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Phylogeography and molecular
species delimitation reveal
cryptic diversity in Potamolithus
(Caenogastropoda: Tateidae)
of the southwest basin
of the Andes
Gonzalo A. Collado1,2, Cristian Torres‑Díaz1,2 & Moisés A. Valladares1,2*
The species of the genus Potamolithus inhabiting the southwestern basin of the Andes are dicult
to distinguish due to small size and similar shell morphology. Only Potamolithus australis and
Potamolithus santiagensis have been traditionally recognized in this region, but the occurrence of
several morphologically similar undescribed populations could increase the regional richness. Here we
delimit described and potentially undescribed cryptic species of the genus using partial sequences of
the mitochondrial cytochrome c oxidase subunit I (COI) gene. Network analysis and diversity indices
inferred six highly dierentiated haplogroups, many of them sympatric and widespread in the study
area. Phylogeographic analyses suggest a scenario of recent diversication and the occurrence of
multiple refuges during the successive Pleistocene glaciations. Phylogenetic analysis also recovered
six major clades that showed no relationship with physiography. Species delimitation analyses
consistently recognized three or four candidate species apart from P. australis and P. santiagensis.
Divergence times indicate that speciation of Chilean Potamolithus began at the end of the Pliocene,
probably driven by climatic rather than geographic events. Considering the high inter‑ and intra‑basin
genetic diversity, conservation eorts should be focused on protecting sympatric taxa in the basins
with the highest species richness.
Species identication is increasingly important in biodiversity studies, conservation biology and natural herit-
age of the countries1–4. e high rate of species extinction as a consequence of habitat loss, pollution and global
change, together with the enormous unknown biodiversity in some groups has led to the development of more
eective and precise methods of taxonomic discrimination, which has been facilitated by technological advances5.
It is now known that a portion of natural diversity is morphologically cryptic, so its identication depends on
integrating molecular tools and traditional morphological approaches6.
A signicant number of studies has shown that the evolutionary history of the biota in southern South
America has been inuenced by dierent geological processes and climatic events such as the Andean orogeny,
marine introgressions, drainage reversals and glaciations7–20. e Andean upli occurred from the Early Miocene
generated the drainage divide between Chile and Argentina signicantly impacting the distribution of genetic
diversity of species, mainly the aquatic fauna10,12,19,21. e Patagonia Region suered successive glaciations, with
more than 10 major cooling events during the Pliocene and Pleistocene22–25. is intermittent melting and cooling
of glaciers also had an impact on the underlying biota, leaving a genetic imprint on the phylogeography of several
species7,12,19. Some of the strongest glacial events were the Great Patagonian Glaciation (GPG) c. 1 million years
ago (Ma), several ancient glaciations collectively called the Pre-GPG occurred in the Lower/Middle Pleistocene
(2.1–1.0Ma) and three other cooling events called the Post-GPG that occurred in the Middle Pleistocene25–28.
e boundary between the Piacenzian and Gelasian glaciations marks the boundary between the Pliocene and
OPEN
Chillán, Chile.
Chile. *email: valladares.moises@gmail.com
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the Pleistocene, 2.59 Ma29. From the end of the Miocene the land south of 37°S was intermittently covered by
ice sheets extending to Antarctica in the successive glaciations and whose greatest extension was reached during
the Last Glacial Maximum (LGM) in the late Pleistocene, c. 0.025 Ma24,25,30,31. e tectonics and environmental
processes generated expansion and retreat of forests, isolation of species into refugia, habitat fragmentation,
extinctions, and the establishment of distributional limits promoted by transverse glacial tongues from the Andes
that le open areas of secondary contact aer melting10,12,15,19,22.
e superfamily Truncatelloidea Gray, 1840 is one of the most diverse groups of Caenogastropoda, with hun-
dreds of genera32–34. Within this superfamily, the family Tateidae iele, 1925 is composed of minute freshwater
snails that have a wide geographic distribution throughout the South Pacic and southern South America35–38.
