J Mammal Evol

DOI 10.1007/s10914-016-9375-4

ORIGINAL PAPER

Biodiversity in the Amazon: Origin Hypotheses, Intrinsic

Capacity of Species Colonization, and Comparative
Phylogeography of River Otters (Lontra longicaudis
and Pteronura brasiliensis, Mustelidae, Carnivora)
and Pink River Dolphin (Inia sp., Iniidae, Cetacea)
Manuel Ruiz-Garca 1 & Pablo Escobar-Armel 1 & Benoit de Thoisy 2,3 &
Maria Martnez-Agero 1,4 & Myreya Pinedo-Castro 1 & Josep Mark Shostell 5

# Springer Science+Business Media New York 2017

Abstract We sequenced mitochondrial genes of otter (Lontra
longicaudis and Pteronura brasiliensis) and dolphin (Inia sp.)
species to provide new systematics data and to test hypotheses
that offer explanations as to the Amazons biodiversity. Four
of the 11 hypotheses tested Paleogeography (PH), Recent
Lagoon (RLH), Hydrogeological Recent Change (HRCH),
and Refugia (RH) support the evolution of these three spe-
cies. As part of this comparative phylogenetic study, we also
considered the degree of water dependence of each species.
For the least water dependent of the three species,
L. longicaudis, only HRCH and RH had an influence on ge-
netic structure, although it was relatively minor. For the more
water dependent otter species, P. brasiliensis, our analyses
stressed the significance of a single PH event along with two
lesser important PH events. However, its gene diversification
basically occurred during the Pleistocene and our analyses did
detect a relatively small influence of HRCH and RH. For the
completely water dependent species, Inia, we detected two
significant PH events. Its genetic structure was considerably
more developed than in either otter species, although the
Pleistocene was a very important period of genetic diversifi-
cation for the pink river dolphins (HRCH and RLH). Each
species has ancestors with different geographical origins and
genomes with different capacities to colonizemaking it dif-
ficult to rely on a generalized hypothesis to understand the
origins of the Amazons extremely rich biodiversity.
Keywords Lontra . Pteronura . Inia . Mitochondrial
markers . Amazon biodiversity . Systematics . Phylogeny

Electronic supplementary material The online version of this article

(doi:10.1007/s10914-016-9375-4) contains supplementary material,
which is available to authorized users.
* Manuel Ruiz-Garca
mruizgar@yahoo.es; mruiz@javeriana.edu.co
1
Laboratorio de Gentica de Poblaciones Molecular y Biologa
Evolutiva, Unidad de Gentica, Departamento de Biologa, Facultad
de Ciencias, Pontificia Universidad Javeriana, Cra 7 No 43-82,
Bogot, DC, Colombia
2
Association Kwata, Cayenne, French Guiana
3
Institut Pasteur de la Guyane, Cayenne, French Guiana
4
Biologa, Facultad de Ciencias Naturales y Matemticas,
Universidad del Rosario, Bogot, DC, Colombia
5
Department of Math Science and Technology, University of
Minnesota Crookston, Crookston, MN 56716, USA
Introduction
The amazing biodiversity of the Amazonian basin has been a
topic that has drawn the attention of numerous naturalists
since the 1800s (e.g., Wallace 1852). Not surprisingly, a series
of hypotheses have been proposed to explain its noteworthy
diversity (Table 1) (Antonelli et al. 2010; Behling et al. 2010;
Espurt et al. 2010; Lovejoy et al. 2010; Ribas et al. 2012).
Herein, we comparatively analyze and present molecular data
of three Neotropical aquatic mammalian taxa (two otter spe-
cies and one dolphin species) to help explain the generation of
the Amazons biodiversity.
The Neotropical otter (Lontra longicaudis; Mustelidae,
Carnivora) is a small- to medium-sized carnivoran with a body
mass generally less than 12 kg (Eisenberg 1989). It has the
widest geographical distribution of the three species studied,

Table 1 Eleven different hypotheses on the origin of the Amazonian biodiversity tested through molecular population genetics of two otter species
(Lontra longicaudis, Pteronura brasiliensis) and the pink river dolphin (Inia sp.)
Name
Stability-time hypothesis, STH
Paleogeography hypothesis, PH
Riverine Barrier hypothesis, RBH
Recent Lagoon hypothesis, RLH
Refugia hypothesis, RH
River Refuge hypothesis, RRH
Canopy-density hypothesis, CDH
Disturbance-Vicariance
hypothesis, DVH
Museum hypothesis, MH
Gradient hypothesis, GH
Hydrogeological Recent Change
hypothesis, HRCH
J Mammal Evol
Main arguments of each hypothesis Authors
Amazonian forests have existed for an extremely long Sanders 1968, 1969; Sanders and Hessler 1969
time (since the Cenozoic). In such a stable
environment, the rate of extinction would be lower
than the speciation rate, and this led a high level
of biodiversity
Three mechanisms for vicariance events: 1- Nuttall 1990; Hoorn 1993, 1994;
1- uplift of the Northern Andes in the late Miocene and Hoorn et al. 1995; Rsnen et al. 1995
the formation of the current Amazon River drainage 2- Emsley 1965; Croizat 1976; Ron 2000
2- island model by recurrent Miocene marine 3- Patton et al. 1994, 2000; Morell 1996;
introgressions caused by fluctuations of Patton and da Silva 1998; Hubert and
global sea-level and the apparition of internal Renno 2006; Roddaz et al. 2006, 2010;
Amazonian lakes Espurt et al. 2007, 2010
3- apparition of geological arches, which changed
the geomorphology of Amazonian basin
The largest rivers of the Amazonian basin formed in Wallace 1852; Bates et al. 2004
the late Miocene as a barrier to the dispersion
of organisms
Most of Amazonia was covered by a huge lake or lagoon Sombroeck 1966; Klammer 1984;
during the Pliocene, and successively smaller portions Marroig and Cerqueira 1997
of Amazonia were covered during a series of assumed
high sea-level stands during the Pleistocene
Dry/humid cycles caused by the Milankovitch cycles. Haffer 1969, 1974, 1982, 1987, 1997, 2008;
During the dry periods rainforest communities split Vanzolini 1970, 1973, 1992; Vanzolini and
into isolated refugia separated by savannah or arid Williams 1970; Brown et al. 1974; Van der
pampas. This hypothesis was originally established for Hammen 1975; Absber 1982; Prance 1982,
the Pleistocene, but later expanded to the 1996; Prance and Lovejoy 1985;
Miocene-Pliocene periods as well Whithmore and Prance 1987; Terborgh 1992;
Fjeldsa 1994
A combination of the RBH and RH Ayres 1986; Ayres and Clutton-Brock 1992;
Haffer 1993a, 1993b
Changes in vegetation type and structure during Cowling et al. 2001
the Last Glacial Maximum, around 18,000 YA,
occurred near the southern margins of the
Amazonian basin. The results also suggest repeated
reductions and increases in forest canopy
density over large areas during glacial-interglacial
cycles. Under this hypothesis, biological separation
of taxa does not necessarily require forest
fragmentation, as in the RH, but only a change
from wet to dry forests. Although this hypothesis
was initially originated for the Pleistocene, it
could be expanded to prior periods
Cold/warm cycles during the Pleistocene period Colinvaux 1993; Bush 1994
As a first step, this hypothesis claimed speciation in Roy et al. 1997; Fjeldsa et al. 1999
very localized stable habitat pockets in mountainous
regions around the periphery of Amazonia due to
climatic fluctuations without major vegetational
changes, followed, in a second step, by range
expansion of new species into the Amazonian
lowlands where they accumulate and are
preserved as in a museum
This model predicts parapatric speciation across Endler 1982
steep environmental gradients, boundaries,
whereas all the other hypotheses are based
on allopatric speciation
This hypothesis considers the role of Montoya-Burgos 2003;
Pliocene-Pleistocene geological changes Hubert and Renno 2006;
in the formation of the Neotropical rivers Ribas et al. 2012;
as more important than the role of the Lynch Alfaro et al. 2015
J Mammal Evol

Table 1 (continued)

Name Main arguments of each hypothesis Authors

Miocene North Andean uplift in the origin

of the Amazon drainage. This BYoung Amazon^
model suggests that the current Amazonian drainage
system was established between 2 and
3 Millions of years ago, MYA, and the major
Amazonian River tributaries originated within the
last 2 MYA. Watershed breakdowns and headwater
captures are important when trying to understand the
diversity of aquatic and non aquatic organisms

extending from northwestern Mexico to Uruguay, Paraguay,
and the Buenos Aires province in Argentina (Rheingantz et al.
2014). Less linked to water compared to the other species, the
Neotropical otter is versatiletolerating a range of habitats
and withstanding environmental changes and even occupying
areas close to human activity (MacDonald and Mason 1990).
Three morphological subspecies are recognized (van Zyll de
Jong 1972): L. l. annectens (Nayarit-Mexico), L. l. enudris
(French Guiana), and L. l. longicaudis (Brazil).
The giant otter (Pteronura brasiliensis; Mustelidae,
Carnivora) is a large carnivoran with a weight of 22
45 kg and a length of 1.51.9 m (Duplaix 1980). It has
the second widest geographical distribution of the three
species. It is distributed in the drainage basins of the
Amazon, Orinoco, and Parana-Paraguay rivers as well
as in the Caribbean Guyana rivers and in the eastern
Brazilian Sao Francisco River (Garcia et al. 2007). It
is the most aquatically adapted of all lutrines and is
not able to travel long distances over land (Pickles
et al. 2011). Two morphological subspecies have been
cited (P. b. brasiliensis, distributed within the Amazon
and Orinoco river basins, and P. b. paranensis, distrib-
uted within the Parana and Paraguay rivers). However,
Garcia et al. (2007) do not support the existence of
these subspecies.
The pink river dolphin (Inia; Iniidae, Cetacea) is the largest of
the river dolphins with a weight of 80170 kg and length of 1.80
to 2.80 m (Best and da Silva 1993; Ruiz-Garca et al. 2006). Of
the three species analyzed, it has the greatest body size, the most
restricted geographical distribution, and it is strictly aquatic.
Three subspecies have been traditionally recognized:
I. geoffrensis humboldtiana (San Fernando de Apur-
Venezuela, Pilleri and Gihr 1977), I. g. geoffrensis (Amazonian
basin; type locality lower Negro River or lower Amazon River),
and I. g. boliviensis (Bolivian sub-basin, Principe da Beira-
Brazil, Guapore-Itenez River). However, the Bolivian taxon has
been elevated to the status of a full species thanks to several
molecular works (Banguera-Hinestroza et al. 2002; Ruiz-
Garca et al. 2008; Ruiz-Garca 2010a). More recently, Hrbek
et al. (2014) described a possible new pink river species,
I. araguaiaensis in the Araguaia-Tocantins River basin.
To bring new insights on these two topics (origins of
the Amazonian biodiversity and clarification of system-
atics of these three species), we used sequences of sev-
eral mitochondrial (mt) genes. The mitochondrial genes
are interesting markers for phylogenetic tasks because
they lack introns and include a rapid accumulation of
mutations, rapid coalescence time, a negligible recombi-
nation rate, and haploid inheritance (Avise et al. 1987).
The Cyt-b gene is commonly used among the molecular
markers relevant for phylogeography, biosystematics, and
genetic structure studies in mammal populations
(Lavergne et al. 2010; Ruiz-Garca et al. 2015). The
COI gene has emerged as the standard barcode region
for animals, including mammals (www.mammaliabol.
org) (Hebert et al. 2003, 2004). The COII gene has been
employed extensively in the phylogenetics of different
mammalian groups (Ashley and Vaughn 1995; Ruiz-
Garca et al. 2010, 2014). The control region of mt
DNA is a portion that evolves particularly rapidly and
thus allows fine-scale resolution of population structure
and microevolutionary divisions (Rosel et al. 1995;
Wang et al. 1996).
There is an intensive debate on what mechanisms are
responsible for the high levels of biodiversity in the
Amazon. Some hypotheses claim the importance of cli-
matic changes during the Pleistocene (last two millions
of years ago, MYA); these are the cases of the RH, RRH,
CDH, and DVH (see Table 1 for abbreviations for the
name of the hypotheses). Contrarily, other hypotheses
(PH, RBH) sustain that geomorphologic and hydrological
changes during the Miocene (255 MYA) are responsible
for these high levels of biodiversity. Recently, other hy-
potheses have empathized the possible importance of
hydrological changes during the Pliocene (52 MYA)
and the Pleistocene (RLH, HRCH). Our results could
give support to some of these hypotheses.
Taking all of this into consideration, the two main aims of
the current work are to: 1) test hypotheses on the origin of the
Amazons biodiversity and 2) offer new molecular population
genetics data and systematics information on these otter spe-
cies as well as the pink river dolphin.

