Molecular Phylogenetics and Evolution 43 (2007) 952–973
www.elsevier.com/locate/ympev
Molecular evidence for a species complex in the patagonian lizard
Liolaemus bibronii and phylogeography of the closely related
Liolaemus gracilis (Squamata: Liolaemini)
Mariana Morando a,¤, Luciano J. Avila a, Cameron R. Turner b,c, Jack W. Sites Jr. b
a
b
CONICET-CENPAT. Boulevard Almirante Brown s/n, 9120, Puerto Madryn, Chubut, Argentina
Department of Integrative Biology, and M.L. Bean Life Science Museum, Brigham Young University, 401 WIDB, Provo, UT 84602, USA
c
Department of Biology, College of Arts and Sciences, Indiana University, Bloomington, IN 47405, USA
Received 31 May 2006; revised 14 September 2006; accepted 18 September 2006
Available online 27 September 2006
Abstract
The lizard genus Liolaemus is endemic to temperate South America and includes 190 species. Liolaemus bibronii has a large geographic
distribution and inhabits a great diversity of habitats, including the Monte, Steppe, and high Andean grassland environments. Liolaemus
gracilis has a similar body size and shape to L. bibronii; the two are parapatrically distributed, and L. gracilis is also widely distributed.
Here we use the mtDNA cytb sequence data of these two species to investigate lizard phylogeographic patterns in southern South America. L. bibronii is paraphyletic with respect to L. gracilis, Liolaemus ramirezae, Liolaemus robertmertensi and Liolaemus saxatilis; it is composed of many genetically diVerent allopatric haploclades, some of which are reciprocally monophyletic. We also found evidence for
introgression between L. bibronii and L. gracilis in the same area that introgression was hypothesized in the Liolaemus darwinii complex.
We discuss the distribution of the major haploclades with inferences of their population histories, the concordance of these clades’ distributions and histories with other lizard complexes studied with the same markers and methods, and taxonomic implications of these
results.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Argentina; Liolaemus bibronii; Liolaemus gracilis; Lizard; mtDNA; Phylogeography
1. Introduction
The Patagonian region of southern South America represents a geographically extensive and ecologically and
topographically diverse area that is biologically poorly
known. It includes two main ecoregions, Patagonian Steppe
and Austral Monte (Xatlands and plateaus), with two other
small ecoregions along its western edges, the Valdivian Forest and southern High Andes (Burkart et al., 1999). Physiographic complexity increases from east to west along an
elevational gradient, and the landscape is dissected from
north to south by several east-Xowing river systems. The
*
Corresponding author. Fax: +54 2965 451543.
E-mail addresses: [email protected] (M. Morando), cartune
@indiana.edu (C. Turner), [email protected] (J.W. Sites).
1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2006.09.012
southern Andean Range and adjacent regions have been
aVected by orogenic and volcanic events, and cyclical glaciations (Simpson-Vuilleumier, 1971; Simpson, 1975, 1979,
1983; Villagrán et al., 1995, 1998; Thorson, 1999; McCulloch et al., 2000). Andean uplift began in the Miocene and
continues to the present (Ramos, 1989), and mountainbuilding has been accompanied by multiple Pleistocene glaciations (Hollin and Schilling, 1981; Clapperton, 1993;
McCulloch and Bentley, 1998; Hulton et al., 2002; Sugden
et al., 2002). Superimposed on these events have been
repeated Pleistocene volcanic disturbances and marine introgressions (Rabassa and Clapperton, 1990), which collectively form a remarkably diverse landscape.
The biogeographic and phylogeographic eVects of
the Pliocene–Pleistocene historical events have been
well documented in Europe and North America (e.g.
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
Hewitt, 1996, 2000; Comes and Kadereit, 1998; Petit
et al., 2003; Taberlet et al., 1998; Avise et al., 1998;
Hewitt, 2004). These events have promoted extensive
shifts in distributions as well as fostered speciation and
intraspeciWc diVerentiation, and refugia have been identiWed in some regions (e.g. Hewitt, 1996, 2000). In contrast,
homogenization of populations through long-distance
postglacial dispersal is also hypothesized (Hewitt, 1996).
Concordant genetic signals of population expansion into
deglaciated areas have been found across taxa with
widely diVerent life histories, perhaps reXecting the common role of glacial vicariant events in shaping community composition at higher latitudes (Lessa et al., 2003).
In southern South America, Miocene to Pliocene paleoclimatic and geological events have also presumably
imposed strong selective forces on the evolutionary histories of the southern temperate-adapted vertebrate fauna
(Baez and Scillato Yané, 1979; Markgraf et al., 1995).
Marine incursions in several regions along coastlines and
regional tectonic uplift may have contributed to habitat
fragmentation during this period, promoting divergence
between isolated populations. During the late Pliocene to
early Pleistocene (0.7–2 Ma), climatic oscillations intensiWed
and produced rapid Xuctuations between cool–dry and
warm–wet conditions across southeastern Patagonia
(Simpson, 1979). The impacts of glaciations have been less
well documented than in the northern hemisphere,
although it is clear that much local glaciation occurred,
which most probably caused extinction or migration northward and to lower elevations for many plant and animal
groups (Simpson-Vuilleumier, 1971; Simpson, 1975, 1979,
1983; Villagrán et al., 1995, 1998; Thorson, 1999; McCulloch et al., 2000). Indeed the major part of Patagonia
appears to have been a bogland (Simpson-Vuilleumier,
1971) dissected by outwash streams and lakes from glacialmelt waters, although there was an overall cooling–drying
trend evident by late Pliocene (Baez and Scillato Yané,
1979; Markgraf et al., 1995), causing dramatic shifts in the
ranges of taxa by latitude and topography.
Although the extensive glaciations, habitat shifts, marine
incursions, and tectonic and volcanic activities in the Patagonia region over the past several million years (Clapperton, 1993) have likely generated interesting biogeographic
patterns, few phylogeographic studies have been conducted
for widely distributed Patagonian vertebrates (Avila et al.,
2006; Kim et al., 1998; Morando et al., 2003, 2004).
Detailed information about the geographical distribution
of population genetic variation within and among diverging taxa is critical for analyses of the evolutionary processes
driving divergence and speciation (Hewitt, 1996, 2000;
Avise et al., 1998; Klicka and Zink, 1999; Knowles, 2000,
2001). Analysis of phylogeographic structure is particularly
important for species with extensive ranges and complex
geographical patterns of polymorphism (Irschick and
ShaVer, 1997; Starkey et al., 2003). Phylogeographic data
can be used to infer the biogeographic and demographic
histories of such organisms (Avise, 2000, 2004), and under-
953
standing the role of historical environmental change in generating current patterns of biodiversity is central to
identifying processes of divergence and speciation and their
implications for conservation (Moritz et al., 2000; Hewitt,
2004).
The lizard genus Liolaemus is endemic to temperate
South America, where it is distributed over a wide geographic region and occupies a large range of latitudinal
(14°30’–52°30’S), altitudinal (0–4500 m), and climatic
regimes; Liolaemus range from the extremely arid Atacama
desert to temperate Nothofagus rainforest (Cei, 1986, 1993;
Donoso Barros, 1966; Etheridge and de Queiroz, 1988;
Lobo, 2001). This genus includes 190 recognized species
and subspecies and new species are being discovered at a
rapid rate (see examples in Abdala, 2002, 2003, 2005; Avila,
2003; Avila et al., 2003; Avila et al., 2004; Espinoza et al.,
2000; Etheridge, 2001; Etheridge and Christie, 2003; Espinoza and Lobo, 2003; Martínez Oliver and Lobo, 2002;
Nuñez et al., 2000; Pincheira-Donoso and Nuñez, 2003;
Verrastro et al., 2003). Some new species are endemic to single mountaintops or recently explored isolated areas (e.g.
Avila, 2003; Avila et al., 2003), whereas others were formerly grouped into single species (“inertial species” of
Good, 1994) and later recognized after detailed studies (e.g.
Abdala, 2002, 2003, 2005; Espinoza and Lobo, 2003; Martínez Oliver and Lobo, 2002; Lobo and Kretzschmar, 1996).
Morando et al. (2003) recently estimated that the actual
number of Liolaemus species could be as high as twice the
number currently recognized.
Although lizard biogeography of these regions is
scarcely known, some historical and demographic
hypotheses have been recently proposed for some Liolaemus species (Avila et al., 2006; Morando et al., 2003,
2004). All of these studies revealed that several widely distributed species constituted species complexes, with
smaller distributions and more genetically structured
clades in the unglaciated northern regions and larger
ranges and lower genetic diversity—signatures for recent
range expansions—in the southern regions. Based on
these results, high levels of genetic diVerentiation are
likely to be uncovered among populations of many other
widespread Liolaemus groups (and by extension, to other
unrelated clades) in the Andean–Patagonian areas. Therefore current taxonomy likely conceals a long history of
multiple evolutionary processes operating throughout this
region (Morando et al., 2003).
Liolaemus bibronii is a species typical of several of the
groups mentioned above; it has a large geographic distribution and inhabits a great diversity of habitats, including the
Monte, Steppe, and high Andean grassland environments.
This species is common in high elevation Andean valleys
(up to 3000 m) in southern San Juan Province (32°S), and
remains common across all environments extending to the
coastline of Santa Cruz Province (48°S). Like most Liolaemus, L. bibronii is insectivorous, and according to Cei
(1986) is oviparous, but Donoso Barros (1966) considered
Chilean populations to be viviparous.
