Molecular Phylogenetics and Evolution 50 (2009) 16–30
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Molecular phylogeny and biogeography of the Cuban genus Girardinus Poey,
1854 and relationships within the tribe Girardinini (Actinopterygii, Poeciliidae)
Ignacio Doadrio a,*, Silvia Perea a, Lourdes Alcaraz a, Natividad Hernandez b
a
b
Department of Biodiversity and Evolutionary Biology, Museo Nacional de Ciencias Naturales CSIC, José Gutiérrez Abascal 2, 28006 Madrid, Spain
Instituto de Medicina Tropical Pedro Kouri, Apdo 601, Marianao 13, Ciudad de La Habana, Cuba
a r t i c l e
i n f o
Article history:
Received 26 March 2008
Revised 16 September 2008
Accepted 19 September 2008
Available online 30 September 2008
Keywords:
Girardinini
Molecular phylogeny
Biogeography
Taxonomy
Caribbean islands
a b s t r a c t
Phylogenetic relationships among members of the freshwater fish tribe Girardinini were inferred to test
existing colonization and diversification hypotheses for this group in the Caribbean. The genetic material
examined was mitochondrial (cytochrome b, 1140 bp) and nuclear (RAG-1 and b-actin, 2450 bp) DNA
from 161 specimens representing 44 ingroup and three outgroup taxa. Our mtDNA and combined data
matrix (mtDNA + nuclear DNA) results rendered a well-supported phylogeny for the tribe Girardinini
and suggest the need to review the group’s current taxonomy. From the data presented here, it may
be inferred that the Girardinini diverged from other poeciliid fishes approximately 62 Mya ago in the Palaeocene period. This estimate, however, conflicts with the hypothesis that today’s vertebrate fauna is the
result of the more recent colonization of the Antillean islands during the Early Oligocene (35–33 Mya
ago). The isolation of western, central and eastern Cuba during the Miocene and that of the Juventud
Island and Guanahacabibes Peninsula during the Pliocene, are the main geologic events that could have
promoted speciation in this group.
! 2008 Elsevier Inc. All rights reserved.
1. Introduction
The tribe Girardinini Hubbs, 1924 was proposed to comprise 6
genera and 15 species and subspecies of endemic freshwater fishes
from the main island of Cuba and Juventud island (former Pinos)
(Rivas, 1958; Barus et al., 1991). However, according to the taxonomy of Rosen and Bailey (1963), the tribe is only considered to
have three genera (Girardinus Poey, 1854; Quintana Hubbs, 1934
and Carlhubbsia Withley, 1951) and a number of species (from
ten to twelve) that depends on the describing author (Rosen and
Bailey, 1963; Parenti and Rauchenberger, 1989; Ghedotii, 2000;
Lucinda and Reis, 2005). The monotypic genus Quintana is represented by the species Quintana atrizona Hubbs, 1934 inhabiting
both Juventud Island and western Cuba Island. The genus Carlhubbsia has two recognized species: C. kidderi (Hubbs, 1936) from Mexico and Guatemala, and C. stuarti Rosen and Bailey, 1959 from
Guatemala and Belize. In turn, the genus Girardinus has eight recognized species (Lucinda, 2003): G. metallicus Poey, 1854; G. uninotatus Poey, 1860; G. creolus Garman, 1895; G. denticulatus
Garman, 1895; G. cubensis (Eigenmann, 1903); G. falcatus (Eigenmann, 1903); G. microdactylus Rivas, 1944 and G. rivasi Barus and
Wohlgemuth, 1994.
* Corresponding author. Fax: +34 915645078.
E-mail address: mcnd147@mncn.csic.es (I. Doadrio).
1055-7903/$ - see front matter ! 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2008.09.014
The genus Girardinus is among the endemic freshwater fishes
that inhabit both the islands of Cuba and Juventud. This genus
shows its highest diversity in western Cuba and is absent from central Cuba south of the Sancti-Spiritus district and from some eastern areas of Cuba Island, such as short rivers south of the Sierra
Maestra and west of Sagua-Baracoa. Compared to other freshwater
fishes of the Antillean islands, Girardinus is a good model for deciphering the evolutionary history of Cuban hydrographical systems
because of its wider range of distribution throughout Cuba, its
higher diversity, its presence on two islands of known geological
age and its ecology, being more restricted to freshwater systems.
Studies on the salt tolerance of representatives of Girardinini have
failed to recover fishes from salt, brackish, or fresh water with a Cl!
content higher than 1000 parts per million (Rivas, 1958, 1986). We
can therefore assume that dispersion in Girardinini occurs only via
connected freshwater systems (Rivas, 1958).
Models of the palaeogeography of Cuba based on biological data
can be useful for geological reconstructions, since Cuba occupies
one of the most geologically complex areas of the planet. Cuba’s
main and Juventud islands boast over 600 fluvial systems mostly
crossing karst landscapes. The country’s karst topography determines that some rivers are fragmented by waterfalls or disappear
for kilometers underground and may also be the cause of local speciation events in freshwater fishes such as Girardinus with a limited
capacity for dispersal. By identifying the barriers that caused the
fragmentation and dispersion of freshwater fishes and the timing
17
I. Doadrio et al. / Molecular Phylogenetics and Evolution 50 (2009) 16–30
of these events in a geological scenario, insight can be gained into
the evolution of organisms and areas. Effectively, studies on the
biogeography of freshwater fishes in Central America and the
Caribbean Islands have contributed significantly to palaeobiogeography models (Rosen 1975, 1978, 1985; Nelson and Rosen, 1981).
The genus Girardinus has also been examined as a means of biologically controlling disease-transmitting mosquitoes (Rodríguez
et al., 2004; Menéndez Díaz et al., 2007); other studies have focussed on its complex taxonomy (Barus et al., 1981, 1986, 1991,
1998; Barus and Libosvarsky, 1986; Barus and Wohlgemuth,
1994). Most of the latter works have focused on basic aspects such
as taxonomy, geographical distribution and ecological requirements. In contrast, there are only a few models of the phylogenetic
relationships and biogeography of this genus. The first and most
developed model is that proposed by Rivas (1958), in which the
ancestor of today’s Girardinus colonized Cuba from the eastern
Yucatan during the Upper Miocene–Pliocene across an intercontinental Yucatan-Cuba landbridge. Western Cuba is considered the
center of origin of Girardinus, where it shows its highest diversity,
and its speciation is thought to have occurred during the Pleistocene. The ancestral species of the genus is considered by Rivas
(1958) to be similar to Girardinus creolus, which is the only species
confined to upper stream reaches (Fig. 1). Rodriguez et al. (1992)
corroborated the model suggested by Rivas (1958) considering Girardinus creolus to be the ancestral species of the genus Girardinus.
An eastern Yucatan-Cuba connection was also supported by the
phylogenetic relationships proposed by Rosen and Bailey (1963),
who considered the genera Quintana from Cuba and Carlhubbsia
from eastern Yucatan and Guatemala, as sister genera of Girardinus.
However, recent studies on poeciliid fishes question the Yucatan-Cuba connection as well as the relationship of Carlhubbsia as
sister group of the genera Girardinus and Quintana (Hrbek et al.,
2007). An alternative model has been developed to explain the
presence of Poeciliidae representatives in the Antilles, including
the tribe Girardinini across GAARlandia (Greater Antilles + Aves
Ridge) (Iturralde-Vinent and MacPhee, 1999; Iturralde-Vinent,
2006). This model of geological evolution of the Caribbean basin
suggests that the Greater Antilles did not permanently emerge until the Eocene-Oligocene boundary (30–35 mya), when they were
connected to northern South America by the Aves Ridge. The three
biogeographical models (Rivas, 1958; Rosen and Bailey, 1963;
Hrbek et al, 2007) are represented in Fig. 2.
The present study was designed to test previous phylogenetic
relationships of the tribe Girardinini (Rivas, 1958; Rosen and Bailey, 1963) and the three biogeographical models proposed for the
Cuba and Juventud islands (Fig. 2) using the most complete molecular dataset for the tribe Girardinini available to date.
Neoheterandria (= Allogambusia)
A
Upper Miocene/Pliocene
~11.6-3.6 Mya
B
Eocene/Oligocene
~30-35 Mya
C
Cretaceous
~65 Mya
Chicxulub
impact
Fig. 2. Main biogeographical hypotheses proposed to explain the colonization of
the Antillean islands. (A) Biogeographical scenario in agreement with Rivas (1958).
(B) Biogeographical scenario in agreement with Iturralde-Vinent and MacPhee
(1999) (C) Biogeographical scenario in agreement with Rosen (1975). (Pictures
modified from original authors).
Toxus serripenis
Toxus creolus
Yucatan-Cuba
split
Girardinus microdactylus
2. Materials and methods
Girardinus metallicus
2.1. Sampling
Dactylophallus denticulatus
Allodontium cubense
Glaridichthys falcatus
Glaridichthys uninotatus
Pliocene
Pleistocene
Fig. 1. Taxonomy and implied phylogenetic hypothesis for the tribe Girardinini
according to Rivas (1958).