Material and Methods
We collected tissue samples from 47 L. longicaudis, 33
P. brasiliensis, and 206 Inia sp. (Table 2 and Fig. 1). For otter
samples we obtained hair from pelts as well as integument,
teeth, muscle, and blood of animals hunted by Indigenous
communities.
The Inia samples were obtained during six expeditions in
20022011 within different areas of the Amazonian and
Orinoco basins in Colombia, Venezuela, Peru, Ecuador,
Bolivia, and Brazil. More than 10,000 km of the Amazonian
and Orinoco rivers were transected searching for river dol-
phins. The 206 dolphins, the largest sample size ever studied,
were captured using special fishing nets each with a length of
400 m and width of 10 mtaking special care to ensure the
physical safety of each dolphin during capture. The individ-
uals were brought on board and their caudal fins were
biopsied. After biopsy, an antibiotic was applied to the wound
and then each animal was measured and then safely released
within 58 min. The biopsies were stored in absolute alcohol
until DNA extraction. To obtain information about sample
permissions, please refer to the Acknowledgment section.
Molecular Analyses
DNA from muscle, caudal fins, and skins were extracted using
the phenol-chloroform procedure (Sambrock et al. 1989),
while DNA samples from hairs and teeth were extracted with
10% Chelex resin (Walsh et al. 1991).
For otters, we sequenced three mitochondrial genes: Cyt-b
(1024 base pairs, bp), COI (657 bp), and COII (693 bp).
Primers and PCR conditions for these three genes are located
in Collins and Dubach (2000), Folmer et al. (1994), Ruiz-
Garcia (2010a, b),and Ruiz-Garca et al. (2012a, 2012b).
These sequences were concatenatedsumming a total of
2374 bp for the analyses herein showed.
In the case of Inia, two mitochondrial genes were se-
quenced. We used primers H16498 and TRO (LeDuc, unpub-
lished data) to analyze the mitochondrial control region gene
(D-loop; 600 bp). The Cyt-b gene (600 bp) was amplified by
using the primers Tglu and MHB2 (Palumbi et al. 1991).
Primers and PCR conditions can be found in Banguera-
Hinestroza et al. (2002). Thus, for the pink river dolphins,
1200 bp were sequenced.
All amplifications, including positive and negative con-
trols, were checked in 2% agarose gels, using the molecular
weight marker 174 DNA digested with Hind III and Hinf I.
The amplified samples were purified using membrane-
binding spin columns (Qiagen). The double-stranded DNA
was directly sequenced in a 377A (ABI) automated DNA
sequencer. The samples were sequenced in both directions
using the BigDye TM kit. In cases of mismatching, the PCR
reaction was repeated and the product sequenced again.
J Mammal Evol
Sequence alignments and editing were performed by using
the Sequencer 3.0 (Gene Codes Corp.) and DNA Alignment
programs (Fluxus Technology Ltd.). The sequences obtained
were deposited in GenBank. For the river dolphins, the acces-
sion numbers and submissions were https://www.ncbi.nlm.
nih.gov/nuccore/AF521113.1-AF521152.1 , https://www.
ncbi.nlm.nih.gov/nuccore/AF521106.1-AF52137.1 , Bankit
ID 1970983 (https://www.ncbi.nlm.nih.gov/WebSub/) and
Bankit 1,972,232 (KY296741-KY296947; https://www.ncbi.
nlm.nih.gov/WebSub/); for L. longicaudis, Bankit ID
1971014 (https://www.ncbi.nlm.nih.gov/WebSub/) and
Bankit ID 1972258 (KY296981-KY297027; https://www.
ncbi.nlm.nih.gov/WebSub/), and for P. brasiliensis, Bankit
ID 1971017 (https://www.ncbi.nlm.nih.gov/WebSub/) and
Bankit ID 1972249 (KY296948-KY296980; https://www.
ncbi.nlm.nih.gov/WebSub/). The data sets generated and
analyzed during the current study are available from the
corresponding author on reasonable request at the e-mails,
mruizgar@yahoo.es and mruiz@javeriana.edu.co and in the
supplementary material.
Population Genetics and Phylogeographic Analyses
Genetic Diversity and Heterogeneity Analyses
We used the following statistical tests to determine genetic
diversity of each species as a whole as well as populations
within each species: haplotypic diversity (Hd), the nucleotide
diversity (), the average number of nucleotide differences
(k), and the statistic by sequence (mutation rate per site
per generation times the number of heritable units in the
population; Hedrick 2005; Hartl and Clark 2006). Additional
statistical procedures (HST, KST, KST*, ST and FST, Hudson
et al. 1992) were applied to the nucleotide sequences to deter-
mine the overall genetic heterogeneity within each species.
Some indirect gene flow estimates were obtained, assuming
an infinite island model (Wright 1965). Two different data sets
were used, by river basins (RB) and by populations (POP), for
each one of the three species. Gene diversity and genetic het-
erogeneity statistics were completed using DNAsp 5.1
(Librado and Rozas 2009) and Arlequin 3.5.1.2 (Excoffier
and Lischer 2010).
We also used the Migrate 3.6 program (Beerli 2009) to
estimate asymmetrical gene flow estimates among the popu-
lations studied of the three species. The program calculated
(= e) for each population and considered Ne as the female
effective size and as the mutation rate per nucleotide base
and generation. M (migration rate) was equal to m/ among
the population pairs analyzed, where m was the chance for a
lineage to immigrate per generation (Beerli 2009). The gene
flow was the product of Ne and m that we obtained from both
of these equations. The average value for the mt D-loop
gene in Inia is 5.81 108 (Caballero et al. 2007; Cunha
J Mammal Evol

Table 2 Number and geographical origins of the samples of Lontra longicaudis, Pteronura brasiliensis, and Inia sp. sequenced in this study

Species Sample sizes Geographical origins River basins Kind of samples

Lontra longicaudis 47 A-3 individuals: from El Banco, Magdalena; Magdalena River basin Pieces of skins and hairs
Portachuelos, North of Santander; Sumapaz
River, Cundinamarca (all in Colombia)
B-8 individuals: Orinoco River basin Pieces of skins
Meta, Guaviare and Vichada Departments
in Colombia
C-29 individuals: 7 from the Colombian- Western Amazon River basin Pieces of skins, hairs,
Peruvian-Brazilian frontier; 21 from the blood, teeth and muscles
Amazon, Nanay, Tigre, Itayari, Pintayacu,
Maraon, Pachitea, Ucayali, Tame and
Ene rivers, in the Peruvian Departments
of Amazonas, Loreto, Ucayali, Hunuco,
Junin and Pasco; 1 from the Payamino
River, Orellana Province, Ecuador;
D- 6 individuals from the Santa Cruz Bolivian sub-basin rivers Pieces of skins and hairs
and Beni Departments (Ibare, Mamore
and Beni rivers);
E-1 individual from the Paran River Parana River basin Hairs
in Paraguay
Pteronura brasiliensis 33 A-6 individuals from the Casanare, Orinoco River basin Pieces of skins and hairs
Vichada, Meta, Guaviare
Departments in Colombia;
B- 25 exemplars: 1 from the Western Amazon River basin Pieces of skin, hairs and blood
Negro River in Brazil; 6 from
the Colombian-Peruvian-Brazilian
frontier; 2 from the Caqueta River
and 3 from the Putumayo River,
both in Colombia; 13 from the
Peruvian rivers: Nanay, Tigre, Curaray,
Mazan, Tambopata and Ucayali;
C- 2 individuals from the Bolivian Bolivian sub-basin rivers Pieces of skin
Mamore River
Inia sp. 206 A-25 individuals: 5 from the Orinoco Orinoco River basin Biopsies from the
River in Colombia, 2 from La Urbana caudal fin
in the Orinoco River in Venezuela,
11 from the Inirida River, 1 from the
Vichada River, 2 from the Guaviare
River, 2 from the Meta River and
2 from the Arauca River;
B- 124 individuals: 38 from the Putumayo Western and Central Biopsies from the
River and 26 from the Caqueta River, Amazon River basin audal fin
both in Colombia; 1 from the Brazilian
Negro River; 4 from the Yavari and
the Amazon rivers at the Colombian-
Peruvian-Brazilian frontier; 4 from
the Pacaya River, 1 from the Samiria
River, 12 from the Ucayali River,
9 from the Maran River and 29
from the Napo-Curaray rivers, all
them in the Peruvian Amazon;
C-57 individuals: 36 from the Mamore Bolivian sub-basin rivers Biopsies from the caudal fin
River, 14 from the Ipurupuru River,
5 from Ibare River and 2
from the Tijamuchi River

et al. 2011). Comparatively, averages of values (6.18 108) Donoghue 2007; Nabholz et al. 2008, 2009). We concatenated
are reportedly higher for the mt Cyt-b, mt COI, and mt COII the genes employed and used an average value. We used a
genes in both otters and Inia (Wayne et al. 1997; Benton and Bayesian procedure (Migrate 3.6 software; Beerli 2006) with

