The Amazon River system as an ecological barrier driving genetic differentiation of the pink dolphin (Inia geoffrensis)

The pink dolphin (Inia geoffrensis) is widely distributed along the Amazon and Orinoco basins, covering an area of approximately 7 million km². Previous morphological and genetic studies have proposed the existence of at least two evolutionary significant units: one distributed across the Orinoco and Amazon basins and another confined to the Bolivian Amazon. The presence of barriers in the riverine environment has been suggested to play a significant role in shaping present‐day patterns of ecological and genetic structure for this species. In the present study, we examined the phylogeographic structure, lineage divergence time and historical demography using mitochondrial (mt)DNA sequences in different pink dolphin populations distributed in large and small spatial scales, including two neighbouring Brazilian Amazon populations. mtDNA control region (CR) analysis revealed that the Brazilian haplotypes occupy an intermediate position compared to three previously studied geographic locations: the Colombian Amazon, the Colombian Orinoco, and the Bolivian Amazon. On a local scale, we have identified a pattern of maternal isolation between two neighbouring populations from Brazil. Six mtDNA CR haplotypes were identified in Brazil with no sharing between the two populations, as well as specific cytochrome b (cyt b) haplotypes identified in each locality. In addition, we analyzed autosomal microsatellites to investigate male‐mediated gene flow and demographic changes within the study area in Brazil. Data analysis of 14 microsatellite loci failed to detect significant population subdivision, suggesting that male‐mediated gene flow may maintain homogeneity between these two locations. Moreover, both mtDNA and microsatellite data indicate a major demographic collapse within Brazil in the late Pleistocene. Bayesian skyline plots (BSP) of mtDNA data revealed a stable population for Colombian and Brazilian Amazon lineages through time, whereas a population decline was demonstrated in the Colombian Orinoco lineage. Moreover, BSP and Tajima's D and Fu's F_s tests revealed a recent population expansion exclusively in the Bolivian sample. Finally, we estimated that the diversification of the Inia sp. lineage began in the Late Pliocene (approximately 3.1 Mya) and continued throughout the Pleistocene. Published 2011. This article is a US Government work and is in the public domain in the USA. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 812–827.     

Looking to the past and the future: were the Madeira River rapids a geographical barrier to the boto (Cetacea: Iniidae)?

In the present study we tested if a series of 18 rapids on the upper Madeira River form an effective barrier to gene flow, and in particular if they delimit the distribution of the boto Inia boliviensis—which it is believed to occurs only in the Bolivian sub-basin, above the rapids—and I. geoffrensis, which occurs throughout the Amazon basin and below the upper Madeira River rapids. We analyzed 125 individuals from the Madeira River basin sampled from upstream and downstream of the rapids. As the two species are morphologically similar, we used diagnostic molecular characters from known reference specimens to assign individuals to species. We observed that all individuals of Inia from the Bolivian sub-basin up to almost the mouth of the Madeira River belong to the species I. boliviensis. Therefore we concluded that the rapids do not delimit the distribution of I. boliviensis upstream and I. geoffrensis downstream of the rapids as previously hypothesized. Since we registered I. boliviensis along almost the entire length of the Madeira River, we estimated gene flow, time of divergence and effective population sizes of the upstream (Bolivian) and downstream (Madeira River) groups of I. boliviensis using IMa2. We concluded that gene flow is uni-directional from the upstream to the downstream group. Divergence time between the two groups was estimated to have occurred ~122 thousand years ago. The coalescent effective population size for the upstream group was estimated at ~131 thousand individuals, while for the downstream group it was estimated at ~102 thousand individuals. Recently two dams have been constructed in the region of the rapids; neither has a mechanism that will maintain connectivity between the upstream and downstream regions, and together with anthropogenic alterations to the hydrodynamic regime and ecology of the river will likely pose serious long-term and short-term consequences for I. boliviensis and other aquatic taxa.     

Population,density estimates,and conservation of river dolphins (Inia and Sotalia) in the Amazon and Orinoco river basins

This study is part of an on‐going effort to evaluate and monitor river dolphin populations in South America. It comprises the largest initiative to estimate population size and densities of Inia and Sotalia dolphins using statistically robust and standardized methods. From May 2006 to August 2007, seven visual surveys were conducted in selected large rivers of Bolivia, Colombia, Brazil, Ecuador, Peru, and Venezuela in the Amazon and Orinoco river basins. Population sizes of Inia and Sotalia were estimated for different habitats (main river, tributary, lake, island, confluence, and channel). A total of 291 line and 890 strip transects were conducted, covering a distance of 2,704 linear kilometers. We observed 778 Inia geoffrensis, 1,323 Inia boliviensis, and 764 Sotalia fluviatilis. High‐density areas were identified (within 200 m from the river banks, confluences, and lakes) and we propose that these constitute critical habitat for river dolphins. High densities of river dolphins seem to coincide with well‐managed freshwater protected areas and should be considered as hot spots for river dolphins in South America.     

