemic
Xantus’ hummingbird (Hylocharis xantusii), and for mainland species (H. leucotis and
ity estimates from MrBayes 3.2 were obtained using Bayesian
Cynanthus latirostris). Numbers of sampling sites correspond to the numbers in Fig. 1. inference (Ronquist et al., 2012). We used two parallel runs, one
‘cold’ and three ‘hot’ chains for 20,000,000 generations, sampling
Sampling sites Species sample size
every 10,000 generations until a standard deviation value less than
Hylocharis Hylocharis Cynanthus 0.01 of split frequencies was reached. Data was partitioned by the
xantusii leucotis latirostris
mtDNA gene, then, using the best-fit model for each one, it was
Peninsula analyzed in jModelTest 2 considering the corrected Akaike infor-
1 Santa Gertrudis 9
mation criteria (Darriba et al., 2012). We used a 25% burn-in, and
2 San Ignacio 5
3 San Zacarías 5 it was calculated a majority rule consensus tree. Each phylogenetic
4 San José de Magdalena 10 analysis included concatenated sequences (Cyt-b, COI, ND2) of five
5 San Nicolás 6 species as outgroup: Hylocharis leucotis (seven haplotypes, this
6 Carambuche 10 study), Cynanthus latirostris (one haplotype, this study), Amazilia
7 San Isidro 3
8 San José 4
versicolor (one haplotype, accession number KF624601), Calypte
9 San Miguel 8 costae (five haplotypes, this study), and A. colubris (one haplotype,
10 San Javier 4 accession number EF532935).
11 La Soledad 9
12 San Blas 3
13 San Dionisio 3 2.3.3. Analysis of molecular variance (AMOVA)
14 Sierra de la Laguna 11 There was no geographical criteria for delineating populations,
15 Santiago 9
so we performed AMOVA’s using Arlequin 3.5 (Excoffier and
Mainland Lischer, 2010), following these hypotheses (1) we considered two
16 Álamos, Sonora 6 4
17 El Fuerte, Sinaloa 4 3
groups, assuming that the vicariant Isthmus of La Paz separated
the south from other regions; (2) three groups, assuming the
 C. González-Rubio et al. / Molecular Phylogenetics and Evolution 102 (2016) 265–277
Isthmus of La Paz, and that the vicariant Mid-peninsular seaway
separated the north from central region; and (3) we considered
11 groups, assuming that each oasis of the eleven oases supports
one phylogeographic lineage. These oases are: Santa Gertrudis,
San Ignacio (including San Ignacio and San Zacarías), San José de
Magdalena, San Nicolás, La Purísima (including Carambuche and
San Isidro), Los Comondús (including San José and San Miguel),
San Javier, La Soledad, San Blas, Sierra La Laguna, and Santiago
(Fig. 1).
2.3.4. Spatial analysis of molecular variance (SAMOVA)
We used SAMOVA 1.0 (Dupanloup et al., 2002) and K2P dis-
tances to detect major genetic discontinuities among sampling
sites, explicitly to test phylogeographic structure in H. xantusii. This
method maximizes the proportion of genetic variance; based on
the differences found within the number of groups defined by
the user (K), assigning localities to populations, and with the
assumption that they must be geographically adjacent and genet-
ically homogeneous (Dupanloup et al., 2002).
In order to find the optimal K value in which FCT (i.e., genetic
variance owing to divergences between groups) was the highest
and most significant, we tested different K values. We established
the hypotheses for AMOVA: K = 2, vicariant event of Isthmus of La
Paz; K = 3, vicariant event of Mid-peninsular seaway, three geo-
graphic regions; and K = 11, oases sampled for H. xantusii. To verify
consistency, we performed the analysis five times for each K value,
with statistical significance of 1000 permutations.
2.3.5. Isolation by distance’s test
We applied the model of Isolation by distance (Wright, 1943) in
order to understand whether it exist a correlation between geo-
graphical and genetic distances between different pairs of Xantus’
hummingbird populations. We carried out the analysis using the
Isolation by Distance web service (http://ibdws.sdsu.edu/; Jensen
et al., 2005), with the same grouping hypotheses described in
AMOVA section.
2.4. Historical events
Phylogeographic patterns related to historical events, either a
geological event or Pleistocene climate change, were tested by per-
forming divergence time estimates which allowed establishing a
time frame for genetic differentiation and the historical demogra-
phy of Xantus’ hummingbird.
