Diversity and biogeography of South American mud turtles elucidated by multilocus DNA sequencing (Testudines: Kinosternidae).

Kinosternon is the most speciose genus of extant turtles, with 22 currently recognized species, distributed across large parts of the Americas. Most species have small distributions, but K. leucostomum and K. scorpioides range from Mexico to South America. Previous studies have found discordance between mitochondrial and nuclear phylogenies in some kinosternid groups, with the current taxonomy following the nuclear-based results. Herein, based on extended molecular, geographic, and taxonomic sampling, we explore the phylogeographic structure and taxonomic limits for K. leucostomum and the K. scorpioides group and present a fossil-calibrated nuclear time tree for Kinosternon. Our results reveal contrasting differentiation patterns for the K. scorpioides group and K. leucostomum, despite overlapping distributions. Kinosternon leucostomum shows only shallow geographic divergence, whereas the K. scorpioides group is polyphyletic with up to 10 distinct taxa, some of them undescribed. We support the elevation of K. s. albogulare and K. s. cruentatum to species level. Given the deep divergence within the genus Kinosternon, we propose the recognition of three subgenera, Kinosternon, Cryptochelys and Thyrosternum, and the abandonment of the group-based classification, at least for the K. leucostomum and K. scorpioides groups. Our results show an initial split in Kinosternon that gave rise to two main radiations, one Nearctic and one mainly Neotropical. Most speciation events in Kinosternon occurred during the Quaternary and we hypothesize that they were mediated by both climatic and geological events. Additionally, our data imply that at least three South American colonizations occurred, two in the K. leucostomum group, and one in the K. scorpioides group. Additionally, we hypothesize that discordance between mitochondrial and nuclear phylogenetic signal is due to mitochondrial capture from an extinct kinosternine lineage.

The importance of cryptic diversity in the conservation of wide-ranging species: The red-footed tortoise Chelonoidis carbonarius in Colombia.

Wide-ranging species are seldom considered conservation priorities, yet they have the potential to harbour genetically deeply differentiated units across environments or ecological barriers, including some that warrant taxonomic recognition. Documenting such cryptic genetic diversity is especially important for wide-ranging species that are in decline, as they may comprise a set of even more endangered lineages or species with small distributions. However, studies of wide-ranging species, particularly when they cross political borders, are extremely challenging. One approach to overcoming these challenges is to conduct detailed local analyses in combination with less detailed, range-wide studies. We used this approach with the red-footed tortoise (Chelonoidis carbonarius), a threatened species likely to contain cryptic diversity given its vast range and the distinctive ecoregions that it inhabits. Previous single-gene molecular studies indicated the presence of at least five lineages, two of which occur in different ecoregions separated by the Andes within Colombia. We used a comprehensive genomic analysis to test the hypothesis of cryptic diversity within the single jurisdiction of Colombia. We used a combination of restriction-site-associated DNA sequencing and environmental niche modelling to provide three independent lines of evidence that support the presence of important cryptic diversity that may deserve taxonomic recognition: allopatric reproductive isolation, local adaptation and ecological divergence. We also provide a fine-scale genetic map with the distribution of conservation units in Colombia. As we complete ongoing range-wide analyses and make taxonomic adjustments, we recommend that the two lineages in Colombia be treated as separate units for conservation purposes.

Las especies con distribuciones amplias rara vez son consideradas prioridades de conservación, sin embargo, tienen el potencial de albergar unidades genéticamente diferenciadas que en algunos casos justifican reconocimiento taxonómico. Documentar dicha diversidad genética críptica es especialmente importante para las especies de rangos amplios que ya están en peligro de extinción, pues pueden comprender un conjunto de linajes o especies aún más amenazadas y con distribuciones más pequeñas. Sin embargo, los estudios de especies de rangos amplios, particularmente cuando cruzan fronteras políticas, son extremadamente desafiantes. Un enfoque para superar estos desafíos es realizar análisis locales detallados en combinación con estudios en todo el rango de distribución menos detallados. Nosotros usamos este enfoque con la tortuga de patas rojas (Chelonoidis carbonarius), una especie amenazada que probablemente contiene diversidad genética críptica dada su amplia distribución y las distintas ecorregiones en las que habita. Estudios moleculares previos de un solo gen indicaron la presencia de al menos cinco linajes, dos de los cuales ocurren en diferentes ecorregiones separadas por los Andes en Colombia. En este estudio utilizamos una combinación de secuenciación de ADN asociada a sitios de restricción (RADseq) y modelamiento de nicho ecológico para proporcionar tres líneas independientes de evidencia que respaldan la presencia de diversidad críptica importante que puede merecer reconocimiento taxonómico: aislamiento reproductivo alopátrico, adaptación local y divergencia ecológica. También proporcionamos un mapa genético a escala fina con la distribución de unidades de conservación en Colombia. Mientras completamos análisis genómicos en todo el rango de distribución y hacemos ajustes taxonómicos, recomendamos que los dos linajes en Colombia se traten como unidades independientes para fines de conservación.

