Hidden diversity in spring snails from the Andean Altiplano, the second highest plateau on Earth, and the Atacama Desert, the driest place in the world

Abstract

Background: The Chilean Altiplano and the Atacama Desert in northern Chile harbor isolated hydrological systems or oases where it is possible to find minute aquatic snails of the genus Heleobia whose taxonomy is uncertain and where many populations remain unknown. Here, we obtained samples from 30 localities distributed in the region and used molecular (12S and 16S mitochondrial genes) and morphological (penis) characters to investigate diversity of this poorly known fauna. Results: Molecular phylogenetic analysis consistently recovered five clades, one of which constitutes a cryptic species previously assigned to a species recognized in the area. Four other clades contained sequences of one nominal species consistent with its type locality and at least two additional candidate species, which were corroborated by a particular penis morphology. Furthermore, some morphological differences in penis morphology were observed in two Altiplano populations not resolved by the DNA sequences, providing support for two additional candidate species in the genus. A molecular clock analysis allowed tracing the origin of lineages back to the Early Pleistocene. Conclusions: We found support for recognizing four nominal species, one undescribed species and at least other four candidate species of the genus Heleobia in northern Chile. We also suggest that the current level of species diversity of Heleobia in the region is underestimated by the use of conchological criteria to recognize species and by the limited sampling conducted to date.

Full text

R E S E A R C H Open Access
Hidden diversity in spring snails from the Andean
Altiplano, the second highest plateau on Earth,
and the Atacama Desert, the driest place in the
world
Gonzalo A Collado
1*
, Moisés A Valladares
2
and Marco A Méndez
2
Abstract
Background: The Chilean Altiplano and the Atacama Desert in northern Chile harbor isolated hydrological systems
or oases where it is possible to find minute aquatic snails of the genus Heleobia whose taxonomy is uncertain and
where many populations remain unknown. Here, we obtained samples from 30 localities distributed in the region
and used molecular (12S and 16S mitochondrial genes) and morphological (penis) characters to investigate
diversity of this poorly known fauna.
Results: Molecular phylogenetic analysis consistently recovered five clades, one of which constitutes a cryptic
species previously assigned to a species recognized in the area. Four other clades contained sequences of one
nominal species consistent with its type locality and at least two additional candidate species, which were
corroborated by a particular penis morphology. Furthermore, some morphological differences in penis morphology
were observed in two Altiplano populations not resolved by the DNA sequences, providing support for two
additional candidate species in the genus. A molecular clock analysis allowed tracing the origin of lineages back to
the Early Pleistocene.
Conclusions: We found support for recognizing four nominal species, one undescribed species and at least other
four candidate species of the genus Heleobia in northern Chile. We also suggest that the current level of species
diversity of Heleobia in the region is underestimated by the use of conchological criteria to recognize species and
by the limited sampling conducted to date.
Keywords: Cochliopidae; Distribution patterns; Semisalsinae; Spring snails; Taxonomy
Background
The practical matter of species delimitation is receiving
increased attention considering the high rate of extinction
of species along with the huge undiscovered biodiversity
in a number of taxa (Sites and Marshall 2003, 2004;
Bickford et al. 2007; Wiens 2007; Puillandre et al. 2011).
In the Andean Altiplano, the second highest plateau on
Earth (Babeyko and Sobolev 2005), the dynamic and
complex geological history of the region has produced
ecosystems with a high degree of endemism (Veloso and
Bustos-Obregón 1982; Dyer 2000; Vargas et al. 2004),
probably related to the extensive terraces and restricted
mountain ranges that have originated since the Miocene
(Wörner et al. 2000; Risacher et al. 2003; Strecker et al.
2007). For example, the diversity of the killifish of the
genus Orestias Valenciennes, 1839 and the lunged aquatic
snails of the genus Biomphalaria Preston, 1910 is hypo-
thesized to be principally a consequence of the frag-
mentation of populations during the Middle and Late
Pleistocene after the regression of several paleolakes that
existed in the area (Lüssen et al. 2003; Vila 2006; Vila
et al. 2011, 2013; Collado et al. 2011a). West of this area,
the Atacama Desert, the driest place in the world (McKay
2002; Vesilind 2003) with thousands of square miles of
* Correspondence: collado.gonzalo@gmail.com
1
Departamento de Biología y Ciencias Ambientales, Facultad de Ciencias,
Universidad de Valparaíso, Gran Bretaña, 1111 Valparaíso, Chile
Full list of author information is available at the end of the article
© 2013 Collado et al.; licensee Springer. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited.
Collado et al. Zoological Studies 2013, 52:50
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arid desert interrupted by occasional oases, is a hyper-
arid system thought to be relatively simple ecologically
and with low productivity (Noy-Meier 1973), but con-
taining endemic species, for example, within the genus
Basilichthys Girard, 1855 (Atheriniformes), that has allo-
patric distribution (Dyer 2000). Both regions harbor re-
stricted and isolated hydrological systems ranging from
springs on the banks of the Pacific coast along the Ata-
cama Desert to watersheds situated at more than 4,000 m
altitude in the Andes. In some of these watersheds, it is
possible to find gilded snails of the genus Heleobia Stimp-
son, 1865 (Courty 1907; Biese 1944, 1947; Hershler and
Thompson 1992; Collado et al. 2011b), subfamily Semisal-
sinae Giusti and Pezzoli, 1980 (Bouchet and Rocroi 2005),
sometimes living in amazing conditions of water stress
and habitat degradation (Collado 2012). The group is
characterized by small species that live in marine, brack-
ish, and freshwater environments, and whose shells gener-
ally are less than 10 mm in length (Biese 1944, 1947;
Hershler and Thompson 1992). The species have a wide
range of ecological tolerances and reproductive strategies
(Marcus and Marcus 1963, 1965; Cazzaniga 1982a; Martín
2002; Neves et al. 2010; Collado and Méndez 2011), and
some of them serve as intermediate hosts in the life cy-
cles of digenean trematodes (Etchegoin and Martorelli
1997; Simões et al. 2008, 2009, 2010; Alda et al. 2010;
Merlo and Etchegoin 2011). Kroll et al. (2012) studied
the phylogenetic relationships of species mainly distrib-
uted in the Altiplano region and its major internal Lake
Titicaca, and this is the only phylogenetic study per-
formed in the genus Heleobia.