2.4.1. Estimating divergence times
We used BEAST 1.8 (Drummond et al., 2012) to estimate the
time of divergence of the Xantus’ hummingbird and phylogenetic
analysis, with three calibration points obtained from fossils and
biogeographical events: (a) the external calibration point was 12
my, which is an estimated divergence time using fossil evidence,
that separates the Central and North American regions from the
South American region (Bleiweiss, 1998), (b) 10 my, which approx-
imately represents the formation of two major volcanic arcs, the
Sierra Madre Occidental and the Trans-Mexican volcanic belt
(Ferrari et al., 2000), events that separate the northern Mesoamer-
ican hummingbird species (bees) from the north-central and
northern American hummingbird species (emeralds), and (c) 5
my, approximate age of separation of the peninsula from mainland
Mexico (Riddle et al., 2000a,b), separating the peninsular species
(Hylocharis xantusii) from the emerald group. The intraspecific sub-
stitution rate of H. xantusii was calibrated according to rates previ-
ously reported for those genes in birds; we used 2.07  10 8
substitutions/site/year for Cyt-b; 1.6  10 8 substitutions/site/year
for COI, and 2.9  10 8 substitutions/site/year for ND2, (Weir and
Schluter, 2008; Qu and Lei, 2009; Sly et al., 2011; Arbeláez-
269
Cortés et al., 2014; Licona-Vera and Ornelas, 2014). We ran the
analyses for 1  108 generations, sampling every 10,000 iterations,
using as prior the Yule speciation tree, a UPGMA starting tree, and
a log-normal relaxed uncorrelated molecular clock (Drummond
and Rambaut, 2007). After the analysis, we used TreeAnnotator
1.7.4 (Rambaut and Drummond, 2012) to generate a tree file com-
piling the data, with a 25% burn-in and a posterior probability limit
of 0.5. We used Tracer 1.6 (Rambaut and Drummond, 2009) to
assess chain convergence and determine that our sample of the
posterior distribution of the likelihoods had reached a sufficiently
effective sample size (ESS), and therefore, stationarity.
2.4.2. Historical demography
Historical demography of the Xantus’ hummingbird was
assessed by separating haplotypes into the most significant num-
ber of populations resulting from SAMOVA analyses. Each popula-
tion was analyzed independently. We sought evidence for sudden
population growth in each population, for this we constructed mis-
match distributions of pairwise nucleotide differences between
haplotypes, using Arlequin 3.5 (Excoffier and Lischer, 2010). Mis-
match distributions are usually ragged in populations at demo-
graphic equilibrium, but are unimodal in populations that have
experienced recent expansion (Rogers and Harpending, 1992).
We estimated the raggedness index (Harpending, 1994), which in
the presence of relatively stable populations shows large values
and exhibits a multimodal distribution. Parameters used in the
spatial expansion model were estimated from the data, assuming
h = 1, population size after expansion, as infinite. Goodness of fit
in spatial expansion was assessed by calculating the significance
of the raggedness index through 1000 simulations that estimated
the probability of obtaining an index less than the observed value
(Harpending, 1994).
Additional evidence to support population expansion was
obtained by applying the neutrality test by the D statistic
(Tajima, 1989). In this test, the significant positive numbers belong
to a population that has recently undergone a bottleneck, whereas
significant negative numbers belong to a population that has
recently begun an expansion. An insignificant number is consistent
with a population at drift-mutation equilibrium. Population expan-
sion was also assessed with Fs (Fu, 1997). A negative number of Fs
evidence an excess number of genetic variants, as it would be
expected from recent population expansion or from genetic hitch-
hiking, while a positive number of Fs evidence a deficiency in
genetic variants, as it would be expected from a recent population
bottleneck or in the presence of population structure (Fu, 1997). All
analyses were performed with Arlequin 3.5.
We analyzed the relationship among sampled haplotypes, in
the program Network 4.6.1.2 performing a network haplotype
reconstruction through the median-joining algorithm (Bandelt
et al., 1999; http://www.fluxus-engineering.com/).
2.4.3. Population dynamics through time
Past population dynamics for the Xantus’ hummingbird were
examined in a coalescence-based framework using Extended Baye-
sian Skyline Plot approach in BEAST (EBSP; Heled and Drummond,
2008). The skyline plot is a piecewise-constant model of popula-
tion size that fits a range of demographic scenarios. It has proved
very useful as a model selection tool used to indicate the most
appropriate demographic model for any given data set. The EBSP
model uses standard Markov chain Monte Carlo (MCMC) sampling
procedures. This allows estimating a posterior distribution of effec-
tive population size through time directly from a sample of gene
sequences, given any specified nucleotide-substitution model. This
method includes credibility intervals for the estimated effective
population size at every point in time, back to the most recent
common ancestor of the gene sequences (Drummond et al.,
270 C. González-Rubio et al. / Molecular Phylogenetics and Evolution 102 (2016) 265–277

2005). Analyses were run using a strict molecular clock, and the
HKY + G + I substitution model. The analysis was performed using
separated mtDNA genes, and concatenated sequences (three times
using the three mutation rates (1.6%, 2.1%, and 2.9% per site per
my) for 50 million iterations. Convergence, stationarity, effective
sample size for each parameter, and appropriate burn-in, were
evaluated in the analysis using Tracer.