Morphometric and molecular characterization of an unpigmented haemosporidian parasite in the Neotropical turnip-tailed gecko (Thecadactylus rapicauda).

Morphological traits from blood stages have been the gold standard for determining haemosporidian parasite species. However, the status of some taxa and the value of such traits in parasites from reptiles remain contentious. The scarce sampling of these species worsens the situation, and several taxa lack molecular data. A survey was performed in the Magdalena Department in Colombia, where 16 species of reptiles were captured. A peculiar haemosporidian parasite was found in the Turnip-tailed gecko Thecadactylus rapicauda. This haemosporidian does not show malarial pigment in blood stages under light microscopy; thus, it fits the Garnia genus's characters belonging to the Garniidae. However, the phylogenetic analyses using a partial sequence of cytochrome b and the mitochondrial DNA placed it within the Plasmodium clade. Our findings suggest that many putative Garnia species belong to the genus Plasmodium, like the one reported here. This study either shows that visible malarial pigment in blood stages is not a diagnostic trait of the genus Plasmodium or malarial pigment might be present in an undetectable form under a light microscope. In any case, the current taxonomy of haemosporidian parasites in reptiles requires revision. This study highlights the importance of using molecular and morphological traits to address taxonomic questions at the species and genus levels in haemosporidian parasites from reptiles.

The genetic and morphological diversity of Haemogregarina infecting turtles in Colombia: Are mitochondrial markers useful as barcodes for these parasites?

Adeleorinid parasites commonly infect turtles and tortoises in nature. Currently, our knowledge about such parasites is extremely poor. Their characterization is based on morphological and molecular approaches using the 18S rDNA molecular marker. However, there is a limitation with the 18S rDNA due to its slow rate of evolution. For that reason, the goals of this study were to 1) design primers for new molecular mitochondrial markers to improve the phylogenetic reconstructions of adeleorinid parasites and 2) to determine the morphological and genetic diversity of Haemogregarina infecting turtles and tortoises in Colombia. Turtles from 16 species representing six families were examined for the presence of haemoparasites. We analyzed 457 samples using PCR, and 203 of them were also analyzed by microscopy. Using a mitochondrial genome of Haemogregarina sequenced in this study, we designed primers to amplify fragments of the cytochrome oxidase I (coxI), cytochrome oxidase III (coxIII), and cytochrome b (cytb) mitochondrial markers in adeleorinid parasites. Lineages obtained from nuclear and mitochondrial molecular markers clustered according to the turtle lineages from which they were isolated. It is noteworthy that we found different evolutionary lineages within the same morphotype, which may indicate heteroplasmy and/or cryptic diversity in Haemogregarina. Due to this situation, we could not make a species delimitation, even when integrating the different lines of evidence we had in this study. However, the primers presented here are useful for diagnosis and, moreover, according to the available information, all three genes retain phylogenetic signals; thereby fragments amplified can be used in reconstructing evolutionary relationships. This effort contributes to the knowledge of the diversity of these parasites infecting continental turtles from Colombia.

Beginning the quest: phylogenetic hypothesis and identification of evolutionary lineages in bats of the genus Micronycteris (Chiroptera, Phyllostomidae).