DNA samples were analyzed in 111 individuals representing all
described species of the genus Girardinus (except G. cubensis). Populations were collected in the wild from 48 localities of the Cuba
and Juventud islands including the type locality when possible (Table 1); (Fig. 3). DNA was extracted from the dorsal muscle, which
was preserved in liquid nitrogen or 70% ethanol. Voucher specimens of these species were deposited in the collections of the
Tropical Medicine Institute Pedro Kouri, La Habana, Cuba and
the Museo Nacional de Ciencias Naturales, Madrid, Spain. The
18
Table 1
Samples of the tribe Girardinini, collection sites, number of populations sampled in the phylogenetic tree and GenBank accession numbers
Locality/Source
Designation in phylogenetic tree
GenBank Acc. No. (CB/RAG1/b-actin)
New taxonomical denomination
Number in map
Alfaro cultratus
Alfaro cultratus
Alfaro cultratus
Anableps anableps
Belonesox belizanus
Brachyrhaphis cascajalensis
Brachyrhaphis hartwegi
Brachyrhaphis hartwegi
Brachyrhaphis parismina
Brachyrhaphis rhabdophora
Brachyrhaphis terrabensis
Carlhubbsia kidderi
Carlhubbsia kidderi
Carlhubbsia stuarti
Cnesterodom decemmaculatus
Crenichthys baileyi
Fluviphylax pygmaeus
Gambusia affinis
Girardinus creolus
Girardinus creolus
Girardinus creolus
Girardinus creolus (G. riddley)
Girardinus creolus
Girardinus creolus
Girardinus creolus
Girardinus creolus
Girardinus creolus (G. serripenis)
Nicaragua Lake. San Carlos. Nicaragua
Nicaragua Lake. San Carlos. Nicaragua
El Monje River. San Miguel. Nicaragua
Hrbek et al., 2007
Hrbek et al., 2007
Costa Rica
Aquarium stock
Hrbek et al., 2007
Costa Rica
Hrbek et al., 2007
Hrbek et al., 2007
Mamantel River. San Antonio. México
Mamantel River. San Antonio. México
Hrbek et al., 2007
Hrbek et al., 2007
Doadrio and Domínguez, 2004
Hrbek et al., 2007
Hrbek et al., 2007
La Legua River. Entronque de Herradura. Cuba
La Legua River. Entronque de Herradura. Cuba
La Legua River. Entronque de Herradura. cuba
San Francisco River. San Cristóbal. Cuba
San Cristóbal River. La Hoya. Cuba
San Cristóbal River. La Hoya. Cuba
San Cristóbal River. La Hoya. Cuba
San Cristóbal River. La Hoya. Cuba
Taco-Taco River. Upstream cave entrance. Rangel.
Cuba
Taco-Taco River. Upstream cave entrance. Rangel.
Cuba
Taco-Taco River. Upstream cave entrance. Rangel.
Cuba
Taco-Taco River. Downstream cave. Rangel. Cuba
Taco-Taco River. Downstream cave. Rangel. Cuba
Taco-Taco River. Upstream cave entrance. Rangel.
Cuba
Guaso River. Argeo Martínez. Cuba
Alfaro cultratus 1
Alfaro cultratus 2
Alfaro cultratus 3
Anableps anableps
Belonesox belizanus
Brachyrhaphis cascajalensis
Brachyrhaphis hartwegi 1
Brachyrhaphis hartwegi 2
Brachyrhaphis parismina
Brachyrhaphis rhabdophora
Brachyrhaphis terrabensis
Carlhubbsia kidderi 1
Carlhubbsia kidderi 2
Carlhubbsia stuarti
Cnesterodom decemmaculatus
Crenichthys baileyi
Fluviphylax pygmaeus
Gambusia affinis
TCRE1
TCRE2
TCRE3
TCRE4
TCRE5
TCRE6
TCRE7
TCRE8
TCRE9
FJ178772*/!/–*
FJ178774*/!/–*
FJ178773*/!/!
EF017511/ EF017408/!
EF017514/ EF017411/!
FJ178734*/ FJ185096*/ FJ194984*
FJ178735*/!/!
FJ178736*/!/!
FJ178737*/!/!
FJ178738*/!/!
FJ178739*/!/!
FJ178740*/ FJ185104*/ FJ194995*
FJ178741*/!/ FJ194996*
FJ178742*/!/!
Alfaro cultratus
Alfaro cultratus
Alfaro cultratus
Anableps anableps
Belonesox belizanus
Brachyrhaphis cascajalensis
Brachyrhaphis hartwegi
Brachyrhaphis hartwegi
Brachyrhaphis parismina
Brachyrhaphis rhabdophora
Brachyrhaphis terrabensis
Carlhubbsia stuarti
Carlhubbsia stuarti
Carlhubbsia stuarti
Cnesterodom decemmaculatus
Crenichthys baileyi
Fluviphylax pygmaeus
Gambusia affinis
Toxus creolus
Toxus creolus
Toxus creolus
Toxus creolus
Toxus creolus
Toxus creolus
Toxus creolus
Toxus creolus
Toxus creolus
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
9
9
9
14
15
15
15
15
12
TCRE10
FJ178743*/!/!
Toxus creolus
12
TCRE11
FJ178744*/!/!
Toxus creolus
12
TCRE12
TCRE13
TCRE14
FJ178745*/!/!
FJ178746*/!/!
FJ178747*/!/!
Toxus creolus
Toxus creolus
Toxus creolus
12
12
12
DRAM
FJ178733*/!/ FJ194993*
Dactylophallus ramsdeni
31
Aguas Azules, Las Chambas. Cuba
Yao River. Nuevo Yao. Cuba
Yao River. Nuevo Yao. Cuba
Yao River. Nuevo Yao. Cuba
Contramaestre River. Contramaestre. Cuba
Laguna River. Pilon. Cuba
Aguacate River. Mayajigua. Cuba
Malnombre River. Macahuabo. Las Tozas. Cuba
Manaquita River. Norte Viñas. Cuba
Manaquita River. Norte Viñas. Cuba
Canoao River. Esmeralda. Cuba
Bautista River. San Juan y Martinez. Cuba
Los Palacios. Cuba
Cañada del Infierno. Las Terrazas. Cuba
San Cristobal. Cuba
Hawa River. Cienaga Zapata. Cuba
Aguas Azules. Las Chambas. Cuba
Guayabo Dam. La Fe. Juventud I. Cuba
DDEN1
DDEN2
DDEN3
DDEN4
DDEN5
DDEN6
DDEN7
DDEN8
DDEN9
DDEN10
GFAL1
GFAL2
GFAL3
GFAL4
GFAL5
GFAL6
GFAL7
GFAL8
FJ178723*/!/!
FJ178724*/ FJ185102*/ FJ194991*
FJ178725*/!/!
FJ178726*/!/!
FJ178727 */!/!
FJ178728*/!/!
FJ178729*/!/!
FJ178730*/!/!
FJ178731*/!/!
FJ178732*/ FJ185100*/ FJ194989*
FJ178749*/!/!
FJ178750*/ FJ185094*/ FJ194981*
FJ178751*/!/!
FJ178752*/!/!
FJ178753*/!/!
FJ178754*/!/!
FJ178755*/!/!
FJ178756*/!/!
Dactylophallus denticulatus
Dactylophallus denticulatus
Dactylophallus denticulatus
Dactylophallus denticulatus
Dactylophallus denticulatus
Dactylophallus denticulatus
Dactylophallus denticulatus
Dactylophallus denticulatus
Dactylophallus sp
Dactylophallus sp
Glaridichthys falcatus
Glaridichthys falcatus
Glaridichthys falcatus
Glaridichthys falcatus
Glaridichthys falcatus
Glaridichthys falcatus
Glaridichthys falcatus
Glaridichthys falcatus
26
34
34
34
33
35
24
25
22
22
27
5
10
20
14
21
26
40
Girardinus creolus (G. serripenis)
Girardinus creolus
Girardinus creolus
Girardinus creolus
Girardinus creolus
Girardinus denticulatus (G.
ramsdeni)
Girardinus denticulatus
Girardinus denticulatus
Girardinus denticulatus
Girardinus denticulatus
Girardinus denticulatus
Girardinus denticulatus
Girardinus denticulatus
Girardinus denticulatus
Girardinus denticulatus
Girardinus denticulatus
Girardinus falcatus
Girardinus falcatus
Girardinus falcatus
Girardinus falcatus
Girardinus falcatus
Girardinus falcatus
Girardinus falcatus (G. atherinoides)
Girardinus falcatus
EF017508/ EF017405/!
EF017519/ EF017416/!
FJ178767*/!/!
FJ178769*/ FJ185090*/ FJ194976*
EF017521/ EF0174198/!
FJ178768*/!/!
EF017522/ EF017419/!
EF017520/ EF017430/!
FJ178777*/!/!
FJ178778*/ FJ185091*/!
EF017532/ EF017417/!
EF017529/ EF017427/!