Seasonality of Reproduction in Amazon River Dolphins (Inia geoffrensis) in Three Major River Basins of South America1

Reproduction of Amazon River Dolphins, Inia geoffrensis, is generally reported to be highly seasonal; however, this conclusion is based on studies from only one area of Inia distribution from throughout the Amazon and Orinoco river basins. Our observations of live dolphins from the Orinoco, Amazon, and Mamoré river basins (in Venezuela, Peru and Bolivia, respectively) indicate that reproduction in Inia often occurs year‐round, with seasonal peaks varying according to geographic location. Inia neonates in Peru and Bolivia were seen in all seasons, and were observed most often in falling water (season was defined by relative water level). Conversely, neonates in Venezuela were seen at the end of low water and in rising water, yet were never observed during falling water. Inia mating behavior in Peru was observed in all seasons, while mating was observed only during falling and low water in Bolivia. Our review of the literature from throughout the range of Inia indicates variation in reproductive seasonality, with year‐round reproduction in some areas. Seasonality of peaks in births varied according to study area, and may be more closely associated with local environmental and prey conditions than with taxonomic relatedness, relative seasonal differences in water levels, or broad geographic distribution.     

Living between rapids: genetic structure and hybridization in botos (Cetacea: Iniidae: Inia spp.) of the Madeira River,Brazil

Until the recent construction of hydroelectric dams, a series of 18 rapids divided the upper and lower Madeira River, and these rapids were thought to separate two species of Amazonian freshwater dolphins (boto): Inia boliviensis (above) and I. geoffrensis (below). Some reports and articles, however, mention the occurrence of botos within the rapids region and that they occasionally cross the rapids, but without mentioning the species concerned. Based on our previous studies, it is likely that I. boliviensis occurs in the region of the rapids. To test this supposition, we sampled 18 individuals from this region, and collected mitochondrial (control region, cytochrome b and cytochrome oxidase I) and nuclear (10 microsatellite loci) DNA data, in order to test if there is connectivity between the dolphins that were found within the rapids region and dolphins collected upstream and downstream of the rapids, and investigate population structuring between these localities. All animals in our study were molecularly identified using three mitochondrial markers as belonging to the species I. boliviensis. Animals upstream of the Teotônio waterfall, the main and highest waterfall of the region, had nuclear genome of I. boliviensis, while most dolphins downstream of the waterfall had nuclear genome of I. geoffrensis. Inia boliviensis collected in the rapids region above the Teotônio waterfall belong to a management unit (MU) distinct from the I. boliviensis MU occupying Bolivian rivers. Downstream of Teotônio waterfall most dolphins are I. boliviensis/geoffrensis hybrids, with remaining individuals being migrant I. boliviensis. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 764–777.     

Aotus from the southwestern Amazon region is geographically and chromosomally intermediate betweenA. azarae boliviensis andA. infulatus

We described the karyotypes detected in 23Aotus specimens captured on Southwestern Amazon region (63°30'W, 9°00'S). This region should be occupied byA. nigriceps. However, the animals described here have a fur pattern and a karyotypes similar to those ofA. azarae boliviensis andA. infulatus. The females presented 50 chromosomes and the males 49 as a consequence of fusion between the chromosome Y and an autosome. This sample presented polymorphisms of G- and C-banding and NOR-staining. These animals are karyotypically intermediate betweenA. a. boliviensis andA. infulatus. The taxonomy and karyotypic evolution of these taxa are discussed.     

Molecular identification of evolutionarily significant units in the Amazon River dolphin Inia sp. (Cetacea: Iniidae)