954
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
Due to its wide geographic distribution, the variety of
habitats it occupies, and conXicting interpretations of parity mode, we hypothesize that L. bibronii may be a species
complex, and that as with the complexes previously studied
by Morando et al. (2003, 2004) and Avila et al. (2006), its
southernmost clades will have wider geographic distributions than northern clades, and southern clades will show
evidence for recent range expansions and less genetic diversity and/or genetic structure than northern clades.
A sister-group relationship of L. bibronii (from its northernmost distribution) to Liolaemus gracilis with Liolaemus
robertmertensi (L. bibronii) (L. gracilis + L. robertmertensi)
was reported by Schulte et al. (2000). However, Morando
et al. (2004) recovered southern populations of L. bibronii
as the sister species of Liolaemus ramirezae, and this clade
as the sister group of the clade ((L. gracilis + Liolaemus
saxatilis) L. robertmertensi). L. gracilis has a similar body
size and shape to L. bibronii; the two are parapatrically distributed, and L. gracilis is also widely distributed latitudinally through a variety of habitats in the Monte region and
the Monte-Patagonia ecotone. Here we use the mitochondrial DNA (mtDNA) sequence (cyt-b) data of these two
species, L. bibronii and L. gracilis, to investigate lizard phylogeographic patterns in southern South America.
2. Materials and methods
2.1. Taxon sampling
Previous experience (Morando et al., 2003, 2004; Avila
et al., 2006) has suggested that the mtDNA cyt-b gene was
suYciently variable for exploratory phylogeographic studies in Liolaemus, and we use this region here. Sequence data
were collected from a total of 150 lizards, of which 138 samples from 41 localities (Table 1, Fig. 1) represented populations under the names L. bibronii (Bell, 1843) (105
individuals) and L. gracilis (Bell, 1843) (32 individuals);
these were the “focal species” (Wiens and Penkrot, 2002) of
this study. L. saxatilis Avila et al., 1992; L. robertmertensi
Hellmich, 1964; L. ramirezae Lobo and Espinoza, 2000; L.
pictus (Dumeril and Bibron 1837), L. cyanogaster (Dumeril
and Bibron, 1837), L. chiliensis (Lesson, 1828), L. neuquensis (Müller and Hellmich, 1938), L. punmahuida Avila et al.,
2003; and L. tenuis Dumeril and Bibron, 1837, were used as
non-focal species (n D 1 each, Table 1). L. lineomaculatus
Boulenger 1885, and L. kingii (Bell, 1843), members of the
Liolaemus clade Eulaemus were used to root the trees, thus
allowing the position of the non-focal species to remain
unconstrained with respect to the “focal species”.
Table 1 summarizes the number of individuals
sequenced per locality and distributional information for
all taxa used in this study (Table 1, Fig. 1), and voucher
specimens are deposited in the LJAMM herpetological collection (now housed in the Centro Nacional Patagónico
[CENPAT], Puerto Madryn, Argentina), Fundación Miguel Lillo (FML, San Miguel de Tucumán, Argentina),
Museo de Ciencias Naturales La Plata (MLP.S, La Plata,
Argentina) and M.L. Bean Life Science Museum (BYU,
Provo, USA). Museum voucher numbers are listed by
taxon and locality in Appendix A, and museum acronyms
follow Leviton et al. (1985).
2.2. Laboratory procedures
Total genomic DNA was extracted from liver/muscle
tissues preserved in 96% ethanol, following the protocol
developed by Fetzner (1999). Three l of extraction product were electrophoresed on 1% agarose gel to estimate
the quality and amount of genomic DNA, and sample
dilutions were performed where necessary prior to polymerase chain reaction (PCR) ampliWcation. The cyt-b
gene region was ampliWed via PCR following Morando
et al. (2003). A 607 bp fragment was ampliWed using the
light-strand primers GluDGL (Palumbi, 1996) and cyt-b 1
(Kocher et al., 1989), and the heavy-strand primer Cyt b 3
(Palumbi, 1996); Cyt b 2 (Palumbi, 1996) and Cyt.F.1
(Whiting et al., 2003) were used as internal sequencing
primers. Double-stranded PCR-ampliWed products were
checked by electrophoresis on a 1% agarose gel (the size
of the target region estimated using a molecular-weight
marker), puriWed using a GeneClean III kit (BIO101, Inc,
Vista, CA), and directly sequenced using the Perkin Elmer
ABI PRISM Dye Terminator Cycle Sequencing Ready
Reaction (PE Applied Biosystems, Foster City, CA).
Excess Dye Terminator was removed with CentriSep spin
columns (Princeton Separations Inc.), and sequences were
fractionated by polyacrylamide gel electrophoresis on an
ABI PRISM 3730XL automated DNA sequencer (PE
Applied Biosystems, Foster City, CA) at the DNA
Sequencing Center at BYU. Sequences are deposited in
GenBank under Accession Numbers DQ989648 to
DQ989791. Individuals previously deposited in GenBank
and included in these analyses: L. bibronii: AY173791; L.
gracilis: AY367804; outgroups: AY367848, AY367803,
AY173828, AY367818.
2.3. Sequence alignments
Sequences were edited and aligned using the program
Sequencher 3.1.1 (Gene Codes Corp. Inc. 1995), and
translated into amino acids for conWrmation of alignment. No indels were present, and missing data were
coded as “?”.
2.4. Phylogenetic analyses
Only non-redundant haplotypes (from the 137
sequences of the ingroup) selected with the program Collapse v.1.1 (http://bioag.byu.edu/zoology/crandall_lab/
programs.htm), were used for the tree-based phylogenetic
methods. ModelTest (version 3.06; Posada and Crandall,
1998) identiWed the best Wt model of evolution for the
complete data set of unique haplotypes (106 individuals 93 haplotypes from the ingroup + 13 outgroup—and
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
955
Table 1
Number of individuals of all ingroup and outgroup taxa, by locality and ordered according to results, used in this study; locality numbers (in parentheses)
match those in Fig. 1 and Appendix A (which provides museum numbers for all specimens)
Province
Department
Focal species
Liolaemus bibronii
Rio Negro
25 de Mayo
Valcheta
Santa Cruz
Lago Buenos Aires
Deseado
Escalante
L. bibronii 1
Chubut
Telsen
L. bibronii 2
Chubut
Cushamen
L. bibronii 3
Rio Negro
Pilcaniyeu
El Cuy
L. bibronii 4
Rio Negro
El Cuy
Pilcaniyeu
L. bibronii 5
Neuquén
Catan Lil
L. bibronii 6
Neuquén
Huiliches
L. bibronii 7
Rio Negro
Ñorquinco
25 de Mayo
L. bibronii 8
Mendoza
Malargüe
San Rafael
L. bibronii 9
Neuquén
Minas
Locality
N
Coordinates
(1) R. Nac. 23, 14 Km W Aguada de Guerra
(2) R. Pcial. 5, 40 Km SE Maquinchao
(4) 7.5 Km W Los Menucos
(3) R. Pcial 60. 10 Km SE Chipauquil
4
3
3
3
41°09’37”S, 68°30’53”W
41°30’07”S68°33’16”W
40°51’06”S, 68°10’48”W
41°00’37”S, 66°44’02”W
(5) R. Pcial 43, 19 Km W Perito Moreno
(57)R.Pcial. 43. El Pluma
(6) R. Pcial 43. 16 Km E Las Heras
(7) R. Nac. 281, 45 Km NW Tellier
(58) Empalme R. Nac. 281 y R. Nac. 3, 7 Km NW Jaramillo
(59) R. Nac 26, 27 Km W Pampa del Castillo
1
1
4
1
1
1
46°36’02”S, 71°09’53”W
46°28’50”S, 70°02’56”W
46°33’50”S, 68°40’16”W
47°28’20”S, 66°33’14”W
47°08’17”S, 67°12’44”W
45°42’57”S, 68°19’27”W
(16) R. Pcial 4. 0.8 Km E Gan Gan
(17) 65.5 Km W Telsen
(19) 9 Km W Telsen
(20) R. Pcial. 4. 41.6 Km W Telsen
(60) R. Pcial. 4. 5.4 Km W Telsen
(61) R. Pcial. 4. 2 Km E Gan Gan
1
1
1
4
2
2
42°31’35”S, 68°15’29”W
42°31’35”S, 68°15’29”W
42°26’55”S, 67°02’42”W
42°22’06”S, 67°24’07”W
42°26’40”S, 67°00’06”W
42°31’44”S, 68°01’06”W
(18) R. Pcial. 13, 8 Km N El Molle
3
42°10’24”S, 69°32’51”W
(26) R. Nac. 40. 15 Km S entrada Alicura