2.5. Identifying refuge areas
Refuge areas are expected to be restricted geographical areas
during Pleistocene climatic fluctuations, where populations main-
tained their genetic diversity; if individuals are restricted to
refuges for a long time it would be expected a genetic structure
of isolated populations (e.g. Reding et al., 2012; Zink et al., 2013).
When conditions are restored, a process of dispersion could pro-
duce a gradient of genetic diversity or isolation by distance pattern
with or without genetic flow (e.g. Hull and Girman, 2004; González
et al., 2011). We analyzed the existence of possible areas based on
high levels of genetic diversity, and presence of ancestral and
recent haplotypes as evidences of long-term refuge areas related
to climatic fluctuations.
3. Results
3.1. Phylogeographic pattern for the Xantus’ hummingbird
3.1.1. Genetic diversity
We sequenced 100 hummingbirds for Cyt-b (811 bp), COI
(804 bp), ND2 (682 bp), and concatenated mtDNA (2297 bp). The
lowest diversity value were obtained for Cyt-b gene, observing
11 haplotypes, in which haplotype diversity = 0.6851, nucleotide
diversity = 0.0012, and mean number of pairwise differences =
0.924. The greatest number of haplotypes and haplotype diversity
were in COI gene (27 and 0.8811, respectively). In nucleotide
diversity and mean number of pairwise differences, the highest
numbers occurred in ND2 gene (0.0097 and 4.107, respectively).
In concatenated sequences, we identified 56 unique haplotypes
at 39 polymorphic sites; haplotype diversity was 0.9682, nucleo-
tide diversity was 0.0048, and mean number of pairwise differ-
ences was 6.529. Genetic diversity for each region determined by
spatial analyses (see below) were similar, with the exception of
north population, with the lowest haplotype diversity (0.908)
and mean number of pairwise differences (6.12) (Table 2).
3.1.2. Phylogenetic analysis
The best-fit DNA evolution model for all mtDNA genes was the
Tamura-Nei model, which allow variable base frequencies, equal
transversion rates, and variable transition rates (Tamura and Nei,
1993), with some invariable and variable sites, following a discrete
gamma distribution with four categories (TrN + I + G).
Based on the analysis of 56 Xantus’ haplotypes defined by con-
catenated sequences, both maximum likelihood and Bayesian phy-
logenetic reconstructions showed the same topology (Appendix A),
and had high statistical support; a main clade included Hylocharis
xantusii, H. leucotis, Cynanthus latirostris, and Amazilia versicolor
(emerald clade). Within the emerald clade, all Xantus’ humming-
bird haplotypes form a well supported (100%) clade by both phylo-
genetic methods. However, a clear phylogeographic pattern was
not observed, as nine (Hxan_01, 04, 05, 07, 45, 09, 16, 17, 03) of
the 56 haplotypes identified in Xantus’ hummingbird are shared
among distant oases. All Xantus’ haplotypes assigned to each indi-
vidual are listed in Appendix B.
Haplotype 01 was found at San Blas and San Javier (340 km
apart) oases; haplotypes 04 and 05 were found at Santiago and
San Ignacio (700 km); haplotype 07 was found at Santa Gertrudis
and La Soledad (600 km); haplotype 45 at Santa Gertrudis and
Sierra La Laguna (800 km); haplotype 09 at Carambuche and San
Dionisio (450 km); haplotype 16 at San Dionisio and San Nicolás
(475 km), and is one of the ancestral haplotypes; haplotype 17 at
San Ignacio and San Isidro (220 km); and haplotype 03 at Santiago
and San Isidro (485 km).
3.1.3. Spatial analysis of genetic variation
AMOVA analyzes showed no significant differences for defining
genetic structure. However, when we incorporate geographic
information to SAMOVA and isolation by distance analyzes, it
was possible to determine a genetic structure related to south, cen-
tral and north regions, as well as relative to the 11 oases distribu-
tion. Variations among groups revealed significant differences
(Table 3), with the highest percentage by K = 3 (39.5%,
FCT = 0.3956, p < 0.05); south region included sites 11–15; central
regions included sites 6–10, while north region included sites
1–5 (Fig. 1). Isolation by distance analysis (Fig. 2), only showed a
significant correlation between geographical and genetic distances
between different oases (K = 11; r = 0.1769, p = 0.002).
Table 3
SAMOVA results and percentage of variation depending of K defined groups.
Source of variation K=3 K = 11
% variation % variation
(fixation index) (fixation index)
Among groups 39.57 (0.3956**) 31.73 (0.3172***)
Among populations 5.98 ( 0.0989 ns) 27.51 ( 0.4029 ns)
within groups
Within populations 66.41 (0.3358 ns) 95.78 (0.0422 ns)
ns = non significant.
**
p < 0.05.
***
p < 0.001.