AF510819/ FJ185089*/ FJ194975*
I. Doadrio et al. / Molecular Phylogenetics and Evolution 50 (2009) 16–30
Species
Carapechibey, Juventud I. Cuba
Carapechibey. Juventud I. Cuba
Pino Alto Lagoon. Mella. Juventud I. Cuba
Cienaga de Lanier. Cayo Potrero Juventud I. Cuba
Guanahacabibes Peninsula. Cuba
La Fe. Pinar del Río. Cuba
El Cayuco. Cuba
Hondo River. Pilotos. Cuba
Surgidero de Batabano. Batabano. Cuba
Cienaga de Lanier. Cayo Potrero. Juventud I. Cuba
San Juan River. San Juan y Martinez. Cuba
Bautista River. San Juan y Martínez. Cuba
Guayabo Dam. La Fe. Juventud I. Cuba
Surgidero de Batabano. Batabano. Cuba
Hawa River. Cienaga de Zapata. Cuba
La Pastora River. Artemisa. Cuba
La Pastora River. Artemisa. Cuba
Surgidero de Batabano. Batabano. Cuba
Cienaga de Lanier. Cayo Potrero. Juventud I. Cuba
Yaguajay. Cuba
Yaguajay. Cuba
Mayajigua. Cuba
Yao River. Nuevo Yao. Cuba
Yao River. Nuevo Yao. Cuba
Guaimaro River. Guaimaro. Cuba
Gascon River. Santiago de Cuba. Cuba
El Sábalo. Cuba
La Fe. Pinar del Río. Cuba
El Cayuco. Cuba
Barranco del Infierno. Las Terrazas. Cuba
San Francisco River. San Cristobal. Cuba
San Francisco River. San Cristobal. Cuba
Taco-Taco River. Aspiro. Cuba
Taco-Taco River. Aspiro. Cuba
Barranco del Infierno. Las Terrazas. Cuba
Barranco del Infierno. Las Terrazas. Cuba
Barranco del Infierno. Las Terrazas. Cuba
San Francisco River. San Cristobal. Cuba
San Juan River. Las Terrazas. Cuba
San Juan River. Las Terrazas. Cuba
Barranco del Infierno. Las Terrazas. Cuba
Barranco del Infierno. Las Terrazas. Cuba
San Cristobal River. La Hoya. Cuba
Cañada del Infierno. Las Terrazas. Cuba
San Juan River. Las Terrazas. Cuba
Mosquito River. Minas. Cuba
Reparto Militar Lagoon. Minas. Cuba
Canoao River. Esmeralda. Cuba
Nueva Gerona River. Nueva Gerona. Juventud I. Cuba
Demajagua River. Demajagua. Juventud I. Cuba
Las Tunas. Juventud I. Cuba
Los Colonos. Juventud I. Cuba
Mazareno River. Nueva Gerona. Juventud I. Cuba
Mazareno River. Nueva Gerona. Juventud I. Cuba
Cienaga de Lanier. Cayo Potrero. Juventud I. Cuba
Curva de la Muerte River. Juventud I. Cuba
La Fe. Juventud I. Cuba
La Fe. Juventud I. Cuba
Granja La Libertad. Juventud I. Cuba
Argelia Libre. Juventud I. Cuba
GFAL9
GFAL10
GFAL11
GFAL12
GFAL13
GFAL14
GFAL15
GMET1
GMET2
GMET3
GMET4
GMET5
GMET6
GMET7
GMET8
GMET9
GMET10
GMET11
GMET12
GMET13
GMET14
GMET15
GMET16
GMET17
GMET18
GMET19
GMET20
GMET21
GMET22
GMIC1
GMIC2
GMIC3
GMIC4
GMIC5
GMIC6
GMIC7
GMIC8
GMIC9
GMIC10
GMIC11
GMIC12
GMIC13
GMIC14
GMIC15
GMIC16
GMIC17
GMIC18
GMIC19
GRIV1
GRIV2
GRIV3
GRIV4
GRIV5
GRIV6
GRIV7
GRIV8
GRIV9
GRIV10
GRIV11
GRIV12
FJ178757*/!/!
FJ178758*/!/!
FJ178759*/ FJ185098*/ FJ194987*
FJ178760*/!/!
FJ178761*/!/ FJ194979*
FJ178762*/!/!
FJ178763*/!/ FJ194983*
FJ178653*/!/!
FJ178654*/!/!
FJ178655*/!/!
FJ178656*/!/!
FJ178657*/!/!
FJ178658*/!/!
FJ178659*/!/!
FJ178660*/!/!
FJ178661*/!/ FJ194994*
FJ178662*/!/!
FJ178663*/!/!
FJ178664*/!/!
FJ178665*/!/!
FJ178666*/!/!
FJ178667*/!/!
FJ178668*/ FJ185103*/ FJ194992*
FJ178669*/!/!
FJ178670*/!/!
FJ178671*/!/!
FJ178672*/ FJ185095*/ FJ194982*
FJ178673*/!/!
FJ178674*/!/!
FJ178675*/!/!
FJ178676*/!/!
FJ178677*/!/!
FJ178678*/ FJ185097*/ FJ194985*
FJ178679*/!/–
FJ178680*/!/–
FJ178681*/!/!
FJ178682*/!/!
FJ178683*/!/!
FJ178684*/!/!
FJ178685*/!/!
FJ178686*/!/!
FJ178687*/!/!
FJ178688*/!/!
FJ178689*/!/–
FJ178690*/!/!
FJ178691*/!/!
FJ178692*/!/!
FJ178693*/ FJ185101*/ FJ194990*
FJ178694*/!/–*
FJ178695*/!/!
FJ178696*/!/!
FJ178697*/!/!
FJ178698*/!/!
FJ178699*/!/!
FJ178700*/!/!
FJ178701*/ FJ185099*/ FJ194988*
FJ178702*/!/!
FJ178703*/!/ FJ194986*
FJ178704*/!/!
FJ178705*/!/!
Glaridichthys falcatus
Glaridichthys falcatus
Glaridichthys falcatus
Glaridichthys falcatus
Glaridichthys falcatus
Glaridichthys falcatus
Glaridichthys falcatus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus metallicus
Girardinus microdactylus
Girardinus microdactylus
Girardinus microdactylus
Girardinus microdactylus
Girardinus microdactylus
Girardinus microdactylus
Girardinus microdactylus
Girardinus microdactylus
Girardinus microdactylus
Girardinus microdactylus
Girardinus microdactylus
Girardinus microdactylus
Girardinus microdactylus
Girardinus microdactylus
Girardinus microdactylus
Girardinus microdactylus
Girardinus sp
Girardinus sp
Girardinus sp
Girardinus rivasi
Girardinus rivasi
Girardinus rivasi
Girardinus rivasi
Girardinus rivasi
Girardinus rivasi
Girardinus rivasi
Girardinus rivasi
Girardinus rivasi
Girardinus rivasi
Girardinus rivasi
Girardinus rivasi
44
44
42
43
1
2
3
8
21
43
6
5
41
20
21
19
19
20
43
23
23
24
34
34
30
32
4
2
3
17
14
14
11
11
17
17
17
14
17
17
17
17
15
17
18
29
28
27
36
47
48
38
37
37
43
41
39
39
46
45
(continued on next page)
19
falcatus
falcatus
falcatus
falcatus
falcatus
falcatus
falcatus
metallicus
metallicus
metallicus
metallicus
metallicus
metallicus
metallicus
metallicus (G. pygmaeus)
metallicus
metallicus
metallicus
metallicus
metallicus
metallicus
metallicus
metallicus
metallicus
metallicus
metallicus
metallicus
metallicus
metallicus
microdactylus
microdactylus
microdactylus
microdactylus
microdactylus
microdactylus
microdactylus
microdactylus
microdactylus
microdactylus
microdactylus
microdactylus
microdactylus
microdactylus
microdactylus
microdactylus
microdactylus
microdactylus
microdactylus
rivasi
rivasi
rivasi
rivasi
rivasi
rivasi
rivasi
rivasi
rivasi
rivasi
rivasi
rivasi
I. Doadrio et al. / Molecular Phylogenetics and Evolution 50 (2009) 16–30
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
Girardinus
20
Table 1 (continued)
Locality/Source
Designation in phylogenetic tree
GenBank Acc. No. (CB/RAG1/b-actin)
New taxonomical denomination
Number in map
Girardinus rivasi
Girardinus rivasi
Girardinus uninotatus
Girardinus uninotatus
Girardinus uninotatus
Girardinus uninotatus
Girardinus uninotatus
Girardinus uninotatus
Girardinus uninotatus
Girardinus uninotatus
Girardinus uninotatus
Girardinus uninotatus
Girardinus uninotatus
Girardinus uninotatus
Girardinus uninotatus
Girardinus uninotatus
Girardinus uninotatus
Heterandria bimaculata
Heterophallus milleri
Limia dominicensis
Limia melanogaster
Limia tridens
Limia vittata
Limia vittata
Neoheterandria tridentiger
Phallopytchus januarius
Phallichthys amates
Phallichthys pittieri
Phallichthys pittieri
Phallichthys tico
Phallichthys tico
Poecilia latipunctata
Poecilia mexicana
Poecilia reticulata
Poeciliopsis fasciata
Priapella compressa
Priapella intermedia
Priapella olmecae
Priapichthys festae
Quintana atrizona
Quintana atrizona
Scolichthys greenwayi
Xenodexia ctenolepis
Xenophalus umbratilis
Xenophalus umbratilis
Xiphophorus helleri
Xiphophorus sp
Guayabo Dam, La Fe. Juventud I. Cuba
La Fe. Juventud I. Cuba
Santiago River. Soroa. Cuba
Santiago River. Soroa. Cuba
Santiago River. Soroa. Cuba
Santiago River. Soroa. Cuba
Guaimaro River. Guaimaro. Cuba
Hawa River. Cienaga de Zapata. Cuba
Barranco del Infierno. Las Terrazas. Cuba
Pinar del Río. Cuba
Pinar del Río. Cuba
Barranco del Infierno. Las Terrazas. Cuba
Santiago River. Soroa. Cuba
San Francisco River. San Cristobal. Cuba
San Francisco River. San Cristobal. Cuba
La Legua River. Entronque de Herradura. Cuba
La Legua River. Entronque de Herradura. Cuba
Hrbek et al., 2007
Hrbek et al., 2007
Hrbek et al., 2007
Hrbek et al., 2007
Hrbek et al., 2007
La Boca Lagoon. Camagüey. Cuba
Abra River. Juventud I. Cuba
Hrbek et al., 2007
Hrbek et al., 2007
Hrbek et al., 2007
Sardinas River. Colon. Nicaragua
Sardinas River. Colon. Nicaragua
Hrbek et al., 2007
Nicaragua Lake. Nicaragua
Hrbek et al., 2007
Col River. Veracruz. Mexico
Hrbek et al., 2007
Hrbek et al., 2007
Hrbek et al., 2007
Hrbek et al., 2007
Hrbek et al., 2007
Hrbek et al., 2007
Hrbek et al., 2007
Guanahacabibes Peninsula. Cuba
Hrbek et al., 2007
Hrbek et al., 2007
Hrbek et al., 2007
El Monje River. San Miguel. Nicaragua
Hrbek et al., 2007
Tabar River. Tabara de abajo. Dominican Republic
GRIV13
GRIV14
GUNI1
GUNI2
GUNI3
GUNI4
GUNI5
GUNI6
GUNI7
GUNI8
GUNI9
GUNI10
GUNI11
GUNI12
GUNI13
GUNI14
GUNI15
Heterandria bimaculata
Heterophallus milleri
Limia dominicensis
Limia melanogaster
Limia tridens
Limia vittata 1
Limia vittata 2
Neoheterandria tridentiger
Phallopytchus januarius
Phallichthys amates
Phallichthys pittieri 1
Phallichthys pittieri 2
Phallichthys tico
Phallichthys tico
Poecilia latipunctata
Poecilia mexicana
Poecilia reticulata
Poeciliopsis fasciata
Priapella compressa
Priapella intermedia
Priapella olmecae
Priapichthys festae
Quintana atrizona 1
Quintana atrizona 2
Scolichthys greenwayi
Xenodexia ctenolepis
Xenophalus umbratilis 1
Xenophalus umbratilis 2
Xiphophorus helleri
Xiphophorus sp
FJ178706*/!/!