Fig. 1 Map of South America with the areas and number of samples obtained for two otter species (Lontra longicaudis and Pteronura brasiliensis) and
one river dolphin species (Inia sp.)
one long Markov chain, with 5000 recorded steps, 100 incre-
ments, one concurrent chain, and a total of 500,000 sampled
genealogies with a burn-in of 10,000.
Spatial Analyses
Two different spatial analyses were carried out. First, we
used the IBD version 1.2 software (Bohonak 2002) to
determine possible relationships among the genetic dis-
tances (Kimura 2P distance; Kimura 1980) and the geo-
graphic distances between population pairs within each
one of the three species. A Mantel test (Mantel 1967)
was used to determine the significance between the ge-
netic distance and geographic distance matrices. The in-
tercept and the slope of this relationship were calculated
using a Reduced Major Axis (RMA) regression (Hellberg
J Mammal Evol
1994). In the case of L. longicaudis, seven populations
were taken into account, six for P. brasiliensis, and eight
for the dolphins (Table 3). Second, we created
distograms based on Gregoriuss genetic distance for all
individuals of the three species studied (Gregorius 1978;
Degen and Scholz 1998; Vendramin et al. 1999).
Ten distance classes (DC) were used for all three species
taken as a whole. In the case of Inia, diverse distograms were
analyzed for the Amazon and Orinoco basins, as well as for
the Orinoco, Amazon, and the Bolivian Mamore River basins
separately. The significances of distograms, and autocorrela-
tion coefficients were calculated by means of 1000 Monte-
Carlo simulations (Manly 1997) with an estimated confidence
interval of 95% (Streiff et al. 1998). We used the SGS version
1.0d software to determine the significance of these autocor-
relation coefficients (Degen et al. 2001).
J Mammal Evol
Demographic Analyses
We used a Bayesian skyline plot analysis (BSP) to determine
possible demographic changes across the natural history of
these three aquatic mammals. The BSP was estimated using
BEAST v. 1.6.2 and Tracer v1.5 software. The Coalescent-
Bayesian skyline option in the tree priors was selected with five
steps and a piecewise-constant skyline model with 40,000,000
generations (the first 4,000,000 discarded as burn-in). The mar-
ginal densities of temporal splits were analyzed in Tracer v1.5
and we selected the Bayesian Skyline reconstruction option for
the trees log file. A stepwise (constant) Bayesian skyline variant
was selected with the maximum time as the upper 95% higher
posterior density (HPD).
Genetic Distances and Phylogenetic Trees
The Kimura 2P distance was used to determine the genetic
differences (%) among POP and RB within each one of these
three mammalian species. We used this genetic distance be-
cause it is standardly used for barcoding gene analyses to
distinguish species (Hebert et al. 2003, 2004).
We used Bayesian trees (BI; Rannala and Yang 1996; Mau
et al. 1999) based on GTR + G + I for each species with the
gamma distributed rate varying among sites, and variable rate
categories. Two trees were obtained, one for L. longicaudis
and P. brasiliensis taken together, and another for Iniataking
into account haplotypes. This Bayesian analysis was complet-
ed with the BEAST v1.6.2 program (Drummond and
Rambaut 2007). Two separate sets of analyses were run, as-
suming a Yule speciation model and a relaxed molecular clock
with an uncorrelated log-normal rate of distribution
(Drummond et al. 2006). Results from the two independent
runs (60,000,000 generations with the first 6,000,000
discarded as burn in and parameter values sampled every
1000 generations) were combined with LogCombiner v1.6.2
software (Drummond and Rambaut 2007). Posterior probabil-
ity values provided an assessment of the degree of support of
each node on the tree. The final tree was estimated using
TreeAnnotator v1.6.2 software and visualized in the FigTree
v1.3.1 program (Drummond and Rambaut 2007).
Additionally, this program was run to estimate the time to
most recent common ancestor (TMRCA) for each of the hap-
lotype lineages. We used a temporal calibration of 6.4 1.0
MYA to determine possible temporal splits within Lontra and
Pteronura (Koepfli et al. 2008; Pickles et al. 2011). For the
pink river dolphins, we used two calibrated points in the
Bayesian tree. We used 22.07 2.5 MYA for between Inia-
Pontoporia and the Delphinoidea (the average from Cassens
et al. 2000; Hamilton et al. 2001; Cunha et al. 2011; Hrbek
et al. 2014). For the calibrated point between Inia and
Pontoporia, we used the temporal estimation 15.11 2.5
MYA (average from Muizon 1983; Cassens et al. 2000;
Hamilton et al. 2001; Cunha et al. 2011; Hollatz et al. 2011;
Hrbek et al. 2014). We plotted the likelihood versus genera-
tion and estimated the effective sample size (ESS > 200) of all
parameters across the two independent analyses.
Following Pennington and Dick (2010), we employed two
different approaches for inferring divergence times: fossil-
calibrated DNA phylogenies (BI) and Bborrowed molecular
clocks^ with direct nucleotide substitution rates inferred from
other taxa. For the second one, we used a median joining net-
work (MJN) with the help of software Network 4.6.10 from
Fluxus Technology Ltd. (Bandelt et al. 1999). The statistic
(Morral et al. 1994) was estimated and transformed into years.
To determine the temporal splits we estimated the mutation rate
per sequence and per million years. For the three mt Cyt-b, COI,
and COII genes, the average mutation rate used was 2.5 106
(we used a molecular clock estimate of 2.5% mutations per
million of years for all the length of the sequences employed,
2374 bp for otters, meanwhile the mutation rates for Migrate
3.6 software were by nucleotide and by generation), which is
equivalent to one mutation every 117,302 years (Nabholz et al.
2008, 2009). In the case of the pink river dolphins for the mt D-
loop region, we used a mutation rate of 1.1 106, which is
equivalent to one mutation every 227,273 years (Caballero et al.
2007; Cunha et al. 2011). The networks are more appropriate
for intraspecific phylogenies than tree algorithms, because they
explicitly allow for the co-existence of ancestral and descendant
haplotypes, whereas trees treat all sequences as terminal taxa
(Posada and Crandall 2001).
Results
Genetic Diversity, Genetic Heterogeneity and Genetic
Distances
The genetic diversity levels for each of the species studied,
both for RB and for POP, are shown in Table 4. Both otter
species have similar overall genetic diversity statistics, al-
though their values of Hd and were considerably greater than
those of Inia. However, the reverse was true for , with Inia
showing the highest value. We also separated I. boliviensis
from I. geoffrensis, and compared their genetic diversities to
the otter species. Inia boliviensis had the lowest genetic diver-
sity levels of the three (two otters and I. geoffrensis). The
genetic heterogeneity values for these species by RB and
POP are provided in Table 5.
The genetic differentiation, by river basin in L. longicaudis,
showed one out of four tests with no significant heterogeneity
between the Orinoco (plus the Magdalena basin) and the
Amazon (plus one animal of the Parana River basin) areas
(Table 5). Likewise, the values of some statistics such as FST
and ST were very small (0.041 and 0.033, respectively) and
the theoretical gene flow values throughout these statistics
 J Mammal Evol

Table 3 Species, number, and populations of three Neotropical aquatic mammals (Lontra longicaudis, Pteronura brasiliensis, and Inia sp.) sequenced
at mitochondrial genes and analyzed for possible spatial structure

Species and number of populations Populations considered

L. longicaudis (7) (1) Orinoco, (2) Magdalena, (3) northern Peruvian and Ecuadorian Amazon,
(4) Colombian-Brazil Amazon frontier, (5) southern Peruvian Amazon plus Pasco and Junin Departments,
(6) Bolivia, and (7) Parana River
P. brasiliensis (6) (1) Upper Orinoco, (2) Lower Orinoco, (3) northern Peruvian Amazon, (4) Colombian-Brazil Amazon
frontier, (5) southern Peruvian Amazon, and (6) Bolivia
Inia sp. (8) (1) Upper Orinoco, (2) Lower Orinoco, (3) Putumayo River, (4) Caqueta River, (5) Colombian-Brazil
Amazon frontier (Amazon and Javari rivers), (6) Ucayali-Maraon rivers in the Peruvian Amazon,
(7) Napo and Curaray rivers in the northern Peruvian Amazon, and (8) Bolivia

were high (11.61 and 14.61, respectively). By POP, the levels
of genetic differentiation slightly increased with two out of
four tests showing significant values, although some statistics
such as FST (0.089) and ST (0.171) showed relatively low
gene heterogeneity. The Bolivian sample was more differen-
tiated from the other populations. The samples from the north-
ern Peruvian Amazon-Ecuador and the Colombian Amazon-
western Brazilian Amazon were undifferentiated. The same
occurred between the samples of the Orinoco basin and the
Magdalena River. The overall mean Kimura 2P genetic dis-
tance for L. longicaudis was 0.018 0.001 and within the
Amazon and Orinoco basins, these values were respectively
0.011 0.001 and 0.009 0.002 (Table 4 and supplementary
data, Appendix 1). The genetic distance between the Orinoco
and Amazon basins was small, 0.010 0.001. Within the
population data set (POP), northern Peruvian Amazon-
Ecuadorian Amazon had the highest diversity
(0.012 0.002), whereas the Orinoco had the lowest value
(0.007 0.001). The Bolivian-Magdalena and Magdalena-
Parana River Basin had the highest genetic distances of all
population pairs (both values: 0.015 0.003).
The genetic differentiation, by RB in P. brasiliensis,
showed a very limited differentiation between the Orinoco
and Amazon rivers (Table 5). Only one test out of four showed
significant genetic heterogeneity. Some statistics, such as FST
(0.024) and ST (0.084), yielded very small values of genetic
heterogeneity and, in turn, very elevated gene flow estimates
between the Amazon and Orinoco basins (20.23 and 5.43,
respectively). The situation was very similar for the popula-
tion data set. Only one out of four tests were significant and
the genetic differentiation statistics were small (FST = 0.009
and ST = 0.174). The overall mean Kimura 2P genetic dis-
tance was 0.009 0.001, which is considerably lower than for
the other otter species (Table 4 and supplementary data,
Appendix 1). Within the Amazonian basin, the genetic dis-
tance was very small (0.005 0.001) as well as in the case of
the Orinoco basin (0.002 0.001). The genetic distance
between the Orinoco and the Amazonian basins was
somewhat higher than for L. longicaudis, 0.015 0.001.
Similar to the previous otter, the northern Peruvian Amazon
had the highest gene diversity (0.015 0.004). The highest
genetic distances for population pairs (POP set; 0.0640.072)
involved those with the individual sampled in the most north-
ern Orinoco area of Paz de Ariporo (Casanare Department).
The lowest genetic distance values of the POP pertained
to the Colombian Amazon-western Brazilian Amazon,
southern Peruvian Amazon-Junin and the Orinoco basin
(0.0040.005).
The genetic differentiation by RB in Inia sp. showed a very
strong genetic heterogeneity (Table 5). Four out of four tests
showed a very striking genetic differentiation among the
Orinoco, Amazon, and Bolivia. FST (= 0.774) and ST (=
0.701) were both extremely elevated and there were very
low levels of gene flow (Nm = 0.15 and 0.21, respectively).
The situation of extreme genetic differentiation was the same
for the population data set. Also, when the Bolivian basin
animals were removed from the analyses, the genetic hetero-
geneity between the Orinoco and Amazon basins was still
significant in seven out of seven tests. However, the values
of FST (= 0.478) and ST (= 0.261) were reduced to 1/31/2 of
those obtained with the Bolivian animals. Similarly, when
only the Amazonian Inia populations were considered, four
out of four tests showed significant genetic heterogeneity with
relatively elevated values of FST (= 0.319) and ST (= 0.149)
and moderated gene flow estimates (Nm = 1.07 and 2.86,
respectively). For example, genetic heterogeneity between
the two different haplogroups detected in the Orinoco basin
(FST = 0.788) was higher compared to that between one of
these haplogroups (Upper Orinoco) and an Amazonian popu-
lation (Napo River) (FST = 0.594). The genetic differentiation
was even higher between some Amazonian population pairs
(for example, between the Putumayo River and the
Colombian Amazon River, FST = 0.726). The Kimura 2P ge-
netic distance (Table 4 and supplementary data, Appendix 1)
had an elevated value (0.026 0.005), for the Orinoco indi-
viduals, whereas the Amazon and Bolivian basins had lower
values of 0.012 0.002 and 0.001 0.001, respectively. The
genetic distance between the Orinoco and the Amazon basins
was considerably higher than that obtained for the two otter
species, 0.036 0.007. We found the greatest genetic
J Mammal Evol

Table 4 Genetic diversity statistics for Lontra longicaudis, Pteronura (Hd), the nucleotide diversity (), the average number of nucleotide dif-
brasiliensis, and Inia sp. for the overall sample, for River basins (RB) and ferences (K), and the statistic (= Ne; Ne = effective female population
for populations (POP) for the mitochondrial genes studied. The statistics size; = mutation rate per generation) by sequence
estimated were the number of haplotypes (NH), the haplotypic diversity

Lontra longicaudis NH Hd K per sequence

Orinoco and northern area of Colombia (RB) 10 0.982 0.046 0.0084 0.0014 8.65 4.33 11.61 4.89
Amazon (RB) 32 0.984 0.015 0.0110 0.0013 11.33 5.26 21.70 6.73
Bolivia (RB and POP) 6 1.000 0.096 0.0100 0.0020 10.27 5.47 11.82 5.89
Magdalena (POP) 2 1.000 0.500 0.0147 0.0073 15.00 10.95 15.00 10.95
Only Orinoco (POP) 8 0.972 0.064 0.0072 0.0016 7.39 3.82 10.30 4.62
Colombian-Brazil Amazon frontier (POP) 8 1.000 0.063 0.0096 0.0026 9.82 5.04 13.11 5.97
Northern Peruvian and Ecuadorian Amazon (POP) 9 0.945 0.066 0.012 0.0028 12.25 6.00 17.07 7.04
Southern Peruvian Amazon plus Pasco and Junin Departments (POP) 10 1.000 0.045 0.0094 0.0017 9.58 4.80 14.49 6.18
Total sample of L. longicaudis 41 0.985 0.012 0.0110 0.0011 10.86 5.03 23.55 6.89
Pteronura brasiliensis NH Hd K per sequence
Orinoco (RB and POP) 6 1.000 0.096 0.0223 0.0110 23.53 6.96 30.22 14.51
Amazon (RB) 21 0.954 0.032 0.0053 0.0012 5.38 2.68 11.67 4.00
Bolivia (RB and POP) 2 1.000 0.500 0.0147 0.0021 15.00 10.95 15.00 10.95
Colombian-Brazil Amazon frontier (POP) 8 0.933 0.077 0.0043 0.0002 4.42 2.38 5.66 2.61
Northern Peruvian Amazon (POP) 8 0.927 0.066 0.0040 0.0003 4.14 2.23 5.46 2.48
Southern Peruvian Amazon (POP) 4 1.000 0.177 0.0060 0.0012 6.17 3.71 6.00 3.56
Total sample of P. brasileinsis 26 0.968 0.022 0.0086 0.0014 8.79 4.16 24.15 7.58
Inia sp. NH Hd K per sequence
Inia geoffrensis (Orinoco + Amazon) 32 0.792 0.033 0.0185 0.0090 7.39 3.47 13.81 3.45
Overall Orinoco (RB) 9 0.787 0.073 0.0246 0.0110 9.85 4.66 7.68 2.79
Amazonas (RB) 23 0.708 0.043 0.0116 0.0070 4.65 2.29 12.42 3.22
Inia boliviensis Bolivia (RB) 6 0.516 0.067 0.0014 0.0003 0.58 0.47 1.08 0.55
Upper Orinoco (POP) 4 0.867 0.098 0.0090 0.0023 3.60 1.42 4.32 1.62
Lower Orinoco (POP) 5 0.637 0.083 0.0112 0.0037 4.49 1.04 6.12 2.97
Putumayo River (POP) 2 0.149 0.033 0.0022 0.0008 0.89 0.22 1.47 0.67
Caqueta River (POP) 8 0.766 0.047 0.0100 0.0028 4.02 1.86 5.87 2.11
Colombian-Brazil Amazon frontier (Amazon and Javari rivers) (POP) 3 0.833 0.112 0.0075 0.0008 3.00 1.43 4.58 2.39
Ucayali-Maran rivers (POP) 8 0.772 0.056 0.0146 0.0071 5.85 2.37 7.24 2.14
Napo-Curaray rivers (POP) 11 0.877 0.053 0.0173 0.0065 6.91 2.33 8.24 2.58