Tateid snails were traditionally included in the family Amnicolidae Tryon, 186335, Hydrobiidae Troschel, 185739
or Lithoglyphidae Troschel, 185737,40, revealing inconsistencies in the classication; the monophyly of the Tateidae
was recovered by Wilke et al41. Two native genera of this family have been recognized in South America, Pota-
molithus Pilsbry, 1896, which contains about 47 species42, and the monospecic genus Strobellitatea Cazzaniga,
201743. A third genus is Potamopyrgus Stimpson, 1865, represented in Chile by the invasive species Potamopyrgus
antipodarum (Gray, 1843)44. Potamolithus is a diverse genus of thick-shelled operculate snails comprising species
that reach between 2.0 and 7.0mm in length35,45–49. Although there are drawings or illustrations of the so parts
in some taxa, species taxonomy has been based mainly on characters of the external shell morphology39,45,46,48,50.
In Chile only two species of the genus have been described based solely on external shell characters and oper-
culum, Potamolithus australis Biese, 1944 and Potamolithus santiagensis (Biese, 1944). Apart from the original
description, P. australis, typical of Llanquihue Lake in Chilean Patagonia, appears mentioned in a few later works
and lists of species47,51–54 while López Armengol55 considered it as nomen dubium. Potamolithus santiagensis,
originally described under the genus Littoridina Souleyet, 1852 from Dehesa Stream36 in central Chile and to
which a population of El Yeso Spring was later added56, was subsequently included in the genus Heleobia Stimp-
son, 1865 but recently transferred to Potamolithus by Collado etal.57. ese authors also added two populations
from central Chile (Lo Carreño and El Colorado) to the range of P. santiagensis.
Mitochondrial DNA sequences generally constitute a powerful tool for species delimitation, particularly
in groups that are dicult to resolve using morphological data58. e advent of species delimitation methods
based on DNA sequences has provided a useful method to validate previously described species and identify new
evolutionary units in a wide variety of taxa59–64. In the present study, based on the current taxonomic knowledge
of the genus Potamolithus, we assessed the hypothesis that only two species exist along its distribution range in
Chile. We performed phylogenetic and network analyses in a phylogeographic framework to study the biodi-
versity of the genus in the southwest basin of the Andes, both within and among species, including potentially
cryptic species. We also used molecular species delimitation approaches to dene species partitions considering
already described species and new lineages in the group.
Methods
Specimen collection. Snails were collected in 2015–2017 from multiple freshwater ecosystems located in
central and southern Chile (Fig.1; TableS1). All animals were collected using a hand sieve and then stored in
absolute ethanol. Snail sampling was authorized by the Subsecretaría de Pesca y Acuicultura, Ministerio de
Economía, Fomento y Turismo, República de Chile (Resolution No. 3285). e procedure for handling animals
was approved by the Bioethics Committee of the Universidad de Valparaíso (Resolution No. 009–2013), institu-
tion with which the rst author was associated at the beginning of the project. All methods were carried out in
accordance with relevant guidelines and regulations proposed. e study was carried out in compliance with the
ARRIVE guidelines (http:// www. nc3rs. org. uk/ page. asp? id= 1357).
Molecular analysis. For DNA extraction, PCR amplications and sequencing of partial sequences of the
mitochondrial cytochrome c oxidase subunit I (COI) gene we followed the methods described by Collado44
and Collado etal.57,65. Forward and reverse strands were corrected for misreads and merged into one sequence
le using Sequencher v5.4.6 (Gene Codes Corporation, Ann Arbor, MI, USA). Sequences were aligned with
MAFFT v7.47066 using the MAFFT online service for multiple sequence alignment67. To estimate genetic diver-
sity of Potamolithus across the landscape we calculated the number of polymorphic sites (S), number of haplo-
types (H), haplotype diversity (Hd) and nucleotide diversity (π) in DnaSP v5.10.168. To visualize the relationships
between haplotypes we constructed a median-joining network69 using the program PopART v1.770. Genetic
distances were estimated in MEGA Xv10.2.671.