The systematics of the Chilean Semisalsinae fauna
has been contentious at different taxonomic levels (De
Francesco and Isla 2004; Cazzaniga 2011; Collado et al.
2011b). While some authors have included the species in
the genus Heleobia Stimpson, 1865 (Davis et al. 1982;
Hershler and Thompson 1992; Kabat and Hershler 1993),
others have preferentially assigned them to the genus Lit-
toridina Souleyet, 1852 (e.g., Pilsbry 1911; Preston 1915;
Biese 1944, 1947; Haas 1955; Hubendick 1955; Stuardo
1961; Weyrauch 1963; Figueroa et al. 2003; Sielfeld 2001;
Valdovinos 1999, 2006, 2008). At the species level, the
knowledge of the group has not been clarified, so alpha
taxonomic work is still needed (see Collado et al. 2011b).
In the Chilean Altiplano and the Atacama Desert, eight
species and seven subspecies of Heleobia have been de-
scribed based on conchological characters: Heleobia
atacamensis (Philippi 1860), Heleobia loaensis (Biese
1947), Heleobia opachensis (Biese 1947), Heleobia striata
(Biese 1944), Heleobia transitoria (Biese 1947), Heleobia
ascotanensis (Courty 1907) with five subspecies, and
Heleobia chimbaensis (Biese 1944) and Heleobia co-
piapoensis (Biese 1944) with one subspecies (Hershler and
Thompson 1992; Collado et al. 2011b). The taxonomic
status of these species and several previously unexamined
populations distributed in the region has never been inves-
tigated. Additionally, the distribution ranges and relation-
ships among the species have been addressed by few
studies (Biese 1944, 1947; Kroll et al. 2012). Considering
that these two geographical areas are difficult to access and
concomitantly poorly explored, it is highly possible that the
biodiversity of Heleobia is underestimated. In the present
study, we use DNA sequence data of the large (16S) subunit
and small subunit (12S) of ribosomal RNA mitochondrial
genes and penis morphology to investigate diversity of
Heleobia, using an extensive sampling covering the majority
of the type localities and distribution range of the genus in
the region and to test the hypotheses of nominal species
proposed until now. We investigate penis morphology con-
sidering that the organ is discriminatory in Heleobia species
(Gaillard and de Castellanos 1976; Cazzaniga 1980, 1982a, b;
Hershler and Thompson 1992; Pons da Silva 1993;
Collado et al. 2011b; Ovando and De Francesco 2011).
Hubendick (1955) described the penis of H. chimbaensis
from northern Chile, and Collado et al. (2011b) studied
this organ in this and other species of the genus.
Methods
The snails were collected from macrophyta or sediment
using a sieve, from 2010 to 2012 from 30 localities situated
in the Chilean Altiplano and the Atacama Desert (Figure 1,
Table 1). The snails were preserved in 70% to 100% etha-
nol. The material included specimens collected from
Quebrada La Chimba, the type locality of H. chimbaensis,
a ravine near Antofagasta city, Vertiente Opache, the type
locality of H. opachensis in San Salvador River, and Las
Cascadas, the type locality of H. loaensis in Loa River. The
last two localities are situated near the city of Calama in
Loa Basin. The snails were also obtained from Tilopozo in
the Salar de Atacama, Chilean Altiplano, the type locality
of H. atacamensis, and from Quebrada Cachina, the type
locality of H. transitoria, a ravine located in the Pacific
coast in the Atacama Desert. Heleobia copiapoensis,H.
copiapoensis costata,andH. striata were described from
the Copiapó River and from its tributary stream Ojancos,
the type locality of the species (Biese 1944, 1947); we sam-
pled the Copiapó River at four sites located over the ma-
jority of its length. We also obtained snails from two
isolated springs from the Salar de Ascotán, the type locality
of H. ascotanensis (see Courty 1907; Collado and Méndez
2012a). Because we assigned snails to H. ascotanensis in a
previous study with samples of Spring 2 from this salt pan,
we have kept this name for a set of snails obtained from
this site which formed a monophyletic group. Additional
internal subclades formed with samples of this site and
from Spring 11 from the Salar de Ascotán were treated as
Heleobia sp. Voucher specimens were deposited in the
Laboratorio de Genética y Evolución (GEVOL), Facultad
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de Ciencias, Universidad de Chile and the malacology sec-
tion of the Museo Nacional de Historia Natural, Santiago,
Chile (MNHNCL).
For morphological data, the shell of adult specimens was
cleaned in 1% sodium hydroxide for 10 h to remove ad-
hered sediment, washed in distilled water, and finally pre-
served in 70% ethanol. The snails were photographed at the
same magnification with a Motic SMZ-168 Stereo Micro-
scope with a Moticam 2000 (British Columbia, Canada) in-
tegrated digital camera. For the study of penis morphology,
we followed the method implemented by Collado et al.
(2011b), dissecting three male specimens for each popula-
tion surveyed when possible. The penis was photographed
using the same equipment. Considering the similarity of
the characters of the shell and penis between some popula-
tions of the same species (see for instance Collado 2012),
we do not show these data for all locations, but only those
representatives of geographic areas or clades, or when it is
required to emphasize the possibility of finding candidate
species. For the molecular analyses, genomic DNA was iso-
lated from the gill and mantle tissue of the snails fixed in
100% ethanol using the cetyltrimethyl ammonium bromide
method (CTAB) (Winnepennickx et al. 1993). The mito-
chondrial 16S rRNA gene was amplified by polymerase
chain reaction (PCR) using the primers 16Sar-L (5′-
CGCCTGTTTATCAAAAACAT-3′) and 16Sbr-H (5′-
CCGGTCTGAACTCAGATCACGT-3′) (see Palumbi 1996).