(27) 2.7 Km S Estancia San Pedro
(28) R. Pcial 67. 19.2 Km NE Mencue
4
4
3
40°40’46”S, 70°42’56”W
40°52’43”S, 70°34’28”W
40°19’31”S, 69°26’18”W
(29) R. Pcial. 67. 20 Km S Mencue
(30) R. Pcial. 67. 3.5 Km N Cañadon Chileno
(31) R. Pcial. 67. 2 Km N Cañadon Chileno
(32) R. Pcial. 23. 4.8 Km SE Comallo
(27) 2.7 Km S Estancia San Pedro
2
6
5
1
1
40°34’04”S, 69°44’59”W
40°51’09”S, 69°58’15”W
40°52’30”S, 70°01’14”W
41°02’40”S, 70°12’56”W
40°52’43”S, 70°34’28”W
(33) R. Pcial. 23. 8 Km N Pilolil
1
39°32’29”S, 70°57’21”W
(34) 2 Km W CEAN. R. Pcial. 61. Valle Chimehuin
1
39°52’00”S, 71°10’00”W
(35) R. Pcial. 6. 1 Km NW Ojo de Agua
(62) R. Nac. 23, 10 Km W Ing. Jacobacci
1
2
41°32’30”S, 69°51’33”W
41°20’16”S, 69°41’42”W
(21) R. Nac. 40. 6 Km S Laguna Coipo Lauquen
(22) R. Nac. 40. 3.2 Km N. Ranquil Norte
(23) R. Nac. 40. 1.5 Km S Bardas Blancas
(24) 3.4 Km W. Bardas Blancas, entrada Paso Pehuenche
(36) R. Pcial. 180, 90 Km S El Nihuil
(25) 22 Km NW El Sosneado
4
3
1
1
2
2
36°37’57”S, 69°50’15”W
36°38’24”S, 69°49’55”W
35°52’11”S, 69°47’43”W
35°52’01”S, 69°46’39”W
35°48’59”S, 68°40’05”W
35°04’47”S, 69°34’54”W
(14) Las Olletas. 7 Km N Aguas Calientes
(15) 28.5 Km NW empalme R. Pcial. 43
4
6
36°39’35”S, 70°35’04”W
36°55’57”S, 70°55’04”W
(continued on next page)
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M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
Table 1 (continued )
Province
L. bibronii 10
Neuquén
Ñorquin
Loncopue
L. bibronii 11
Neuquén
Minas
L. bibronii 12
Neuquén
Zapala
L. gracilis
Rio Negro
Gral. Roca
San Antonio
Neuquén
Zapala
Añelo
Loncopue
La Pampa
Chical Co
Chubut
Biedma
Mendoza
Malargüe
San Rafael
Nonfocal species
L. saxatilis
L. robertmertensi
L. ramirezae
L. pictus argentinus
L. pictus
L. cyanogaster
L. chiliensis
L. neuquensis
L. punmahuida
L. monticola
L. tenuis
Outgroups
L. lineomaculatus
L. kingii
Locality
N
Coordinates
(10) R. Pcial. 21, 15 KmN Loncopue
(11) R. Pcial33.23.9 KmSE Loncopue
5
1
37°58’19”S, 70°38’38”W
38°08’27”S, 70°26’38”W
(12) R. Pcial. 57. 2.4 Km S Nahueve
(13) R. Pcial. 57. Rio Riñileuvu
5
1
37°12’35”S, 70°44’24”W
37°20’24” S, 70°45’24”W
(8) K s/n. 6 Km S Covunco Centro
(9) R. Pcial. 46. 22 Km SW Zapala
3
2
38°49’55”S, 70°01’58”W
39°02’37”S, 70°13’22”W
(37) Chichinales
(38) 25 Km N Cervantes, around Salitrosa Lagoon
(41) 38 Km N R. Pcial. 22 a Gral. Roca desde cruce a Casa de Piedra
(42) Las Grutas. Playa Piedra Colorada
1
1
4
1
39°06’00”S, 66°56’12”W
38°49’05”S,67°21’52”W
38°39’38”S, 67°35’28”W
40°50’26”S, 65°07’04”W
(39) 6 Km NW La Amarga
(8) K s/n. 6 Km S Covunco Centro
(40) R. Pcial. 7. 28.7 N Añelo
(56) R. Nac. 40, 8 Km N Rio Salado
1
3
6
1
39°04’07”S, 69°37’31”W
38°49’55”S,70°01’58”W
38°11’04”S,69°01’22”W
38°15’45”S, 70°03’56”W
(44) Camino a Chos Malal. 54 Km W empalme R. Nac. 151
1
36°42’31”S, 67°57’00”W
(43) R. Pcial. 42 11 Km NE Pto. Madryn. El Doradillo
1
42°39’25”S, 64°59’32”W
(45) R. Pcial. 180. 12.5 Km S La Salinilla
(46) R. Pcial. 190. 16.6 Km S El Toscoso
(47) R. Pcial. 186. 20 Km W Mina Ethel
(48) R. Pcial 190. 12 Km S El Toscoso
(49) R. Pcial. 180. 28.1 Km N entrada sur a La Matancilla
(50) R. Pcial. 180. 15 Km S La Cortadera
(51) R. Pcial. 180. 59 Km W Gobernador Ayala
(52) R. Pcial. 190. 2 Km N Agua Escondida
(53) 5 Km NE La Salinilla. 1268 m
(54) R. Pcial. 180. 30 Km S El Nihuil
(55) El Nihuil
1
1
1
1
1
1
1
1
1
1
2
36°23’04”S, 68°35’07”W
35°49’31”S, 68°02’56”W
35°58’06”S,69°01’54”W
35°47’19”S,68°01’48”W
36°37’17”S, 68°36’38”W
36°39’44”S, 68°40’32”W
37°17’56”S, 68°33’12”W
36°08’19”S, 68°17’23”W
36°13’47”S, 68°31’09”W
35°17’09”S, 68°41’52”W
35°01’59”S,68°39’55”W
Córdoba. Rio Cuarto. R. Pcial 1, 4.5 Km W Achiras
Catamarca. Tinogasta. R. Nac. 60 & Rio La Puerta. Km 1298
Tucumán. TaW del Valle. R. Pcial. 307, 21.7 Km E Amaicha del Valle
Rio Negro. Bariloche. Near Bariloche town
Chile. Termas de Chillan, VIII Región
Chile. Tucapel, VIII Region
Chile. Termas de Chillan, VIII Región
Neuquén. Picunches. R. Pcial. 13, Primeros Pinos
Neuquén. Chos Malal. R. Pcial. 37. Volcán Tromen
Región Metropolitana. Stgo. de Chile
Chile. Termas de Chillan, VIII Región
1
1
1
1
1
1
1
1
1
1
1
33°10’07”S, 65°02’20”W
28°14’44”S, 67°27’11”W
26°40’51”S, 65°48’45”W
41°08’00”S, 71°19’00”W
36°32’00”S, 71°57’00”W
37°37’00”S, 73°20’00”W
36°32’00”S, 71°57’00”W
38°51’43”S, 70°33’04”W
37°06’16”S, 70°08’27”W
33°26’00”S, 70°39’00”W”
36°32’00”S, 71°57’00”W
Rio Negro. Bariloche, PN Nahuel Huapi, Piedra del Condor, Co. Catedral
Río Negro. Valcheta. R. Pcial. 4, 84 km S junction R. Nac. 250, extremo W
Gran Bajo del Gualicho
1
1
41°12’00”S, 71°17’00”W
40°06’20”S, 66°00’26”W
Numbers under the N column give the number of lizards sequenced from each locality.
607 bp without missing data) as GTR + I + (Yang, 1994;
Gu et al., 1995). With parameters calculated under this
model we obtained a neighbor-joining distance tree using
PAUP that recovered several monophyletic groups (see
Results). The same data set was run four times in MrBayes (Huelsenbeck and Ronquist, 2001) for 2 £ 106 genera-
tions and sample frequency D 500 with the same model of
evolution. We determined when stationarity was reached
(to discard the “burn-in” samples) by plotting the log likelihood scores of sample points against generation time;
when the values reached a stable equilibrium, before
10,000 generations in all cases, stationarity was assumed.
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
957
Fig. 1. Geographic distributions of the taxa and populations sampled in this study. Symbols denote the following: numbers with cross D localities for L.
gracilis, and squares and circles D localities for the diVerent clades of L. bibronii. Locality numbers correspond to those in Table 1 and Appendices I & II,
and symbols correspond to those in Fig. 2.
The Bayesian analysis recovered the same clades as did
the NJ distance method; thus, one individual from each
clade (the one with the most complete sequence) was
selected for further phylogenetic analyses to study the
relationships between these clades and outgroups (27 individuals and 762 bp).
A maximum parsimony (MP) search with all characters equally weighted was performed with 10,000
replicates and 10,000 pseudoreplicates for non-parametric bootstrap analyses (Felsenstein, 1985). A maximumlikelihood (ML) search was performed with the
GTR + I + (Yang, 1994; Gu et al., 1995) model of evolution (selected with ModelTest as described above) with
10 replicates and 100 pseudoreplicates for bootstrap
analyses. The MP and ML searches were implemented
using PAUP* 4b5 (SwoVord, 2002), and Bayesian analyses were performed with this reduced dataset using Mr
Bayes 2.0 (Huelsenbeck and Ronquist, 2001) for 2 £ 106
generations and sample frequency D 500. The equilibrium
samples (3961 trees) were used to generate a 50% majority-rule consensus tree. The percentage of samples that
recover any particular clade on this tree represents that
clade’s posterior probability; we consider p 7 95% as evidence of signiWcant support for a clade (Huelsenbeck and
Ronquist, 2001). Recent simulation analyses indicate that
although Bayesian support values are usually higher than
958
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
those from non-parametric bootstrap, they provide a
much closer estimate of the phylogenetic accuracy (Wilcox et al., 2002). We also used a genetic algorithm as
implemented in MetaPIGA (Lemmon and Milinkovitch,
2002) with diVerent settings for population size (n D 4
and 10) and with diVerent replicates (n D 10 and 100).