Table 2
Summary of genetic diversity and demographic statistics of the Xantus’ hummingbird endemic of the BCP, based on mtDNA haplotypes (Cyt-b, COI, ND2, concatenated
sequences).

Groups n S H h p Mean no. pairwise differences Raggedness index D’Tajima (ns) Fu’s Fs
Cyt-b (811 bp) 100 8 11 0.6851 0.0012 0.924 0.1249 ns 1.0876 6.197*
COI (804 bp) 100 15 27 0.8811 0.0033 2.442 0.0242 0.4396 17.786**
ND2 (682 bp) 100 17 19 0.7846 0.0097 4.107 0.0439 ns 0.709 2.665*
Concatenated (2297 pb) 100 39 56 0.9682 0.0048 6.529 0.0084 ns 0.2698 45.004**
South 35 29 27 0.9802 0.0063 8.498 0.0109 ns 0.3094 7.417**
Central 29 35 21 0.9872 0.0093 12.542 0.0421 ns 0.7884 3.050*
North 35 24 23 0.9077 0.0045 6.123 0.0366 ns 0.0964 5.298*

ns = non significant.
n = sample size; S = segregating sites; H = number of haplotypes; h = haplotype diversity; p = nucleotide diversity.
South, north and central = regions identified by SAMOVA results and confirmed by Isolation by distance analyses.
*
p < 0.05.
**
p < 0.001.
 C. González-Rubio et al. / Molecular Phylogenetics and Evolution 102 (2016) 265–277 271

3.2. Historical events: vicariance versus Pleistocene climate changes

3.2.1. Temporal framework of genetic differentiation of the Xantus’

Fig. 2. Isolation by distance analysis showing a significant correlation between
geographical and genetic distances between different oases (r = 0.4769, p = 0.002).
hummingbird
BEAST results suggest that H. xantusii diverged from its sister
species (H. leucotis) during the Pliocene, 3.6 mya (Appendices C
and D), after the formation and separation of the peninsula from
the mainland (about 5 mya). Sequences belonging to Calypte costae,
a co-distribute species with H. xantusii, suggest divergence at 3.5
my after the separation of the peninsula.
For genetic differentiation of Xantus’ hummingbird haplotypes,
the BEAST analysis suggests separation among groups of haplo-
types during the late Pliocene and early Pleistocene, 3–2 mya
(Fig. 3); however, for almost 90% of the haplotypes, divergence
started within the last 500,000 years, and approximately 40% of
the haplotypes are less than 100,000 years old.
Three main Xantus’ haplotype groups coincide with the three
geographic regions, with few shared haplotypes between
populations.
In the southern region, the time to the most recent common
ancestor (tMRCA) is 2.14 my, which is the age of one of the ances-
tral haplotypes occurring in the Sierra La Laguna area. The more
recent haplotypes are about 16,200 years old and were found at

Fig. 3. Divergence time estimates in years (HPD and 95% confidence interval) for the Xantus’ hummingbird, based on mtDNA concatenated sequences (Cyt-b, COI, ND2), using
log-normal relaxed uncorrelated molecular clock and UPGMA starting tree implementing in BEAST (Drummond et al., 2012). Calibration points were: (a) ancestral divergence
time estimation using fossil calibration (12 mya; Bleiweiss, 1998), (b) formation of volcanic arcs (10 mya; Ferrari et al., 2000), and (c) complete separation of the BCP (5 mya;
Riddle et al., 2000a,b). The gray dotted line indicates the divergence time for most of the haplotypes (500,000 years ago). Only ancestral and recent haplotype are show as
terminal tips including their divergence time and location in parenthesis.
272 C. González-Rubio et al. / Molecular Phylogenetics and Evolution 102 (2016) 265–277

Santiago, and also in central region (San José and San Javier sites).
Haplotypes of La Soledad (site above the Isthmus of La Paz) began
to diverge 460,000 years ago. The southern region shows evidence
that it began to diverge after the formation of the water channel
formed in the Isthmus of La Paz (3 mya), this indicates that this
vicariant event served as a barrier to isolated the Cape region.
In the Central region, the tMRCA is 922,000 years old. The oldest
haplotype in this region is 780,000 years, and it is found in Caram-
buche, but it is also found in north region (San José de Magada-
lena). The second oldest has 480,000 year and it is found at San
José.
The Northern region (particularly San Nicolás oasis) contains
the most ancestral haplotype, 2.5 my, which also was found at
the Sierra La Laguna (southern region); the second ancestral haplo-
types have 2.06 my and they were found at San Nicolás and Sierra
La Laguna. For the other haplotypes in this region, the tMRCA is 1.6
my; and the oldest haplotype is 879,500 years, it was found at
Santa Gertrudis; this region also includes the most recent haplo-
types, 12,500 years old, which also were found in the southern
region.