PCR conditions were described in Collado and Méndez
(2012b) for this molecular marker. The mitochondrial 12S
rRNA gene was amplified by PCR using the primers L1091
(5′-AAAAAGCTTCAAACTGGGATTAGATACCCCACT
AT-3 ′)andH1478(5′-TGACTGCAGAGGGTGACGGG
CGGTGTGT-3′) (Kocher et al. 1989). The PCR reaction
cycle was 94°C for 3 min followed by 40 cycles of 94°C
for 30 s, 45°C for 45 s, and 72°C for 60 s. Nucleotide se-
quences were obtained from the Macrogen Company
(South Korea), edited in BioEdit (Hall 2001), and aligned
in Clustal X (Thompson et al. 1997), with final visual in-
spection. Phylogenetic analyses were performed using
maximum parsimony (MP) and Bayesian inference (BI)
for the separate and combined analyses. Alternative ana-
lyses were performed using the neighbor-joining (NJ)
method in Mega 5 (Tempe, AZ, USA) (Tamura et al.
2011). The MP analysis was performed with PAUP* 4.0
(MA, USA) (Swofford 2003) using a heuristic search with
the TBR algorithm and the addition of random sequences.
Characters not informative were excluded from the ana-
lyses. The statistical confidence of the nodes was evaluated
Argentina
Bolivia
Chile
Pacific
Ocean
Col
Par
Is
Car
As S2
As S11
Tat
LC
Cz
Tot
CP
Pab
Le
Cho
Hum Ho
Cop
Cc
Br
Cb
LP
Fi
Ca Ch
Pe
Tm
Tp
20°S
70°W
30°S
40°S
50°S
70°W
18°S
28°S
20°S
22°S
24°S
26°S
N
Chilean
Altiplano
Atacama
Desert
0 200 400 km 0 50 100 km
VO
Op
Ve
Figure 1 Collection sites for the genus Heleobia in northern Chile. As S2: Salar de Ascotán (Spring 2), As S11: Salar de Ascotán (Spring 11), Br:
Las Breas (Quebrada de Taltal), Ca: Las Cascadas, Car: Salar de Carcote (Spring 1), Cb: Quebrada Cascabeles, Cc: Quebrada Cachina, Ch: Chiu-Chiu,
Cho: Aguada de Chorrillos, Col: Colpa, Cop: Copiapó, Copiapó River, CP: Carrera Pinto, Cz: Quebrada Carrizo (= Quebrada La Negra), Fi: La Finca,
Ho: Hornitos, Copiapó River, Hum: Humedal, Copiapó River, Is: Isluga, LC: Quebrada La Chimba, Le: Quebrada El León, LP: Los Perales (Quebrada
Paposo), Op: Laguna Opache, Pab: Pabellón, Copiapó River, Par: Parinacota, Pe: Peine, Tat: El Tatio, Tm: Tilomonte, Tot: El Totoral, Tp: Tilopozo, Ve:
Las Vertientes, Vo: Vertiente Opache.
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Table 1 Characteristics of water systems and identification of snails sampled used in the present study
System, altitude (m) Latitude (S), longitude (W) Voucher GenBank accession number
Locality GEVOL MNHNCL 12S rDNA 16S rDNA
Caquena Basin
Colpa Stream-swamp, 4,384 18°03′29.00″, 69°13′52.00″2 5900 KF285868 KF285827
Lauca River Basin
Parinacota Stream-swamp, 4,399 18°12′51.00″, 69°18′05.00″2 5901 KF285869 KF285828
Isluga Basin
Isluga River-swamp, 3,776 19°15′10.00″, 68°42′15.00″1 5902 KF285870 -
2 5903 KF285871 -
Salar de Carcote Basin
Salar de Carcote Spring 1, 3,688 21°16′04.00″, 68°19′21.00″9 V1 5904 KF285872 KF285829
11 V1 5905 KF285873 KF285830
Salar de Ascotán Basin
Salar de Ascotán Spring 2, 3,716 21°29′07.00″, 68°15′21.00″67 MG 5906 KF285874 KF285831
68 MG 5907 KF285875 KF285832
70 MG 5908 KF285876 -
84 MP 5909 KF285877 KF285833
86 MP 5910 KF285878 -
Salar de Ascotán Spring 11, 3,734 21°41′13.90″, 68°12′54.00″1 V11 5911 KF285879 -
6 V11 5912 KF285880 -
7 V11 5913 KF285881 -
Loa River Basin
El Tatio Stream-geysers, 4,264 22°20′10.00″, 68°00′59.00″8 5914 KF285882 -
17 5915 KF285883 -
Las Cascadas River, 2,260 22°29′54.00″, 68°58′18.00″2 5916 KF285889 KF285838
5 5917 - KF285839
8 5918 - KF285840
11 5919 - KF285841
Las Vertientes Spring, 2,181 22°13′15.20″, 68°58′20.00″1 5920 KF285890 KF285845
2 5921 KF285891 KF285846
La Finca River, 2,099 22°30′34.62″, 68°59′27.90″1 5922 - KF285842
2 5923 KF285892 KF285843
Chiu Chiu River, 2,470 22°20′02.00″, 68°38′57.00″13 5924 KF285893 -
14 5925 KF285894 -
Vertiente Opache Spring-river, 2,184 22°29′02.50″, 69°00′08.00″1 5926 KF285895 -
2 5927 - KF285847
3 5928 KF285896 KF285848
Laguna Opache Lagoon, 2,100 22°30′20.50″, 68°59′43.30″1 5929 - KF285844
Pacific coastal basins
Quebrada La Chimba Spring, 500 23°32′22.05″, 70°21′36.40″1-1 5930 KF285897 KF285849
2-2 5931 KF285898 KF285850
Quebrada Carrizo Spring-stream, 72 25°41′56.72″, 70°24′42.51″1 5932 KF285899 KF285851
2 5933 KF285900 KF285852
Quebrada Cascabeles Spring, 42 25°17′33.10″, 70°26′45.40″1 5934 - KF285853
2 5935 KF285901 -
Quebrada El León Spring, 243 26°57′34.70″, 70°44′15.00″1 5936 KF285902 KF285854
2 5937 - KF285855
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using 100 bootstrap pseudoreplicates (Felsenstein 1985).