differences to be between these two (Orinoco/Amazon) and
the Bolivian basin (0.0720.089). Within the population
data set, the Ucayali and Maraon rivers
(0.015 0.003) and Napo River (0.018 0.004), all
in the Peruvian Amazon, had the highest levels of gene
diversity. The analysis by POP showed that there were
really two highly differentiated populations in the
Orinoco: the Upper and Lower. Also, the genetic dis-
tance between them was higher (0.049 0.010) than
between either of them and any other Amazonian pop-
ulation (0.0310.042). All the populations studied
showed very pronounced differences with the Bolivian
population. These genetic distances are higher than
those found for the entire range of the two otter species.
There was a unique molecular population of L. longicaudis
in the Orinoco and Magdalena basins (north of the Amazon).
Within the Orinoco basin we found two different molecular
populations of P. brasiliensis and I. geoffrensis. We found the
highest gene diversity levels in the Peruvian Amazon rivers
for all three aquatic species.
The Bayesian gene flow estimates with Migrate 3.6
can be seen in Table 6. In the case of L. longicaudis,
the Amazon and the Orinoco basins were considered.
Although both Nm estimates indicated high levels of
gene flow, clearly the largest gene flow was from the
Amazon to the Orinoco (Nm = 272.12 vs. Nm = 5.21).
For P. brasilensis, three basins were considered: Bolivia,
Amazon, and Orinoco. All the Nm estimates were in a
context of high gene flow, but the largest Nm estimates
were from the Amazon basin towards Bolivia and
Orinoco (Nm = 28.09 and Nm = 22.62, respectively).
The lowest Nm estimates were from the Orinoco towards the

other two basins. For Inia, the gene flow levels were consid-
erably lower. The Amazon basin towards Bolivia and Orinoco
had Nm estimates greater than 1 (Nm = 5.14 and Nm = 4.85,
respectively). The gene flow estimates from Bolivia and the
Orinoco towards the Amazon basin and between them were
always lower than 1.
Spatial Genetic Structure
The IBD analysis of L. longicaudis within the population data
set showed that geographic and genetic distances were not sta-
tistically correlated (r = 0.523 and p = 0.069). In the best case
scenario, the geographic distance only explained around 27.3%
of the genetic distances. In the case of P. brasiliensis, the geo-
graphic distance explained only 13% of the genetic distances
(r = 0.361, p = 0.201). Again, there was no statistical signifi-
cance. For Inia, the geographic distance significantly explained
around 51.9% of the genetic distances found (r = 0.720,
p = 0.005). If the Bolivian pink river dolphin population was
removed from the analysis, the percentage dropped to 17.8 and
was no longer significant. Thus, the Bolivian population created
a globally significant isolation by distance model for Inia.
We obtained distograms to determine possible genetic
structure among the individuals of the three aquatic mammal
species studied (Figs. 2, 3, and 4). In the case of
L. longicaudis, two distance classes (DC) showed positive
spatial autocorrelation (1 DC: 0306 km, p = 0.000 and
3 DC: 611917 km, p = 0.022). The remaining DC did not
show any spatial autocorrelation trend. Therefore, some
patches of individuals had a closer genetic relationship than
expected by chance at distances of around 900 km (Fig. 2).
In the case of P. brasiliensis, the situation is relatively sim-
ilar to L. longicaudis. The first three DCs showed significantly
positive spatial autocorrelation: 1 DC (0187 km, p = 0.000),
2 DC (187374 km, p = 0.002), and 3 DC (374562 km,
p = 0.002). The remaining DC did not show any evidence of
significant autocorrelation. Thus, individuals separated by
600 km showed mitochondrial similarity significantly larger
than that expected only by chance (Fig. 3). For Inia, when all
206 individuals were used, the distogram showed a very strik-
ing spatial genetic structure (Fig. 4a). The ten DCs obtained
were significant. Thus, all the DC showed significantly posi-
tive spatial autocorrelation up until around 2900 km and then
it became a negative spatial autocorrelation from 3900 km to
9650 km. We also detected significant spatial structure for
pink river dolphins within the Amazon and Orinoco basins
(Fig. 4b). All of the DCs we estimated were significant with
the exception of the 5 DC (27643456 km). Within the
Orinoco basin, there was also significant spatial structure
(Fig. 4c), with the first two DC showing positive spatial struc-
ture (1 CD: 0114 km, p = 0.000; 2 CD: 114228 km,
p = 0.000) and the last four DCs yielding significant negative
spatial autocorrelation (7 DC: 685799 km, p = 0.004; 8 DC:
J Mammal Evol
799913 km, p = 0.000; 9 DC: 9131027 km, p = 0.022;
10 DC: 10271141 km, p = 0.000). Thus, Inia in the
Orinoco basin showed very significant genetic structure. The
same occurred in the Amazon basin, with positive significant
autocorrelation in the first 2300 km and negative significant
autocorrelation from 2900 km to 5700 km (Fig. 4d). In con-
trast, the distogram was clearly not significant in the Bolivian
rivers areashowing an absence of spatial genetic structure
(Fig. 4e). Therefore, Inia presented more spatial structure than
the two otter species.
Demographic Evolution
The BSP analyses are shown in Fig. 5. For L. longicaudis
(Fig. 5a), there was a continuous female population expansion
throughout the last 1.5 MYA and a population decline in the
last 10,00015,000 years. For P. brasiliensis (Fig. 5b), the
population experienced a striking female population expan-
sion starting around 250,000 YA continuing until about
20,000 YA. For Inia, three different BSP analyses were un-
dertaken. For the Amazon, Orinoco, and Bolivian basins
(Fig. 5ce), the results were very similar. The female popula-
tions were more or less constant, until approximately 15,000
30,000 YA when the populations declined. Thus, all three
species populations declined in numbers of females during
the last phase of the Pleistocene, but the two otter species
showed population expansions during some phases of the
Pleistocene but not Inia.
Phylogenetic Inferences
The DNA phylogenies (fossil calibrated, BI) (Fig. 6a) for
L. longicaudis and P. brasiliensis show a clear split around
4.78 MYA (HPD 95% confidence interval: 3.616.44 MYA;
probability, P = 1). For L. longicaudis, the first temporal split
occurred approximately 2.7 MYA (1.54.6 MYA). Our analyses
show that the animals from the Orinoco and Amazon basins were
intermixed, but that two big clusters formed. There were individ-
uals of the Orinoco, Amazon, and Bolivian basins within the first
cluster and significant temporal splits occurred between 0.23 to
2.22 MYA (Pleistocene times). In the second cluster, there were
also animals from the Magdalena, Orinoco, Amazon, Bolivian,
and Parana basins. The significant temporal splits occurred from
0.09 to 2.52 MYA (Pleistocene times). We also detected several
small clusters containing individuals of related geographical
areas. For example, one cluster had two Orinoco animals from
the Colombian Meta Department and a second contained animals
from Bolivia. These findings agree with those of the distogram
with some significant spatial autocorrelation in the first distance
classes. The first temporal split for P. brasiliensis was the sepa-
ration of the individuals ancestor from Paz de Ariporo (Casanare
Department, Colombia) from the remaining individuals of this
species, around 3.35 MYA (1.795.51 MYA; P = 1). The second
J Mammal Evol

Table 5 Genetic heterogeneity and gene flow (Nm) statistics for Lontra Table 6 Asymmetric gene flow (Nm) estimates obtained by Migrate
longicaudis by River basin (RB) (A) and by populations (POP) (B), for 3.6 software among populations of Lontra longicaudis (A), Pteronura
Pteronura brasiliensis by RB (C) and POP (D), and for Inia sp. by RB (E) brasiliensis (B), and Inia sp. (C). For l. longicaudis: 1 = Orinoco basin;
and POP (F). *Significant Probability (P < 0.05). NS = Not significant. 2 = Amazon basin. For P. brasiliensis: 1 = Orinoco basin; 2 = Amazon
df = degree of freedom basin; 3 = Bolivia basin. For Inia sp. : 1 = Orinoco basin; 2 = Amazon
basin; 3 = Bolivia basin
Genetic differentiation estimated P Gene flow
(A)
(A) 1 2
2 = 42.351 df = 40 NS 1 0 272.12
HST = 0.0016 NS ST = 0.0331 Nm = 14.61 2 5.21 0
KST = 0.0145 0.0204* (B)
KST* = 0.0041 NS FST = 0.0413 Nm = 11.61 1 2 3
(B) 1 0 22.62 13.54
2 = 210.843 df = 195 NS 2 5.15 0 8.98
HST = 0.0047 NS ST = 0.1716 Nm = 2.41 3 2.42 28.09 0
KST = 0.0603 0.0034* (C)
KST* = 0.0432 0.0032* FST = 0.0889 Nm = 5.12 1 2 3
(C) 1 0 4.85 0.07
2 = 29.639 df = 25 NS 2 0.93 0 0.15
HST = 0.0073 NS ST = 0.0844 Nm = 5.43 3 0.44 5.14 0
KST = 0.0125 NS
KST* = 0.0280 0.0456* FST = 0.0241 Nm = 20.23
third largest cluster. It contained exemplars of neighboring geo-
(D)
graphical areas, which coincided with the spatial autocorrelation
2 = 118.150 df = 100 NS
analysis.
HST = 0.0201 NS ST = 0.1739 Nm = 2.38
The BI (Fig. 6b) for Inia (with haplotypes) showed a
KST = 0.0153 NS
temporal divergence between the ancestors of Sotalia
KST* = 0.0783 0.0458* FST = 0.0089 Nm = 55.44
guianensis and Pontoporia blainvillei and those from
(E)
Inia that occurred 11.49 MYA (13.8423.16 MYA;
2 = 412.000 df = 74 0.0001*
P = 1) and 11.38 MYA (1017.27 MYA; P = 1), respec-
HST = 0.2232 0.0001* ST = 0.7013 Nm = 0.21
tively. Within Inia, the first split separated the Bolivian
KST = 0.6974 0.0001*
haplotypes from the remaining haplotypes (P = 1), with a
KST* = 0.4995 0.0001* FST = 0.7736 Nm = 0.15
temporal divergence of around 3.85 MYA (2.875.28
(F) MYA). Within the I. geoffrensis clade, there were two
2 = 1150.845 df = 296 0.0001* significant clades (P = 1), which split around 3.35
HST = 0.3572 0.0001* ST = 0.7838 Nm = 0.14 MYA (2.414.82 MYA). One had haplotypes from the
KST = 0.7744 0.0001* Maraon, Napo, and Caqueta rivers and the second clade
KST* = 0.6004 0.0001* FST = 0.7932 Nm = 0.13 contained the remaining haplotypes. We observed three
clusters in this second clade, although only two yielded
high posteriori probabilities. The first one (P = 0.83) had
temporal split occurred about 1.53 MYA (1.083.5 MYA; P = 1) two sub-clusters, one with all the haplotypes found in the
and separated the ancestor of one individual from the Mamore Upper Orinoco River (P = 0.94) and the other with
River in Bolivia and the ancestor of the remaining individuals. Amazonian haplotypes (P = 0.82). The Upper Orinoco
The next split separated some haplotypes residing in Peruvian cluster was related to a group of individuals from a great
Amazon rivers (Madre de Dios, Ucayali, and Napo rivers) from variety of Amazonian rivers (Putumayo, Caqueta,
the remaining haplotypes in the Amazon and Orinoco basins Amazonas, Javari, Ucayali, Maraon, and Napo). The
(P = 1). This split occurred around 1.08 MYA (0.752.65 temporal split between these two sub-clusters occurred
MYA; Pleistocene times). The remaining haplotypes constituted around 1.39 MYA (1.23.79 MYA). The mitochondrial
a large cluster. The significant temporal splits within this large diversification within the Upper Orinoco clade began
cluster occurred around 0.030.96 MYA (Pleistocene times). It is around 0.96 MYA (0.622.88 MYA). The second cluster
noteworthy to remark that another individual from the Mamore had two other sub-clusters, one comprised of Amazonian
River (Alejandra) in Bolivia was within this last cluster and it haplotypes from Caqueta, Ucayali, Napo, Samiria, and
was not linked with the other more divergent Bolivian individual. Negro rivers. The other contained one Napo River hap-
Also, as in L. longicaudis, we found minor clusters within the lotype and all of the haplotypes from the Lower Orinoco.
 J Mammal Evol