3.2.2. Demographic history
Mismatch distribution of pairwise differences among
haplotype-concatenated sequences for each region contain marked
bimodal curves (Fig. 4), contrasting with expected unimodal pat-
tern in populations that experienced recent demographic expan-
sion. Demographic parameters, such a Harpending’s Raggedness
index (Table 2) indicated that the observed mismatch distribution
did not differ significantly from the distribution expected under
population expansion. On the other hand, neutrality test showed
differing results: Tajima’s D statistic did not show significant devi-
ations of strict neutrality in all regions (Table 2), suggesting demo-
graphic stability; however, Fu’s FS statistic showed negative and
significant differences, suggesting an excess of low frequency hap-
lotypes, which is expected if H. xantusii has experienced recent
demographic expansion.
The results of Median-joining network for each individual gene
(Appendix E) show a complex pattern on the relationships between
haplotypes. A network of two haplotypes with high frequency
interconnects with other lower frequency, each forming a star con-
figuration. This strongly suggests that, in each network, the two

Fig. 4. Mismatch distribution (left), and Extended Bayesian skyline plots (right; dashed lines indicate 95% HPD intervals) are displayed for each region for the Xantus’
hummingbird, using mtDNA concatenated genes (Cyt-b, COI, ND2). In skyline plots the south and north region underwent and expansion beginning 80,000 years ago,
whereas central region expanded to a lesser degree 1,000,000 years ago.
 C. González-Rubio et al. / Molecular Phylogenetics and Evolution 102 (2016) 265–277
groups of haplotypes evolved independently for different time
periods. This haplotype spatial pattern is also consistent with the
bimodal mismatch distribution (Fig. 4). This pattern remains, but
with more complexity for sequences concatenated (Appendix E).
The EBSP analyses, based on mtDNA concatenated genes (Fig. 4)
were similar to each other when using different mutation rates,
suggesting that the south and north regions underwent an
expansion process that began 80,000 years ago. Central region
expanded to a lesser degree, 1,000,000 years ago. Because the tim-
ing for the expansion are dependent on the calibration data used,
we do not interpret dates precisely, but only found that there was
at least one expansion process in the central region, and potentially,
a more recent one in the southern and northern regions, where we
found that there is a greater number of unique haplotypes.
3.3. Identifying refuge areas of the Xantus’ hummingbird
Based on genetic diversity (high haplotype diversity; Table 2
and Appendix F) and presence of ancestral haplotypes (Fig. 3),
there are two main refuge areas suggested, the northern and
southern regions, which are also regions of diversification within
the species, based on the presence of recent haplotypes (Fig. 3).
4. Discussion
The deserts in the Baja California peninsula act as areas of ende-
mism, resulting in some clear patterns of diversification that are
mainly explained by vicariant events (Riddle et al., 2000a, 2000b;
Zink, 2002). Although regional differentiation of birds of the penin-
sula has been explained by vicariance, no clear phylogeographic
divisions had been observed as expected for highly vagile birds
(Zink et al., 2013, 2000), yet it has been described there is high
genetic diversity and ancestral haplotypes in the southern region
(Zink et al., 2013, 2001; Zink, 2002). This work is the first example
of phylogeographic structure in an endemic species of bird, Xantus’
hummingbird, in the Baja California Peninsula. Our work showed a
phylogeographic pattern emerged explained by vicariant events
followed by climatic changes during the Pleistocene.
4.1. Phylogenetic analyses
4.1.1. Using mtDNA as a single locus
Patterns of divergence and relationships inferred from different
types of data (e.g. mtDNA versus nuclear DNA) can disagree (e.g.
Crews and Hedin, 2006), reflecting dissimilarities in modes of
inheritance or relative rates of evolution (Avise, 2000). Mitochon-
drial DNA in animals evolves faster than nuclear DNA, and depend-
ing of the event that has caused divergence and speciation, the
ability to detect this event depends upon rates of evolution of these
types of DNA (Avise, 2000). For example, if vicariance has caused
speciation, mitochondrial genes may be the only partition
expected to reveal divergence due its faster substitution rate
(Avise, 2000). However, mitochondrial history may not reflect the
true history of species because mtDNA gene variation may be
caused by introgression (Funk and Omland, 2003). Mitochondrial
genes are less constrained by linkage to selected loci and hence
are expected to introgress deeper than their nuclear counterparts
(Funk and Omland, 2003). Mitochondrial introgression has been
reported in hummingbird species (e.g. Rodríguez-Gómez and
Ornelas, 2015; Jiménez and Ornelas, 2016); however, introgression
of maternally inherited mtDNA should be unlikely to happen for
the Xantus’ hummingbird because all phylogenetic trees using sep-
arated mtDNA genes and concatenate sequences, showed all spe-
cies’ haplotypes in a single 100% supported clade, separating H.
xantusii from the other five species.