The BI was performed in the MrBayes v. 3.1.2 program
(Ronquist and Huelsenbeck 2003), selecting the best evo-
lutionary model with jModelTest (Posada 2008) for each
data partition based on the Bayesian information criterion.
This analysis was run three times for three million genera-
tions for the combined analyses. Trees were sampled every
1,000 generations, and posterior probabilities were ob-
tained after a burn-in period of 10%.
Divergence times were estimated using the BEAST pro-
gram (version 1.5.3, GNU Lesser General Public License,
Boston, MA, USA) (Drummond and Rambaut 2007). We
used a nucleotide substitution rate of 1.9% per million
years for the 16S gene, the average divergence rate of this
gene estimated for invertebrates (Cunningham et al. 1992;
Patarnello et al. 1996) and previously used in gastropods
(DeJong et al. 2001; Collado and Méndez 2012b). The ana-
lysis was performed using a lognormal molecular clock,
the general time reversible (GTR) substitution model
plus the gamma distribution and the Yule process of
speciation. Convergence of posterior distributions was
analyzed in Tracer (version 1.5, GNU Lesser General
Public License) (Rambaut and Drummond 2007), and
the Bayesian tree was obtained after removing burn-in
(10%) with TreeAnnotator (version 1.5.3, GNU Lesser
General Public License).
Original 16S rRNA and 12S rRNA sequences obtained
in this study were deposited in the National Center for
Table 1 Characteristics of water systems and identification of snails sampled used in the present study (Continued)
Aguada de Chorrillos Spring, 5 27°12′32.40″, 70°57′03.30″1 5938 KF285907 KF285858
6 5939 KF285908 KF285859
El Totoral Stream, 200 27°53′50.70″, 70°54′01.50″3 5940 KF285922 KF285863
4 5941 KF285923 KF285864
Los Perales, Quebrada Spring-stream, 332 25°01′45.60″, 70°27′17.90″1 5942 KF285924 KF285865
Paposo 4 5943 KF285925 KF285866
6 5944 KF285926 KF285867
Salar de Atacama Basin
Tilomonte Stream, 2,365 23°47′24.40″, 68°06′34.20″1-4 5945 KF285884 KF285834
Tilopozo Pool, 2,313 23°47′05.00″, 68°14′12.30″21 5946 KF285885 KF285835
22 5947 KF285886 KF285836
Peine Stream, 2,440 23°41′00.00″, 68°03′31.00″1 5948 KF285887 -
2 5949 KF285888 KF285837
Taltal Basin
Las Breas, Quebrada de Spring-pool, 588 25°30′10.10″, 70°24′40.20″1 5950 KF285903 -
Taltal 2 5951 KF285904 -
Pan de Azúcar Basin
Quebrada Cachina Spring, 321 25°54′03.40″, 70°36′47.90″1 5952 KF285905 KF285856
2 5953 KF285906 KF285857
Copiapó River Basin
Hornitos, Copiapó River River, 826 27°46′04.40″, 70°09′43.60″8 5954 KF285909 -
10 5955 KF285910 -
12 5956 KF285911 KF285860
Copiapó, Copiapó River River, 465 27°26′25.90″, 70°16′02.60″19 5957 KF285912 -
20 5958 KF285913 -
21 5959 KF285914 -
Pabellón, Copiapó River River, 693 27°39′54.90″, 70°13′55.80″13 5960 KF285915 -
15 5961 KF285916 -
18 5962 KF285917 -
Humedal, Copiapó River River-swamp, 17 27°19′14.80″, 70°55′08.90″2 5963 KF285918 -
3 5964 KF285919 -
Carrera Pinto Spring, 1,565 27°06′52.50″, 69°53′52.00″1 5965 KF285920 KF285861
2 5966 KF285921 KF285862
GEVOL: specimen voucher number deposited in the Laboratorio de Genética y Evolución; MNHNCL: sample of tissue (or DNA) deposited in the Museo Nacionalde
Historia Natural, Santiago, Chile.
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Biotechnology Information database [GenBank: KF285827
to KF285867 for 16S; KF285868 to KF285926 for 12S]
(Table 1). The 12S rRNA and 16S rRNA sequences of the
Lithoglyphidae Lithoglyphus naticoides (Pfeiffer, 1828)
[GenBank: AF445341 and AF445351, respectively] and the
Pyrgulidae Pyrgula annulata (Linnaeus, 1758) [GenBank:
AF445340 and AF445350, respectively] were used as
outgroup in the molecular analyses (Hausdorf et al.
2003). The matrix used in the combined analysis in-
cluded taxa for which we did not have sequences for
some of the two loci; in this case, the taxa were coded
as “missing data”, which has shown not to unduly in-
fluence the phylogenetic resolution (Wiens and Reeder
1995; Wiens 1998; Collado and Méndez 2012b).
Results
Morphology
With few exceptions, the morphology of the penis showed
differences between populations and species of Heleobia
from northern Chile (Figure 2). Conversely, the shell of
the snails presented little differentiation (Figure 3), with a
size always greater than 3.5 mm (and less than 8 mm), so
they belong to the large-sized species group proposed in
the genus (see Biese 1944, 1947).
12S rRNA gene
We amplified 369 bases of the 12S rRNA gene from 59
Heleobia specimens, the final length of the alignment
including the two outgroup sequences. The base com-
position was A = 0.41, C = 0.14, G = 0.16, and T = 0.29.