Fig. 2 Distogram, using the Distogram using mean Genetic Distance (Gregorius 1978)
Gregorious (1978)s distance and
1.00
10 distance classes, to analyze the
spatial genetic structure in Lontra
longicaudis observed
0.98

0.96 95% CI
D
0.94

95% CI

0.92

Reference/ Mean
0.90
306 611 917 1223 1529 1834 2140 2446 2752 3057
Spatial distance

In this Orinoco cluster (P = 1), the haplotype diversifi-
cation occurred around 0.55 MYA (0.332.5 MYA). The
third cluster (P = 0.98) began its diversification around
0.272.64 MYA. It had haplotypes from the Peruvian
Ucayali and Napo rivers. Thus, this analysis showed
the Bolivian population to be the most differentiated tax-
on within Inia. Furthermore, two different Inia popula-
tions present in the Orinoco basin were separated by the
same rapids, which isolated one mitochondrial lineage in
P. brasiliensis.
The MJN analysis for L. longicaudis, P. brasiliensis, and
Inia are shown in Supplementary data (Appendix 2) and the
temporal splits are provided in Table 7. For both otter species,
the Orinoco haplotypes (with the exception of the Paz de
Ariporo individual for P. brasiliensis) were clearly younger
than the Amazonian haplotypes, similar to what the
Bayesian trees showed. For Inia, all the temporal divergence
times of I. boliviensis were significantly lower than those ob-
tained with the BI. However, the temporal divergence esti-
mates within I. geoffrensis were inside the ranges obtained
with the BI. It is interesting to note that one individual we
sampled in the Upper Negro River was more linked, by means
of the MNJ, to the Upper Orinoco group than to the rest of the
Amazon haplotypes (not in the BI).

Fig. 3 Distogram, using the Distogram using mean Genetic Distance (Gregorius 1978)
Gregorious (1978)s distance and
10 distance classes, to analyze the 1.00
spatial genetic structure in
Pteronura brasiliensis
observed
0.98

0.96 95% CI
D

0.94

95% CI

0.92

Reference/ Mean
0.90
187 374 562 749 936 1123 1311 1498 1685 1872

Spatial distance
J Mammal Evol

Distogram using mean Genetic Distance (Gregorius 1978)

a 1.00 b Distogram using mean Genetic Distance (Gregorius 1978)
1.00

observed observed
0.95 0.95

0.90 0.90
95% CI 95% CI
D 0.85 D 0.85

0.80 95% CI 0.80 95% CI

0.75 0.75

Reference/ Mean Reference/ Mean

0.70 0.70
965 1930 2895 3860 4825 5790 6755 7720 8685 9650 691 1382 2073 2764 3456 4147 4838 5529 6220 6911
Spatial distance Spatial distance

Distogram using mean Genetic Distance (Gregorius 1978)

c Distogram using mean Genetic Distance (Gregorius 1978) d 1.0

1.0
observed
observed
0.9
0.9

95% CI
95% CI
D
D 0.8
0.8

95% CI 95% CI
0.7 0.7

Reference/ Mean Reference/ Mean

0.6 0.6
114 228 342 456 571 685 799 913 1027 1141 574 1147 1721 2294 2868 3441 4015 4589 5162 5736
Spatial distance Spatial distance
Distogram using mean Genetic Distance (Gregorius 1978)
e 1.00
observed
0.98

0.96 95% CI
D
0.94
95% CI

0.92

Reference/ Mean
0.90
451 902 1353 1805 2256 2707 3158 3609 4060 4512
Spatial distance
Fig. 4 Distogram, using the Gregorious (1978)s distance and 10 dis- b only the samples from Orinoco and Amazon; c only the samples from
tance classes, to analyze the spatial genetic structure in Inia sp. consider- Orinoco; d only the samples from Amazon; e only the samples from
ing: a all the samples analyzed together (Orinoco, Amazon, and Bolivia); Bolivia

Discussion most important aquatic mammals in Neotropical rivers to un-

Evolutionary History and Systematics of L. longicaudis,
P. brasiliensis, and Inia sp.
Our work is the first to compare (with the same gene set and
analytical methods) molecular genetics results of three of the
derstand the generation of biodiversity in the Amazon.
Lontra longicaudis. Our results agreed with Trinca et al.
(2007), who reported high levels of haplotype diversity and no
signal of genetic structuring in populations from southern
Brazil for the mitochondrial control region. Our results also
align with those of Weber et al. (2009). They analyzed

microsatellites to study two populations of L. longicaudis in
southern Brazil and found high gene diversity and an impor-
tant gene flow among populations. Similar to Trinca et al.
(2012), we did not find any significant relationship between
geographic and genetic distances. They suggested the exis-
tence of four distinct evolutionary lineages within this species,
which expanded in the last 0.58 MY. Also, both studies
showed relatedness of haplotypes sampled in very distant
areas, which indicates important levels of historical gene flow.
In contrast, we did not find any clear geographic phylogroup
and we reported an earlier mitochondrial divergence (roughly
2.7 MYA for BI or 0.9 MYA for MJN). Both studies supported
a population expansion during the Pleistocene.
It is easy to explain the differences between both works. All
of the samples in the Trinca et al. (2012) study came from
southern and southeastern Brazil, the central-eastern
Brazilian Amazon, and French Guiana. They only studied
one Colombian sample from the Magdalena Valley, one sam-
ple from the Peruvian Amazon, and one sample from the
Bolivian Amazon. Thus, their samples were highly skewed
to the most recent and derived L. longicaudis populations in
South America. In contrast, our samples mainly came from
areas they did not sample. That is, our sample basically rep-
resented the oldest and original populations of L. longicaudis
in South America. The three samples from Colombia, Bolivia,
and Peru that they used were the basal ones in their tree. The
oldest populations surely had more time to exchange genes
than the more derived populations and for this reason we did
not detect any important geographical phylogroup in our anal-
ysis. Regarding this species, our study is the first to include a
large sample from the Western Amazon and the first to include
data for the Neotropical otter from the Orinoco Basin.
Kartavtsev (2011) analyzed sequences of mt COI from
20,731 vertebrate and invertebrate animal species and obtain-
ed 0.89% 0.16% for populations within species,
3.78% 1.18% for subspecies or semispecies, and
11.06% 0.53% for species within a genus. At mt COII,
Collins and Dubach (2000), Ascunce et al. (2003), and Ruiz-
Garca et al. (2014) reported, an average genetic distance of
around 6% among species within a genus, and around 24%
for subspecies. Bradley and Baker (2001) claimed, for mt Cyt-
b, that values <2% would equal intra-specific variation, values
between 2% and 11% would merit additional study, and
values >11% would be indicative of specific recognition.
Avise (1994) determined 57% of differences at the mt control
region for different species and around 2% for subspecies in
mammals. If we take an average value for all of these genes,
its clear, in L. longicaudis, that the highest percentage was
1.5%. Therefore, our results do not agree with the traditional
taxonomic studies of L. longicaudis based on variation in the
rhinarium shape (Cabrera 1957; Harris 1968), which sug-
gested the existence of three subspecies in Latin America. In
the current study, we included samples of these three putative
J Mammal Evol
Ba priori^ subspecies and examples from them were
intermixed in the molecular trees we obtained.
The beginning of the mitochondrial diversification in
L. longicaudis was 2.7 MYA (BI) and 0.9 MYA (MJN), re-
spectively. These values are in the range obtained by Koepfli
et al. (2008) for the splitting between L. longicaudis and
L. felina (1.8 MYA, 95% HPD: 0.63.2 MYA). The 2.7
MYA date is roughly within the range in and after formation
of the Panamanian land bridge, around 23 MYA (Marshall
1985). The oldest fossil record of L. longicaudis dates back to
1.80.98 MYA and was found in the Ensenadan of Buenos
Aires (Soibelzon et al. 2005). This agrees with our molecular
time estimations. Thus, all the diversification genetics pro-
cesses in this otter were during the Pleistocene and, although
no important vicariance events affected this species, its genetic
diversification should be moderately driven by typical
Pleistocene events such as RH and/or HRCH.
Pteronura brasiliensis. Pickles et al. (2011) published the
most complete work on population genetics of P. brasiliensis.
Similar to our findings, they found high levels of gene diver-
sity and Pleistocene population expansions for the main
groups they studied with temporal population expansions
around 0.670.41 MYA. They also detected four phylogroups
distributed in four different regions: Madre de Dios-Madeira
River basins, the Itenez-Uru River basins, the Pantanal, and
the Amazon-Orinoco-Guianas. They estimated a temporal
split of the Madre de Dios-Madeira rivers and Itnez-Uru
rivers from the rest of the giant otter populations that occurred
around 1.241.69 MYA. One of the individuals from Mamor
River we analyzed split from the rest of the Amazon-Orinoco
individuals around 1.53 MYA (BI) agreeing quite well with
the temporal split of Pickles et al. (2011). They estimated
splitting processes for the Amazon and Orinoco populations
around 0.88 MYA, agreeing quite well with our estimated
temporal splits in Western Amazon and Orinoco, 1.08 MYA
(BI) and 0.7 MYA (MJN), respectively. However, we detected
some differences from the findings of Pickles et al. (2011). We
detected one exemplar from the Casanare River (Lower
Orinoco after the Atures-Maipures rapids) with a temporal
divergence from the rest of the giant otter populations studied
of around 3.35 MYA (BI)- 3.87 MYA (MJN). Pickles et al.
(2011) did not include Orinoco individuals from this area of
the Colombian Orinoco. If more individuals from this area
show this same differentiation there is probably a new, non-
considered, subspecies or ESU within Pteronura. All the ge-
netic distances among the P. brasiliensis populations studied
were less than 1.1% (no existence of subspecies), whereas the
percentage of genetic differentiation of the exemplar of the
Casanare River was around 6%. A very recent published work
also reported this new molecular lineage within Pteronura
(Caballero et al. 2015). We also detected the phylogroup they
named Itnez-Uru in our sample (one of the two individuals
sampled) at the Mamore River in Bolivia. But we also
J Mammal Evol
Fig. 5 Bayesian skyline plot analysis (BSP) to determine possible demo-
graphic changes across the natural history of the three aquatic mammals
herein studied for mitochondrial genes: a for Lontra longicaudis; b for
Pteronura brasiliensis; c for Inia geoffrensis for the Amazon basin; d for
detected another exemplar in the same river that belonged to
the Western Amazon and Orinoco cluster. Thus, the Mamore
River area could contain mixed individuals from two of the
phylogroups detected by Pickles et al. (2011). Thus, the area
may not contain just one of the phylogroups as they conclud-
ed. The same occurred with a specimen that we sampled in the
Tambopata River (an affluent of the Madre de Dios River).
This specimen did not belong to the differentiated Madre de
Dios-Madeira river group detected by Pickles et al. (2011).
Inia geoffrensis for the Orinoco basin; e for Inia boliviensis in the
Bolivian basin. On the x-axis, time in millions of years; on the y-axis,
size of the effective female population
Instead, it belonged to the Western Amazon-Orinoco group.
Therefore, disagreeing with Pickles et al. (2011), within the
Madre de Dios river basin one can detect two different
intermixed groups, not just one. Therefore, hybrid zones not
previously detected for different phylogroups of this species
could exist in the Mamore and Madre de Dios rivers.
With regard to the fossil record of Pteronura, there is less
evidence than for L. longicaudis. The oldest giant otter fossil
in South America is dated to be about 0.13 MYAoriginating
 J Mammal Evol

a 0.98/2.06
LL702 Puerto Nario Amazonas

LL41 Payamino Ecuador

1/1.17

LL198 Bolognesiup Peru

LL305 Ener River Junin Peru

0.33/ 2.22 0.7/1.25

LL401 OcuneVichada River Colombia

0.77/1
LL44 Ariari River Meta Colombia
0.96/0.92
0.15/1.41 LL33 Atalaia Javari River Brazil
0.85/0.46