Thirteen species of Neotropical bats of the genus Micronycteris are currently recognized and are allocated to four subgenera Leuconycteris, Micronycteris, Schizonycteris, and Xenonectes. Despite that, the presence of polyphyletic clades in molecular phylogenies suggests that its diversity is underestimated. Additionally, the incorrect identification of some genetic sequences, the incorrect assignation of available valid names, and restricted geographic sampling have biased the identification of independently evolutionary lineages within Micronycteris. In this study, several unknown genealogical lineages in the genus are identified and an updated phylogenetic hypothesis is proposed using mitochondrial and nuclear DNA fragments. The phylogenetic analyses congruently showed all individuals in four well-supported subgenera, but M. schmidtorum was revealed as the sister taxon of M. brosseti in the subgenus Leuconycteris. Twenty-seven different genealogical lineages were identified. These included eight confirmed species: M. brosseti, M. buriri, M. giovanniae, M. matses, M. schmidtorum, M. simmonsae, M. tresamici, and M. yatesi. Nineteen either allopatric or parapatric candidate species were also confirmed, two within the M. hirsuta complex, nine within the M. megalotis complex, seven within the M. minuta complex, and one corresponding to "M. sp.". These results revealed an extensive undescribed diversity within each subgenus of Micronycteris. Nevertheless, the evolutionary processes associated with the specific radiations are poorly understood. This is just the beginning of the assessment of the taxonomy and systematics of Micronycteris, which requires additional integrative taxonomical approaches for its advance.

Molecular and morphological description of the first Hepatozoon (Apicomplexa: Hepatozoidae) species infecting a neotropical turtle, with an approach to its phylogenetic relationships.

Haemogregarines (Adeleorina) have a high prevalence in turtles. Nevertheless, there is only one Hepatozoon species described that infects Testudines so far; it is Hepatozoon fitzsimonsi which infects the African tortoise Kinixys belliana. Colombia harbours a great diversity of chelonians; however, most of them are threatened. It is important to identify and characterize chelonian haemoparasite infections to improve the clinical assessments, treatments and the conservation and reintroduction programs of these animals. To evaluate such infections for the Colombian wood turtle Rhinoclemmys melanosterna, we analysed blood from 70 individuals. By using the morphological characteristics of blood stages as well as molecular information (18S rRNA sequences), here we report a new Hepatozoon species that represents the first report of a hepatozoid species infecting a semi-aquatic continental turtle in the world. Although the isolated lineage clusters within the phylogenetic clades that have morphological species of parasites already determined, their low nodal support makes their position within each group inconclusive. It is important to identify new molecular markers to improve parasite species identification. In-depth research on blood parasites infecting turtles is essential for increasing knowledge that could assess this potential unknown threat, to inform the conservation of turtles and for increasing the state of knowledge on parasites.

A phylogenetic study of Haemocystidium parasites and other Haemosporida using complete mitochondrial genome sequences.

Haemosporida are diverse vector-borne parasites associated with terrestrial vertebrates. Driven by the interest in species causing malaria (genus Plasmodium), the diversity of avian and mammalian haemosporidian species has been extensively studied, relying mostly on mitochondrial genes, particularly cytochrome b. However, parasites from reptiles have been neglected in biodiversity surveys. Reptilian haemosporidian parasites include Haemocystidium, a genus that shares morphological features with Plasmodium and Haemoproteus. Here, the first complete Haemocystidium mitochondrial DNA (mtDNA) genomes are studied. In particular, three mtDNA genomes from Haemocystidium spp. sampled in Africa, Oceania, and South America, are described. The Haemocystidium mtDNA genomes showed a high A + T content and a gene organization, including an extreme fragmentation of the rRNAs, found in other Haemosporida. These Haemocystidium mtDNA genomes were incorporated in phylogenetic and molecular clock analyses together with a representative sample of haemosporidian parasites from birds, mammals, and reptiles. The recovered phylogeny supported Haemocystidium as a monophyletic group apart from Plasmodium and other Haemosporida. Both the phylogenetic and molecular clock analyses yielded results consistent with a scenario in which haemosporidian parasites radiated with modern birds. Haemocystidium, like mammalian parasite clades, seems to originate from host switches by avian Haemosporida that allowed for the colonization of new vertebrate hosts. This hypothesis can be tested by investigating additional parasite species from all vertebrate hosts, particularly from reptiles. The mtDNA genomes reported here provide baseline data that can be used to scale up studies in haemosporidian parasites of reptiles using barcode approaches.