For MP, 48 characters were informative, and 321 were
excluded from the analysis. This analysis recovered 100
trees (not shown) with a length of 65 steps, consis-
tency index = 0.83, retention index = 0.93, and rescaled
consistency index = 0.78. For the BI (tree not shown),
the best model of evolution for this data set was the gen-
eral time reversible model (Rodríguez et al. 1990) and
gamma distribution rate heterogeneity (GTR + G). The
mean genetic divergence among all the populations and
species of Heleobia from northern Chile was 1.20% using
the two-parameter model of Kimura (1980) (K2P). The
greatest distance among the nominal species occurred
between H. ascotanensis and H. atacamensis (1.95%), and
between the latter species and H. chimbaensis (1.95%, data
not shown).
16S rRNA gene
We amplified 497 to 499 bases of the 16S rRNA gene
from 41 Heleobia specimens. The alignment was 499 nu-
cleotide sites in length considering the two outgroup se-
quences. The base composition was A = 0.34, C = 0.14,
G = 0.19, and T = 0.33. For MP, 54 characters were in-
formative, and 445 were excluded from the analysis. This
analysis recovered two trees with the same topology (not
shown) and a length of 88 steps, consistency index = 0.76,
retention index = 0.89, and rescaled consistency index =
0.68. For the BI, the best evolutionary model was GTR + G.
The mean genetic divergence among all populations and
species of Heleobia was 1.30% using the K2P model. Genetic
distance analysis was performed between Heleobia species
included in the present study plus 16S sequences of the
Semisalsinae taxa, Semisalsa dalmatica Radoman, 1974
[GenBank: AY676119] (Wilke 2005) and Semisalsa stag-
norum Gmelin, 1791 [GenBank: JX970535] (Wilke et al.
Figure 2 Penis morphology of species and populations of Heleobia from northern Chile. (A) Los Perales, (B) Salar de Carcote, (C) Isluga,
(D) Aguada de Chorrillos, (E) Heleobia transitoria,(F) Heleobia ascotanensis,(G) El Tatio, (H) Tilomonte, (I) Heleobia atacamensis. Left, dorsal view;
right, ventral view. Scale bar = 0.5 mm.
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2013) for comparative purposes (Table 2). The greatest dis-
tance occurred between H. atacamensis and H. transitoria
(2.63%), followed by H. atacamensis and H. chimbaensis
(2.40%). The percentage of genetic divergence between
Semisalsa dalmatica and Semisalsa stagnorum was 0.43%,
much lower than several pair-wise comparison values ob-
tainedinthepresentstudy(Table2).TheBEASTanalyses
showed that the origin of the lineages occurred in the Early
Pleistocene, with an average of 2.1 million years to the most
ancestral node of Heleobia.
Combined analysis
For the combined phylogenetic analyses, 5 sequences were
left as missing data within the 12S rRNA and 16 within
the 16S rRNA data partition for which these sequences
were unavailable (the alignment data are available from
the corresponding author). The data matrix included 55
Heleobia sequences and 868 characters, of which 102 were
parsimony informative. This analysis recovered 100 trees
(one of them shown in Figure 3) with a length of 148
steps, consistency index = 0.82, retention index = 0.92, and
rescaled consistency index = 0.75. These indexes were
slightly lower than those obtained with the 12S gene but
considerably higher than those obtained with the 16S
gene. Base frequencies were A = 0.38, C = 0.13, G = 0.17,
and T = 0.31. Most of the major clades retained in the sep-
arate analyses were recovered in the combined MP analysis,
withsimilarsupportvaluesformostnodes.Weusedthe
GTR + G model to perform the combined BI analyses (tree
not shown).
MP analysis of the combined data set and the BI re-
covered five major clades, four of which were composed
Figure 3 One of 100 equally most parsimonious trees obtained by the combined MP analysis. The tree was constructed using
concatenated 12S and 16S mitochondrial sequences. Numbers above the nodes indicate bootstrap values obtained under the MP analysis
(only values above 50% are shown), followed by the posterior probability values obtained in the BI (only values equal to or above 0.95 are shown).
To the right of the figure are shown some shells of nominal species and previously unknown populations of Heleobia from northern Chile.
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Table 2 Percentage sequence divergence (K2P) between pairs of mtDNA 16S sequences of Heleobia populations from northern Chile
Taxa 1 23456789101112131415161718192021222324
1. S. stagnorum
2. S. dalmatica 0.43
3. H. ascotanensis 2.20 2.20
4. H. atacamensis 2.67 2.67 2.21
5. H. chimbaensis 2.41 1.97 1.52 2.24
6. H. loaensis 1.97 1.52 1.52 1.97 1.30
7. H. opachensis 1.97 1.52 1.52 1.97 1.30 0.00
8. H. transitoria 1.97 1.52 1.30 2.67 1.52 1.08 1.08
9. Ag. de Chorrillos 1.75 1.31 0.09 2.20 1.08 0.65 0.65 0.43
10. Ascotán (S2) 2.20 2.20 0.00 2.21 1.52 1.52 1.52 1.30 0.86
11. Carrera Pinto 1.75 1.31 0.09 2.20 1.08 0.65 0.65 0.43 0.00 0.86
12. Carcote (S1) 1.97 1.52 1.52 1.97 1.30 0.00 0.00 1.08 0.65 1.52 0.65
13. Carrizo 2.41 1.97 1.52 2.42 0.00 1.30 1.30 1.52 1.08 1.52 1.08 1.30
14. Cascabeles 2.41 1.97 1.52 2.42 0.00 1.30 1.30 1.52 1.08 1.52 1.08 1.30 0.00
15. Colpa 1.97 1.52 1.52 1.97 1.30 0.00 0.00 1.08 0.65 1.52 0.65 0.00 1.30 1.30
16. El León 2.19 1.75 1.52 2.88 1.74 1.30 1.30 2.15 0.65 1.52 0.65 1.30 1.74 1.74 1.30
17. Hornitos 1.97 1.52 1.52 2.42 0.43 0.86 0.86 1.08 0.65 1.52 0.65 0.86 0.43 0.43 0.86 1.30
18. La Finca1 1.74 1.31 1.74 1.75 1.52 0.21 2.14 1.30 0.86 1.74 0.86 0.21 1.52 1.52 0.21 1.52 1.08