0.22/0.23 LL407 Chachapoyas Peru

LL11 Rivera Alta Beni River Bolivia

LL23 Islandia Yavari River

LL 184 Sumapaz Cundinamarca Colombia

0.17/0.95

1/0.1 LL37 Meta River Meta Colombia

1/2.7
LL46 El Banco Magdalena Colombia
0.33/1.15

LL19 Amacayacu Amazons Colombia

1/0.34

LL18 Nanay River Peru

1/0.58 1/0.24

LL371 Itaya River Peru

LL16 Nanay River Peru

0.84/0.47 LL28 Pintayacu River Peru

0.34/0.57
LL34 Islandia Yavari River Peru

LL1 Ibare River Bolivia

LL27 El Tigrillo Lake Atacuari Amazons Peru

0/1.37 LL310 Ene River Junin Peru

0.12/0.87
0.34/2.52 0/2.04
LL205 Masanghai Ucayali River Peru
0/1.3
0.74/0.37 LL49 Portachuelo Santander Colombia

LL502 Santa Rosa Guaiviare River Colombia

0.03/0.93

LL25 Villavicencio Meta Colombia

1/0.09
0.01/1.85
LL24 Orocue Meta River Colombia

0.04/0.32
LL306 Nanay River Peru

LL501 Oxa Pampa Pasco Peru

LL23 El Tuparro National Park Vichada Colombia

0/0.46
LL203 Pucallpa Ucayali River Peru
0.98/0.09
0/0.76
LL26 Sucuaro Vichada River Colombia
0.03/2.15 0/0.5
0.03/0.34
LL17 Tigre River Peru

LL20 Nauta Maraon River Peru

0.02/0.12

LL412 Tame River Junin Peru

0/0.71
0.09/0.1
LL2 Nanay River Peru
1/4.78

LL1973 Amacayacu National Park Amazonas Colombia

0.45/0.12 LL10 Santa Cruz Bolivia

0.72/0.53
LL6 Santa Rosa Beni Bolivia

0.98/0.58
LL7 Beni Bolivia
0.24/0.84

LL9 Santa Ana de Yacumo Mamore River Bolivia

LL210 Ene River Junin Peru

0.06/1.46

LL32 San Pablo Amazosn River Peru

0.34/1.12 LL29 Pachitea River Peru

0.74/0.29

0.76/0.4 LL14 Parana River Paraguay

0.13/0.75
LL206 Pucallpa Ucayali River Peru

LL3 Nanay River Peru

 J Mammal Evol
Fig. 6 Bayesian trees of mitochondrial genes for a the two otter species
studied (Lontra longicaudis and Pteronura brasiliensis) and b for Inia sp.
In the case of the otters, the relationships among individuals are shown
whereas for the river dolphins, the haplotype relationships are shown.
Different colors represent the different river basins where the samples
were obtained. In nodes, the numbers are the posterior probabilities and
the divergence times in millions of years. In the case of the B tree, in each
one of the haplotypes, the number of individuals are indicated
out of the Lujanian of Entre Rios province (Argentina)
(Prevosti and Ferrero 2008). Given this time line, we cannot
search for connections between the fossil record and the mo-
lecular results obtained.
In the case of Pteronura, the Casanare River individual
showed the influence of a vicariance event related to the PH
(divergence 34 MYA). Its possible that the origins of the
Atures-Maipures rapids are related to the formation of several
arches and to the orogeny of the Merida Andes. The Baul
Arch (Pindell et al. 1998) and the Arauca Arch could have
uplifted around 85 MYA (late Miocene-Pliocene) and creat-
ed barriers within the Orinoco basin and thus be responsible
for the isolation of some of the Pteronura lineage. However,
the role of PH in the formation of the Fitzcarrald Arch (4
MYA) for the Pteronura diversification (1.530.4 MYA), is
extremely moderate. Such a finding is supported by Rossetti
et al. (2005) and Wesselingh and Salo (2006) who suggest that
many arches could have been buried under subsequent sedi-
mentary deposits and thus not be responsible for the differen-
tiation among taxa.
Inia sp. There are some published papers on population
genetics, systematics, and evolutionary biology of Inia, al-
though none have provided insights as to the origin of the
Amazons biodiversity (Banguera-Hinestroza et al. 2002;
Ruiz-Garca et al. 2006, 2007, 2008; Ruiz-Garca 2010a, b;
Hollatz et al. 2011; Hrbek et al. 2014; Gravena et al. 2014,
2015). Pilleri and Gihr (1977) concluded that the Bolivian
form is a separated species (I. boliviensis) from the animals
of the rest of the Amazon and Orinoco basins (I. geoffrensis).
They determined significant differences at the morphological
and morphometric levels. Pilleri and Gihr (1977) identified
two significant morphometric differences that are fundamental
to understanding the origins of Inia. Inia boliviensis has 33
teeth on each side of its jaws, whilst I. geoffrensis has 2627
teeth and the average neurocranial volume is significantly less
in I. boliviensis (558 cm3) than in I. geoffrensis (665 cm3).
Following Willistons law, I. boliviensis should have a more
primitive form than the current I. geoffrensis. Pilleri and Gihr
(1977) claimed an allopatric speciation process (vicariance
barrier) split the Inia forms. There is a 350 km section of the
Madeira River (from Guayaramern to Porto Velho) that con-
tains 18 rapids and a collective drop of 60 m due to falls such
as Teotnio and San Antonio (Cella-Ribeiro et al. 2013). This
is the same vicariance event that only partially isolated one of
the haplogroups of P. brasiliensis. Pilleri and Gihr (1977)
claimed that I. boliviensis did not originate in the Pacific.
The original Pacific drainage of the Beni area in Bolivia was
closed due to the increased folding of the Andean chain,
which stopped long before the appearance of Inia.
Following these authors, the split between I. geoffrensis and
I. boliviensis began at the end of the Pliocene and at the
beginning the Pleistocene throughout the Abua Pass.
Coming from the Amazon, the ancestor of I. boliviensis
reached Beni Lake. Later in the Pleistocene, Beni Lake was
gradually broached to form the rapids between Guayaramern
and Porto Velho in the Madeira River. In contrast, Grabert
(1984a, 1984b) showed another perspective. A Pacific coastal
Iniidae penetrated to a large Bolivian lake (sub-Andean
freshwater molasses) during the Miocene (15 MYA). Inia
boliviensis then later appeared in this molasse lake during
the Pliocene around 5 MYA. It became highly adapted to the
turbidity of these waters. Sometime afterwards, approximately
1.8 MYA, I. boliviensis penetrated the Amazon basin through
the Purus or the Iquitos gates eventually causing the appear-
ance of I. g. geoffrensis. Later, about 10,000 YA, I. g.
humboldtiana formed from I. g. geoffrensis throughout the
Cassiquiare channel. Our data more strongly support the point
of view of Pilleri and Gihr (1977) than that of Grabert (1984a,
1984b). Our data show that the Bolivian haplotypes were
completely different and not shared but derived from the
Amazonian ones. Furthermore, our data support a more recent
diversification of the Bolivian haplotypes (BI: 0.58 MYA and
MJN: 0.126 MYA) compared to that of the Amazonian hap-
lotypes. Microsatellite results by Ruiz-Garca (2010a) clearly
indicated that the Bolivian population passed through a strong
bottleneck, as it shows considerable lower gene diversity in
both nuclear and mitochondrial genes. Therefore, it could not
be the founding population of gene variability found in the
other Inia populations. Thus, the molecular data reject
Willistons law applied to the evolution of Inia. The differ-
ences between both Inia forms is due to gene drift and founder
events in the Bolivian population. Our estimated BI split be-
tween the ancestor of I. boliviensis and I. geoffrensis (3.85
MYA; Pliocene) could agree quite well with a reported PH
vicariance event (similar result to that obtained by Hollatz
et al. 2011: 3.1 MYA, and Hrbek et al. 2014: 2.87 MYA).
Espurt et al. (2007, 2010) demonstrated that the Nazca
Ridge subduction imprint had a significant influence on the
eastern side of the Andes by means of the Fitzcarrald Arch.
This uplift is responsible for the atypical three-dimensional
shape of the Amazonian foreland basin. Related to Nazca
Ridge subduction, arc volcanism in the Peruvian Andes
ceased around 4 MYA (Rosenbaum et al. 2005). Thus, the
time estimate of the Fitzcarrald Arch uplift is not older than
4 MYA (Pliocene). This could explain changes in the relation-
ship of the Mamore-Beni rivers (or Beni Lake) with other
drainage river systems, which isolated the Bolivian dolphins.
This first split within Inia could be the result of a PH event.
 J Mammal Evol

Fig. 6 (continued)
J Mammal Evol

b
Sotalia guianensis 1

Pontoporia blainvillei 1

Inia Haplo 38 Mamore River 1 Bolivia

0.11/0.21

Inia Haplo 35 Mamore River 3 Bolivia

0.23/0.33

1/11.49
Inia Haplo 37 Mamore River 1 Bolivia
0.16/0.14

Inia Haplo 33 Mamore and Ipurupuru rivers 38 Bolivia

0.06 / 0.33

0.95/0.58

Inia Haplo 36 Mamore and Iruyaez rivers 2 Bolivia

Inia Haplo 34 Ipurupuru and Tijamuch rivers 12 Bolivia

1/11.38

Inia Haplo 30 Maraon River 2 Peru

0.6/1.91
Inia Haplo 28 Maraon River 2 Peru

0.25/1.12

0.51/2.4 Inia Haplo 29 Maraon River 2 Peru

Inia Haplo 23 Napo River 1 Peru

0.45/2.67
Inia Haplo 10 Putumayo, Caqueta, Pacaya, Ucayali, Napo and Maraon rivers 65 Colombia and Peru
0.41/0.19

Inia Haplo 15 Caqueta River 5 Colombia

0.41/0.36
1/3.85
Inia Haplo 16 Tefe River 1 Brazil

0.94/0.05

Inia Haplo 18 Tefe River Brazil

Inia Haplo 4 Inrida and Orinoco rivers 11 Colombia

1/0.3

Inia Haplo 2 Orinoco River 1 Colombia

1.0/0.6

Inia Haplo 3 Guaviare and Orinoco rivers 4 Colombia

0.94/0.96

Inia Haplo 1 Inrida and Orinoco rivers 2 Colombia

0.9/0.58

Inia Haplo 8 Vichada River 1 Colombia

0.83/1.39

Inia Haplo 14 Tarapoto Amazon River 1 Colombia

Inia Haplo 17 Caqueta River 1 Colombia

1/3.35
0.17/0.71
0.82/1.34 Inia Haplo 22 Napo River 1 Peru

0.11/0.7
Inia Haplo 11 Putumayo, Caqueta, Ucayali, Maraon and Napo rivers 15 Colombia and Peru

0.19/0.51
0.02/0.83 Inia Haplo 12 Yavari River 1 Brazil

0.21/2.91

Inia Haplo 13 Tarapoto Amazon River 2 Colombia

Inia Haplo 19 Caqueta and Napo rivers 5 Colombia and Peru

0.24/0.45
Inia Haplo 24 Napo River 1 Peru

0.86/0.29

0.07/1.48 Inia Haplo 21 Ucayali and Napo rivers 5 Peru

Inia Haplo 31 Samiria River 1 Peru

0.83/0.8
0.1/1.55
Inia Haplo 32 Negro River Brazil

Inia Haplo 25 Napo River 2 Peru

0.17/2.94

Inia Haplo 6 Meta River 1 Colombia

0.42/0.93
1/0.5
Inia Haplo 7 Meta River 1 Colombia

0.93/0.26

1/0.55 Inia Haplo 5 Arauca River 2 Colombia

Orinoco
Inia Haplo 9 La Urbana Orinoco River 2 Venezuela

Inia Haplo 27 Napo Ri ver 2 Peru Boli vi a

0.98/2.9 Amazon
Inia Haplo 20 Napo and Ucayali ri vers 4 Peru

0.9/0.77

Inia Haplo 26 Napo River 1 Peru

Fig. 6 (continued)
 J Mammal Evol

Table 7 Temporal splits among different haplotypes within Lontra longicaudis, Pteronura brasiliensis, and Inia sp. by means of a Median Joining
Network (MJN). YA = Years Ago