FJ178707*/!/!
FJ178708*/!/!
FJ178709*/!/!
FJ178710*/!/!
FJ178711*/!/!
FJ178712*/!/!
FJ178713*/!/!
FJ178714*/!/!
FJ178715*/!/!
FJ178716*/!/!
FJ178717*/!/!
FJ178718*/!/!
FJ178719*/ FJ185093*/ FJ194980*
FJ178720*/!/!
FJ178721*/!/!
FJ178712*/!/!
EF017533/ EF017431/!
EF017517/ EF017414/!
EF017533/ EF017431/!
EF017523/ EF017420/!
EF017535/ EF017433/!
FJ178765*/!/!
FJ178766*/!/!
EF017526/ EF017423/!
EF017530/ EF017428/!
EF017513/ EF017410/!
FJ178770*/!*/ FJ194977*
FJ178771*/!/!
Girardinus rivasi
Girardinus rivasi
Glaridicthys uninotatus
Glaridicthys uninotatus
Glaridicthys uninotatus
Glaridicthys uninotatus
Glaridicthys uninotatus
Glaridicthys uninotatus
Glaridicthys uninotatus
Glaridicthys uninotatus
Glaridicthys uninotatus
Glaridicthys uninotatus
Glaridicthys uninotatus
Glaridicthys uninotatus
Glaridicthys uninotatus
Glaridicthys uninotatus
Glaridicthys uninotatus
Heterandria bimaculata
Heterophallus milleri
Limia dominicensis
Limia melanogaster
Limia tridens
Limia vittata
Limia vittata
Neoheterandria tridentiger
Phallopytchus januarius
Phallichthys amates
Phallichthys pittieri
Phallichthys pittieri
Phallichthys tico
Phallichthys tico
Poecilia latipunctata
Poecilia mexicana
Poecilia reticulata
Poeciliopsis fasciata
Priapella compressa
Priapella intermedia
Priapella olmecae
Priapichthys festae
Quintana atrizona
Quintana atrizona
Scolichthys greenwayi
Xenodexia ctenolepis
Xenophalus umbratilis
Xenophalus umbratilis
Xiphophorus helleri
Xiphophorus sp
40
39
16
16
16
16
30
21
17
7
7
17
16
14
14
9
9
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1
–
–
–
–
–
–
EF017512/ EF017409/!
AF412127/!/!
EF017539/ EF017436/!
FJ178776*/!/!
EF017536/ EF017434/!
EF017546/ EF017443/!
EF017554/ EF017451/!
EF017553/ EF017450/!
EF017555/ EF017452/!
EF0175543/ EF017440/!
EF017556/ EF017453/!
FJ178764*/ FJ185092*/ FJ194978*
EF017541/ EF017438/!
EF017557/–/–
EF017527/ EF017424/!
FJ178775*/!/!
EF017548/ EF017445/!
FJ194974*/ !/ !
GenBank accession numbers with an * are new sequences obtained in this study. In the first column the taxonomic identification of specimens collected in the field are indicated. Column five indicates the new taxonomical
denomination proposed after this study.
I. Doadrio et al. / Molecular Phylogenetics and Evolution 50 (2009) 16–30
Species
21
I. Doadrio et al. / Molecular Phylogenetics and Evolution 50 (2009) 16–30
22
.
. ..
.
1
20
26 27
28
29
30
21
Sancti-Spiritus
25
13
16 17 18
10
9
24
23
.
34
.
..
Sagua-Baracoa
33
Sierra Maestra
5
35
. .
32
31
14 19
11
15
12
2
6
4
7
8
48
47
1
3
46
45
44
38 37 36
39
40
41
43
42
Fig. 3. Map of sampling localities and areas lacking Girardinini representatives in the Cuba and Juventud islands. Numbers of localities correspond to those indicated in
Table I.
following non-Girardinus species of Poeciliidae were also analyzed:
Alfaro cultratus (Regan, 1908); Belonesox belizanus Kner, 1860; Brachyrhaphis cascajalensis (Meek and Hildebrand, 1913); Brachyrhaphis
hartwegi Rosen and Bailey, 1963; Brachyrhaphys rhabdophora (Regan, 1908); Brachyrhaphis terrabensis (Regan, 1907); Carlhubbsia kidderi (Hubbs, 1936); Carlhubbsia stuarti Rosen and Bailey, 1959;
Cnesterodon decemmaculatus (Jenyns, 1842); Gambusia affinis (Baird
and Girard, 1859); Heterandria bimaculata (Heckel, 1848); Heterophallus milleri Radda, 1987; Limia dominicensis (Valenciennes,
1846); Limia melanogaster (Günther, 1866); Limia tridens (Hilgendorf, 1889); Limia vittata (Guichenot, 1853); Neoheterandria tridentiger (Garman, 1895); Pamphorichthys hollandi (Henn, 1916);
Phallichthys pittieri (Miller, 1907); Phallichthys tico Bussing, 1963;
Phalloceros caudimaculatus (Hensel, 1868); Phalloptychus januarius
(Hensel, 1868); Poecilia latipunctata Meck, 1904; Poecilia mexicana
Steindachner, 1863; Poecilia reticulata Peters, 1859; Poeciliopsis fasciata (Meck, 1904); Priapella compressa (Alvarez, 1948); Priapella
intermedia Alvarez and Carranza, 1952; Priapella olmecae Meyer
and Espinosa Pérez, 1990; Pseudopoecilia festae (Boulenger, 1898);
Quintana atrizona Hubbs, 1934; Scolicthchys greenwayi Rosen,
1967; Tomeurus gracilis Eigenmman, 1909; Xenodexia ctenolepis
Hubbs, 1950; Xenophallus umbratilis and Xhipophorus helleri (Meek,
1912). The outgroups selected for phylogenetic analysis were the
species Fluviphylax pygmaeus (Myers and Carvalho, 1955) of the subfamily Fluviphylacinae considered one of the sister subfamilies of
Poeciliinae (Parenti, 1981) and Anableps anableps (Linnaeus, 1758),
which belongs to the family Anablepsidae, considered the sister
family of Poeciliidae. We also included a more distantly related outgroup, the goodeid species Crenichthys baileyi (Gilbert, 1893). The
choice of these outgroups was based on published molecular phylogenetic hypotheses for the Poeciliidae (Hrbek et al., 2007).
With the exception of Girardinus cubensis (Eigenmann, 1903),
we included in our molecular analysis all Girardinus species recognized as valid by Lucinda (2003): G. creolus Garman, 1895; G. denticulatus Garman, 1895; G. falcatus (Eigenmann, 1903); G. metallicus
Poey, 1854; G. microdactylus Rivas, 1944; G. uninotatus Poey, 1860
and G. rivasi Barus and Wohlgemuth, 1994. Girardinus serripenis
was considered a junior synonym of G. creolus, as proposed by
Rodriguez et al. (1992).
The original description of Girardinus cubensis was based on
three female specimens from Los Palacios and Pinar del Río. This
was followed by a description of the G. cubensis male based on
fishes from the Taco-Taco River in Aspiro (Howell-Rivero and Rivas,
1944), and recently Barus et al. (1998) found individuals of G. cubensis in La Pastora River between Artemisa and Las Mangas.
Despite our sampling efforts in Los Palacios, Pinar del Río, TacoTaco River in Aspiro and La Pastora River between Artemisa and
Las Mangas, we only found the species G. metallicus, G. microdactylus and G. falcatus in these localities.