19. Laguna Opache 1.97 1.52 1.52 1.97 1.30 0.00 0.00 1.08 0.65 1.52 0.65 0.00 1.30 1.30 0.00 1.30 0.86 0.21
20. Las Vertientes 1.97 1.52 1.52 1.97 1.30 0.00 0.00 1.08 0.65 1.52 0.65 0.00 1.30 1.30 0.00 1.30 0.86 0.21 0.00
21. Los Perales 2.43 1.98 1.75 1.99 1.52 1.08 1.08 1.74 1.30 1.75 1.30 1.08 1.52 1.52 1.08 1.97 1.52 1.08 1.08 1.08
22. Parinacota 1.97 1.52 1.52 1.97 1.30 0.00 0.00 1.08 0.65 1.52 0.65 0.00 1.30 1.30 0.00 1.30 0.86 0.21 0.00 0.00 1.08
23. Peine 2.67 2.67 2.20 0.00 2.43 1.97 1.97 2.65 2.20 2.21 2.20 1.97 2.42 2.42 1.97 2.88 2.42 1.76 1.97 1.97 1.99 1.97
24. Tilomonte 2.89 2.89 1.98 0.21 2.65 2.20 2.20 2.42 1.97 1.98 1.97 2.20 2.65 2.65 2.20 2.66 2.65 1.97 2.20 2.20 2.21 2.20 2.14
25. El Totoral 2.19 1.75 1.74 2.66 0.65 1.08 1.08 1.30 0.86 1.74 0.86 1.08 0.65 0.65 1.08 1.52 2.15 1.30 1.08 1.08 1.75 1.08 2.66 2.88
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exclusively of one each of the nominal species recog-
nized in the region: H. ascotanensis,H. chimbaensis,H.
transitoria, and H. atacamensis. The first clade (clade A,
Figure 3) was integrated by H. atacamensis from Tilopozo
and the allopatric snails from Peine and Tilomonte. This
clade was inferred as the sister group of all the rest of the
species and populations of Heleobia sequenced (100%
bootstrap, 1.00 posterior probability). However, the sister
group position of all Heleobia snails from northern Chile
was occupied by a different taxon or lineage in the 12S
and 16S analyses performed, suggesting that further
work is needed to resolve this issue. Likewise, relation-
ships among the remaining species were not resolved.
The monophyly of the sequences of H. atacamensis and
those from Peine was supported by 91% bootstrap and
0.96 posterior probability. Within this clade, the snails
from Tilomonte were recovered as the sister group of
these sequences with high support values (99% boot-
strap, 1.00 posterior probability). The sequences of the
snails from Salar de Ascotán formed a monophyletic
group (clade B in Figure 3) with the sequences from the
allopatric locality of El Tatio with moderate bootstrap
values (67%). Within this clade, two subclades were in-
ferred, one containing snails from Spring 2 from this
salt pan (assigned to H. ascotanensis) plus a group of
snails from this same spring, which was supported by 54%
bootstrap, and another composed of snails from Spring 11
from Salar de Ascotán and those from El Tatio, with mo-
derate bootstrap values (60%).
The monophyly of the sequences of H. transitoria
(clade C in Figure 3) from its type locality Quebrada
Cachina plus snails from Quebrada El León was sup-
ported by high bootstrap values (85%), but low posterior
probability. The snails from Carrera Pinto and Aguada
de Chorrillos were recovered as the sister group to this
subclade but without node support.
The sequences of H. chimbaensis from the type locality
Quebrada La Chimba nested in a clade together with
snails from the Atacama Desert (74% bootstrap, 0.98 pos-
terior probability) (clade D in Figure 3). Within this clade,
the sequences from El Totoral formed a monophyletic
group with a 78% bootstrap and high posterior probability
values (0.99). The sequences of snails from Los Perales
(clade red in Figure 3) formed a monophyletic group sup-
ported by 100% bootstrap and 1.00 posterior probability.
The sequences of snails from the Salar de Carcote in
the Chilean Altiplano nested in a clade together with se-
quences from the Copiapó River but without support.
The penis morphology of these snails, however, showed
similarities with those of the species H. loaensis,H. opa-
chensis, and H. atacamensis, in the latter case suggesting
convergence of characters (Figure 2).
The sequences of snails from Las Cascadas, the type
locality of H. loaensis and Vertiente Opache, the type
locality of H. opachensis, were not resolved by the com-
bined analysis, as well as the sequences of the snails
from Chiu-Chiu, La Finca, Laguna Opache, and Las
Vertientes in the Loa basin, and those from Colpa,
Parinacota and Isluga in the Chilean Altiplano. However,
we found the penis morphology of snails from Isluga to
be different from those of other localities (Figure 2).
Discussion
The combined and separate phylogenetic analyses recov-
ered the populations of Heleobia from Los Perales as a
monophyletic group with high support values. These snails
were originally assigned to H. chimbaensis by Biese (1947)
based on external shell morphology, but our results show
that they constitute an independent lineage phylogenetic-
ally separated from this species, and the male copulatory
organ (Figure 2) is markedly different (see Hubendick
1955; Collado et al. 2011a; Collado 2012); it is apparent
that a description and a new scientific name is required
for this cryptic species. Apart from this clade, our mo-
lecular analyses show that the populations of Heleobia
from northern Chile form four other generally well sup-
ported clades. The division of the clades suggests that a
relatively long amount of time elapsed to produce gen-
etic lineage diversification in the region, which is not
reflected in the morphology of the shell (see Figure 3).
This is not infrequent in rissooidean snails, a taxon with a
large number of genera, many of which include species
morphologically similar but genetically divergent (e.g., Liu
et al. 2003; Hershler et al. 1999; Hershler et al. 2003;
Bichain et al. 2007; Hershler et al. 2007; Falniowski et al.
2012). The percentage sequence divergence between the
Heleobia species observed in the present study was gener-
ally greater than those found in other rissooidean taxa.