MetaPiga implements a new algorithm (the metapopulation genetic algorithm) based on a heuristic search that
improves the speed with which maximum likelihood
(ML) trees are found. It also estimates the posterior
probability distribution of possible trees, with clade frequencies closely approximating their posterior probabilities (Lemmon and Milinkovitch, 2002). To obtain a
consensus tree based on this algorithm, we ran MetaPiga
for 10£ with 10 populations each and generated a consensus tree from the 100 recovered trees (as suggested by
Lemmon and Milinkovitch, 2002).
2.5. Nested clade phylogeographic analyses
The complete set (no missing data) of 138 short cyt-b
sequences (607 bp) was used for the nested clade phylogeographic analyses (NCPA). We have suggested elsewhere
(Avila et al., 2006) that the NCPA is useful when the
absence of climatic, geological, or paleoecological data
preclude the a priori formulation and statistical testing of
multiple alternative hypotheses, as advocated by some
(Knowles and Maddison, 2002). We are aware that uncritical use of the NCPA for a single gene tree may lead to
over-interpretation (Irwin, 2002; Knowles, 2004 (Table
1)), but our position here is that the NCPA with the mitochondrial locus is ideal for generating hypotheses of evolutionary and demographic histories that are then
amenable to further testing (Templeton, 2004), and we use
this method (Templeton et al., 1995; Templeton, 1998,
2001, 2004) to infer the population history of L. bibronii
and L. gracilis.
The program TCS version 1.13 (Clement et al., 2000;
available at http://bioag.byu.edu/zoology/crandall_lab/
programs.htm), was used to construct the haplotype network, and nesting categories were assigned following
Templeton et al. (1995) and Templeton and Sing (1993).
The networks were then used for NCPA, which was
implemented with the GeoDis program (version 2.0,
Posada et al., 2000) available from the same web site. All
statistical analyses were performed using 10,000 Monte
Carlo replications. A single ambiguous connection (loop)
in the L. gracilis network was resolved using predictions
from coalescent theory (reviewed in Nordborg, 2001 and
Rosenberg and Nordborg, 2002), as validated with
empirical data sets (Crandall and Templeton, 1993; see
also Pfenninger and Posada, 2002 and Morando et al.,
2004; for recent examples). The rule used here, that high
frequency haplotypes in a population are expected to be
found on the interior of the cladogram (i.e., ancestral)
while lower frequency haplotypes should be found at the
tips, was used to resolve the loop in the L. gracilis net-
work in favor that the network shown here (Fig. 4). However, we compared the other equally parsimonious
resolutions for this connection, and no diVerences were
found between them. Statistically signiWcant results were
interpreted following the inference key of Templeton
(2004, also available from the same website).
2.6. Neutrality tests and molecular-diversity analysis
To check the assumption that cyt-b sequence evolution
approximates neutral expectations (Ballard and Whitlock,
2004), we used the McDonald and Kreitman (1991) test
(hereafter M–K) of neutrality as implemented in the program DNASP vs. 3.99.4 (Rozas and Rozas, 1999), and to
assess population equilibrium independent of the NCPA
inferences, we implemented Tajima’s (1989) D and Fu’s
(1997) Fs tests. These tests diVer in their statistical power
(Wayne and Simonsen, 1998; Ford, 2002) but both assume
that populations are in mutation-drift and migration-drift
equilibrium. SigniWcant values for either may indicate that
the populations are not evolving in a neutral manner (i.e.,
they are not in mutation-drift equilibrium), or that they
were previously subdivided and/or have experienced past
population growth (i.e., they are not in migration-drift equilibrium). The Fs test appears to be more sensitive to detection of population expansion (Fu, 1997), and we evaluated
signiWcance of this test by comparing the Fs statistic against
a distribution generated from 1000 random samples under
the hypotheses of selective neutrality and population
equilibrium.
For the main clades identiWed in the phylogenetic and
NCPA analyses (see below), we calculated the uncorrected average pairwise genetic distances using PAUP¤
4b5 (SwoVord, 2002). We also estimated gene diversity
(Nei, 1987, p. 180) and nucleotide diversity (, the mean
of pairwise sequence diVerences, Nei, 1987, p. 257) for
these same clades. The parameter can be estimated
using or the number of segregating sites (S). If evolution is neutral then both estimates give the same value of
, and this can be compared via the Tajima’s test (Tajima,
1989) to assess whether impacts of selection or population change can be detected (see below). As alternative
evidence of gene Xow we calculated M (2 Nm) as a relative estimate of the number of migrants between identiWed clades (see below). According to the phylogenetic
results we performed three AMOVAs (ExcoYer, 2001)
to: (1) estimate the genetic structure between the southern and northern clades of L. bibronii (see below); (2)
estimate the genetic structure among three groups within
the southern clade (group 1: L. bibronii, L. bibronii 1, 2;
group 2: L. bibronii 3, 4; group 3: L. bibronii 5, 6); and (3)
estimate the genetic structure between two groups (group
1: L. bibronii 9, 10, 11; group 2: L. bibronii 12) within the
northern clade. As a Wnal assessment of population
demographic histories, we performed mismatch analyses
(1,000 permutations) and calculated the raggedness index
(Harpending, 1994) for several clades of L. bibronii and
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
L. gracilis (see below). This index takes larger values
(Rgg > 0.05) for multimodal distributions expected in stationary populations, relative to unimodel and smoother
distributions typical of expanding populations
(Rgg < 0.05). We calculated the probability of observing
by chance a higher value of the raggedness index than the
observed value p (Ragobs), under the hypothesis of population expansion. The nucleotide diversity, population
structure, estimates of migration rates, Tajima and Fu
tests, and uncorrected and corrected average pairwise
genetic distances were performed with the program Arlequin vs. 2.00 (Schneider et al., 2000). This combination of
methods permits us to evaluate speciWc NCPA inferences
tied to population growth—including dispersal or range
expansions—by statistical tests based on completely
diVerent assumptions.
2.7. Coalescent estimation of population divergence times
and migration rates
We used the program MDIV (Nielsen and Wakeley,
2001) to evaluate whether shared mtDNA variation
between L. gracilis and L. bibroni 8 (see below) likely
reXected gene Xow or recent isolation. The method provides a non- equilibrium approach that uses a Bayesian
framework to estimate both parameters by calculating
their posterior probabilities given the genealogy (Nielsen
and Wakeley, 2001), under a Wnite sites model of
sequence evolution (Hasegawa et al., 1985). MDIV uses
Markov-Chain Monte Carlo simulations to estimate the
likelihood of the parameters (2Nef), M (Nef m), and T
(t/Nef) (where Nef is the female eVective population size, m
is the migration rate and is the mutation rate per locus
per generation). We ran 5,000,000 generations with burnin of 500,000. We made several runs with diVerent priors
for M and T (from 0 to 10 for M, and from 1 to 10 for T).
When possible, we estimated credibility intervals for the
estimates as the interval that contains 95% of the posterior probability distribution. In general for iguanid
mtDNA (Macey et al., 1998, 1999), and cyt-b from other
reptiles (Zamudio and Greene, 1997; Malhotra and
Thorpe, 2000; Giannasi, 1997) a pairwise sequence divergence has been estimated in the range of 1.3–2%. According to this, we calculated Nef from the estimate of theta
for a range of values of D 3.94 £ 10¡6–6.07 £ 10¡6 per
lineage per locus (607 bp in this case) per generation,
assuming a generation time of one year. This estimate of
Nef was used to obtain an estimate of the population
divergence time and the gene divergence time (Time-toMost-Recent-Common-Ancestor; TMRCA).
3. Results
3.1. Phylogenetic relationships of cyt-b haplotypes
A total of 68 diVerent haplotypes were identiWed
among the 105 L. bibronii specimens and 25 diVerent
959
haplotypes among the 32 L. gracilis individuals. The
neighbor-joining tree for the non- redundant haplotypes
set is depicted in Fig. 2. Almost identical results were
obtained in the four Bayesian runs, and posterior probabilities for each clade are given in Fig. 2. Eight of the
eleven non-focal taxa were phylogenetically outside the
focal group, but L. ramirezae was recovered as the sister
taxon of one of the two main clades recovered for the
ingroup taxa (northern clade) with strong support (posterior probability [PP] D 1.0). The non-focal taxa L. robertmertensi and L. saxatilis are nested within the
northern clade, both with strong support (Fig. 2). All of
the L. bibronii samples were grouped into two main
clades (northern and southern) with strong support
(Fig. 2, PP D 1.0), but there is extensive structure within
each. All L. bibronii haploclades were numbered (L.
bibronii 1) to facilitate description and discussion, except
for the group that includes all haplotypes from animals
collected from the type locality (Puerto Deseado in Santa
Cruz Province); these are called L. bibronii.
The northern clade includes two well-supported groups;
one recovers four haploclades (Fig. 2 : L. bibronii 9, 10, 11,
12; PP D 1.0) from central and northern areas of the Neuquén Province (Fig. 1), and the second clade includes L.
robertmertensi as the sister taxon of (L. gracilis + L. bibronii
8) (PP D 0.99). Two haplotypes corresponding to L. bibronii
8 and the sequence from L. saxatilis are nested within the
group that includes all sequences from L. gracilis (PP D 1.0).