Species delimitation in Potamolithus was studied using the Automatic Barcoding Gap Discovery (ABGD)
method72 (a non-tree-based method)59, the multi-rate Poisson tree processes (mPTP) analyses73 and a Bayes-
ian general mixed Yule coalescent (bGMYC) approach74. e ABGD method is based on genetic distances and
automatically detects the gap between intra and interspecic divergence, which is then used recursively to delimit
species hypotheses. e mPTP method delimits species trying to determine the transition from a between‐ to a
within‐species process, but also incorporates dierent levels of intraspecic genetic diversity deriving from dier-
ences in either the evolutionary history or sampling of each species73. e GMYC method requires an ultrametric
tree (or multiple trees sampled from a posterior distribution in the Bayesian implementation) and attempts to
detect the transition between the branching pattern attributed to speciation and intra-species coalescent process.
e ABGD method was performed on the online web server (https:// bioin fo. mnhn. fr/ abi/ public/ abgd/ abgdw
eb. html) and was run with the default settings (relative gap width of 1.5; intraspecic divergence: between 0.001
and 0.1). For the mPTP and bGMYC analyses, phylogenetic reconstructions were performed previously using
the Maximum Likelihood (ML) and Bayesian Inference (BI) algorithms, respectively. Tatea huonensis (Tenison-
Woods, 1876) (GenBank accession number: JX97061941) was used as outgroup in both reconstructions. In
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addition, four species of Potamolithus from Argentina were included in the reconstructions: Potamolithus aga-
petus Pilsbry, 1911 (GB: KM22091075), Potamolithus buschii (Frauenfeld, 1865) (GB: KM22090975), Potamolithus
elenae de Lucía & Gutiérrez Gregoric, 2016 (GB: KX397599, KX39760076) and Potamolithus lapidum supersulcatus
Pilsbry, 1896 (GB: KX15884376). e ML reconstruction was performed in RAxML v8.2.1277 using the GTRGAM-
MAI model of nucleotide substitution and the node support was obtained by performing a bootstrap analysis of
1000 pseudo-replicates. e Bayesian reconstruction for the bGMYC method was performed in BEAST v2.6.378
using the Relaxed Clock Log Normal model and a Coalescent Constant Population tree prior.
We applied two phylogeny‐aware approaches to delimit species in Potamolithus. First, mPTP analyses were
performed using the online server (https:// mcmc- mptp.h- its. org/ mcmc/) and the ML tree from RAxML. For the
mPTP method, aer removing the outgroup, we performed two independent runs, each of 1,000,000 generations,
sampling every 1000 generations and with a 10% burn-in. e second method was a GMYC model implemented
in the package bGMYC v1.0.274 in R v3.5.2 soware79. For the analyses we used 100 random trees from the BEAST
reconstruction (aer removing outgroups). Simulations considered 50,000 generations, discarding 40,000 rep-
licates, and setting a thinning every 100th generation. Aer preliminary analyses with varying parameters on a
single tree, the upper and lower bounds of the Yule and coalescent rate change parameters were set to 1.0and
0.5, respectively, and the upper prior threshold for the number of species was set at 100. Finally, all results were
visualized using the package phytools v0.6–2080.
Divergence times were estimated in BEAST using a reduced data set of the haplotypes retrieved by DnaSP.
For this analysis, a strict clock model was implemented using a substitution rate of 1.7% with a standard devia-
tion of 0.34%81. For allBEAST analyses the site model was specied using bModelTest v1.2.182, performing
three independent analyses with 50 million generations each to infer a Maximum Clade Credibility Tree using a
burn-in of 25%. A non-ultrametric Bayesian tree was reconstructed for the complete COI dataset using MrBayes
v3.2.783. e analysis used four parallel runs with 20 million generations each using a burn-in period of 25%.
ML and BI reconstructions were performed in the CIPRES cluster of the San Diego Supercomputer Center84.
Figure1. Sampling localities of Potamolithus in central and southern Chile ofthe present and
other studies44,57,65. e areas of Maule-Ñuble and Llanquihue Lake-Puelo River are enlarged. e locality of
El Yeso, historically assigned to Potamolithus santiagensis, is indicated by a white triangle. e type locality of
Potamolithus australis, Puerto Chico in Llanquihue Lake, is indicated by a white square. e limits of the ice
sheet during the Last Glacial Maximum in Patagonia were generated following dierent authors24,30,31. e map
was created using QGIS Geographic Information System v3.4.9 (http:// www. qgis. org). (Map: M.A. Valladares).