A new Terrarana frog of genus Pristimantis from an unexplored cloud forest from the eastern Andes, Colombia.

A new species of Pristimantis (Craugastoridae, subgenus Pristimantis) is described from a relict and unexplored cloud forest in the western slope from Cordillera Oriental of the Colombian Andes. The specific name was chosen by consensus expert scientists and local people. Pristimantis chamezensis sp. nov. is easily distinguished from congeneric species by having a gray iris with black reticulations in life, subconical tubercles on the upper eyelid, the chin edged with irregular, dark-brown blotches, and conical heel tubercles. The phylogenetic analyses suggest that the origin and radiation of its clade may have occurred in the highlands. With the description of P. chamezensis sp. nov., we identify 14 species distributed throughout the eastern slope of the Andes that are associated with the Orinoco Basin.

Accediendo al pasado: uso de especímenes de colección como fuentes de información genética para el género Bombus (Hymenoptera: Apidae)

Introducción: Recientemente ha tomado relevancia el uso de especímenes de museo como fuente de información genética para desarrollar estudios que resuelven preguntas taxonómicas, ecológicas, demográficas y evolutivas a diversas escalas temporales y geográficas. Sin embargo, material genético obtenido a partir de ejemplares depositados en colecciones biológicas es poco usado, debido al deterioro natural del ADN preservado en dichos ejemplares, de manera que la obtención de material genético de calidad es demandante en términos de tiempo y dinero. Objetivo: Usando material de museo, identificar una secuencia mini-barcode que pueda ser empleada en la determinación taxonómica, y que a su vez suministre información que permita la estimación de relaciones filogenéticas de especies del género Bombus. Métodos: Se estandarizó el protocolo de extracción de ADN a partir de la extremidad mesotoracica derecha y/o una muestra de músculo torácico de 96 especímenes depositados en la colección LABUN entre 7 y 38 años atrás. Las diferentes combinaciones de oligonucleótidos evaluadas permitieron amplificar fragmentos de 152 a 407 pares de bases (pb) del gen mitocondrial Cytochrome Oxidase I (COI). Usando como plantilla un grupo de 31 secuencias amplificadas a partir de especímenes recolectados recientemente, los fragmentos obtenidos de los especímenes del museo fueron ensamblados y analizados en un marco filogenético. Además, se realizó un análisis de red de haplotipos para evaluar en detalle las relaciones entre los haplotipos mitocondriales resultantes. Resultados: Se determinó un mayor éxito de extracción de ADN a partir de muestras de extremidad depositadas a partir del año 1982.Entretanto, la amplificación exitosa de fragmentos de más de 300 pares de bases (pb) se logró principalmente en muestras depositadas en fechas posteriores a 1999, lo que indica una mayor integridad del material genético recuperado de individuos de 19 años de recolección en adelante. Aunque todos los fragmentos evaluados pueden ser empleados como mini-barcode, solo con uno se obtiene una topología similar a la observada con el fragmento completo. Se detectó una gran variacion genética, particularmente al interior de las especies Bombus atratus y B. funebris, en las que se reveló una clara estructura filogeográfica. Conclusiones: Se obtuvieron nuevas secuencias de códigos de barras mediante extracción de ADN y protocolo de amplificación de muestras de museos. Además, se generó nueva información sobre la variabilidad genética intraespecífica, detectando la presencia de haplotipos mitocondriales únicos que podrían constituir Unidades Significativas Evolutivas sujetas a conservación. Dicha información es de vital importancia para formular estrategias de conservación para estos polinizadores en Colombia.