Temporal splits

Lontra longicaudis
Central haplotype (simultaneously in the Amazon and Orinoco basins) relative to the remaining Amazon haplotypes 851,525 90,090 YA
Central haplotype (simultaneously in the Amazon and Orinoco basins) relative to the remaining Orinoco haplotypes 483,871 82,945 YA
Pteronura brasiliensis
Central haplotype (exclusively Amazonian) relative to the Lower Orinoco haplotype (Casanare River) 3,870,966 185,471 YA
Central haplotype (exclusively Amazonian) relative to the Mamore River haplotypes 645,161 58,651 YA
Central haplotype (exclusively Amazonian) relative to the other Amazonian haplotypes 425,991 85,547 YA
Central haplotype (exclusively Amazonian) relative to the other Orinoco haplotypes 254,154 123,647 YA
Inia sp.
Central Amazonian haplotype relative to the central Bolivian haplotype 1,666,665 75,756 YA
Haplotype diversification within the Bolivian sub-basin 126.263 56,466 YA
Upper Orinoco haplotypes relative to those diverged from Amazonian haplotypes 808,080 214,275 YA
Lower Orinoco haplotypes relative to those diverged from the Amazonian haplotypes. 959,595 290,129 YA
Central Amazonian haplotype relative to those diverged from the Amazonian haplotypes. 778,236 148.671 YA

However, the other temporal split that we found (MJN) was
quite small (1.7 MYA) and related with Pleistocene events.
Following Ribas et al. (2012), the Madeira River was formed
around 12 MY. If the MJN estimate was more realistic than
the BI estimate, the Bolivian Inia split was probably more of a
direct consequence of HRCH than the result of PH.
Our results could also help to provide new ideas regarding
the entry of the ancestor of Inia into continental South
American rivers. However, our results clearly do not support
the hypothesis of Grabert (1984a, 1984b). During the middle
Miocene (1512 MYA), significant marine transgressive-
regressive cycles occurred globally (Haq et al. 1987), which
could have created shallow epi-continental seas in a large
fraction of South America. Following Cassens et al. (2000)
and Hamilton et al. (2001), the mixing of riverine and marine
waters could have created a large diversity of food resources
to be exploited by the ancestor of Inia. The main question of
interest is in regard to what geographical area the ancestor of
Inia arose. There are four possibilities: 1) they came from
more northern Pacific gates than that proposed by Grabert
(1984a, 1984b); 2) they came from the Caribbean area, which
was connected with the Amazon basin (Hoorn 1994; Hoorn
et al. 1995); 3) they arose from the mouth of the Amazon
Basin itself; or 4) they arose through a connection between
the Paran and Amazon basins. Some data may support this
last hypothesis. During the highest global sea levels of the
Miocene, the Paranense Sea formed and was connected to
the Amazon basin (Von Ihering 1927). The current sister taxon
of Inia is Pontoporia, which lives in the region of La Plata
estuary (part of the Paran basin) near the coastal zone.
Additionally, some iniid fossils, such as Saurocetes and
Ischyrorhynchus, have been found along the banks of the
Paran River. Thus, the ancestor of Inia could have penetrated
the Amazon basin from the Paranense Sea. However, there
seems to be more support for the first and second
hypotheses than for the third and fourth. The Inia population
at the Napo River in the northern Peruvian Amazon had the
highest gene diversity and the greatest variety of haplotypes.
In fact, both Orinoco populations were related with different
haplotypes detected in the Napo River. Dobzhansky (1971)
showed that ancestral populations contained higher levels of
gene diversity. The nearest sea area to the current Napo River
was the Guayaquil Gate on the Pacific coast of Ecuador where
the Paleo-Amazon drainage emptied during the middle
Miocene. This could be one possible entry point of the ances-
tor of Inia. However, it is also possible that the ancestor of Inia
entered via the Caribbean area during the Miocene (205
MYA). Both Pebas Lake and the hypothetical Pebasian Sea
(both in the current western Amazon) were closely related to
marine introgressions from the Caribbean Sea (Hoorn 1993,
1994; Hoorn et al. 1995; Lundberg et al. 1998). Even the most
recent marine transgressions during the last Miocene-Pliocene
sea rise (75 MYA; Haq et al. 1987) could have a Caribbean
origin in the origin of Inia. The recent discovery of Isthminia
panamensis, a new fossil inioid from the Caribbean Chagres
Formation of Panama, supports this hypothesis (6.15.8
MYA; Pyenson et al. 2015).
Our genetics results show that the systematics of the
Orinoco dolphins is more complex than claimed by Pilleri
and Gihr (1977), who defined the existence of a subspecies,
I. g. humboldtiana. There are at least two significantly differ-
ent haplogroups of Inia in the Orinoco basin apparently sep-
arated by a set of rapids and falls named Atures (Mapara) and
Maipures (Kituna). The Atures rapids are distributed in an
8 km section of the Orinoco River. Going up the Orinoco
River, there is a 30 km section of fairly calm waters prior to
J Mammal Evol
the Maipures barrier with its 14 rapids over a course of 6 km
and drop of 12 m. These rapids prevent the navigation of that
area of the Orinoco River (Domnguez 1998). The Upper
Orinoco haplogroup is related to different haplotypes dis-
persed across different Western Amazon rivers (Napo,
Amazonas, Javari, Putumayo, and Caqueta), with a temporal
split around 1.39 (BI)-0.8 (MJN) MYA. On the other hand, the
Lower Orinoco haplogroup is closely related to a haplotype
distributed in the Napo River that arose from a temporal split
around 1.0 (MJN)-0.9 (BI) MYA. The current Amazon and
Orinoco basins are connected by the Casiquiare Channel
(250 km), which links the Upper Orinoco River to the Negro
River by means of an anastomosing phenomenon named
stream capture. Recent elevation changes favor the capture
by the Negro River (Stern 1970; Edwards and Thornes
1970), although this elevation increase is only about 20 m
between its source at the Orinoco and its mouth at the Negro
River (Mago-Leccia 1972). At the separation point from the
Orinoco River, this channel is 100 m wide and takes one-
eighth to one-quarter of the Upper Orinocos main channel
volume (Sternberg 1975). The main problem is that the ap-
pearance of the Casiquiare Channel occurs more recently than
the temporal splits of the different Orinoco dolphin
haplogroups from their respective Amazon sources. Stern
(1970), indirectly, and Grabert (1984a, 1984b), directly,
commented that the appearance of the Casiquare Channel oc-
curred during the Holocene, within the last 10,000 years.
Thus, the appearance of the two independent Inia haplogroups
(1.40.8 MYA) in the Orinoco basin occurred before the es-
tablishment of the current connection between the Orinoco
and Amazon basins. Following Hoorn (1993, 1994) and
Hoorn et al. (Hoorn et al. 1995; Hoorn and Wesselingh
2010), both basins were isolated around 810 MYA
(Miocene) after the uplift of the Vaupes Arch in the foreland
of the Andes. Clearly, this Miocene event (PH) is not the cause
of the split of the Orinoco and Amazon Inia populations. More
recent data put forward by Ribas et al. (2012) indicate that the
Lower Negro River could have been formed around 1.00.7
MYA. Also, the appearance of the Upper Negro River is even
more recent and the split of Inia between Orinoco and
Amazon preceded the formation of this river. Two hypotheses
emerge to explain this. The first is the existence of other river
systems (now disappeared) in the Western and/or Central
Amazon that connected the Orinoco and Amazon basins
around 12 MYA. Some evidence in favor of this comes from
geological studies. Assumpo and Surez (1988) and Lima
et al. (1997) claimed that regional neotectonic activity within
the Negro River area could have been responsible for control-
ling the river courses of this area. Almeida-Filho and Miranda
(2007) confirmed this idea. They detected the relicts of a large
ancient drainage system hidden by a tropical rain forest. It was
not visible by optic or synthetic aperture radar images. This
indicates that the current lower course of the Negro River was
produced from a mega capture driven by neotectonics during
the Neogene-Quaternary limits. Other studies support RLH,
that is, with the existence of an internal lake or lagoons
(Amazon Lake) during the last Pliocene and Pleistocene
(Klammer 1984; Frailey et al. 1988; Campbell 1990;
Marroig and Cerqueira 1997; Nores 1999, 2004; Rossetti
et al. 2005). These aquatic structures connected the Orinoco
and Amazon basins after their separation during the Miocene.
RLH is caused by eustatic changes in sea level of 100 m or
more, during the late Pliocene and Pleistocene. Aleixo and de
Fatima (2007) claimed that the midwestern Amazon may be
the most dynamic area for new colonization, due to a relative-
ly recent reduction of the Amazon Lake and subsequent flood-
plains in that area. Although some authors have resisted this
view (Tuomisto et al. 1992; Haffer 2008), Inia data regarding
the splitting of the Orinoco and Amazon populations support
RLH and HRCH.
Recently, I. araguaiaensis has been proposed as a new Inia
species in the Araguaia-Tocantins River basin (Hrbek et al.
2014). Following these authors, this Inia form split from the
common ancestor of I. geoffrensis around 2.08 MYA.
Currently, the Araguaia-Tocantins River basin is disconnected
from the Amazon River basin because it directly pours its
waters into the Atlantic Ocean. However, it seems to have
not been completely disconnected during the transition of
the Pliocene to the Pleistocene (Rossetti and Valeriano
2007). The authors suggested this new Inia species based on
three sets of data. First, a set of microsatellites clearly differ-
entiated the Inia population of the Araguaia-Tocantins River
basin from that of the Brazilian Central Amazon River.
Second, no mitochondrial haplotypes were determined to be
shared between I. araguaiaensis and I. geoffrensis. Third,
there were some minor (not statistically significant) cranial
differences between Inia forms. Nevertheless, some criticism
may be valid regarding this new species. There are also sig-
nificant genetic differences in microsatellites among Inia pop-
ulations of different lagoons in the same river, or neighboring
rivers (Ruiz-Garca et al. 2007; Ruiz-Garca 2010b; Hollatz
et al. 2011) within the Amazon River basin. This is without the
existence of apparent geographical barriers (due to a high
degree of fidelity; Martin and da Silva 2004) and no one
claims that each one of these populations are different species.
Also, there were no shared mitochondrial haplotypes between
Orinoco groups and among them and the Amazon groups as
we have demonstrated in this study, but they are not different
species. Additionally, the Orinoco specimens show some mi-
nor morphological differences compared to the Amazon ani-
mals but not sufficiently enough to support the existence of
two species. An additional criticism is that the current
Tocantins River (Ribas et al. 2012) formed approximately
0.80.5 MYA. This is more recent than the supposed split
between I. araguaiaensis and I. geoffrensis. Thus, its highly
improbable that I. araguaiaensis is a new species. It may be a