All fishes were captured by electrofishing and hand nets. Besides the seven Girardinus species examined, we also analyzed
specimens from the terra typica of species described as Girardinus
but considered invalid (Table 1): G. riddlei (Eigenmann, 1903) synonym of G. creolus from San Cristobal; G. atherinoides Rivas, 1944
synonym of G. falcatus from Mabuya; G. serripenis, synonym of
G. creolus from the Taco-Taco River in Rangel (Rodriguez et al.,
1992); G. ramsdeni, synonym of G. denticulatus from Rio Guaso
Guantanamo; G. pygmaeus (Rivas, 1944), synonym of G. metallicus
from the Rio Negro Hatiguanico system (Rosen and Bailey, 1963);
and G. torralbasi (Eigenmann, 1903) synonym of G. uninotatus from
Pinar del Río (Rosen and Bailey 1963).
2.2. DNA extraction, amplification and sequencing
Total cellular DNA was extracted from tissues by standard proteinase K and phenol/chloroform procedures (Sambrook et al.,
1989). Two overlapping fragments of the mitochondrial cytochrome b gene (total of 1140 bp) were amplified via polymerase
chain reaction (PCR) from each individual DNA sample. In all species, the primers used for cytochrome b were those mentioned in
Machordom and Doadrio (2001). The amplification process was
conducted as follows: 94 "C (2 min), 35 cycles at 94 "C (45 s),
48 "C (1 min), 72 "C (1.45 min). PCR mixtures were prepared in
25 ll volumes with a final concentration of 0.5 mM each primer,
0.2 mM each dNTP, 1.5 mM MgCl2, and 1 unit of Taq DNA polymerase (Invitrogen).
We also sequenced two nuclear genes to compare maternal and
paternal lineages of the tribe Girardinini. To this end, we amplified
22
I. Doadrio et al. / Molecular Phylogenetics and Evolution 50 (2009) 16–30
Fig. 4. Phylogenetic tree (50% majority rule consensus tree) rendered by Bayesian analysis of the mitochondrial cytochrome b dataset. Numbers above nodes indicate
posterior probabilities; numbers below nodes indicate bootstrap values for the MP analysis.
I. Doadrio et al. / Molecular Phylogenetics and Evolution 50 (2009) 16–30
23
Fig. 5. Phylogenetic tree (50% majority rule consensus tree) rendered by Bayesian analysis of the mitochondrial cytochrome b plus nuclear (RAG1 and b-actin) dataset.
Numbers above nodes indicate posterior probabilities values; numbers below nodes indicate bootstrap values for the MP analysis.
24
I. Doadrio et al. / Molecular Phylogenetics and Evolution 50 (2009) 16–30
the nuclear genes RAG-1 (1452 bp) and b-actin (998 bp aligned
including gaps, although PCR products varied in size from 929 bp
in Girardinus metallicus, G. microdactylus and G. rivasi to 977 bp in
the outgroup Crenichthys baileyi) in a subset of mitochondrial samples. The primers used for RAG-1 and b-actin were those described
in Perdices et al. (2005) and Robalo et al. (2006), respectively. For
nuclear RAG-1, the amplification process was conducted as follows: 94 "C (5 min), 35 cycles at 94 "C (1 min), 54 "C (1 min),
72 "C (1.30 min), and for nuclear b-actin as: 95 "C (5 min), 35 cycles
at 95 "C (30 s.), 55 "C (40 s.), 72 "C (1.30 min).
After checking the PCR products on 1.5% agarose gels, these
were sequenced using the Big Dye Deoxy Terminator cyclesequencing kit (Applied Biosystems Inc.). All samples were sequenced using an Applied Biosystems 3700 DNA sequencer according to the manufacturer’s instructions. Chromatograms and
alignments were visually inspected and verified.
All sequence data were deposited in GenBank under the Accession numbers: cytochrome b (FJ178653-FJ-178778, FJ194974);
RAG1 (FJ185089-FJ185104); b-actin (FJ194975-FJ194996).
2.3. Phylogenetic analysis
Nucleotide saturation was assessed by plotting the absolute
number of transitions and transversions against patristic distance
values. Analyses were performed independently on complete gene
datasets (cytochrome b, RAG-1, or b-actin) and on the total number
of base pairs sequenced (3590 bp) (‘‘Total evidence”, Kluge, 1989).
Nucleotide composition for all genes was determined using the v2
homogeneity test of base frequencies implemented in PAUP*
4.0b10 (Swofford, 2002). To establish divergences between lineages, we calculated uncorrected p distances and represented them
as percentages.
Modeltest version 3.07 (Posada and Crandall, 1998) was used to
infer the best-fit model of evolution based on the Bayesian Information Criterion (BIC) (Schwarz, 1978). Model selection was performed for each individual gene and for the combined dataset, in
which each gene partition was allowed to follow its own model.
The aligned data were analyzed independently by Bayesian inference (BI) and maximum parsimony (MP) methods. BI analysis
was performed using MrBayes 3.1.2 (Ronquist and Huelsenbeck,
2003), simulating two simultaneous Markov chain analyses
(MCMC) for 3,000,000 generations each to estimate the posterior
probability distribution. Topologies were sampled every 100 generations and a majority-rule consensus tree was estimated after
eliminating the first 104 generations in each analysis. MP analysis
was performed with the PAUP* 4.0b10 package (Swofford, 2002)
through heuristic searches with 10 random stepwise additions
and TBR branch swapping. Maximum parsimony results were
based on a 6:1 Ti/Tv weighting, according to the empirically determined ti/tv ratio (ratio=5.85). MP trees obtained using different
weighting schemes (10:1, 8:1 or equal weights) were similar and
congruent (trees not shown). Confidences for this analysis were
estimated by bootstrapping (1000 repetitions) (Felsenstein, 1985).
The incongruence length difference test (Farris et al., 1995) as
implemented in PAUP* 4.0b10 was used to determine if different
gene partitions had significantly different signals, for their use in
the combined dataset analyses. Since no significant differences
were found (partition-homogeneity test, p > 0.05), all analyses
were performed on the combined data set.
To determine whether a particular tree topology corresponded
to a significantly better or worse interpretation of the data than
an alternative tree, we used the Shimodaira–Hasegawa test (Shimodaira and Hasegawa, 1999), as implemented in PAUP* 4.0b10.
Our phylogenetic analyses were conducted in two successive
steps. First, we analyzed the mitochondrial cytochrome b datasets,
which represented most of the species and populations of the tribe
Girardinini. From this analysis, we inferred phylogenetic relationships within the tribe Girardinini. Second, we selected a subset of
the mitochondrial dataset and combined it with the corresponding
taxonomic sampling of the nuclear genes RAG1 and b-actin. From
this, we inferred relationships between Girardinini and other
American poeciliids. We also tried to resolve phylogenetic relationships among the lineages of Girardinini unresolved by cytochrome
b analysis.
2.4. Molecular clock and divergence times
A semi-parametric penalized likelihood (PL) analysis was
conducted using the r8s package (Sanderson, 2002) to estimate
the divergence times of taxa. The PL method maximizes the log
likelihood of a model with different rates on each branch, but
includes a roughness penalty that costs the model more as
the magnitude of changes in rates increases. The relative contribution of two components (log likelihood and roughness penalty) is determined by increases in a smoothing parameter.
The TN (truncated Newton) algorithm was implemented, and
smoothing parameters were chosen by cross-validated assessment (Sanderson, 2002) ranging from 100 to 108 in exponential
increments of 0.5. The optimal smoothing value was 1.0. The
branches that constituted a polytomy in the tree were collapsed. Divergence times were estimated using the calibration
point of separation between Limia vittata from Cuba Island
and L. dominicensis and L. tridens from Hispaniola. This calibration point was based on the sister relationship proposed for
representatives of the group Limia domicensis from Hispaniola
and Limia vittata from Cuba by Hamilton (2001). The separation
time assumed between the islands of Hispaniola and Cuba
(wind passage) was 14!17 Mya, as the most recent estimate
(Iturralde-Vinent, 2006). Confidence intervals for divergence
dates are based on the curvature of the likelihood surface (Cutler, 2000) as implemented in r8s.
3. Results
3.1. Phylogenetic analysis
The mitochondrial (cytochrome b) and two nuclear (RAG-1 and
b-actin) genes sequenced rendered 3590 characters (1140 mitochondrial and 2450 nuclear). Base frequencies were homogeneous
across all sites (cytochrome b: v2 = 122.22, df = 384, p = 1.0; RAG-1:
v2 = 16.68, df = 72, p = 1.0, b-actin: v2 = 4.30, df = 72, p = 1.0). For
cytochrome b, 665 sites were variable and 538 were parsimony
informative (47.2%). According to codon position, the most informative was the third (335 parsimony informative characters), followed by the first (94 characters). Plots of the absolute number of
sites of each codon position against patristic distances indicated an
absence of nucleotide saturation at any position (plots not shown).
In the joint mitochondrial and nuclear analysis, 1105 sites were
variable, and 392 were parsimony informative. MP cytochrome b
analysis generated one most parsimony tree with the following
parameters: length = 5239, CI = 0.209, HI = 0.791, RI = 0.708. MP
analysis of all genes (mitochondrial plus nuclear) produced three
trees (length = 4144, CI = 0.371, HI = 0.629, RI = 0.584).