For example, there is no variation between 12S sequences
of the species of the family Tateidae Tatea huonensis
(Tenison-Woods, 1876) and Tatea rufilabris (A. Adams,
1862) [GenBank: FJ619852 and FJ619856, respectively]
(see Colgan and da Costa 2009), while divergence between
the Lithoglyphidae Benedictia baicalensis (Gerstfeld) and
Benedictia maxima (W. Dybowski) [GenBank: AF445349
and AF445348, respectively] (Hausdorf et al. 2003) is 0.83%
(K2P), and the divergence between Potamopyrgus anti-
podarum (Gray, 1843) and Potamopyrgus estuarinus
(Winterbourn, 1971) [GenBank: HQ875146 and GQ996415,
respectively] (Neiman et al. 2010) is 2.25%. In the case
of 16S sequences, the divergence between the Hydrobii-
dae Sulawesidrobia bonnei (Abbott, 1945) and Sulawe-
sidrobia botak Haase and Bouchet, 2006 [GenBank:
HM587413 and HM587411, respectively] (Zielske et al.
2011) is 0.19%, while the divergence between the Tatei-
dae Potamopyrgus antipodarum and Potamopyrgus oppi-
danus Haase, 2008 [GenBank: AY634104 and AY634090,
respectively] (Haase 2005) is 0.21%.
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In the genus Heleobia, several studies, including ori-
ginal descriptions of species, show overlapping charac-
ters in the external morphology of the shell (e.g., Bavay
1904; Courty 1907; Pilsbry 1911, 1924; Biese 1944, 1947;
Haas 1955; Preston 1915; Weyrauch 1963). In fact, the
Heleobia fauna from Lake Titicaca and closely related
taxa from the surrounding areas form a species flock, in
some cases including morphologically parallel species
(Kroll et al. 2012). The clades inferred by our molecular
analyses are concordant with the distribution pat-
terns, and some of them include candidate species or
new lineages within the genus not detected previ-
ously and whose copulatory organs show a particular
morphology.
The Salar de Atacama contains snails from Tilopozo,
Peine, and Tilomonte (clade A, Figure 3). Philippi (1860)
described H. atacamensis from Tilopozo, and since then,
it has not been addressed in the literature, with the excep-
tion of checklists of the species (Stuardo 1961; Sielfeld
2001; Valdovinos 1999, 2006, 2008) or the description of
its penis (Collado et al. 2011a). The monophyly of the se-
quences of H. atacamensis from Tilopozo and Peine was
well supported in the MP and BI analyses, while the se-
quence from Tilomonte appears as the sister to this group
in all analyses performed. Likewise, the penis morphology
shows differences between H. atacamensis and snails from
Tilomonte (Figure 2), suggesting that this population rep-
resents a candidate species of the genus. These three local-
ities are located on the east bank of the Salar de Atacama
in the homonymous basin. Tilopozo is separated from
Peine by about 20 km, while Tilomonte is located in the
middle, at a distance of about 13 km from each of these
locations. The Tilomonte oasis includes a small stream
that dries at a distance of approximately 6.5 km of the
shore of the Salar de Atacama, while the oasis of Peine is
formed by a stream with a greater flow of water that flows
at irregular intervals into the salt pan. It is apparent that
seasonal flooding could connect Tilopozo and Peine, pro-
ducing dispersal of the snails between these locations,
while the partial isolation of Tilomonte could explain the
divergence of these populations as a product of vicariance.
Clade B (Figure 3) clusters populations from Springs 2
and 11 in Salar de Ascotán and the allopatric snails from
the locality of El Tatio. Heleobia ascotanensis was de-
scribed by Courty (1907) (under the genus Paludestrina
d’Orbigny, 1840) from the Salar de Ascotán together
with five subspecies and two species of the genus Bythi-
nella Moquin-Tandon, 1856 based on shell morphology
(see Collado and Méndez 2012a). The monophyly of the
sequences and support values suggest that two distinct
lineages co-occur in Spring 2 from this salt pan, but we
have no evidence of reproductive isolation between them.
Additionally, the snail sequences from Spring 11 form a
subclade together with the snails from El Tatio. In the 12S
analysis, the separation of this subclade from the sympat-
ric snails from Spring 2 was supported by a moderate
bootstrap value in the MP analysis (66%) but with a high
posterior probability value in the BI (0.97). Furthermore,
the penis morphology of H. ascotanensis (Collado et al.
2011b; present study) is different from those of the snails
of El Tatio (Figure 2), suggesting the occurrence of an
additional candidate species in our data set. At present,
we cannot establish if the snails from Spring 11 from Salar
de Ascotán constitute a different species from those of El
Tatio, although the support values for their monophyly re-
covered in the 12S separate analysis seem to indicate this.
There is no evidence of subterranean or surface water
connection between Spring 2 in the north of the Salar de
Ascotán and Spring 11 in the south; these two springs are
separated by a distance of approximately 21.5 km. Struc-
turing of populations of the killifish Orestias ascotanensis
Parenti, 1984 in Salar de Ascotán was recently reported,
including those fish located in Spring 11, a process prob-
ably stimulated by habitat fragmentation occurring from
the Late Pleistocene to the beginning of the Holocene
(Morales et al. 2011). Keller and Soto (1998) suggested
that in this salt pan, there is no evidence of major changes
in the water level in the last 12,000 years. Recently, we also
detected microvicariance processes structuring the popu-
lations of the aquatic gastropod Biomphalaria crequii
(Courty 1907) inhabiting the southernmost springs of the
system (Collado and Méndez 2013). There is no evidence
either of subterranean or surface water connection be-
tween Spring 11 and El Tatio, a geyser field located 75 km
to the south in a different basin.