Introduction: The use of museum specimens as a source of genetic information to develop studies that resolve taxonomic, ecological, demographic, and evolutionary questions at various temporal and geographic scales, has recently become relevant. However, genetic material obtained from specimens deposited in biological collections is not used frequently due to the natural deterioration of the DNA preserved in these specimens. Getting quality genetic material is demanding in terms of time and money. Objective: By using museum material,to identify a mini-barcode sequence that can be used in the taxonomic determination and provides information that allows the estimation of phylogenetic relationships of species of the genus Bombus. Methods: The DNA extraction protocol for museum samples was standardized using the mesothoracic right leg and / or a sample of thoracic muscle of 96 specimens deposited in the LABUN collection between 7 and 38 years ago. Different combinations of oligonucleotides allowed to amplify fragments from 152 to 407 base pairs (bp) of the mitochondrial gene Cytochrome Oxidase I (COI). Using as a template a group of 31 sequences amplified from recently collected specimens, the fragments obtained from the museum specimens were assembled and analyzed in a phylogenetic framework. Additionally, a haplotype network analysis was performed in order to evaluate in detail the relationships between the resulting mitochondrial haplotypes. Results: The greatest success of DNA extraction was achieved from limb samples deposited since the year 1982 on. Meanwhile, successful amplification of fragments longer than 300 base pairs (bp) was achieved mostly in samples deposited on dates after 1999, which indicates greater integrity of the genetic material recovered from individuals of 19 years of collection and onwards. Although all the fragments evaluated can be used as mini-barcode, only with one primer pair, it was possible to obtain a topology similar to that observed with the complete fragment. A large genetic variation was detected, particularly within the Bombus atratus and B. funebris species, in which a clear phylogeographic structure was revealed. Conclusions: New barcode sequences were obtained through DNA extraction and amplification protocol from museum samples. Furthermore, new information on intraspecific genetic variability was generated, detecting the presence of unique mitochondrial haplotypes that could constitute management units subject of conservation. Such information is of vital importance to formulate conservation strategies for these pollinators in Colombia.

Genomic analyses reveal two species of the matamata (Testudines: Chelidae: Chelus spp.) and clarify their phylogeography.

The matamata is one of the most charismatic turtles on earth, widely distributed in northern South America. Debates have occurred over whether or not there should be two subspecies or species recognized due to its geographic variation in morphology. Even though the matamata is universally known, its natural history, conservation status and biogeography are largely unexplored. In this study we examined the phylogeographic differentiation of the matamata based on three mitochondrial DNA fragments (2168 bp of the control region, cytochrome oxidase subunit I, and the cytochrome b gene), one nuclear genomic DNA fragment (1068 bp of the R35 intron) and 1661 Single Nucleotide Polymorphisms (SNPs). Our molecular and morphological analyses revealed the existence of two distinct, genetically deeply divergent evolutionary lineages of matamatas that separated in the late Miocene (approximately 12.7 million years ago), corresponding well to the time when the Orinoco Basin was established. As a result of our analyses, we describe the genetically and morphologically highly distinct matamata from the Orinoco and Río Negro Basins and the Essequibo drainage as a species new to science (Chelus orinocensis sp. nov.). Chelus fimbriata sensu stricto is distributed in the Amazon Basin and the Mahury drainage. Additionally, the analyses revealed that each species displays phylogeographic differentiation. For C. orinocensis, there is moderate mitochondrial differentiation between the Orinoco and the Río Negro. For C. fimbriata, there is more pronounced differentiation matching different river systems. One mitochondrial clade was identified from the Amazon, Ucayali, and Mahury Rivers, and another one from the Madeira and Jaci Paraná Rivers. The C. orinocensis in the Essequibo and Branco Rivers have haplotypes that constitute a third clade clustering with C. fimbriata. Phylogenetic analyses of the R35 intron and SNP data link the matamatas from the Essequibo and Branco with the new species, suggesting past gene flow and old mitochondrial introgression. Chelus orinocensis is collected for the pet trade in Colombia and Venezuela. However, neither the extent of the harvest nor its impact are known. Hence, it is crucial to gather more information and to assess its exploitation throughout its distribution range to obtain a better understanding of its conservation status and to design appropriate conservation and management procedures. RESUMEN: La matamata es una de las tortugas más carismáticas del mundo, ampliamente distribuida en el norte de Sudamérica. Debido a su variación morfológica geográfica, se debate sobre el reconocimiento de dos subespecies o especies. A pesar de que la matamata es universalmente conocida, su historia natural, estado de conservación y biogeografía han sido muy poco estudiados. En este estudio examinamos la diferenciación filogeográfica de las matamatas en base ​​a tres fragmentos de ADN mitocondrial (2168 pb de la región de control, la subunidad I del citocromo oxidasa y el gen del citocromo b), un fragmento de ADN genómico nuclear (1068 pb del intrón R35) y 1661 polimorfismos de nucleótido único (SNPs). Nuestros análisis moleculares y morfológicos revelaron la existencia de dos linajes evolutivos distintos de matamatas, genéticamente divergentes que se separaron en el Mioceno tardio (hace aproximadamente 12.7 millones de años), correspondiendo al tiempo en que se estableció la cuenca del Orinoco. Como resultado de nuestros análisis, describimos las genéticamente y morfológicamente distintas matamatas de las cuencas del Orinoco, Río Negro y Essequibo como una especie nueva para la ciencia (Chelus orinocensis sp. nov.). Chelus fimbriata sensu stricto se distribuye en la cuenca del Amazonas y en el drenaje del Mahury. Adicionalmente, los análisis revelaron que cada especie muestra diferenciación filogeográfica. Para C. orinocensis, hay una moderada diferenciación mitocondrial entre el Orinoco y el Río Negro. Para C. fimbriata, hay una diferenciación más pronunciada, concordando con los diferentes sistemas fluviales. Se identificó un clado de los ríos Amazonas, Ucayali y Mahury y otro de los ríos Madeira y Jaci Paraná. Las C. orinocensis de los ríos Essequibo y Branco tienen haplotipos que constituyen un tercer clado que se agrupa con C. fimbriata. Los análisis filogenéticos del intrón R35 y los datos de SNP asocian las matamatas de Essequibo y Branco con la nueva especie, sugiriendo flujo de genes pasado ​​e introgresión mitocondrial antigua. Chelus orinocensis se colecta para el comercio de mascotas en Colombia y Venezuela. Sin embargo, ni se conoce el alcance de las colectas ni su impacto. Por lo tanto, es crucial recopilar más información y evaluar su explotación en todo su rango de distribución, comprender mejor su estado de conservación y para diseñar acciones apropiadas de conservación y manejo.