273
Incomplete lineage sorting can also lead to pronounced differ-
ences between geographic distribution and mtDNA or nuclear
DNA variation. Following the formation of two daughter species
from an ancestor, lineage sorting is more rapid in the case of
mtDNA due to its lower effective population size (Funk and
Omland, 2003). Therefore, incomplete lineage sorting in young
species can potentially result in differences in population struc-
tures inferred from the two kinds of markers. Sex-biased dispersal
may also lead to such differences (Galtier et al., 2009).
The mitDNA genes used in this work (Cyt-b, COI, and ND2) pro-
vide sufficient resolution for identifying intraspecific differentia-
tion in some endemic birds (Arbeláez-Cortés et al., 2014). Our
phylogenetic analyses supported the relationships between Xan-
tus’ hummingbird and their close relatives (McGuire et al., 2014,
2007; Hernández-Baños et al., 2014), suggesting that the genetic
resolution in our study was robust for describing the interspecific
genetic variation of the six species examined in our study (Appen-
dix A).
4.2. Phylogeography of the Xantus’ hummingbird
Analyses of intraspecific lineages of H. xantusii indicate neither a
well-resolved topology tree nor a structure that was completely
concordant with geographic distribution. Some authors suggest
that trees may appear conflicting if they have unequal numbers
of terminal, which can result from a lack of differentiation in
response to a barrier or because some lineages have experienced
extinctions (Zink et al., 2000). For many species adapted to xeric
conditions, such as the birds of the Baja California Peninsula, which
originated later than their relatives on the mainland, climatic
changes of the Pleistocene erased biogeographical records in phy-
logenies (Zink et al., 2013, 2001; Zink, 2002). This seems to be the
case for the phylogenetic pattern of the Xantus’ hummingbird,
since 90% of haplotypes diverged during the last 500,000 years.
Furthermore, endemic species with a restricted distribution and
high genetic variation, such as H. xantusii which has high levels
of haplotype and nucleotide diversity compared to other hum-
mingbirds and other birds in general (for details see Appendix F),
retain high levels of mitochondrial genetic variation in local areas,
and this could represent early stages of the speciation process
(González et al., 2011).
SAMOVA analysis defined three genetically homogeneous and
geographically significantly different groups (south, central and
north regions), but also a significant correlation between genetic
and geographic distances was observed (Fig. 2). Together with
divergence time estimates, we suggested three ancestral popula-
tions occurred during Pliocene and Pleistocene: south, central,
and north (Fig. 3). Actually the oases and the Sierra La Laguna share
ecological features of mesic habitats that seem to be environments
more stable than the rest of the arid peninsula, therefore serving as
contemporary refuges for this hummingbird and other peninsular
species (Grismer and McGuire, 1993). In fact, the Xantus’ hum-
mingbird is more abundant in the Sierra La Laguna and oases in
the Sierra de la Giganta than in other areas, mainly sites where
water ponds are present in arroyos (Rodríguez-Estrella et al.,
2005; Arriaga et al., 1990). For many endemic birds, phylogeo-
graphic patterns suggest that at least some common factors were
extremely influential at the regional level; therefore, phylogeo-
graphic patterns could depend more on climate changes than geo-
logical events to link species behavior and ecology (e.g. phylopatry,
habitat, foraging niche, sexual selection), which are important fac-
tors relevant for species of recent origin with high genetic diversity
(Qu and Lei, 2009; González et al., 2011; Qu et al., 2011; Lara et al.,
2012; Arbeláez-Cortés et al., 2014; Licona-Vera and Ornelas, 2014).
The distance between oases along the mid-peninsula may also be
influencing the current distribution pattern of the species, generat-
274 C. González-Rubio et al. / Molecular Phylogenetics and Evolution 102 (2016) 265–277
ing a degree of genetic structure at a local level, which may not be
totally defined using mitochondrial genes. In this sense, the use of
genetic markers with higher mutation rate (e.g. microsatellite loci)
will define changes in population structure at a finer temporal-
space scale.
4.2.1. Vicariant events versus Pleistocene climatic changes
Contemporary spatial patterns of birds and other vertebrates
cannot be understood without knowing how species responded
to the geologic and climate history of their regions. For example,
the vicariant event of separation of the peninsula (5 mya) was an
important factor that promoted genetic differentiation for H. xan-
tusii and for other peninsular vertebrates (Riddle et al., 2000a,b;
Grismer, 2000). The time of separation of H. xantusii from its main-
land sister species (H. leucotis), began after the separation of the
peninsula (3.6 mya), which was also confirmed for its co-
distributed species, Calypte costae (3.5 mya). The second vicariant
event supported in our study was the seaway at the Isthmus of
La Paz (3 mya; Riddle et al., 2000b), where the Xantus’ humming-
bird in the southern region has the tMRCA at 2.1 mya, confirming
that this seaway isolated the region and promoted genetic differ-
entiation from the central and northern regions. Therefore, the
phylogeographic history for the Xantus’ hummingbird is related
primarily to the historical vicariant event of the seaway in the Isth-
mus of La Paz; secondly and more recently, related with climate
change at the end of the Pleistocene, observing that the 90% of hap-
lotypes diverged during the last 500,000 years.