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Results
e amplication of the COI gene in the Chilean Potamolithus (176 individuals) produced a fragment of 510
nucleotides in length. e mtDNA COI sequence alignment recovered a total of 86 polymorphic and 69 parsi-
mony-informative sites, dening 47 haplotypes from 196 individuals considering our original sequences and
those obtained from GenBank. e nucleotide composition in the complete dataset (excluding outgroup) was
38.7% A, 19.1% C, 16.1% G and 26.1% T. e best-tting model of nucleotide substitution, as determined by
bModelTest, was the HKY + I + G. e sequences showed a pattern of high haplotype diversity (Hd = 0.928) and
nucleotide diversity (π = 0.0382). e diversity indices showed the co-occurrence of several genetic entities in the
same locality (considering sites with N > 2). is was reected by a large number of mutational steps and high
nucleotide diversity within localities (TableS1). e median-joining network recovered six well-dierentiated
haplogroups without any evident latitudinal segregation (Fig.2). Haplogroup 1 was composed of 16 haplotypes
(89 individuals) detected in 33 out of the 48 localities analyzed, representing the largest latitudinal distribution
ranging from Central Chile to the southernmost locality sampled in Patagonia (32° to 53°S). Haplogroup 2 was
composed of ve haplotypes (33 individuals) distributed in 17 localities from central Chile to Tierra del Fuego
(32° to 52°S). Haplogroup 3 contained two haplotypes (7 individuals) presents in three localities in the south-
central area of Chile (35° to 41°S). Haplogroup 4 recovered ve haplotypes (15 individuals) presents in seven
localities in the south-central area (35° to 53°S). Haplogroup 5 was composed of two haplotypes (5 individuals)
presents in two neighboring localities from the south of Chile (41°S), representing the narrowest distribution
among all haplogroups. Haplogroup 6 was composed of 17 haplotypes (42 individuals) presents in 16 localities in
the southcentral area of Chile (37° to 51°S). All haplogroups showed high degrees of geographic overlap with no
clear physiographic separation between or within groups. Only haplogroups 1 and 6 showed a star-like structure,
Figure2. e median-joining haplotype networks of Potamolithus populations obtained in the present study.
e areas of Maule-Ñuble and Llanquihue Lake and Puelo River are enlarged. e number of mutational steps
among haplogroups is indicated by small bars. e limits of the ice sheet during the Last Glacial Maximum
in Patagonia were generated following dierent authors24,30,31. e map was created using QGIS Geographic
Information System v3.4.9 (http:// www. qgis. org). (Map: M.A. Valladares).
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however, this can be attributed to the fact that these groups contain most sampled individuals, and that the rest
of the haplogroups could have been underrepresented.
Both ML and BI methods recovered congruent tree topologies inferring six clades with high values of node
support (Fig.S1), which correspond to the six haplogroups described by the haplotype network. ese clades
conform two major clades, one including haplogroups 1 to 4 and the other haplogroups 5 and 6. e species P.
elenae (Estero Valcheta, Argentinean Patagonia) was recovered as the sister lineage of all Chilean Potamolithus
populations. Potamolithus lapidum supersulcatus (Uruguay River, Argentina), P. agapetus and P. bu sc hi i (Buenos
Aires, Argentina) formed a more distant lineage from the Chilean species. In addition to the Chilean populations
and GenBank sequences, the phylogenetic reconstructions included two individuals from Bariloche, Argentinean
Patagonia, which were recovered in Haplogroup 1 (star in Figs.3 and S1). Genetic distances among Potamolithus
species or haplogroups ranged from 3.1 to 11% (TableS2).
e three dierent species delimitation approaches were partially congruent detecting ve (ABGD and mPTP)
or six (bGMYC) species partitions (Fig.3). Potamolithus snails from El Yeso, originally assigned to P. santiagensis
by Biese56 (triangles in Figs.1, 3 and S1), were consistently identied as a partition with the three methods of spe-
cies delimitation employed here, although the ABGD and mPTP analyses included haplogroups 1 and 2 whereas
the bGMYC only Haplogroup 1. Samples from Puerto Chico (Llanquihue Lake), type locality of P. australis, were
recovered in haplogroups 1 and 6 (square in Fig.1, 3 and S1); most samples sequenced from this locality were
haplogroup 6. Following Biese36, individuals of Haplogroup 1 were assigned to P. santiagensis and Haplogroup
6 to P. australis. e ABGD and mPTP analyses also identied haplogroups 3 to 5 as separate partitions, while
the bGMYC method haplogroups 2 to 5. Divergence times estimated in BEAST suggest that the splitting of a
Potamolithus ancestor from T. huonensis, a closely related tateid, occurred about 12.4 (7.4–18.1) Ma (Fig.S2).
e splitting of Chilean Potamolithus species occurred between 2.7 and 0.68Ma. Potamolithus santiagensis,
circumscribed to the distribution area covered by Haplogroup 1, split from Haplogroup 2 approximately 0.68
(0.39–0.98) Ma whereas P. australis from Haplogroup 5 approximately 1.5 (0.89–2.2) Ma (Fig.S2).