The best-fit models selected following the Bayesian Information
Criterion (BIC) were the TrN + G + I model for cytochrome b, and
the K80 + G model for both RAG-1 and b-actin. Rate matrices for
cytochrome b were R(a) = 1.00, R(b) = 19.78, R(c) = 1.00,
R(d) = 1.00, R(e) = 8.53, R(f) = 1.00. Base frequencies were: cytochrome b, A = 0.28, C = 0.35, G = 0.11, T = 0.26. RAG1 and b-actin
had equal base frequencies. Among site variation was approached
by the gamma distribution shape parameter a (cytochrome b:
25
I. Doadrio et al. / Molecular Phylogenetics and Evolution 50 (2009) 16–30
Crenichthysbaileyi
Fuviphylaxpygmaeus
Anablepsanableps
Xenodexiactenolepis
Poeciliopsisfasciata
Poeciliareticulata
Pamphorichthys hollandi
Poeciliamexicana
Poecilialatipunctata
Limiam elanogaster
Limiat ridens
Limia dominicensis
Limia vittata1
Limia vittata 2
Phalloptychus januarius
Cnesterodomdecemmaculatus
Xiphophorushelleri
Heterandriabimaculata
Belonesoxbelizanus
Gambusia affinis
Heterophallus milleri
Scolichthys greewayi
Neoheterandria tridentiger
Carlhubbsiastuarti
Carlhubbsiakidderi1
Carlhubbsiakidderi 2
GMIC1
GMIC2
GMIC3
GMIC4
GMIC5
GMIC6
GMIC7
GMIC8
GMIC9
GMIC10
GMIC11
GMIC12
GMIC13
GMIC14
GMIC15
GMIC16
GRIV9
GRIV10
GRIV11
GRIV12
GRIV13
GRIV14
GRIV1
GRIV2
GRIV3
GRIV4
GRIV5
GRIV6
GRIV7
GRIV8
GMIC17
GMIC18
GMIC19
GMET20
GMET21
GMET22
GMET19
GMET18
GMET17
GMET16
GMET15
GMET14
GMET13
GMET12
GMET11
GMET10
GMET9
GMET8
GMET7
GMET6
GMET5
GMET4
GMET3
GMET2
GMET1
TCRE4
TCRE5
TCRE6
TCRE7
TCRE8
TCRE1
TCRE2
TCRE3
TCRE9
TCRE10
TCRE11
TCRE12
TCRE13
TCRE14
TCRE15
GUNI1
GUNI11
GUNI12
GUNI13
GUNI2
GUNI3
GUNI4
GUNI5
GUNI14
GUNI15
GUNI6
GUNI7
GUNI8
GUNI9
GUNI10
GFAL1
GFAL8
GFAL9
GFAL10
GFAL11
GFAL12
GFAL2
GFAL3
GFAL4
GFAL5
GFAL6
GFAL7
GFAL13
GFAL14
GFAL15
DRAM1
DDEN9
DDEN10
DDEN1
DDEN2
DDEN3
DDEN4
DDEN5
DDEN6
DDEN7
DDEN8
Quintanaatrizona 1
Quintanaatrizona 2
Priapellacompressa
Priapellaolmaceae
Priapellaintermedia
Priapichthysfestae
Alfarocultratus 1
Alfarocultratus 2
Alfarocultratus 3
Brachyrhaphis cascajalensis
Brachyrhaphis parismina
Phallichthyspittieri 1
Phallichthyspittieri 2
Phallichthyspittieri 3
Phallichthys tico 1
Phallichthys tico 2
Xenphalusumbratilis 1
Xenphalusumbratilis 2
Xenphalusumbratilis 3
Brachyrhaphi s terrabensis
Brachyrhaphis rhabdophora
Brachyrhaphis hartwegii 1
Brachyrhaphis hartwegii 2
Girardinus microdactylus
Girardinus rivasi
Girardinus sp.
Girardinus-Toxus
split
Girardinus metallicus
Glaridichthys
split
Toxus creolus
Dactylophallus
split
Glaridichthys uninotatus
Quintana
split
Glaridicthys falcatus
Girardinini
split
Dactylophallus ramsdeni
Dactylophallus sp
Dactylophallus denticulatus
Mya
Geological time
Cretaceous
Paleocene
Eocene
Oligocene
Miocene
Pleistocene
Pliocene
Fig. 6. Divergence times of the major cladogenetic events occurring in the evolutionary history of the tribe Girardinini. The scale bar below tree shows the time scale
indicated by a relaxed molecular clock.
26
I. Doadrio et al. / Molecular Phylogenetics and Evolution 50 (2009) 16–30
a = 0.75; RAG-1: a = 0.30 and b-actin: a = 0.16). For cytochrome b,
the proportion of invariable sites was I = 0.34. These parameters
were used in the subsequent phylogenetic analyses.
Considering the parameters estimated and the empirical frequencies of the nucleotides, MP and BI of the mitochondrial and
mitochondrial/nuclear datasets rendered congruent topologies
that in no case supported the monophyly of the tribe Girardinini
(Rosen and Bailey, 1963; Parenti and Rauchenberger, 1989; Ghedotii, 2000; Lucinda and Reis, 2005) yet did support major lineages
and population divergences (Figs. 4 and 5). In the mitochondrial
and mitochondrial/nuclear analyses, the genus Quintana appeared
as sister group of Girardinus whereas Carlhubbsia did not appear
in the monophyletic clade Girardinus-Quintana.
Relationships between the clade Girardinus-Quintana and the
remaining poeciliids examined were not fully resolved. The combined data analysis grouped with low support (81% posterior probability) the clade Girardinus-Quintana with another two clades. The
first clade was comprised of species of the genera Brachyrhaphis,
Xenophallus and Phallichthys, all of them from lower Central America. The second clade clustered the species of the genera Poecilia,
Limia, Cnesterodom and Pamphorichthys showing a wide distribution range across America and Greater Antilles. Relationships
among the three clades were not resolved.
Within the genus Girardinus, four strongly supported evolutionary independent lineages were identified: Group I included the
populations of G. metallicus, G. microdactylus and G. rivasi; Group
II was composed of populations of the single species G. creolus;
Group III included the populations of G. uninotatus and G. falcatus;
and finally, Group IV contained the populations of G. denticulatus.
When we analyzed the combined data matrix, intergroup relationships were not fully resolved and we could only appreciate the sister-taxon relationship of Group IV with respect to the remaining
three groups of Girardinus (Fig. 5). In contrast, our mitochondrial
gene analysis resolved the relationships among these three other
clades but with low support (85% posterior probability) (Fig. 4)
and assigned Group III a sister position with respect to Groups I
and II.
For cytochrome b, genetic uncorrected ‘‘p” distances among the
four lineages were x = 9.6 ± 1.2 (7.1–12.9). The minimum distance
was found between Group I and Group II (x = 8.29 ± 0.52 (7.1–
9.6)) and the maximum between Group III and Group IV
(x = 11.7 ± 0.52 (10–12.9)).
Monophyletic Group I corresponded to the genus Girardinus as
defined by Rivas (1958). Within this group, we identified two different clades, one corresponding to the populations of G. metallicus
and the other to the populations of G. rivasi from Juventud Island
plus G. microdactylus from Cuba Island. Within the clade formed
by the G. metallicus populations, the western populations close to
the Guanahacabibes Peninsula formed a lineage well separated
from the others. Uncorrected ‘‘p” distances according to cytochrome b of this western population were x = 2.6 ± 0.1 (2.4–2.8).
When we considered all G. metallicus populations except the one
from the Guanahacabibes Peninsula, uncorrected ‘‘p” distances
for populations of a wide distribution range in Cuba Island were
x = 0.3 ± 0.05 (0.26–0.35). Uncorrected ‘‘p” distances for cytochrome b found between individuals from the terra typica of
G. pygmaeus and the populations of G. metallicus, x = 0.4 ± 0.3
(0.09–1.1), fell within the intra-population divergence range
shown by G. metallicus.
Within the clade formed by the populations of G. rivasi and
G. microdactylus, we found that some of these populations of
G. microdactylus from eastern Cuba Island were more closely related to G. rivasi from Juventud Island than to G. microdactylus from
western Cuba Island. Uncorrected ‘‘p” distances based on mitochondrial cytochrome b were as follows: x = 1.4 ± 0.2 (1.1–1.8)
between populations of G. microdactylus from eastern Cuba and
G. rivasi; x = 2.6 ± 0.1 (2.4–3) between the two populations of G.
microdactylus, and x = 2.8 ± 0.2 (2.6–3.2) between populations of
G. rivasi and G. microdactylus from western Cuba.
Group II corresponded to the genus Toxus as proposed by Rivas
(1958). This group includes the single species Girardinus creolus.
We found three different clades formed by G. creolus populations
from Taco-Taco River, La Legua River in western Cuba and the
San Francisco-San Cristobal rivers. San Cristobal is the terra typica
of G. riddlei (Eigenmann, 1903) and is considered by Rosen and Bailey (1963) a synonym of G. creolus. Relationships among the three
clades were not resolved. The Shimodaira–Hasegawa test for cytochrome b revealed no significant differences (p > 0.05) among the
three possible hypotheses, but indicated a better relationship for
the San Cristobal population as sister group to the La Legua populations (–ln L = 2622.33403) than the San Cristobal population as
sister to the Taco-Taco populations (–ln L = 2622.99649) or TacoTaco as sister to the La Legua populations (–ln L = 2626.78896).
Within the three clades, mitochondrial genetic uncorrected ‘‘p” distances were X = 0.1 ± 0.00 (0.00–0.06). The minimum distance was
detected between populations of G. creolus from La Legua and the
San Francisco–San Cristobal rivers x = 1.4 ± 0.2 (1.1–1.9) and the
maximum distance between the La Legua and Taco-Taco populations x = 2 ± 0.2 (1.7–2.4). Populations of the Upper Taco-Taco in
Rangel, terra typica of G. serripenis, did not differ from populations
downstream of the Taco-Taco falls. Genetic distances within the
Taco-Taco populations were x = 0.05 ± 0.08 (0.00–0.18).