4.2.2. Historical demography
Evidence of population expansion is provided by Fu’s FS test,
suggesting a significant excess of low frequency haplotypes in H.
xantusii in the three populations. However, differences in results
of Fu’s and Tajima’s neutrality tests can be related to their statisti-
cal properties. Fu’s test is more powerful in detecting demographic
changes and genetic hitchhiking (Fu, 1997).
Another indication of recent population expansion is provided
by the results of the divergence time estimates and EBSP analysis,
which suggested a recent expansion, where 90% of haplotypes
diverged in the last 500,000 years (Fig. 3), and more recently, a
population expansion that began 80,000 years ago (Fig. 4).
The presence of the same haplotypes found in distant oases
could reflect historical connectivity because some of them are
ancestral haplotypes at low frequency, or incomplete linage sort-
ing. If migration is interrupted for several generations, bottleneck
events could result in the elimination of haplotypes in local popu-
lations. In this sense, strong genetic drift after reduction might
explain the occurrence of haplotype divergence at some sites
(Avise and Walker, 1998). We would expect that pairs of individu-
als taken at random from the population differ, on average, by
many nucleotide substitutions, causing a mismatch distribution
with two or more modes at large values of pairwise differences
(Rogers and Harpending, 1992). Divergent haplotypes within the
same population can occur in species that have undergone severe
bottleneck processes; however, the presence of haplotypes with
low pairwise differences in bimodal mismatch distribution also
suggests a population that experienced a recent expansion
(Rogers and Harpending, 1992). This is highly important because
a population with a complex demographic history, including both
ancient population bottleneck and recent expansion, with the pres-
ence of ancestral haplotypes, could promote retention of novel
mutations and speciation processes (Avise and Walker, 1998;
Scoble and Lowe, 2010). Our results point to a scenario where
the genetic composition of the Xantus’ hummingbird could be a
result of a recent diversification process. Phylogeographical studies
in species in arid environments provide evidence that range expan-
sions during the Late Pleistocene and Holocene played a major role
in shaping spatial patterns of intraspecific diversity of vertebrate,
plants, and invertebrate species on the peninsula (Crews and
Hedin, 2006; Garrick et al., 2013, 2009; Zink et al., 2013).
4.3. Defining refuge areas
North American deserts provide a useful context to explore
methods in phylogeography (Zink et al., 2013, 2001; Zink, 2002;
Devitt, 2006; Garrick et al., 2013, 2009). Many phylogeographic
patterns of species in an arid region are a consequence of the arid-
ification during Pleistocene interglacial periods, after which spe-
cies expanded from isolated refuge (Hull and Girman, 2004;
Mapelli et al., 2010; Reding et al., 2012; Zink et al., 2013, 2000).
Pleistocene environments must have permitted continued differ-
entiation of populations whose separations often had been initi-
ated earlier (Avise and Walker, 1998). Sequences of mtDNA
concatenate genes show that the ancestral haplotypes are
restricted to the northern and southern regions, and are less wide-
spread than derived haplotypes and likely are centered on the ori-
gins of expansion, suggesting potential refuge areas. This has been
suggested in other birds of the peninsula: The California gnat-
catcher (Polioptila californica; Zink et al., 2013, 2001; Zink, 2002)
and the California quail (Callipepla californica; Zink, 2002), but only
in the southern region. Our study shows that southern and north-
ern regions with populations of the Xantus’ hummingbird were
refuges (ancestral refuges), and therefore deserve special attention
because they support birds with high genetic variation that
includes all ancestral haplotypes.
Several studies of Neotropical birds document phylogeographic
structure for species with discontinuous ranges resulting from
topographical barriers that restricted gene flow and drove genetic
divergence (González et al., 2011; Arbeláez-Cortés et al., 2014). In
these cases, the explanation of phylogeographic patterns is rela-
tively straightforward because it is concordant with topographical
heterogeneity. The Xantus’ hummingbird does not appear to be
tied to distributions with clear geographical barriers; however, this
peninsula includes a heterogeneous array of landscapes, with arid,
warm, and temperate regions (Grismer, 2000). The peninsula has
distinct desert subregions, the Desierto del Vizcaíno in the north
and the Desierto de Magdalena in the south (Grismer, 2000) that
are separated by volcanic mountain ranges (the Sierra San Fran-
cisco and Volcán las Tres Vírgenes). Extensive lava flows over the
past 1.2 my and high aridity produced low-quality habitats. Thus,
these arid areas could represent a topographical barrier to disper-
sal and gene flow in the Xantus’ hummingbird. Northern and
southern regions should be recognized as long-term refuge, not
just because the hummingbird in the Sierra La Laguna and San
Nicolás have ancestral haplotypes, but these oases are a source of
high haplotype diversity and many unique haplotypes.