The Wrst phylogenetic split within the southern clade
separates L. bibronii 7 (localities 35 and 62, west of Rio
Negro Province, Fig. 1) from a group containing the
remaining species, which are divided into two haploclades: one consists of samples north of L. bibronii 7
(localities 35 and 62, Fig. 1) with L. bibronii 3, L. bibronii
4, L. bibronii 5 and L. bibronii 6 (PP D 0.78), and the second contains samples south of L. bibronii 7 and including
L. bibronii, L. bibronii 1 and L. bibronii 2 (PP D 1.0). The
MP search recovered one tree (L D 1063, CI D 0.420,
RI D 0.563), which was almost identical to the consensus
Bayesian tree, the genetic algorithm tree, and the ML tree
(¡ln D 5527.89411). We present only the ML tree here
(Fig. 3), but all recovered phylogenetic relationships are
similar to those depicted in Fig. 2. Exceptions are relationships between haploclades L. bibronii 9, 10 and 11 in
the northern clade.
3.2. Nested-clade phylogeographic analyses
Application of the Templeton et al. (1992) algorithm
(as implemented in TCS) to the 66 cyt-b haplotypes of L.
bibronii (Appendix B) showed that sequences diVering by
up to 10 substitutions have at least a 0.95 probability of
being parsimoniously connected. L. bibronii 2, 5, 6, and 7
are represented by unique haplotypes, and haplotype
networks of L. bibronii and L. bibronii 1, 3, 4, 8, 9, 10, 11,
and 12 are represented in Fig. 4. The limits of individual
networks were largely congruent with the haploclades
960
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
Fig. 2. Neighbor joining distance tree including all non-redundant haplotypes. Numbers above branches represent posterior probabilities. Terminal symbols correspond with those in Fig. 1.
from the phylogenetic analyses (Figs. 2 and 3). A single
network (Fig. 5) linked all haplotypes of L. gracilis
(Appendix B), two haplotypes from L. bibronii 8 (loc. 36),
and the haplotype from L. saxatilis, in agreement with the
phylogenetic results. Small sample sizes for most of the L.
bibronii networks precluded NCPA analyses, but these
were performed for L. bibronii nested clade 2–1 (Fig. 4)
and the total cladogram and for the L. gracilis clade
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
961
Fig. 3. Maximum likelihood tree based on single haplotypes from each of the major haploclades shown in Fig. 2. Numbers above branches represent MP
and ML bootstrap support, respectively, and numbers below branches represent Bayesian and genetic algorithm generated posterior probabilities.
(Fig. 5), and inferences from these analyses are summarized in Table 2. For L. bibronii N (clade 2–1, Fig. 4, Table
2) it was not possible to discriminate isolation by distance
and long-distance colonization, and due to a geographic
sampling gap between the northern and southern haplogroups of L. bibronii (sensu estricto) (Fig. 1, white squares
in Río Negro correspond to L. bibroni N and white
squares in Sta. Cruz and Chubut correspond to L. bibroni
S), it was not possible to discriminate between contiguous
range expansion, long distance colonization, and past
fragmentation. Two clades (clade 3–5 and clade 4–1,
Fig. 5, Table 2) of L. gracilis showed evidence for a contiguous range expansion, and for clade 4–2 restricted gene
Xow with isolation by distance was inferred.
3.3. Neutrality tests and molecular diversity analysis
Results of the McDonald–Kreitman tests are consistent with neutral evolution expectations in all sister-clade
comparisons within the ingroup, and for the ingroup as a
whole compared with the outgroups. Estimations of haplotype and gene diversity and , as well as results of Tajima and Fu tests and the raggedness index, are
summarized in Table 3. All the M ( D Nm) values between
the identiWed clades were <1.00, and results of the
AMOVA analyses are summarized in Table 4. In the Wrst
AMOVA most of the genetic variation was strongly partitioned between the northern and southern clades (72%).
In the southern and northern clades (second and third
AMOVAS) the within-population variation was very low
(9.54 and 2.97%, respectively), and genetic diversity was
approximately equally distributed among groups (46.46
and 58.14%, respectively) and among populations within
groups (44 and 38.89%, respectively).
The average pairwise genetic distances (uncorrected
and corrected) between L. bibronii, L. gracilis and all
other recovered clades are summarized in Table 5. Group
comparisons within the northern clade had higher pairwise p values (min. 2.94–11.44% max) than those within
the southern clade (1.81–7.29%). All comparisons
962
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
Fig. 4. Nine TCS networks obtained for Liolaemus bibronii (L. bibronii, 1, 3, 4, 8, 9, 10, 11, 12). Designations of diVerent haplotypes within these, as well as
their frequencies and geographic locations, are summarized in Appendix B.
between the northern and southern groups are above
9.17% and up to 15.22%.
3.4. Coalescent estimation of population divergence times
and migration rates
The MDIV-based migration estimates (M) between L.
gracilis and L. bibroni 8, when Mmax was set to 1, 5 or 10
and Tmax set to 5 or 10, ranged from 0.12 to 0.20, and the 95%
conWdence intervals did not include zero nor one (0.04–0.98).
In all these cases, the estimated T had a peak of maximum
likelihood ranging from 1.25 to 1.52 but the density curve did
not go to zero oV to the right. This means that other higher
values of T, except those close to zero, are also compatible
with the data. When we set Mmax D 0 and Tmax D 1 or 10, estimates of T ranged from 0.49 to 0.52 with conWdence intervals
(0.27–0.89). Estimation of TMRCA in all cases ranged from
1.8524 to 1.9312, as expected these values are bigger than the
estimates of T. Although migration estimates are small, these
results are consistent with limited dispersal. To further
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
963
Fig. 5. Network of Liolameus gracilis. Haplotype 9 (star) corresponds to a L. saxatilis individual shared with one L. gracilis individual. Haplotypes 19 and
20 (circles) correspond to L. bibronii 8 individuals.
examine the migration hypothesis we compared the likelihood value at M D 0.002 with the maximum-likelihood estimate of M in a log likelihood ratio. If the log likelihood ratio
is suYciently large, we can reject the hypothesis of M D 0. We
obtained a value of ¡0.84 that based on the simulations presented in Nielsen and Wakeley (2001), are consistent with
small amounts of gene Xow, probably due to migration
events occurring at or near the original divergence time. Con-
sidering migration, one of the estimates of T was 1.46, which
gives an estimate of 2.6–4.0 mys of divergence between these
two populations, consistent with the estimates derived from
the genetic distance (4.7%, Table 5) comparison (2.3–3.6 mys).
If we make the same estimation of years but considering the
estimates of T without migration (T D 0.522), then the estimated divergence time between these populations ranges
between 917,000 years to 1.4 mys; considerably younger than
964
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
4.2. Population histories of L. bibronii haploclades and L.
gracilis
Table 2
Clades identiWed by resolved haplotype network/nesting design (Figs. 4
and 5) for which statistically signiWcant clade distances (Dc) and/or nested
clade distances (Dn) were obtained
Clade
L. bibroni
Clade 2-1 (north)
Total Cladogram
L. gracilis
Clade 3-5
Clade 4-1
Clade 4-2
2
p
Inference chain
29.25
21
0.0002
0.0004
1-2-3-5-6-7-8: IbD or LDD
1-19: CRE, LDC or PF
48
16
35
0.048
0.0785
0.0232
1-2-11: RE-12: CRE
1-19-20-2-11 :RE -12: CRE
1-19-20-2-11-17-4: RGFwIbD
The mtDNA gene tree for L. bibronii is clearly paraphyletic; it is composed of many allopatric haploclades, some
of which are closer to haplotypes of other species than to
some other L. bibronii haplotypes. With respect to genetic
diVerences, the levels of mtDNA sequence divergence in
most of these haplotypes are similar to those found among
species in other lizard genera (Carranza et al., 2000; Harris,
2002). The L. bibronii complex includes reciprocally monophyletic northern and southern haploclades separated by
the central region of Neuquén Province (Fig. 6; Catan Lil
Mountains). This is an area in which the geological and
topographic character of the Andes changes dramatically,
and mean elevation declines by some 2000 m (Rabassa and
Clapperton, 1990), evidently coincident with a thinner and
younger portion of continental crust (Clapperton, 1993).
The southern clade includes L. bibronii (sensu stricto),
which contains two haploclades (Fig. 2), L. bibronii S (the
area of the type locality) and L. bibronii N (corrected pairwise genetic diVerence 0.93%), but with a considerable
unsampled area between them (LbN and LbS; Fig. 6); most
probably this is why despite statistical signiWcance NCPA
was unable to distinguish range expansion, long colonization or a past fragmentation (Table 2) as explanations of
this pattern. Estimates of gene and nucleotide diversity for
clade 2–1 (L. bibronii N) are almost double those from the
south (L. bibronii S); the Fu test was signiWcant (Table 3),
and isolation-by-distance or long-distance colonization
were inferred as likely causes for this structure (Table 2).
We favor the Wrst of these two interpretations because the
second may be an artifact of insuYcient sampling and/or
extinction of intermediate populations (Masta et al., 2003).
Also included in the southern clade are L. bibronii 3 and
L. bibronii 4. These sister clades are geographically close
The nested phylogenetic clade analysis (NCPA) inferences are: IbD, isolation
by distance; LDD, long distance dispersal; RE, range expansion; CRE, continuous range expansion; LDC, long distance colonization; RGFwIbD,
restricted gene Xow with isolation by distance; and PF, past fragmentation.
expected for the level of genetic divergence exhibited by these
populations. These results also favor the hypothesis of small
amount of gene Xow between these populations.