Monophyletic Group III corresponded to Glaridichthys as was
defined by Rivas (1958). We distinguished two different clades,
one formed by G. uninotatus and the other clade comprised of the
populations of G. falcatus. This G. falcatus clade showed a geographical population-structure, with four different lineages. These four
lineages corresponded to populations of G. falcatus from Juventud
Island, Guanahacabibes Peninsula, central Cuba Island and western
populations from Cuba Island. The most differentiated lineage was
that formed by the populations of the Guanahacabibes Peninsula.
For cytochrome b, uncorrected ‘‘p” distances for these Guanahacabibes populations with respect to the other three lineages were
x = 3.4 ± 0.2 (3.0–3.7). The other three populations exhibited less
divergence. Minimum distances x = 1.0 ± 0.2 (0.6–1.4) were those
among populations of G. falcatus from western Cuba. Genetic
uncorrected ‘‘p” distances between the populations of G. falcatus
from central Cuba versus the western Cuban populations were
x = 1.6 ± 0.19 (1.3–1.7) and between central Cuba and Juventud Island were x = 2 ± 0.28 (1.7–2.4). No significant genetic differences
were found between individuals of the terra typica of G. atherinoides (Mabuya-Las Chambas) and individuals of G. falcatus from
the central and western area of Cuba Island (x = 0.6 ± 0.24 (0.3–
0.9)). Specimens of G. uninotatus lacking the typical black spot from
several localities of Pinar del Rio province, described as G. torralbasi
(Eigenmann, 1903), and typical individuals of G. uninotatus showed
no genetic differences according to their cytochrome b sequences.
Monophyletic Group IV corresponded to Dactylophallus as described by Rivas (1958). This group showed three different clades.
The first clade was composed of the individuals from the Guaso
River, terra typica of G. ramsdeni. The second clade contained specimens from central Cuba Island in Viñas district and the third clade
was comprised of the remaining populations from central and eastern Cuba. For cytochrome b, minimum uncorrected ‘‘p” distances
were observed between the populations from Viñas and those from
central and eastern Cuba (x = 1.9 ± 0.1 (1.7–2.1). Maximum distances were those between the Guaso River populations (terra
typica of G. ramsdeni) and those of central and eastern Cuba
(x = 2.4 ± 0.1 (2.3–2.5). Distances between Guaso and Viñas were
x = 2.2 ± 0.06 (2.2–2.3). The relationships among the three clades
were unresolved by both the mitochondrial and combined mitochondrial/nuclear datasets. The Shimodaira–Hasegawa test
I. Doadrio et al. / Molecular Phylogenetics and Evolution 50 (2009) 16–30
revealed no significant differences among the three different
hypotheses (p > 0.05) but indicated a better relationship for the
Guaso population as sister group to the central and eastern populations of G. denticulatus (–ln L = 2692.61030) than the Guaso populations as sister group to Viñas (–ln L = 2693.27613) or than the
Viñas population as sister group to the central and eastern populations (–ln L = 2696.47910).
3.2. Molecular clock and divergence times
A likelihood ratio test rejected the hypothesis of evolutionary
rate constancy across all taxa analyzed (–lnL with
clock = !11638.74, –lnL without clock = !11481.81, df = 127,
p < 0.000). In the absence of a constant rate of evolution, we used
the semi-parametric penalized likelihood method (Sanderson,
2002) to estimate the divergence times of taxa. Splitting of the Girardinini tribe from other poeciliids occurred approximately 62 mya
(CI 95%, 44–77 mya), in the Paleocene (Fig. 6). Within the tribe Girardinini, splitting of the genus Quintana was dated in the Eocene
(approximately 50 mya, CI 95% 32–67 mya). The separation of
the different genera formerly considered Girardinus was ascribed
to the Oligocene–Eocene periods. The first genus to split was Dactylophallus (29 mya, CI 95% 18–39 mya), followed by Glaridicththys
(21 mya, CI 95% 12–27 mya) and Girardinus from Toxus (18 mya, CI
95%, 10–25 mya).
4. Discussion
4.1. Phylogeny of Girardinini and taxonomical implications
Our data resulted in a well-resolved phylogeny for the tribe Girardinini, although relationships between Girardinini and other
poeciliids could not be resolved using our mitochondrial and nuclear gene data. Our analyses did not support the monophyly of
the tribe Girardinini as proposed by most authors (Rosen and Bailey, 1963; Parenti and Rauchenberger, 1989; Ghedotii, 2000; Lucinda and Reis, 2005). Also contrary to the findings of previous
morphological studies (Rosen and Bailey, 1963; Ghedotii, 2000; Lucinda and Reis, 2005), while the genus Quintana emerged as the
sister group of Girardinus, the genus Carlhubbsia seems more related to other poeciliids than to the genera Girardinus and Quintana. These data are in agreement with a previous molecular
phylogeny of the poeciliids in which Carlhubbsia was assigned to
a clade comprised of the genera Xiphophorus, Heterandria, Belonesox, Gambusia, Scolichthys and Priapella (Hrbek et al., 2007). Our
phylogenetic analysis recovered this clade based on mitochondrial
data but our nuclear gene data could not resolve the relationships
between the genera Priapella and Scolichthys and the clade comprised of Carlhubbsia, Xiphophorus, Belonesox, Heterandria and Gambusia. The results of this study indicate that the tribe Girardinini
should only contain species of Girardinus and Quintana, contrasting
with the findings of previous morphological approaches based on
the morphology of the gonopodium and on osteological data (Rosen and Bailey, 1963; Ghedotii, 2000; Lucinda and Reis, 2005). Suggestions that Girardinus is closely related to Allogambusia (=
Neoheterandria) and Phalloceros (Rivas, 1958) were rejected by
our analyses.
The sister group of Girardinini was unresolved in our analyses
because of low posterior probability support. Previous molecular
phylogenies of poeciliids have established that Girardinus is a basal
group to a high number of species from North and Central America
(Hrbek et al., 2007). However, some of the clades recovered by our
phylogeny suggest a high level of homoplasy at the morphological
level has probably confounded poeciliid classification. This is obvious for traditional characters such as the gonopodium, which prob-
27
ably evolved multiple times in response to similar ecological
pressures or maybe the result of sexual selection (Hankison and
Morris, 2002; Langerhans et al., 2005; Meyer et al. 2006). In effect,
as for the Girardinini, recent morphological and molecular studies
are now beginning to reject the monophyly of these poeciliid tribes
(Ghedotii, 2000; Lucinda and Reis, 2005; Hrbek et al., 2007).
Within the tribe Girardinini, the phylogenetic relationships defined here are in disagreement with those proposed by Rivas
(1958) on the basis of morphological characters. Notwithstanding,
our monophyletic clades corresponded to the four genera: Girardinus Poey, 1854; Dactylophallus Howell-Rivero and Rivas, 1944;
Glaridichthys Garman, 1896; Toxus Eigenmann, 1907 recognized
as Girardinini by Rivas (1958) along with the genus Quintana Hubbs, 1934. These four genera were monophyletic and their genetic
divergence was similarly high to that found in other Cyprinidontiform genera (8–11% (x = 10%)) such as those of the Goodeidae family (Doadrio and Domínguez, 2004). Hence, the genera recognized
by Rivas (1958) and corroborated by our molecular phylogeny,
are of evolutionary significance and strongly supported by morphological and genetic characters. The tribe Girardinini should thus
comprise the four genera recognized by Rivas (1958): Dactylophallus, Girardinus, Glaridichthys and Toxus plus the genus Quintana. The
genus Carlhubbsia should be excluded from the tribe Girardinini on
the basis of our molecular phylogeny and that reported by Hrbek
et al. (2007).
The species Toxus creolus has been considered the ancestor of all
other Girardinini representatives (Rivas, 1958; Rodriguez et al.,
1992). Our phylogeny based on sister groups rather than ancestor–descendant histories revealed that the genus Dactylophallus
was the one that first split from the remaining three genera (Girardinus, Glaridichthys and Toxus). Girardinus and Glaridichthys are
sister taxa. This idea that Dactylophallus was the first to break away
is consistent with the separation of the islands of Cuba and Juventud into two endemic regions on the basis of their freshwater
fishes (Rauchenberger, 1988). Dactyllophalus is the only Girardinini
genus that inhabits easternmost Cuba and has a population in central Cuba, while Girardinus, Glarydichthys and Toxus inhabit central
and western Cuba Island and Juventud Island yet not the easternmost part of Cuba.
Our phylogenetic hypothesis has further major taxonomic
implications. The described species Girardinus pygmaeus, Toxus
riddlei, T. serripenis and Glaridichtys atherinoides showed no significant genetic differences with respect to Girardinus metallicus, Toxus creolus and Glaridichthys falcatus respectively. These results are
in agreement with a previous morphological study that failed to
consider the former four species as valid (Rosen and Bailey,
1963). In contrast, Dactyllophalus ramsdeni varied sufficiently (genetic differences of 2.2% and 2.4% versus the populations of G. denticulatus) to be assigned its own identity and should be considered
a different species to G. denticulatus and not its subspecies.