Description of a new diminutive Hyloxalus (Anura: Dendrobatidae: Hyloxalinae) from the Magdalena Valley of Colombia.

Using different sources of evidence (i.e., integrative taxonomy), we describe a new species of Hyloxalus, Hyloxalus arliensis sp. nov. The new species occurs in the middle Magdalena River valley on slopes between the Cordillera Central and Cordillera Oriental of the Andes of Colombia. Previously, the new species was referred to as Hyloxalus "Ibagué" but was not formally described. Phylogenetic re-analyses of 2440 base pairs (bp) of mitochondrial DNA (mtDNA), revealed the species to be a sister taxon of H. lehmanni, within a monophyletic clade also comprising H. delatorreae lineage 1, H. pulchellus and H. vertebralis. The adult males of H. arliensis sp. nov. have a pale arm gland, which differentiates it from all other species of the genus, except for H. saltarius. The new species also differs from other species of Hyloxalus by its smaller size, disc on Finger III not expanded, toes unwebbed, dorsolateral stripe absent, oblique lateral stripe present, white spots on ventrolateral flanks in life and cloacal tubercles absent. The advertisement call consists of long trains of a single note repeated at a rate of 89-132 notes/min with a dominant frequency ranging from 4808-6387 Hz. The new species is a tiny frog that inhabits the sheltered areas around streams in sub-Andean forests. Aspects of its natural history are described.

Haemocystidium spp., a species complex infecting ancient aquatic turtles of the family Podocnemididae: First report of these parasites in Podocnemis vogli from the Orinoquia.