The Amazon river dolphin, genus Inia, is endemic to the major river basins of northern South America. No previous studies have focused on the genetic structure of this genus. In this work, 96 DNA samples from specimens of this genus were collected in the Orinoco basin (four rivers), the Putumayo River, a tributary of the Colombian Amazon and the Mamoré, and the Tijamuchí and Ipurupuru rivers in theBolivian Amazon. These samples were used to amplify a fragment of 400 bp of the mitochondrial DNA (mtDNA) control region. In addition, 38 of these samples were also used to sequence 600 bp of the mitochondrial cytochrome b gene. The analysis of the population structure subdivision with an analysis of molecular variance (AMOVA) revealed important aspects about the genetic structure of Inia groups fromthese three geographically separate regions. By comparing the control region DNA and cytochrome b sequences, distinct types of nonshared haplotypes were observed. The net genetic divergence of control region sequences was 6.53% between the Orinoco and Bolivian rivers, 5.32% between the Putumayo and Bolivian rivers, and 2.50% between the Orinoco and Putumayo rivers. For the cytochrome b gene, these values were 2.48%, 2.98%, and 0.06%, respectively. The nucleotide sequences were analyzed phylogenetically using several genetic distance matrices and applying neighbor-joining, maximum likelihood, and maximum parsimony procedures. The results support the proposal to subdivide the Inia genus into at least two evolutionarily significant units: one confined to the Bolivian river basin and the other widely distributed across the Amazon and Orinoco basins.     

Deciphering the diet of a wandering spider (Phoneutria boliviensis; Araneae: Ctenidae) by DNA metabarcoding of gut contents

Arachnids are the most abundant land predators. Despite the importance of their functional roles as predators and the necessity to understand their diet for conservation, the trophic ecology of many arachnid species has not been sufficiently studied. In the case of the wandering spider, Phoneutria boliviensis F. O. Pickard‐Cambridge, 1897, only field and laboratory observational studies on their diet exist. By using a DNA metabarcoding approach, we compared the prey found in the gut content of males and females from three distant Colombian populations of P. boliviensis. By DNA metabarcoding of the cytochrome c oxidase subunit I (COI), we detected and identified 234 prey items (individual captured by the spider) belonging to 96 operational taxonomic units (OTUs), as prey for this wandering predator. Our results broaden the known diet of P. boliviensis with at least 75 prey taxa not previously registered in fieldwork or laboratory experimental trials. These results suggest that P. boliviensis feeds predominantly on invertebrates (Diptera, Lepidoptera, Coleoptera, and Orthoptera) and opportunistically on small squamates. Intersex and interpopulation differences were also observed. Assuming that prey preference does not vary between populations, these differences are likely associated with a higher local prey availability. Finally, we suggest that DNA metabarcoding can be used for evaluating subtle differences in the diet of distinct populations of P. boliviensis, particularly when predation records in the field cannot be established or quantified using direct observation.     

Potentially infanticidal behavior in the Amazon river dolphin (<Emphasis Type="Italic">Inia geoffrensis</Emphasis>)

Infanticide by males is a phenomenon common in species in which the reproductive output of large numbers of females can be monopolized by a small number of males. It is thought to increase a male’s fitness, at the expense of the fitness of the infant’s parents, by bringing females into season more quickly. Infanticide by males has been recorded in just three cetacean species. We report aggressive behavior suggestive of infanticide in a fourth, the Amazon river dolphin (Inia geoffrensis). We observed and photographed a series of attacks on a neonate Amazon river dolphin by a large male, with apparent protective behavior by the mother. Although infanticide was not confirmed, the forceful, aggressive behaviors were highly suggestive of infanticidal behavior and represent another important data point for comparative studies of infanticide in mammals. Amazon river dolphins may have a polygynous, polyandrous, or promiscuous mating system, the latter two of which are not the norm in species in which the reproductive output of large numbers of females are monopolized by a small number of males. However, sexual dimorphism, high rates of aggression by males, socio-sexual object-carrying displays by males, and a long interbirth interval suggest that successful male Amazon river dolphins may be able to monopolize a large proportion of mating opportunities, and it is plausible that male dolphins can improve their reproductive success by bringing females into estrous sooner by killing the offspring of other males.     

Conflicts with fisheries and intentional killing of freshwater dolphins (Cetacea: Odontoceti) in the Western Brazilian Amazon

We report three cases of conflicts with fishing activities of freshwater dolphins Inia geoffrensis and Sotalia fluviatilis in the Western Brazilian Amazon. The animals presented several cuts produced by perforating and cutting objects, especially on the dorsum, sides and flukes. The wounds were probably caused by harpoons and machetes, gear commonly used by local inhabitants for fishing and agricultural practices in the Amazon. The carcasses had not been subsequently used in any way, which suggests that the animals were not killed for consumption. Conflicts with fishermen and persistent cultural taboos may have led to the deaths. These records are an indication of a growing situation of conflict with fishing activities that should be taken into consideration in the conservation policy planning of aquatic mammals in the Amazon.     