Discussion
Using dierent molecular analyses, in the present study we showed that the biodiversity of the genus Potamo-
lithus in Chile is underestimated, with more species than the two previously recognized. Our results suggest
that there is cryptic diversity in the group and at least three species partitions would require validation and
formal description. However, besides relying on single locus analyses, an integrative approach is recommended
considering multiple loci and combined with other characters such as morphology, internal anatomy, radula,
larval development and ecology, among others59. e phylogenetic analysis inferred at least six well-supported
monophyletic groups, depicting several phylogenetic species, which is congruent with the haplogroups recovered
by the network analysis.
Two of the three species delimitation methods were congruent in the number and conformation of the spe-
cies partitions recovered. Five partitions were delimited using ABGD and mPTP and six with bGMYC. Previous
studies have shown that the Generalized Mixed Yule Coalescent model is useful in detecting incipient specia-
tion events85, when the threshold of intra- and interspecic distances is low, but on certain occasions it can
overestimate the number of species due to pronounced intraspecic genetic variability86,87. e tree recovered
in our analysis shows large branch lengths between the main clades, but short within each lineage, which is also
noticeable in the haplotype network. ese results suggest that the evolutionary process of the species is consist-
ent with a recent diversication scenario (see below). Considering this, it is likely that the model implemented
in the mPTP method is more appropriate to the Potamolithus pattern, mainly since it considers dierent levels
of intraspecic diversity within a species as a result of their evolutionary history or uneven sampling73. Using
a conservative approach88, the species delimitation analyses were consistent with the denition of ve species
partitions in Potamolithus in Chile, three of them previously undetected.
Potamolithus australis and P. santiagensis were described in the same article36, and partly on the same sheet,
using characters from the external shell morphology and operculum, which are not entirely clear, despite they
were described in dierent genera. Although these species have not been evaluated until now with independent
morphological evidence, recent phylogenetic analyses based on COI sequences performed with samples of P.
australis from Puerto Chico and P. santiagensis from El Yeso showed that they are dierent species42,57,65. ree
methods of species delimitation in the present study identied these morphologically cryptic taxa as separate
units, with P. santiagensis covering from Valparaíso Region to Tierra del Fuego in southern Patagonia, including
Chiloé Island. Potamolithus australis has a narrower range, covering from Trupán Lake in the Bío-Bío Region
to Sofía Lake in the southern section of western Patagonia in continental territory. Apart from these two spe-
cies, the ABGD and mPTP methods identied three additional partitions in Potamolithus whereas the bGMYC
method four. One of these lineages (Haplogroup 2) has a similar range to that of P. santiagensis, although it was
not found on Chiloé Island. A second lineage (Haplogroup 4) ranges from the Maule Region to the Strait of
Magellan in southern Patagonia, covering continental territory. A third lineage (Haplogroup 3) is distributed
from Putagán in the Maule Region to Llanquihue Lake in Los Lagos Region, while a fourth lineage (Haplogroup
5), found inhabiting a small waterfall whose waters ow into the Puelo River in Los Lagos Region, is restricted
to that single location.