The genus Haemocystidium was described in 1904 by Castellani and Willey. However, several studies considered it a synonym of the genera Plasmodium or Haemoproteus. Recently, molecular evidence has shown the existence of a monophyletic group that corresponds to the genus Haemocystidium. Here, we further explore the clade Haemocystidium spp. by studying parasites from Testudines. A total of 193 individuals belonging to six families of Testudines were analyzed. The samples were collected in five localities in Colombia: Casanare, Vichada, Arauca, Antioquia, and Córdoba. From each individual, a blood sample was taken for molecular analysis, and peripheral blood smears were made, which were fixed and subsequently stained with Giemsa. The prevalence of Haemocystidium spp. was 1.55% (n = 3/193); all infected individuals belonged to Podocnemis vogli (Savanna Side-necked turtle) from the department of Vichada. This is the first report of Haemocystidium spp. in Colombia and in this turtle species. The phylogenetic analysis of a mitochondrial cytb fragment revealed Haemocystidium spp. as a monophyletic group and as a sister taxon of Haemoproteus catharti and the genus Plasmodium. Haemocystidium spp. are difficult to identify by morphology only. As a result, it is possible that some of the taxa, such as Haemocystidium (Simondia) pacayae, represent a species complex. The parasite found in our study is morphologically indistinguishable from Haemocystidium (Simondia) pacayae reported in Peru. However, the new lineage found in P. vogli shows a genetic distance of 0.02 with Hae. pacayae and 0.04 with Hae. peltocephali. It is proposed that this divergent lineage might be a new species. Nevertheless, additional molecular markers and ecological features could support this hypothesis in the future.

Landscape genomic signatures indicate reduced gene flow and forest-associated adaptive divergence in an endangered neotropical turtle.

Human-induced transformations of ecosystems usually result in fragmented populations subject to increased extinction risk. Fragmentation is also often associated with novel environmental heterogeneity, which in combination with restricted gene flow may increase the opportunity for local adaptation. To manage at-risk populations in these landscapes, it is important to understand how gene flow is changing, and how populations respond to habitat loss. We conducted a landscape genomics analysis using Restriction-site Associated DNA sequencing to investigate the evolutionary response of the critically endangered Dahl's Toad-headed turtle (Mesoclemmys dahli) to severe habitat modification. The species has lost almost all of its natural habitat in the southwestern part of its range and about 70% in the northeast. Based on least cost path analysis across different resistance surfaces for 3,211 SNPs, we found that the landscape matrix is restricting gene flow, causing the fragmentation of the species into at least six populations. Genome scans and allele-environment association analyses indicate that the population fragments in the deforested grasslands of the southwest are adaptively different from those in the more forested northeast. Populations in areas with no forest had low levels of adaptive genetic diversity and the fixation of ancestrally-polymorphic SNPs, consistent with directional selection in this novel environment. Our results suggest that this forest-stream specialist is adapting to pond-grassland conditions, but it is also suffering from negative consequences of habitat loss, including genetic erosion, isolation, small effective population sizes, and inbreeding. We recommend gene flow restoration via genetic rescue to counteract these threats, and provide guidance for this strategy.

Red-headed Amazon River Turtles in Venezuela and Colombia: population separation and connection along the famous route of Alexander von Humboldt.

Podocnemis erythrocephala, the Red-headed Amazon River Turtle, is distributed in the Amazon and Orinoco basins where it predominantly inhabits blackwater and has never been found in whitewater. The only permanent river connection between the habitats in the different river basins features considerable whitewater proportions and, therefore, is hypothesised to be a dispersal barrier for this turtle species. By using variable neutral nuclear and mitochondrial markers (microsatellite loci and control region sequences), the present study assessed the genetic structure, genetic diversity, gene flow and historical biogeography of P. erythrocephala populations in Venezuela and Colombia in the Brazo Casiquiare (Amazon basin) and in the Ríos Atabapo and Inírida (Orinoco basin). The results clearly indicate a pronounced genetic differentiation between the Amazon basin populations and the Orinoco basin populations suggesting that a vicariant event separated the ancestors of the Orinoco basin populations. Several identified private mitochondrial DNA haplotypes and microsatellite alleles as well as a high genetic diversity observed in the Orinoco basin populations suggest that the Orinoco basin represents a historical blackwater refugium of P. erythrocephala. Analyses of past demographic processes revealed old bottlenecks and recent expansion. Historical and contemporary non-directional gene flow between the populations in the different river basins were identified and are supposed to have taken place via ephemeral inter-basin blackwater connections that might have formed at various locations, one of them being located on the famous travelling route of Alexander von Humboldt. The P. erythrocephala populations in the Ríos Atabapo and Inírida should together be considered as an evolutionarily significant unit, and each of them should be classified as an independent management unit for conservation.