To solve the problem of paraphyly in the genera Girardinus and
Dactyllophallus, we proposed the existence of taxa not formally described. Unexpectedly, we found populations of Girardinus microdactylus in central Cuba outside its distribution range in western
Cuba. These populations showed less divergence with respect to
G. rivasi from Juventud Island than to coespecific populations from
the western area of Cuba. Divergence between G. rivasi and G.
microdactylus was sufficiently high but the latter is not considered
a recent introduction. To avoid the paraphyly problem, we provisionally considered G. microdactylus from central Cuba as a different species. Similarly, within Dactylophallus denticulatus, we
recognized the species D. ramsdeni from the Guaso basin, the same
locality of the endemic cichlid Nandopsis ramsdeni (Fowler, 1938),
and a non-formally described species from central Cuba (Viñas
district).
28
I. Doadrio et al. / Molecular Phylogenetics and Evolution 50 (2009) 16–30
Further, our phylogenetic analysis suggests the existence of several well-differentiated populations that could be considered as
different species after a morphological study. Girardinus metallicus
and Glaridichthys falcatus from Guanahacabibes Peninsula showed
a high degree of differentiation similar to that of valid species such
as Girardinus microdactylus and G. rivasi. The populations of Toxus
creolus showed high divergence, probably due to isolation of the
species in highland streams. These observations prompt the need
for a taxonomic revision of all these populations. A provisional taxonomy based on the results of this genetic study is provided in Table 1.
4.2. Divergence times and biogeography
The evolution of the Antillean islands has puzzled biogeographers for over two centuries (Wallace, 1881). Research on the origins of Antillean fauna has rendered three main models with
implications for the origin and evolution of poeciliids. The classic
vicariant model was essentially proposed by Rosen (1975, 1985).
This model hypothesized that poeciliids inhabited the proto-Antillean arc during the Cretaceous (144–65 Mya), when interchange
between North America and South America was possible, and became isolated when the arc was displaced eastwards. The dispersalist hypothesis as proposed by Myers (1966), essentially involved
recent colonizations of Central America during the Neogene by
freshwater fish fauna. This author indicated that Central America
has been the Poeciliidae family’s center of origin since the Neogene. The third model combines dispersal and vicariant hypotheses
to suggest that the current Antillean fauna came from South America, reaching the islands through GAArlandia (Greater Antilles + Aves Ridge) in the Eocene–Oligocene (33–35 Mya)
(Iturralde-Vinent and MacPhee, 1999; Iturralde-Vinent, 2006).
For poeciliid fishes, this last model has recently been proposed as
the most plausible based on molecular clock approaches (Hrbek
et al., 2007).
The GAArlandia model, nevertheless, poses some inconsistencies for freshwater fish fauna. An example is the presence of populations of the genus Ophisternon in western Cuba and Juventud
Island since its sister group occurs in the Yucatan Peninsula and
is absent from other Central and South American regions (Perdices
et al., 2005). According to the GAArlandia model, there was no connection between the Yucatan and western Cuba and Juventud Island during the Cenozoic (Iturralde-Vinent and MacPhee, 1999;
Iturralde-Vinent, 2006). For mammals, the presence of the insectivore Solenodon in Cuba and Hispaniola with a divergence with respect to other insectivore mammals dated during the Cretaceous
(Roca et al., 2004) created conflict between the GAArlandia model
and molecular clock datings. In other vertebrates, the dispersalist
model recovered the inconsistencies between molecular clocks
and biogeographical models (Matthew et al, 2007). However, for
fishes such as Girardinus with a low dispersal capacity restricted
to contact between different freshwater systems, the dispersion
by flotsam proposed by dispersalist models (Matthew et al.,
2007) is highly unlikely. If inconsistencies between molecular
clock and palaeogeographical data exist, explanations must be
looked for in the concepts of these doctrines rather than trying
to fit models to the wide dispersion range of freshwater fishes.
The estimated divergence time of the endemic genera of Cuba
Girardinus and Quintana from the other poeciliids was approximately 62 Mya (CI 95%, 44–77 Mya), in the Upper Cretaceous-Middle Eocene. This is in agreement with datings provided by other
vertebrates such as Solenodon (Roca et al., 2004) and Cricosaura
(Hedges et al., 1991, 1992). Several arguments have been proposed
to reject the ancient colonization during the Upper CretaceousMiddle Eocene, of current fauna of the West Antillean islands.
One such theory is that the extinction of sister taxa could affect
the molecular clock calibration (Roca et al., 2004). A further explanation for this estimated divergence could be the use of dispersal
by flotsam models (Hedges 1996).
Based on our data, we cannot rule out the extinction of a probable sister group of endemic Cuban taxa on the main or other Antillean islands. The use of the geological event of separation between
Cuba and Hispaniola for the genus Limia could also be a possible
source of error. The model of Hrbek et al. (2007) for poeciliid fishes
is more congruent with the GAArlandia theory (Iturralde-Vinent
and MacPhee, 1999) in terms of colonization of the Caribbean islands. The divergence time for the main radiation of poeciliid fishes
has been estimated at 44 Mya (CI 95% 40.78–48.97 Mya) (Hrbek
et al, 2007), which is a little earlier than the date of 35–33 Mya assigned to the formation of GAArlandia (Iturralde-Vinent, 2006).
The molecular clock calibration for poeciliids (Reznick et al.,
2002; Hrbek et al., 2007) uses the formation of the Trans-Mexican
Volcanic Belt to calculate the maximum age for the separation of
several species of Poeciliopsis. However, the Trans-Mexican Volcanic Belt formation process was a long and intermittent one involving several pulses. For instance pulses dated at 10, 23 and 30 Mya
ago have been reported (Ferrari et al., 1999). The use of this type of
barrier to date the separation of organisms has the limitation that
it is difficult to establish a fixed date for a given area. We believe
that the separation between the Caribbean islands is more reliable
to determine calibration points. The use of the genus Limia as a calibration point, which shares its habitat with Girardinus and is similar in body size to this species, avoids the problems of calculating
substitution rates based on organisms with different metabolic
rates (Estabrook et al., 2007).
The lack of congruence between the separation date of the Girardinini inhabiting Cuba from sister groups on the mainland could
be attributable to the extinction of some sister groups (see Iturralde-Vinent and MacPhee, 1999). Otherwise, we would have to refute the GAArlandia hypothesis and freshwater fishes could have
inhabited western Cuba since 60 Mya (Cretaceous period). Fossil
data are essential for verifying palaeogeographical data or molecular clock calibrations. However, freshwater fish fossils from the
Cenozoic of Cuba have not been examined.
Within the tribe Girardinini, the separation between Girardinus
and the other Cuba and Juventud island endemism Quintana occurred approximately 50 Mya ago (CI 95%, 32–67 Mya). Girardinus
is widely distributed throughout Cuba and Juventud Island. Quintana exhibits a more reduced distribution area than Girardinus, restricted to western Cuba and Juventud Island. This distribution
cannot be explained by current palaeogeographical models if we
accept that speciation is explained by allopatry.
Radiation of the main lineages of the tribe Girardinini took place
in the late Oligocene-Early Miocene, Dactylophallus being the first
genus to split (29 Mya, CI 95% 18–39 Mya). This radiation is consistent with the end of the GAArlandia stage and the isolation of different portions of land on the islands of Cuba and Juventud
(Iturralde-Vinent and MacPhee, 1999).
Populations of clades such as G. metallicus and G. falcatus from
the Guanahacabibes Peninsula diverged from other Cuban populations in the Pliocene (approximately 5–3 Mya). This date suggests
that Guanahacabibes became isolated from the rest of Cuba Island
during the Pliocene. However, there is insufficient palaeogeographical information for this period to explain the divergence times of
these populations.
The palaeogeographical data on Juventud Island suggest its ancient isolation from Cuba Island at least since the Oligocene, and
then a more recent isolation during the Plio-Pleistocene. It is not
possible to infer the ancient isolation of Juventud Island from the
current distribution of freshwater fishes. However, the presence
of Quintana, the sister group of former Girardinus, in Juventud Island could be explained by this ancient event of isolation followed
I. Doadrio et al. / Molecular Phylogenetics and Evolution 50 (2009) 16–30
by dispersion to the few localities inhabited by the species in Cuba
Island. This theory, nevertheless, awaits the support of population
genetics studies. The possibility of a more recent isolation of Juventud Island could be examined by determining the presence of the
endemic species G. rivasi or the well-differentiated populations of
G. falcatus. The data for these freshwater fishes indicate the isolation of Juventud Island during the late Pliocene (2–1.3 Mya). This
date must be considered with caution since the calibration point
used to calculate the molecular clock is older. A 1–2 Mya time
frame has been described as critical for modifying molecular rates
(Ho and Larson, 2006) and could overestimate divergence times
(Waters et al., 2007).
The palaeogeographical data on Girardinus seem to be adequate
for testing the different biogeographical models. Divergence times
for these vertebrates seem to support Rosen’s vicariant ideas to explain the high divergence between the tribe Girardinini and its relatives from the mainland. However, the high extinction rate
assumed for the Chixchulub impact (Izzet et al. 1991; Swisher
et al, 1992), could confound the data since the sister group of Girardinini could be extinct. The absence of Girardinini from the other
Antillean islands, especially Hispaniola, and its high diversity in
western Cuba does not explain its passage from South America
through GAArlandia such that the origin of the tribe remains
unknown.
Acknowledgments
We are grateful to R. Fimia Duarte, M. Díaz Pérez, A. Chamizo, V.
Rivalta, A. de Sostoa, P. Garzon and O. Dominguez for help in field
collection. D. McAllister and K. de Jong sent us valuables specimens. A. Burton reviewed the English of the manuscript. We also
thank L. Rojas Rivero for her support and M. Iturralde-Vinent for
helpful suggestions while preparing the manuscript. This study
was financed by project A/9998/07.
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