Notes on Oenocarpus (Palmae) in the Colombian Amazon

Five species ofOenocarpus from the Río Caquetá, in the Colombian Amazon, are discussed. Two of themOenocarpus simplex andOenocarpus makeru are described as new.Oenocarpus bacaba var.parvus is shown to be a synonym ofO. balickii; this species andO. minor are recorded for the first time in Colombia. Some comments on the poorly knownO. circumtextus are also given. The new findings support the inclusion ofJessenia inOenocarpus.     

Vocalizations of Amazon River Dolphins,Inia geoffrensis: Insights into the Evolutionary Origins of Delphinid Whistles

Oceanic dolphins (Odontoceti: Delphinidae) produce tonal whistles, the structure and function of which have been fairly well characterized. Less is known about the evolutionary origins of delphinid whistles, including basic information about vocal structure in sister taxa such as the Platanistidae river dolphins. Here we characterize vocalizations of the Amazon River dolphin (Inia geoffrensis), for which whistles have been reported but not well documented. We studied Inia at the Mamirauá Sustainable Development Reserve in central Brazilian Amazônia. During 480 5‐min blocks (over 5 weeks) we monitored and recorded vocalizations, noted group size and activity, and tallied frequencies of breathing and pre‐diving surfaces. Overall, Inia vocal output correlated positively with pre‐diving surfaces, suggesting that vocalizations are associated with feeding. Acoustic analyses revealed Inia vocalizations to be structurally distinct from typical delphinid whistles, including those of the delphinid Sotalia fluviatilis recorded at our field site. These data support the hypothesis that whistles are a recently derived vocalization unique to the Delphinidae.     

Ecological factors influencing group sizes of river dolphins (Inia geoffrensis and Sotalia fluviatilis)

Living in groups is usually driven by predation and competition for resources. River dolphins do not have natural predators but inhabit dynamic systems with predictable seasonal shifts. These ecological features may provide some insight into the forces driving group formation and help us to answer questions such as why river dolphins have some of the smallest group sizes of cetaceans, and why group sizes vary with time and place. We analyzed observations of group size for Inia and Sotalia over a 9 yr period. In the Amazon, largest group sizes occurred in main rivers and lakes, particularly during the low water season when resources are concentrated; smaller group sizes occurred in constricted waters (channels, tributaries, and confluences) that receive an influx of blackwaters that are poor in nutrients and sediments. In the Orinoco, the largest group sizes occurred during the transitional water season when the aquatic productivity increases. The largest group size of Inia occurred in the Orinoco location that contains the influx of two highly productive whitewater rivers. Flood pulses govern productivity and major biological factors of these river basins. Any threats to flood pulses will likely have an effect on the functionality of these ecosystems and the species living in them.     

Morphometric characterization of thalassohaline Artemia franciscana populations from the Colombian Caribbean

Aim To establish possible interpopulation relationships among Colombian Artemia franciscana (Crustacea, Anostraca) populations. Location Colombian Caribbean coast (Manaure, Galerazamba, Salina Cero and Tayrona) and a similar thalassohaline reference population from San Francisco Bay (SFB‐USA). Methods Morphometric characters of male and female cultured individuals of A. franciscana were measured. The populations were grouped according to: (1) population type (populations grouped according to two broad regions of origin: North America and the Caribbean coast), and (2) specific geographical origin (populations selected according to five specific local origins: Manaure, Galerazamba, Salina Cero, Tayrona and SFB) and evaluated using forward stepwise discriminant analysis (SPSS, Ver. 10). Results Optimal discriminant variables for males grouped by the type of population were left setae and antenna length, and for females they were abdominal length and antenna length. However, for males grouped by their specific geographical origin, the optimal variables were furca length, left setae, antenna length, eye separation, abdominal width and abdominal length, and for the females, they were furca length, abdominal length, left setae and eye separation. Male and female Colombian Caribbean populations were separated from the North American populations. However, our results show that the classification based on male characters provides better group membership than females. Main conclusions Male morphometric characters separated the type of population groups more clearly than the female characters, because all Colombian populations were correctly positioned in the Caribbean coast region and the SFB population in the North American region, with no overlapping between the two types, as was the case for the female individuals. Likewise, male individuals correctly position the Salina Cero population to its neighbouring Galerazamba population and to the other Colombian populations. In contrast, female individuals from Salina Cero did not cluster with the other Colombian coast populations (Galerazamba, Tayrona and Manaure) or with the SFB population.     