new subspecies of I. geoffrensis created by a vicariance barri-
er. If this is confirmed, this Inia form could be the product of
recent hydrogeological change (HRCH).
The effects of riverine vicariance barriers are clearly more
important for dolphins than for otters. However, it may be less
important for Inia than previously considered (Pilleri and Gihr
1977; Banguera-Hinestroza et al. 2002; Hollatz et al. 2011).
Gravena et al. (2014) showed that the rapids between
Guayaramern and Porto Velho in the Upper Madeira River
do not create a complete barrier to the dispersion of
I. boliviensis. All the dolphins they sampled from the Upper
Madeira River to the area of Borba, relatively near the conflu-
ence of the Madeira River with the Amazon River, showed
molecular characteristics of I. boliviensis. The uni-directional
gene flow follows a downstream path from the Bolivian sub-
basin. The divergence between the I. boliviensis upstream and
downstream populations was estimated to have occurred
around 0.122 MYA, which agrees quite well with our estima-
tion for mitochondrial diversification within I. boliviensis in
the Bolivian sub-basin (MJN: 0.126 MYA). This could mean
that some hydrological changes occurred at the beginning of
the fourth great glaciation of the Pleistocene. These changes
led to the dispersion of I. boliviensis from Beni Lake to the
Madeira River, which reinforces the role of HRCH. Also
Gravena et al. (2015) determined the existence of hybrid spec-
imens from I. geoffrensis and I. boliviensis in the Madeira
River, downstream of the rapids. This means that the
Madeira rapids were only a partial barrier for pink river dol-
phins as we also demonstrated for P. brasiliensis and no re-
productive isolation mechanisms developed between both
Inia forms. This finding has important consequences for the
systematics of Inia. The Bolivian form should again be named
as a sub-species, I. g. boliviensis, disagreeing with our previ-
ous findings of this taxon being a full species (Ruiz-Garca
et al. 2006, 2008; Ruiz-Garca 2010a). This would go against
I. araguaiaensis as a new full species, because the temporal
split with I. geoffrensis is considerably minor compared to that
between I. geoffrensis and I. boliviensis.
Which Amazon Diversity Hypotheses Best Explains
the Population Genetics Results for L. longicaudis,
P. brasiliensis, and Inia?
The fact that these three species are aquatic, or semi-aquatic,
allows us to discard the following four of the eleven proposed
hypotheses on the origin of Amazons biodiversity: STH,
RBH, CDH, and DUH. STH hinges on the ancestors of the
three species arriving to the Neotropics in relatively recent
times. RBH suggests that rivers are not barriers to aquatic
and semi-aquatic species, but rather they are dispersion routes.
CDH focuses on species not strongly linked to the composi-
tion of floral species. Finally, DUH states that cold/warm
J Mammal Evol
cycles do not significantly affect the water bodies with which
these species are strictly or semi-strictly associated. Thus, we
turn to the remaining hypotheses (PH, RH, RRH, MH, GH,
RLH, and HRCH).
Both L. longicaudis and P. brasiliensis experienced gene
diversity and haplotype differentiation during the Pleistocene.
The only exception to this was the P. brasiliensis individual
from the Casanare River that showed differentiation during
the Pliocene in a PH event. If we consider BI in the case of
Inia, the split between I. boliviensis and I. geoffrensis occurred
during the Pliocene by a PH event (Fitzcharrald Arch). The
rest of the time splits were basically during the Pleistocene. If
the MJN results are considered, all the temporal splits within
Inia occur during the Pleistocene. This means that some of the
previous quoted Amazonian diversity hypotheses could have
very limited influence on the three taxa analyzed. In the cases
of the two otters, it seems clear that only one PH event affected
the giant otter but not the Neotropical otter. When individuals
were analyzed for possible spatial structure, in L. longicaudis
and P. brasiliensis, some patches of genetic similarity were
detected (about 900 km and 600 km, respectively). This
should favor the occurrence of GH in the generation of genetic
differentiation, especially, in the case of L. longicaudis, be-
cause no vicariance events were found for this species. There
was also a gradient pattern in genetic distances. However, the
molecular markers we employed are of neutral behavior and
probably are not affected by selective geographical patterns.
Likely, the asymmetrical gene flow seems to be most impor-
tant from the Western Amazon towards the Orinoco and the
Bolivian sub-basin. This could rest importance to GH. Also,
the population expansion we detected during the Pleistocene
agrees better with RH and HRCH. In fact, these genetic
patches could be related to refugium size, with posterior ex-
tensive gene flow and mixture. In the case of P. brasilensis, the
spatial structure is closely related with the fact that the two
paleogeographic events (PH) affecting this species are in the
periphery of the geographical area analyzed. This creates a
significant spatial structure. But within the Amazon and
Orinoco basins, the generation of gene diversity is related to
available refugia (RH) and recent hydrogeologic changes
(HRCH). The recent analyses of Amazonian stratigraphy
and its paleoenvironment (Campbell et al. 2006; Latrubesse
et al. 2010; Nogueira et al. 2013) favoring HRCH may be
especially important. One example is the Beni River in
Bolivia, which has experienced anticlockwise movement over
the last 0.01 million years. This has led to a change in flow
orientation of 45 degrees and is a response to the continuous
uplifting of the Andes piedmont (Dumont 1996). Recent
hydrogeologic changes could cause fragmentation and a de-
gree of isolation of riparian populations due to the physical
behavior by rivers and periodic river-switching events
(Wilkinson et al. 2006). However, an effect on otter species
could be elevated gene flow. Also, for both otter species, river
J Mammal Evol
clusters were not well associated with river refuges (RRH).
Otters did not have the highest gene diversity levels in popu-
lations on the periphery of the Amazon basin when associated
with MH. Thus, GH, RRH, and MH do not seem to play
important roles for either otter species.
In the case of Inia, two PH events seem to be important for
generating gene diversity (directly the Fitzcharrald Arch and
indirectly the El Baul-Arauca-Mrida arches). However, al-
though significant differences within Amazonian clades were
detected, none could be related to other Amazonian arches
(such as Iquitos-Florencia, Caravari, and Purus arches;
Roddaz et al. 2005; Hubert and Renno 2006). Different hap-
lotypes were intermixed in rivers at both sides of these arches.
Also the Vaupes Arch, which separated the Orinoco and
Amazon basins around 108 MYA, did not play a role in the
splitting process of Inia. Thus, PH events are neither the only
nor the most frequent events creating mitochondrial diversifi-
cation in Inia. The Plesitocene influence also seems funda-
mentally significant, but in a different way than in otter spe-
cies. Although the Napo River in the Western Amazon
showed greater gene diversity and a greater number of differ-
ent haplotypes, RHs influence on exclusively aquatic organ-
isms, such as the dolphins, is certainly limited. Notably, we
did not detect population expansions during the Pleistocene
for dolphins as we did for otters. Population expansion is
highly related with the RH and it was not found in Inia. A
very significant spatial structure was recorded for the global
Inia sample, but barriers in the extreme geographical areas
studied (like in P. brasiliensis) could have had a greater influ-
ence than did the GH. Indeed, the GH has been refuted in the
Neotropics by Cracraft and Prum (1988). The more peripheral
populations of Inia within the Amazon basin did not present
the highest levels of variability, thus excluding the importance
of MH. No haplotypes were segregatedly clustered by rivers
(except to those affected by PH events), thus excluding the
importance of RRH. Henceforth, HCRH and RLH (different
in their origins but highly related in practice) could be the
main factors that generated gene diversity in Inia during the
Pleistocene. The existence of a relatively recent Amazon Lake
in the late Pliocene and middle Pleistocene and/or changes and
appearance of the current Amazon rivers during the
Pleistocene by recent tectonic (no Miocene) activity could
provide a reasonable explanation as to the origins of the two
different Orinoco populations coming from two different
Amazonian river sources. There is an additional piece of evi-
dence. The separated and differentiated I. boliviensis could
have again returned to the Middle and Lower Madeira River
(Gravena et al. 2014) at the beginning of the last big
Pleistocene glaciation event. Another point supporting RLH
is the existence of a Pliocene (5 MYA) sea rise of 100 m for a
duration of 0.8 MY (Haq et al. 1987). Thus marine transgres-
sions could have had a great influence on biotic diversifica-
tion. Our data agree quite well with some recent studies that
indicate that the wetland system may have persisted until the
Pliocene or Pleistocene (Rossetti et al. 2005; Campbell et al.
2006; Latrubesse et al. 2010). Additional Pleistocene influ-
ence can be seen in the Last Glacial Maximum (LGM;
15,00030,000 YA), with female population declines evident
in all three species. The RLH could also explain the histori-
cally elevated levels of gene flow throughout the Amazon and
Orinoco basins of both otter species.
Its interesting to note that for the three species, the Western
Amazon, especially in northern Peru, Colombia, and western
Brazil, showed the highest levels of gene diversity. This coin-
cides with the Napo Refugium (or island) stated by the RH
(Whithmore and Prance 1987) and the RLH hypotheses
(Nores 1999, 2004). The Napo Refugia have been determined
to be of importance for butterflies (Racheli and Racheli 2004),
characiform fishes (Hubert and Renno 2006), amphibians and
lizards (Ron 2000), and birds (Prum 1988).
However, each species has a different capacity to avoid
barriers or vicariance events. The smallest and least water-
dependent species (L. longicaudis) was not affected by vicar-
iance events. Pteronura. brasiliensis, a larger and more water
dependent otter relative to L. longicaudis, was clearly affected
by one PH event in the Pliocene and it was also partially
affected by two other vicariance events: the Fitzcharrald
Arch and the Upper Madeira rapids. In contrast, Inia, the
largest and the most linked to the water of the three species
studied, showed very high levels of genetic differentiation,
influenced by Pliocene PH events and also vicariance events
during the Pleistocene. Our study shows that each species was
affected by different events and this contributes to the long
controversy as to which of the Amazonian diversity hypothe-
ses is the most important (Lomolino et al. 2006)
(Supplementary data for this controversy, Appendix 3).
Many of these controversies have not taken into account three
processes. First, the geographical origin of each taxa that gen-
erated the current species under study should be included.
Each colonization process ought to be viewed as a
Markovian process, where the first step conditions the follow-
ing step. For instance, most of the current mammalian species
in South America are thought to be the evolutionary
byproduct that followed the formation of the Isthmus of
Panama, about 3 MYA. In contrast, organisms whose ances-
tors evolved Bin situ^ in the Amazonian basin since
Cretaceous periods (fishes; Lundberg et al. 1998, 2010;
plants; Hooghiemstra and van der Hammen 1998) could have
been influenced more significantly by Miocene paleogeo-
graphic or Bin situ^ older events. Second, each species (or
genome) has different capacities and adaptability to colonize
new areas. For the genome of L. longicaudis, no rapids were
barriers for its colonization process, whereas, rapids represent-
ed considerable barriers for the genome of Inia. Likely, the
orogeny of the Eastern Cordillera of Colombia and isolation of
the Magdalena drainage basin (1211 MYA) (Hoorn et al.

1995; Guerrero 1997) did not influence L. longicaudis but it
was a complete barrier for P. brasiliensis and Inia. Third, the
studies that showed more influence of Miocene diversification
hypotheses are those that centered above the level of genus or
species. Those that detected more Pleistocene influence are
those at or below the level of species. However, the intra-
specific generation of gene diversity is a first step, in many
cases, for new speciation in the future, vindicating the impor-
tance of the Pleistocene period.
Acknowledgments Economic resources to carry out this study were
obtained from Colciencias (Grant 1203-09-11239; Geographical popula-
tion structure and genetic diversity of two river dolphin species, Inia
boliviensis and Inia geoffrensis, using molecular markers) and the
Fondo para la Accion Ambiental (US-Aid) (120108-E0102141;
Structure and Genetic Conservation of river dolphins, Inia and Sotalia,
in the Amazon and Orinoco basins). Many thanks go to Dr. Diana Alvarez
(Colombia), Luisa Castellanos (Colombia), Ariel Rodriguez (Colombia),
Esteban Payn (Colombia), Carlos Vergara (Colombia), Maria Fernanda
Gmez (Colombia), Nathal Romero (Colombia), Mariana Escovar (La
Paz, Bolivia), Juanito, and Angelito (Iquitos, Peru), and to Isaias, and his
sons (Requena, Peru), who participated in the capture of the pink river
dolphins herein studied. Also, diverse Peruvian Indian communities col-
laborated with our pink river dolphin and otter captures throughout the
Peruvian rivers (Bora, Ocaina, Shipigo-Comibo, Capanahua, Angoteros,
Orejn, Cocama, Kishuarana, and Alamas) and also to diverse Bolivian
communities, who helped to capture dolphins and otters throughout the
Mamor river and other Bolivian Amazon tributaries (Sirion,
Canichana, Cayubaba and Chacobo). Dr. Fernando Trujillo (Omacha
Foundation) gently offered some dolphin samples from the Colombian
Orinoco, and Amazon. Additional thanks go to Hugo Glvez (Iquitos,
Per), and Armando Castellanos (Quito, Ecuador) to collaborate in col-
lection permits in both countries. Similarly, many thanks go to the
Bolivian, Peruvian and Ecuadorian Ministry of Environment, to the
Direccin General de Biodiversidad and CITES from Bolivia,
PRODUCE, Direccin Nacional de Extraccin and Procesamiento
Pesquero and to the Instituto Nacional de Recursos Naturales
(INRENA) from Per for their role in facilitating the obtainment of the
collection permits. Special thanks goes to the Coleccin Boliviana de
Fauna (Dr. Julieta Vargas) in La Paz (Bolivia). Thanks also to the
Fundacin Sociedad Portuaria de Santa Marta (Colombia) for its logisti-
cal support.
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