4. Discussion
4.1. General patterns
We have produced a mtDNA phylogeny for lizards of
the L. bibronii and L. gracilis species groups in southern
South America. A substantial phylogenetic break separates
L. bibronii into two main clades, each of which is further
split into geographically isolated haploclades. The following discussion highlights the main features of the phylogeography of L. bibronii and L. gracilis, speciWcally
examining: (4.2) the distribution of the major haploclades
with inferences of their population histories; (4.3) the concordance of these clades’ distributions and histories with
other lizard complexes studied with the same markers and
methods; and (4.4) taxonomic implications of these results.
Table 3
Estimates of gene and nucleotide diversity () and two diVerent estimates of the parameter theta ( and s) for the diVerent clades identiWed in the phylogenetic analyses
L. bibroni
L. b.N
L. b. S
L. bibroni 1
L. bibroni 3
L. bibroni 4
L. bibroni 8
L. bibroni 9
L. bibroni 10
L. bibroni 11
L. bibroni 12
L. gracilis
N
Gene diversity
Nucleotide
diversity (in %)
s
Tajima’s D
Fu’sF
SSD’p
Rgg
21
12
9
11
8
17
11
10
6
6
5
32
0.900 (0.0022)
0.9242 (0.0575)
0.5556 (0.1653)
0.782 (0.0086)
0.893 (0.0124)
0.904 (0.0033)
1.000 (0.0015)
0.378 (0.0329)
0.933 (0.0148)
1.000 (0.0093)
0.900 (0.0259)
0.974 (0.0003)
0.8378 (0.4726)
0.4967 (0.3135)
0.1739 (0.1436)
0.6290 (0.3865)
0.9120 (0.5585)
0.3004 (0.2033)
1.3120 (0.7456)
0.0659 (0.0751)
0.3295 (0.2480)
0.6370 (0.4292)
0.2636 (0.2172)
1.4877 (0.7823)
5.08571 (2.8684)
3.0151 (1.9029)
1.0555 (0.8715)
3.8181 (2.3458)
5.5357 (3.3900)
1.8235 (1.2342)
7.9636 (4.5256)
0.4000 (0.4556)
2.0000 (1.5055)
3.8666 (2.6054)
1.6000 (1.3185)
9.0302 (4.7486)
5.28109 (2.0900)
3.6425 (1.7222)
1.4717 (0.9054)
4.4384 (2.0730)
5.7851 (2.8223)
3.8453 (1.6730)
7.8525 (3.4213)
0.7069 (0.5387)
2.6277 (1.5530)
4.8175 (2.5951)
1.9200 (1.2666)
13.1603 (4.3174)
¡0.1383 N.S.
¡0.71664 N.S.
¡1.14944 N.S.
¡0.6116N.S.
¡0.2205 N.S.
¡1.9925¤
0.0646 N.S.
¡1.4008 N.S.
¡1.3673 p D 0.055
¡1.1936N.S.
¡1.0938 N.S.
¡1.1605 N.S.
¡1.4478 N.S.
¡2.54831¤
0.45295 N.S.
1.1618N.S.
¡0.2733 N.S.
¡7.6912¤¤¤
¡5.0070¤¤
¡1.1639¤
¡2.1119¤
¡2.6960¤
¡1.4047 N.S.
¡10.4781¤¤¤
0.38
0.08
0.39
0.26
0.07
0.40
0.68
0.36
0.39
0.50
0.62
0.21
0.0373 N.S.
0.1099 N.S.
0.166 N.S.
0.1626 N.S.
0.07 N.S.
0.092 N.S.
0.023 N.S.
0.182 N.S.
0.20 N.S.
0.079 N.S.
0.15 N.S.
0.014 N.S.
Tajima’s D-statistic and Fu’s Fs test with associated level of signiWcance. SSD’p, sum of square deviations’ probability for mismatch analyses. Rgg, raggedness index with its associated probability, in bold probabilities <0.05. N.S., not signiWcant.
¤
p < 0.05.
¤¤
p < 0.01.
¤¤¤
p < 0.001.
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
965
Table 4
AMOVA for L. bibroni (group 1: southern clade; group 2: northern clade); South clade (group 1: L. bibroni, L. bibroni 1, L. bibroni 2; group 2: L. bibroni
3, L. bibroni 4; group 3: L. bibroni 5, L. bibroni 6); North clade (group 1: L. bibroni 9, L. bibroni 10, L. bibroni 11; group 2: L. bibroni 12)
Source of variation
L. bibroni
Among populations
Within populations
South clade
Among groups
Among populations within groups
Within populations
North clade
Among groups
Among populations within groups
Within populations
d.f
Sum of squares
Variance components
Percentage of variation
Fixation indexes
1
19
20
30.052
20.806
50.857
2.8152
1.09503
3.91025
72
28
Fst: 0.71996, p < 0.000
2
4
55
442.886
273.880
103.911
9.20190
8.71313
1.88930
46.46
44.00
9.54
Fct: 0.46464, p < 0.01
Fsc: 0.82181, p < 0.01
Fst: 0.90460, p < 0.01
1
2
23
199.322
160.715
19.667
16.75915
11.21189
0.85507
58.14
38.89
2.97
Fct: 0.58139, p D 0.32
Fsc: 0.92914, p < 0.01
Fst: 0.97034, p < 0.01
Table 5
Above the diagonal uncorrected average pairwise distances in percentages (Pi xy)
Diagonal: percentage of divergence within each haploclade. Below the diagonal: corrected average pairwise distances (Pi xy ¡ (Pi x + Pi y)/2). Light shading: pairwise comparisons within the southern clade. Dark shading: pairwise comparisons within the northern clade. (Bold) Largest and smallest values for
comparisons within the north and south clades.
(Fig. 6) and are the only haploclades not completely allopatric; haplotypes from both groups are sympatric at locality 27 (Fig. 1). The ancestral haplotypes of L. bibronii 4 are
from localities 30 and 31 (Figs. 1 and 4), and its network
presents the typical star design of population expansion.
Neutrality tests were signiWcant, and haplotype diversity is
high but nucleotide diversity low (Table 3). All of these
observations suggest a range expansion for L. bibronii 4,
which may have brought this group into contact with L.
bibronii 3 in locality 27. The pairwise diVerence between
these clades is 3.27 % (corrected) suggesting that these may
represent diVerent lineages, but more sampling in the overlapping area and independent markers are necessary to
assess the interactions between these two lineages.
Liolaemus bibronii 5 and L. bibronii 6 are geographically
close and represented by only one individual each. In the
phylogeny (Fig. 3) they are hypothesized to be the sister
clade of the (L. bibronii 3 + L. bibronii 4) clade, from which
they are separated by the Limay River. They are geographically close to the samples from L. bibronii 12 (part of the
northern clade), but isolated by the north-south axis of the
Catan Lil Mountains (Fig. 6). Further sampling mainly
south of this area is needed to study the relationships
between these samples and among these and the other L.
bibronii clades.
The last lineage included in the southern clade of the
complex is L. bibronii 7, represented only by three individuals from localities 35 and 62 (Fig. 1). It is the sister lineage
to the rest of the southern clade (Figs. 2 and 3) of the
complex, although its phylogenetic position is not strongly
supported. The geographic area between these localities
and L. bibronii 3 and L. bibronii 4 in the north includes the
Mesaniyeu Mountains (Fig. 6) and several adjacent plateaus, which most probably are barriers separating these
populations. The corrected pairwise diVerence of L. bibronii
7 is very high compared with any of the other groups of the
southern clade (5.21–7.29%, Table 5), suggesting that it may
represent a distinct species.
Three outgroup species (L. ramirezae, L. saxatilis & L.
robertmertensi) are distributed farther north than any of the
966
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
Fig. 6. Approximate geographic distribution of L. gracilis and main groups of the L. bibronii complex (in blue) and some concordant haplogroups from
other Liolaemus complexes. Provincial codes, MZA D Mendoza; SLS D San Luis; LPA D La Pampa; BAS D Buenos Aires; NQN D Neuquén; RNG D Rio
Negro; CHT D Chubut; STA D Santa Cruz.
L. bibronii complex haploclades identiWed in this study.
Also, all samples of L. gracilis fall within the northern clade
of the L. bibronii complex as the sister group to L. bibronii
8, the northernmost populations of the L. bibronii complex
(Fig. 6). These two clades have the highest haplotype and
nucleotide diversities, but only for L. bibronii 8 was Fu’s
test signiWcant (Table 3). In L. gracilis the genetic structure
returns NCPA inferences of range expansions and
restricted gene Xow with isolation by distance. It is probable that a combination of diVerent factors generated these
inferred processes, including marine ingressions that intermittently bisected the current distribution, and climatic
changes associated with glacial periods.