Heleobia transitoria integrates a clade (clade C, Figure 3)
with allopatric Atacama populations that are separated by
arid desert. After its original description, this species has
not been addressed in the literature, with the exception of
local checklists. The type locality of the species, a small
spring in Quebrada Cachina, is separated by approximately
151 km from the Carrera Pinto oasis, another small south-
western spring, 117 km from a small southern spring in
Quebrada El León and 147 km from the southernmost
spring Aguada de Chorrillos. Within this clade, the mono-
phyly of H. transitoria and the snails from Quebrada El
León was well supported in the MP 16S and the combined
analyses; they are probably conspecific. The snails from
Aguada de Chorrillos (and Carrera Pinto) never formed a
monophyletic group with H. transitoria in the analyses per-
formed, and the penis morphology shows conspicuous dif-
ferences between them (Figure 2).
Clade D (Figure 3) clusters the sequences of H. chim-
baensis from Quebrada La Chimba and snails from other
Atacama localities, almost all of them with allopatric dis-
tribution. The monophyly of snails from Las Breas in
Quebrada de Taltal and Quebrada Cascabeles was weakly
supported in the combined analysis, but was recovered
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with relatively high support values in the 12S analysis
(65% bootstrap, 0.97 posterior probability), suggesting
the presence of a single species within this assemblage.
The monophyly of the sequences from El Totoral and
the Atacama populations from Copiapó River was sup-
ported by 70% bootstrap, suggesting some phylogenetic
structure. This is consistent with the location of El
Totoral, an intermittent stream in the Atacama Desert
63 km south of the Copiapó River. The snails dissemi-
nated within the Copiapó River (Humedal, Copiapó,
Hornitos, and Pabellón) never clustered together within
the subclade integrated by H. chimbaensis or another de-
scribed species of the genus. The lack of monophyly sug-
gests the presence of more than one taxon in this
hydrological system, for which the names H. copiapoen-
sis,H. copiapoensis costata, and H. striata are available
(Biese 1944, 1947).
From the taxonomic point of view, Biese (1947) con-
sidered H. loaensis and H. opachensis as different species
based on external shell morphology. The sequences of
these species were not resolved by any analyses per-
formed in the present study, with the exception of the
NJ analysis (tree not shown), which recovered these taxa
in a moderately well-supported clade (65% bootstrap).
Considering that these taxa have similar shell and penis
morphology (Biese 1944, 1947; Collado et al. 2011a),
their validity needs to be tested using additional mor-
phological characters or a distinct, faster-evolving DNA
locus. Apart from snails from Salar de Carcote and
Isluga, which probably represent other candidate species
judging from penis morphology, the identity and system-
atic position of snails from Colpa and Parinacota in the
Chilean Altiplano and La Finca, Laguna Opache, Chiu-
Chiu, and Las Vertientes from the Loa basin in the Ata-
cama Desert need to be clarified.
Heleobia carinata (Biese), a name that does not meet
the criteria of availability according to the Code (Inter-
national Commission on Zoological Nomenclature ICZN
1999) (see Collado et al. 2011b), was found in Pleistocene
lacustrine facies in northern Chile (Biese 1961; Ochsenius
1974). Regardless of the availability of the name, these
Heleobia record fossil data agree with our molecular cali-
bration that places the origin of the Heleobia in the region
in the Early Pleistocene, implying a recent origin of the
group. Kroll et al. (2012) also found Pleistocene divergence
using the mitochondrial cytochrome c oxidase subunit 1
(COI) gene among the Heleobia populations distributed in
northern Chile and the Altiplano region.
According to our results, the speciation of Heleobia fauna
in the Chilean Altiplano and the Atacama Desert is sug-
gested as being allopatric from the Early Pleistocene. We
consider the diversity of Heleobia at the species level in the
region to be underestimated. We suggest that this is a con-
sequence of the use of conchological criteria to delineate
species boundaries and the limited sampling conducted to
date. The discovery of a cryptic lineage in Los Perales and
other candidate species in different localities in the region
supports this statement.
Conclusions
The present results support the conclusion that the snails
from Tilomonte, El Tatio, Aguada de Chorrillos, Isluga, and
Salar de Carcote (Spring 1) represent candidate species of
the genus Heleobia. The molecular analysis provides sup-
port for recognizing the nominal species H. chimbaensis,H.
atacamensis,H. transitoria,andH. ascotanensis as valid
taxa, while the specific status of H. opachensis and H. loaen-
sis could not be resolved. The phylogenetic analysis and
penis morphology revealed that the snails from Los Perales
constitute a cryptic species of the genus. We also suggest
that vicariance may largely explain the distribution patterns
of Heleobia populations disseminated in the Chilean Alti-
plano and the Atacama Desert. A more intensive effort will
be necessary to test the cryptic species boundaries thor-
oughly in Heleobia.
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
GAC participated in the field trips for sampling, obtained the sequences,
performed the analyses, and wrote the manuscript. MAV performed the
morphological study, made the figures and reviewed the manuscript. MAM
made intellectual contributions, reviewed the manuscript and provided
technical and financial support. All authors read and approved the final
manuscript.
Acknowledgments
This study was supported by a grant from Fondo Nacional de Desarrollo
Científico y Tecnológico (FONDECYT, 3110072) to G. Collado. We also thank
Claudio Correa, Francis Miño, and Cristian Araya for specimen collection, and
the anonymous reviewers who improved the original manuscript.
Author details
1
Departamento de Biología y Ciencias Ambientales, Facultad de Ciencias,
Universidad de Valparaíso, Gran Bretaña, 1111 Valparaíso, Chile.
2
Laboratorio
de Genética y Evolución, Facultad de Ciencias, Universidad de Chile, Las
Palmeras, 3425 Santiago, Chile.
Received: 10 July 2013 Accepted: 21 November 2013
Published: 5 December 2013
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doi:10.1186/1810-522X-52-50
Cite this article as: Collado et al.:Hidden diversity in spring snails from
the Andean Altiplano, the second highest plateau on Earth, and the
Atacama Desert, the driest place in the world. Zoological Studies
2013 52:50.
Collado et al. Zoological Studies 2013, 52:50 Page 13 of 13
http://www.zoologicalstudies.com/content/52/1/50