A relationship seems to exist between the major climatic
changes of the Late Pleistocene and Holocene on the peninsula
and the phylogeographic pattern and historical changes in demog-
raphy, inferred for the genetics of the Xantus’ hummingbird. Given
these associations, we suggest that the current oases wetlands and
their natural resources are the key habitat factor responsible for
the current phylogeographic pattern of the Xantus’ hummingbird.
This suggestion is also supported with the IBD analysis (Fig. 2),
resulting a significant correlation only between geographical and
genetic distances among the eleven oases. Thus, the phylogeogra-
phy of the endemic Xantus’ hummingbird shows a different history
of vicariance for the fauna in the Baja California peninsula.
4.4. Conservation implications
Phylogeographic analyses and the time frame of genetic differ-
entiation of Hylocharis xantusii suggest two ancestral populations
 C. González-Rubio et al. / Molecular Phylogenetics and Evolution 102 (2016) 265–277
that began to spread in the Late Pleistocene and early Holocene,
promoting genetic differences between distant oases. Although
this species show high genetic diversity at regional level, probably
due to the high environmental, geological, climatic, and ecological
heterogeneity, populations and their genetic diversity may be
threatened (Grismer, 2000; Riddle et al., 2000b; Rojas-Soto et al.,
2003). In this sense, incorporating information from nuclear DNA
sequences (e.g. microsatellites) will allow a more comprehensive
history of the contemporary genetic structure, and together with
the information generated in this work, will help to establish better
conservation plans. Conservation of areas should include ancestral
populations as independent units with their environmental char-
acteristics. It has been observed that only one ecological variable,
such as habitat (e.g. oases in the peninsula), can explain much of
the variation in diversification rate in birds, reptiles and terrestrial
mammals (Wiens, 2015), and it should be considered at local-scale
for explaining patterns of genetic differentiation.
For the Xantus’ hummingbird, refuge areas contain high genetic
diversity and retention of ancestral variation. Thus, conservation of
these areas should be prioritized at a regional scale in the Baja Cal-
ifornia peninsula. These oases are considered wetlands and human
activity is strongly affecting and changing them, and it can even
destroy and disappear some of the oases (Rodríguez-Estrella
et al., 2005, 1999). It is clear that if desertification continues, local
extinctions may be inevitable (Gutierrez-Elorza, 1998), but human
activity can act more than synergistically, because it certainly
change the environment and reduces natural resources that are
already increasingly scarce and vulnerable in desert ecosystems.
Oases are small-size, vulnerable habitats. Thus, oases are priority
regions for conservation and from an evolutionary perspective
should include not only the conservation of species richness but
of the whole oases as they contain sources of genetic variation that
support long-term species conservation because of speciation
process.
5. Conclusions
This study presents the first research of phylogeography of the
Xantus’ hummingbird, an endemic species of the Baja California
Peninsula. Unlike most bird phylogeographical studies in the
peninsula, the present study, using a saturated sampling strategy
and three mitochondrial gene sequences, found that Xantus’ hum-
mingbird showed an ancestral phylogeographic structure with two
different populations along its distribution that began to spread
during the last 500,000 years. Two different phenomena could
explain this phylogeographic pattern: (1) vicariant events such as
the separation of the peninsula from the mainland, separating H.
xantusii from its sister species H. leucotis, and the temporary isola-
tion of the southern region at the Isthmus of La Paz, which were
supported in this work; and (2) Late Pleistocene climate changes
promoting population demographic changes (e.g. expansion
event), and together with environmental features and geographic
distribution of oases, leading an incipient genetic differentiation.
This is also the first study that shows two main ancestral refuges
for a peninsular endemic species, which highlighted the impor-
tance of defining areas of evolutionary and conservation relevance
based on genetic and phylogeographic studies.
Acknowledgements
We thank Franco Cota and José Abelino Cota of CIBNOR and Ser-
gio Sánchez-González of Universidad Autónoma de Sinaloa for field
assistance, and Ira Fogel of CIBNOR and Mabilia Urquidi for edito-
rial services. We also thank Juan Francisco Ornelas Rodríguez and
Borja Milá for their valuable comments on the use and interpreta-
275
tion of methods that improved the manuscript. The Secretaría de
Medio Ambiente y Recursos Naturales (SEMARNAT) issued the col-
lecting permit (SGPA/DGVS/10182/11, 06983/13). Funding was
provided by the Consejo Nacional de Ciencia y Tecnología (CONA-
CYT project CB-2008-01-106925) granted to F.J.G.L. and (CONACYT
project 155956) granted to R.R.E. C.G.R.S. is a recipient of a fellow-
ship (CONACYT 8492).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ympev.2016.05.
039.
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