The L. saxatilis haplotype and two haplotypes of
L. bibronii 8 (Fig. 1, loc. 36) are recovered within the
L. gracilis samples. In the L. gracilis network the L. bibronii
8 haplotypes are in a terminal position, and these haplotypes were sampled from the middle of the L. gracilis distribution. These observations together with the coalescent
results that detect small amounts of gene Xow, suggest that
introgression (probably historical) between L. gracilis and
L. bibronii 8 may be the process responsible for this pattern
(vs. incomplete sorting or selective sweeps). In contrast, the
L. saxatilis haplotype recovered within the L. gracilis samples is from a locality further north than our L. gracilis
sampling distribution (Fig. 1), and in the L. gracilis
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
network it is an internal haplotype shared with an individual of L. gracilis from locality 8 (Fig. 1), the most southwestern locality of the L. gracilis distribution (»650 km
from the nearest L. saxatilis location). Although one individual of L. gracilis was found near the locality of L. saxatilis (Morando and Avila, 1999), which precludes
discarding an old introgression event, our observations are
consistent with expectations of incomplete lineage sorting
and retention of ancestral haplotypes (L. saxatilis) in
L. gracilis.
Populations of L. bibronii from central and northern
Neuquén (Lb9–Lb12) are recovered in a monophyletic
group within the northern clade, with L. bibronii 12 from
central Neuquén falling outside a group containing the
remaining haplotypes (Figs. 1 and 2). The corrected pairwise
diVerence of L. bibronii 12 relative to the three other haploclades in the north is 8.37–10.07% (Table 5), while between
L. bibronii 9, 10 and 11, it ranges 2.94– 4.02% (Table 5). In
northern Neuquén there are several high mountain chains
(to 4,000 m) running N–S between the areas of these three
haploclades, for which the Fu test was signiWcant and the
nucleotide diversity was low. However, the gene diversity
was very low only for L. bibronii 9, but sample sizes are the
lowest of the entire L. bibronii complex (Table 1). It is probable that these mountains act as barriers to these populations, but more sampling and independent markers are
needed to identify geographic limits of these three.
4.3. Concordance of L. bibronii haploclades’ distributions
and histories with other lizard complexes
Liolaemus bibronii S has a similar distribution to some
populations of L. Wtzingerii (Fig. 6; Avila et al., 2006),
which are also characterized by very low haplotype and
nucleotide diversities. These concordant distributions for
this area in these phylogenetically unrelated complexes
support the hypotheses proposed for L. Wtzingeri (Avila
et al., 2006); the central area of Chubut Province has been
an historical isolating barrier for multiple taxa. For small
rodents of the genus Phyllotys, Kim et al. (1998) found
evidence that the Chubut River (Fig. 6) may have been
such a barrier; this river Xows eastward from the Andean
glacial Welds (Flint and Fidalgo, 1968) and was an important historical drainage as well as the latitudinal boundary between tundra (to the south) and wetter, warmer
conditions to the north (Iriondo and Garcia, 1993;
Clapperton, 1993). Pleistocene landscape divisions are
still undeWned in this part of Patagonia, but presumably
the warm-adapted biota shifted from west-to-east and
contracted from south-to-north during periods of glacial
advance. These boundaries almost certainly shifted westward and southward again during glacial retreat and
warming; further sampling of L. bibronii and other taxa
in this region will allow more precise testing of this population-expansion hypothesis.
Liolaemus bibronii N has a similar approximate distribution with the ranges of two other two Liolaemus com-
967
plexes previously studied with mtDNA markers
(L. petrophilus N (Morando et al., 2003); and L. melanops
N (Avila et al., 2006), (Fig. 6), and for which range expansions were hypothesized. Between L. bibronii N and
L. bibronii 1 is the Somuncura Plateau (Fig. 6), a 2000 m
high tableland that most probably acts as a barrier, and
the connection between L. bibronii N and S may have
been through the east, as hypothesized for a secondary
zone of contact in the L. Wtzingerii complex (Avila et al.,
2006). South of the Somuncura Plateau, the distribution
of L. bibronii 1 is similar to L. melanops S (Avila et al.,
2006) and L. petrophilus S (Morando et al., 2003); all
three groups have average pairwise distances between
their northern and southern populations of »3.5%. For
the petrophilus and melanops complexes range expansion
was inferred, and although small sample sizes for many
L. bibronii clades precluded any NCPA inferences, it was
not possible to reject the range-expansion hypothesis on
the basis of mismatch-distribution tests (Table 3). These
concordant geographic patterns north and south of the
Somuncura Plateau, population histories of range expansion and similar genetic distance estimates suggest that
these three phylogenetically unrelated complexes were
aVected in similar ways, and possibly at similar points in
time (see below), by a shared set of environmental and
orogenic factors.
There are two possible areas for the ancestral population
of the southern clade: 1-south west of Rio Negro province
and diVerent expansions towards the north (L. b. 3, L. b. 4,
L. b. 5, L. b. 6) and the south (L. b., L. b. 1, 2), which promoted the diversiWcation of these diVerent lineages; or 2 central/south Neuquén (locs. 33–34, L. bibronii 5 and 6,
Fig. 1), with diVerent expansions towards the south and
subsequent isolation and divergence of the groups in the
southern clade of the complex. In the elongatus and kriegi
complexes evidence suggests that ancestral populations also
inhabited this area in Neuquén, and later expansions
allowed extension of these groups to more southern areas
(Morando et al., 2003; unpublished). Thus there also seems
to be a concordant pattern between these complexes and
some groups of the L. bibronii southern clade.
The geographical separation between the northern and
southern clades of the L. bibronii complex is in central
Neuquén Province, speciWcally north and south of the
Catan Lil Mountain Range (Fig. 6); this range seems to be
a barrier that separates L. bibronii 5 (southern clade) from
L. bibronii 12 (northern clade). It is very interesting to
note that in the elongatus and kriegi complexes this mountain chain also constitutes a geographic barrier, separating two diVerent groups in both complexes (Morando
et al., 2003; Morando et al., unpublished). In this case,
both L. elongatus and L. kriegi (sensu stricto) are widely
distributed to the south, while geographically more
restricted and strongly diVerentiated clades of both species are isolated to the north of Catan Lil Mountains. In
this region, L. bibronii 12 has a concordant distribution
with some diVerentiated populations in the elongatus,
968
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
petrophilus and kriegi complexes (Morando et al., unpublished), suggesting that this area had enough separation in
space and time to promote diVerentiation in all of these
complexes. Furthermore, in northern Neuquén province
in the areas of L. bibronii 9, 10, and 11, there are also
diVerentiated clades from the elongatus and kriegi complexes (Morando et al., unpublished).
In the same geographic area where we hypothesized
introgression between L. gracilis and L. bibronii 8, we
also found strong evidence for introgression in the
Liolaemus darwinii complex (a species complex included
in the Eulaemus clade and phylogenetically very distant
from the bibronii complex; Fig. 6 in Morando et al.,
2004); speciWcally, some L. grosseorum individuals are
characterized by L. darwinii mtDNA haplotypes. Part of
the distribution of L. gracilis mapped in this study overlaps L. grosseorum (Lb8 in Fig. 6; and see Morando et al.,
2004), for which a range expansion was also inferred and
the average genetic divergence estimates between
L. gracilis-L. bibronii 8 and L. grosseorum-L. darwini, are
very similar (4.7 and 5.2%, respectively). The L. darwinii
complex and the L. bibronii–L. gracilis complexes belong
to phylogenetically distantly related groups (Schulte
et al., 2000), and this evidence suggests that their populations may have been aVected in similar ways by the processes that inXuenced their distributional shifts in the
past.
In the diVerent clades of the L. bibronii complex we
have found concordance with other Liolaemus complexes
(elongatus, kriegi, petrophilus, darwinii, grosseorum, and
melanops), both in geographic distributions (allopatric
clades) as well as in inferred processes (range expansions,
secondary contact and possible hybridization/introgression, incomplete lineage sorting), and current or historical isolation by geographic barriers. Although these are
crude qualitative observations, they suggest that the geological and environmental factors that aVected these
landscapes may have produced similar eVects on population histories of these complexes. We acknowledge that
interpretation of geographically structured single-locus
genetic variation is complicated by inXuences from a
variety of processes, including demographic and coalescent stochasticity, incomplete lineage sorting, sampling
eVects, and phylogenetic uncertainty (Carstens et al.,
2005). Reciprocal monophyly for the mtDNA locus alone
will, for example, lead to overestimates of divergence
times (Edwards and Beerli, 2000), and diVerences in
ancestral population sizes may produce phylogeographic
structure that is not reXected in other loci (Avise, 2000).
However, these exploratory investigations are crucial to
provide starting points for testing speciWc hypotheses
with approaches that can accommodate some of this
uncertainty. As another example, coalescent methods
based on unlinked loci permit reWned estimates of ancestral population sizes and divergence times (Jennings and
Edwards, 2005; Doman and Moritz, 2006). New
approaches also oVer means to testing multi-species phy-
logeographic divergence in space (LaPointe and Rissler,
2005) and time (Hickerson et al., 2006), and are being
implemented in our group for co-distributed Andean species of Liolaemus (Victoriano et al., in prep).
4.4. Species boundaries and divergence times
This analysis reveals deep mtDNA subdivisions within
L. bibronii (most of which are morphologically cryptic),
until now considered one species. Do these genetically distinct groups represent independently evolving populational
lineages, or could some of them simply represent single
gene sorting of local populations that may otherwise be
inter-connected by gene Xow (Irwin, 2002) and subject to
merger on secondary contact (Avise et al., 1998; Daniels
et al., 2005; Wake and Schneider, 1998)? In some cases large
geographic distances separate haploclades, and outgroup
species are deeply nested within the focal taxa; in these
cases distinct mtDNA haploclades may actually represent
diVerent species (e.g. L. bibronii, L. bibronii 7, L. bibronii 8,
L. bibronii 12). Other cases are far less certain (L. bibronii 1
& L. bibronii 2; L. bibronii 3 & L. bibronii 4; L. bibronii 5 &
L. bibronii 6; L. bibronii 9, L. bibronii 10 & L. bibronii 11),
but in all cases detailed morphological studies and independent nuclear markers (currently being developed) are
needed to delimit species.