e COI phylogenetic tree and network analysis of Potamolithus populations showed a deep genetic diver-
gence but weak phylogenetic structure regarding the landscape physiography. Haplogroup 1 (P. santiagensis),
Haplogroup 6 (P. australis), Haplogroup 2, and to a lesser degree haplogroups 3 and 4, have a wide geographic
distribution in the country so the hypotheses of vicariance and speciation by basins do not explain the diversity
pattern observed in the group. Moreover, we did not nd evidence that phylogenetic structure corresponds to the
faunistic units proposed by Stuardo & Vega89 for the land mollusks in continental Chile, i.e., a “northern fauna” in
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the north of the country and a “forest fauna” from around 38° S to Cape Horn together with a transitional zone of
overlap between the units from 31 to 38° S. Furthermore, we did not nd evidence of any congruence between the
haplotype distribution and the biogeographic regions proposed by Dyer90 for Chilean shes. Similar studies per-
formed in dierent faunal groups have shown contradictory results14, but in all cases population dierentiation
was due to a mixture of evolutionary processes. For instance, in an area similar to the one studied here, a rather
Figure3. Species delimitation of Chilean Potamolithus populations using the COI gene. Delimitation of
molecular clusters was performed using ABGD, mPTP and bGMYC. Posterior probability (> 0.9) values
obtained in the Bayesian COI tree are shown at the nodes (BEAST reconstruction). Scale bar next to the shells
represents 1mm.
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weak inuence of geographic barriers in the dierentiation of several species of the crustaceans and catshes
has been reported 7,12,19,91, although vicariance has also contributed to divergence in several sh species10,12,21.
e splitting of Chilean Potamolithus populations in the two major clades 2.73 (1.8–3.69) Ma is congruent
with the Pliocene–Pleistocene limit about2.59Ma (ICS, 2008). Subsequent pulses of speciation are also con-
gruent with the Pre-GPG in the Lower/Middle Pleistocene 2.1–1.0Ma and the GPG in the Middle Pleistocene.
e origin of P. australis is congruent with both dates whereas P. santiagensis with the last one; the coldest
Pleistocene glaciation occurred 0.7 Ma23. We did not nd association between contemporary levels of genetic
diversity of Potamolithus species and the LGM. e haplotypes distributed in the north of the studied area are
also found in southern Patagonia, and ve of the haplogroups occur in Llanquihue Lake, a large waterbody that
was completely glaciated during the early Pleistocene23. e presence of P. santiagensis, P. australis and several
candidate species in southern Patagonia, an area that was covered by ice during the last ice age, may be due to
the presence of refuge areas that remained in northern and/or southern Chile from which the species could have
recolonized the dierent waterbodies once the ice melted. Colonization signals were detected in several species of
sigmodontine rodents from lower latitudes in southern Patagonia14, with local dierentiation also contributing
to species diversity. Divergence times in Potamolithus also suggest persistence through the Pleistocene glacial
cycles, similar to the pattern inferred in the sigmodontine rodents of the area14.
Several species of Potamolithus from Argentina, Uruguay and Brazil have been proclaimed with some degree
of threat, mainly due to their restricted range, habitat loss, water pollution, tourism and overcollection37,49,50,92,93,
although only seven appear classied in the IUCN Red List of reatened Species as Data Decient or Least
Concern (e.g. Pastorino & Darrigran94,95). e conservation status of P. santiagensis and P. australis has not been
evaluated until now. Although the wide range that both species occupy would mean classifying them in a lower
conservation category, the presence of invasive species constitutes a serious threat, considering that in the type
locality of P. santiagensis the species is probably extinct, possibly linked to the presence of the invasive P. a nt ip o-
darum44,57,65. e occurrence of Physa acuta Draparnaud, 1805 in several freshwater ecosystems in Chile96 also
constitutes a potential threat to these species, in addition to those already mentioned for other South American
congeners.
Data availability
Original mitochondrial sequences obtained in the present study were submitted to GenBank (accession num-
bers MW916963–MW917138) (TableS1). Voucher specimens are housed at the Laboratorio de Malacología y
Sistemática Molecular, Universidad del Bío-Bío, Chillán, Chile.
Received: 16 April 2021; Accepted: 21 June 2021
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Acknowledgements
is paper was supported by Proyecto DIUBB 193309 3/R. MAV acknowledges a Postdoctoral Fellowship (VRIP
UBB Nº 352/6786/2020 and 352/1941/2021). GAC and CT-D acknowledges the Grupo de Investigación en
Biodiversidad y Cambio Global (GI 170509/EF) of the UBB. We also thank Nicolás Villalobos and Francis Miño
for their assistance during samplings and Darío Farías for molecular amplications.
Author contributions
e three authors designed the study. G.A.C. collected samples. M.A.V. carried out molecular analyses and pre-
pared gures. G.A.C. and M.A.V. wrote the paper with input from C.T-D. All authors reviewed and discussed
the manuscript.
Competing interests
e authors declare no competing interests.
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