Ultrastructure of enamel and dentine in extant dolphins (Cetacea: Delphinoidea and Inioidea)

Longitudinal and cross sections of teeth from 17 species of the Recent dolphins (Delphinoidea and Inioidea) were examined under scanning electron microscope to study the arrangement and ultrastructure of dental tissues with reference to phylogenetic and functional constraints. For most species, enamel had a simple bi-layered structure of radial enamel and an outer layer of prismless enamel. The outer prismless layer varied from 5 to 30 % of enamel thickness. The enamel of Burmeister’s porpoise (Phocoena spinipinnis) was entirely prismless. The prisms had an open sheath; tubules and tuft-like structures were common at the enamel-dentine junction. Cetacean dentine was characterized by irregularly distributed dentinal tubules in a relatively homogenous dentinal matrix. Radial enamel was observed in all Delphinoidea and in the franciscana (Pontoporia blainvillei), whereas the Amazon river dolphin (Inia geoffrensis) had prisms organized in Hunter–Schreger bands. HSB in enamel are regarded as a device for resisting propagation of cracks. These may occur due to increased functional demands, possibly related to the hardness of the species diet. Simplification in tooth shape and reduced biomechanical demands plausibly explain the primitive radial organization among delphinoids and Pontoporia. The HSB structure in the Amazon river dolphin, similar to those of extinct archaeocetes, seems to have secondary functional implications. However, the distribution of HSB in more-basal odontocetes is too poorly known to judge whether the HSB of Inia are a retained plesiomorphic feature or convergence.     

Phylogeography and Sex-Biased Dispersal across Riverine Manatee Populations (Trichechus inunguis and Trichechus manatus) in South America

Phylogeographic patterns and sex-biased dispersal were studied in riverine populations of West Indian (Trichechus manatus) and Amazonian manatees (T. inunguis) in South America, using 410bp D-loop (Control Region, Mitochondrial DNA) sequences and 15 nuclear microsatellite loci. This multi-locus approach was key to disentangle complex patterns of gene flow among populations. D-loop analyses revealed population structuring among all Colombian rivers for T. manatus, while microsatellite data suggested no structure. Two main populations of T. inunguis separating the Colombian and Peruvian Amazon were supported by analysis of the D-loop and microsatellite data. Overall, we provide molecular evidence for differences in dispersal patterns between sexes, demonstrating male-biased gene flow dispersal in riverine manatees. These results are in contrast with previously reported levels of population structure shown by microsatellite data in marine manatee populations, revealing low habitat restrictions to gene flow in riverine habitats, and more significant dispersal limitations for males in marine environments.     

Evidence for the reproductive isolation ofDermacentor marginatus andDermacentor reticulatus (Acari: Ixodidae) ticks based on cross-breeding, morphology and molecular studies

The species status ofDermacentor marginatus andDermacentor reticulatus was evaluated by scanning electron microscope (SEM) examination of adult ticks, cross-breeding experiments and molecular biological analysis of eggs derived from transspecific pairings. The SEM investigations including the morphometric quantification of phenotypic features, resulted in an unequivocal differentiation of adultD. marginatus andD. reticulatus ticks. The cross-breeding experiments demonstrated that irrespective of whether female ticks of both species were applied with con- or transspecific male ticks or without males to sheep, they engorged and laid eggs. The larvae, however, developed only in eggs which originated from conspecific matings. A nested polymerase chain reaction (PCR) of the second internal transcribed spacer (ITS2) using the DNA of eggs from transspecific pairings and sequencing of the PCR products revealed two different genotypes. The genotypes of eggs originating fromD. marginatus andD. reticulatus females of these pairings differed. However, the eggs deposited byD. marginatus always possessed the same two genotypes as did the eggs produced byD. reticulatus. These results argue for a strict reproductive isolation ofD. marginatus andD. reticulatus and, therefore, for a separate species status.     

Morphometric study of two species of wood miceApodemus sylvaticus andA. flavicollis (Rodentia: Muridae): traditional and geometric morphometric approach

Skull shape and size were investigated in 311 specimens ofApodemus sylvaticus (Linnaeus, 1758) andA. flavicollis (Melchior, 1834), using traditional and geometric morphometric approach. We searched for potential morphometric diagnostic criteria between these morphologically similar mice and we an alyzed the interpopulation morphometric variation. Specific status of all individuals was identified by scoring four diagnostic allozyme markers. Discriminant analysis based on 16 cranial measure ments as well as that based on land mark data for ventral side of the skull discriminated in dividuals with 99.7% accuracy. The classification functions useful for practical discrimination of unknown specimens are given. Our data showed neither clear intraspecific geographical pattern among studied populations nor evidence for character displacement in syntopic populations ofA. sylvaticus andA. flavicollis.