Although we do not know the exact number of diVerent
lineages that L. bibronii may include, there is no doubt that
it contains many genetically distinct units. We have no
direct evidence for rates of cyt-b evolution in Liolaemus,
but if we use the estimate of 1.3–2% sequence divergence
per million years for iguanid mtDNA (Macey et al., 1998,
1999), and cyt-b from other reptiles (Zamudio and Greene,
1997; Malhotra and Thorpe, 2000; Giannasi, 1997), we can
place the divergences within these groups into a crude temporal framework. The genetic diVerences in cyt-b
sequences suggest that the two main clades of the complex
(south and north) diverged from each other between 7.8
and 11.5 million years ago (9.17–15.22% corrected p values,
Table 5) with the 1.3% rate, or between 4.6 and 7.6 million
years ago (with 2% rate), placing this split in the middle
Miocene. During this period a climatic deterioration
started with cooler climates. A stressing of climatic zonation occurred (Simpson, 1975; Solbrig, 1976) and, as fossil
evidence suggests (Pascual, 1984; Pascual and Ortiz Jaureguizar, 1990), the Patagonian Andes may have exerted an
appreciable rain-shadow eVect on the North Patagonian
massif, turning it into a more arid region. Most of the
divergences within the northern clade also started in the
Miocene, with the most recent between L. bibronii 10 and
L. bibronii 11 approximately 1.5–2.3 m.a. (2.94% uncorrected p, Table 5), placing this split in the very late Pliocene. Divergences in the southern clade have been far more
recent, most of them in the Pliocene or Pleistocene; the
oldest, between L. bibronii and L. bibronii 3 and L. bibronii
4, was approximately 5.6 m.a. ago, placing it at the
Miocene-Pliocene boundary. The most recent split
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
between L. bibronii 5 and L. bibronii 6, 900.000 years–
1.4 m.a. ago (1.81%, Table 5); this is the only split was
placed unambiguously in the Pleistocene. The relatively
recent climatic events of late Pliocene and Quaternary
(Simpson, 1975, 1986) might have driven (some) diversiWcation in this complex. Although mitochondrial lineages
may have predated the formation of species (biasing our
rates toward over-estimates of divergence times), if most of
the divergences in this group occurred in the Miocene,
some in the Pliocene and only one in the Pleistocene, we
suspect that there should be a general agreement between
the more distinct of these groups and actual species boundaries. Following Morando et al. (2003) we refer to these
distinct haploclades as “candidate species”, pending further study based on independent molecular and morphological data (Johnson et al., 2004). Better distributional
data and support for species boundaries will permit more
rigorous assessments of speciation rates through time
(Kozak et al., 2005, 2006), further tests of possible speciation mechanisms (Cicero, 2004; Graham et al., 2005), and
more precise estimates of Patagonia’s globally ranked
endemism (Lamoreaux et al., 2006).
Acknowledgments
We thank C. H. F. Perez, F. Torres, K. Dittmar, D.
Perez, N. Frutos, and M. Christie for assistance in Weld
collections or provision of tissues samples. Financial support was provided by Grants (PEI 0178/98, PEI 6397/01;
L. Avila), a postdoctoral fellowship (M. Morando), and a
postdoctoral fellowship (L. Avila) from Consejo Nacional de Investigaciones CientíWcas y Técnicas (CONICET), various sources from Brigham Young University
(the Kennedy Center for International Studies, the
Department of Integrative Biology, and the M. L. Bean
Life Science Museum), and NSF awards DEB 98-15881
and DEB 01-32227 to J. W. Sites, Jr. We also acknowledge the NSF “Partnership for International Research
and Education” award (OISE 0530267) for support of
collaborative research on Patagonian biodiversity,
granted to the following institutions (listed alphabetically): BYU, CENPAT, Dalhousie Univ., Darwinion
Botanical Inst., Univ. Austral de Chile, Univ. de Comahue, Univ. de Concepción, and Univ. of Nebraska. We
thank fauna authorities from Neuquén and Chubut provinces for collection permits.
Appendix A. Voucher numbers for individuals used as
outgroups
L. pictus argentinus: BYU47193. L. robertmertensi:
LJAMM 1961. L. punmahuida: FML 11958. L. neuquensis:
FML 09465. L. kingi: LJAMM 3040. L. ramirezae: BYU
47180. L. saxatilis: LJAMM 5044. L. gracilis: LJAMM
5060. L. chiliensis: chil362. L. cyanogaster: cyano380. L.
monticola villaricensis 117. L. tenuis: L-306 Chile. L. pictus
chiliensis: L-367. L. lineomaculatus: SDSU4268.
969
Appendix B
DiVerent haplotypes included in the NCPA analyses, with frequencies for
each (N) and voucher numbers with locality number in parentheses (locality numbers match those in Table 1 and Fig. 1)
Haplotype
number
N
Collection numbers
L. bibronii
1
2
3
4
5
6
7
8
9
1
2
1
1
1
3
1
2
6
10
11
2
1
fn 142 (1)
LJAMM 5903-5904 (1)
BYU 47184 (2)
BYU 47183 (1)
BYU 47182 (2)
fn 41 - LJAMM 5900-5901 (3)
LJAMM 5907 (4)
LJAMM 5908-MLP.S 2577 (4)
LJAMM 5918 (5) -5905 (57) -5912
(58) -5916 (59) - BYU 47185 - LJAMM 5913 (6)
BYU 47186-47188 (6)
LJAMM 5909 (7)
L. bibronii 1
12
13
14
15
16
1
4
1
4
1
LJAMM 5529 (17)
LJAMM 5406 (16) -5602 (20) - 5511-5692 (61)
LJAMM 5695 (20)
LJAMM 5474 (19) -5601 (20) -5490-5491 (60)
LJAMM 5600 (20)
L. bibronii 2
17
3
LJAMM 5681-5682-5693 (18)
L. bibronii 3
18
19
20
21
22
23
1
3
1
1
1
1
LJAMM 5423 (26)
LJAMM 5403-5457 (26) -5421 (27)
LJAMM 5418 (27)
LJAMM 5402 (26)
LJAMM 5538 (28)
LJAMM 5540 (28)
L. bibronii 4
24
25
26
27
28
29
30
31
32
33
34
5
1
1
1
1
1
1
1
1
3
1
LJAMM 5615-5619-5620 (30) -5648-5653 (31)
LJAMM 5647 (31)
LJAMM 5588 (32)
LJAMM 5649 (31)
LJAMM 5651 (31)
LJAMM 5654(31)
LJAMM 5617 (30)
LJAMM 5419 (27)
LJAMM 5551 (29)
LJAMM 5552 (29) -5616-5618 (30)
LJAMM 5646 (31)
L. bibronii 5
35
1
fn 933 (33)
L. bibronii 6
36
1
MIC 1645 (34)
3
3
LJAMM 2339 (35) - 5574-5575 (62)
1
1
1
1
1
1
1
LJAMM 5724 (22)
LJAMM 1387 (21)
LJAMM 5723 (22)
LJAMM 1391 (21)
LJAMM 1203 (21)
LJAMM 5725 (22)
LJAMM 1388 (21)
L. bibronii 7
37
L. bibronii 8
38
39
40
41
42
43
44
(continued on next page)
970
M. Morando et al. / Molecular Phylogenetics and Evolution 43 (2007) 952–973
Appendix B (continued )
Haplotype
number
45
46
47
48
N
Collection numbers
1
1
1
1
MIC 1615 (25)
MIC 1614 (25)
BYU 47177 (24)
BYU 47176 (23)
L. bibronii 9
49
50
1
8
51
1
LJAMM 5290 (15)
LJAMM 5258-5274-5276 (14) -5279-5280-52915292-5293 (15)
LJAMM 5275 (14)
L. bibronii 10
52
53
54
55
56
1
2
1
1
1
LJAMM 5340 (10)
LJAMM 5341-5342 (10)
LJAMM 5343 (10)
LJAMM 5344 (10)
LJAMM 5387 (11)
L. bibronii 11
57
58
59
60
61
62
1
1
1
1
1
1
LJAMM 5319 (12)
LJAMM 5321 (12)
LJAMM 5317 (12)
LJAMM 5320 (12)
LJAMM 5318 (12)
LJAMM 5323 (13)
L. bibronii 12
63
64
65
66
1
2
1
1
LJAMM 5364 (8)
LJAMM 5362 (8) - 5396 (9)
LJAMM 5395 (9)
LJAMM 5363 (8)
L. gracilis
1
2
1
4
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1
1
1
1
1
1
2
1
1
1
1
1
1
2
1
1
1
1
1
1
4
1
1
1
1
LJAMM 2640 (39)
FML10360 (37) - LJAMM 5178 (41) - 5366 (8) MIC1609 (56)
LJAMM 5367 (8)
LJAMM 5170 (38)
BYU 47169 (44)
LJAMM 5084 (45)
LJAMM 5107 (46)
LJAMM 5177 (41)
LJAMM 5368 - L. saxatilis: LJAMM 5044 (8)
LJAMM 5713 (40)
LJAMM 5701 (40)
LJAMM 5175 (41)
LJAMM 5176 (41)
LJAMM 5700 (40)
LJAMM 5699 (40)
LJAMM 5711-5712 (40)
LJAMM 2572 (42)
LJAMM 2620 (43)
L. bibronii 8: BYU 47178 (36)
L. bibronii 8: BYU 47179 (36)
BYU 47170 (53)
LJAMM 5108 (48)
BYU 47166 (47) - 47168 (54) - fn 411-412 (55)
LJAMM 5103 (52)
BYU 47167 (49)
LJAMM 5060 (50)
LJAMM 5128 (51)
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