ABUNDANCIA, ESTRUCTURA POBLACIONAL Y CONSERVACIÓN DE Podocnemis lewyana (PODOCNEMIDIDAE) EN EL RÍO PRADO, COLOMBIA / Abundance, Population Structure and Conservation of Podocnemis lewyana (Podocnemididae) at the Prado River, Colombia

En el río Prado, localizado en el sur oriente de Colombia existe una población de Podocnemis lewyana, una tortuga endémica y en peligro de extinción. Para esta población se determinó la abundancia relativa, la estructura poblacional y las amenazas a la conservación a partir de información de campo obtenida en los años 2007 y 2009. La abundancia relativa se estimó por medio de captura de tortugas por unidad de tiempo. Adicionalmente, solo en el 2009, se realizó un conteo de individuos por kilómetro recorrido para contrastarlo con poblaciones del norte del país. La estructura poblacional se determinó por la frecuencia de individuos en varias clases de tamaño y con la proporción de sexos de los animales capturados. Se examinó el dimorfismo sexual en animales adultos con base en su morfometría. En total se capturaron 110 tortugas el año 2007 y 72 tortugas en el 2009. Se tuvo un promedio de 54,46 avistamientos/km (2009), siendo hasta el momento la población más abundante del país. La estructura de la población se caracterizó por una mayor frecuencia de individuos de 21-30 cm de largo recto máximo del caparazón (LRC) y ningún individuo de menos de 10 cm LRC ni mayor a 40 cm LRC. La proporción de sexos fue de 2,52:1 en el 2007 y de 2,75:1 en el 2009, siendo mayor para hembras. Las principales amenazas identificadas para la población del río Prado fueron (i) las alteraciones de su hábitat, (ii) cambios en el caudal natural del río, (iii) uso de artes de pesca inadecuados, y (iv) la probable interrupción de sus migraciones. Se propone el río Prado como lugar prioritario para la investigación y conservación de Podocnemis lewyana en la cuenca alta del río Magdalena.

Along the Prado River, at southeast of Colombia, there is a population of Podocnemis lewyana, an endemic and endangered river turtle. Relative abundance, population structure and conservation threats were determined using field data obtained in 2007 and 2009. Relative abundance was estimated by turtle catch per unit of time, which was used to compare between the two sampling years. Additionally, turtles per kilometer were counted in 2009 alone, to compare with other populations distributed in the north of the country. The population structure was determined by the frequency of individuals of several size classes and sex ratio of captured animals. Sexual dimorphism was examined in adult animals by morphometry. One hundred and ten turtles were captured in 2007 and 72 in 2009. The relative abundance of individuals observed was an averaged of 54.46 sightings/ km representing the most abundant population of the country so far. The population's structure was characterized by a higher frequency of individuals of 21-30 cm maximum straight carapace length SCL and absence of individuals of less than 10 cm SCL or greater than 40 cm SCL. Sex ratio was 2.52:1 for 2007 and 2.75:1 for 2009, being higher for females. The main identified threats to the population of P. lewyana at Prado River were (i) alterations of habitat, (ii) changes in the natural flow of the river, (iii) the use of inappropriate fishing arts and (iv) probable interruption of migrations. Prado River is hereby proposed as priority area for further research and conservation of Podocnemis lewyana in the upper Magdalena river basin.

A revision of African helmeted terrapins (Testudines: Pelomedusidae: Pelomedusa), with descriptions of six new species.

Using nearly range-wide sampling, we analyze up to 1848 bp of mitochondrial DNA of 183             helmeted terrapins and identify a minimum of 12 deeply divergent species-level clades. Uncorrected p distances of these clades equal or clearly exceed those between the currently recognized species of Pelusios, the genus most closely related to Pelomedusa. We correlate genetic discontinuities of Pelomedusa with data on morphology and endoparasites and describe six new Pelomedusa species. Moreover, we restrict the name Pelomedusa subrufa (Bonnaterre, 1789) to one genetic lineage and resurrect three further species from its synonymy, namely P. galeata (Schoepff, 1792), P. gehafie (Rüppell, 1835), and P. olivacea (Schweigger, 1812). In addition to these ten Pelomedusa species, we identify two further clades from Cameroon and Sudan with similar levels of genetic divergence that remain unnamed candidate species. We also note that some problematical terrapins from South Africa and Somalia may represent two additional candidate species. Some of the Pelomedusa species are morphologically distinctive, whilst others can only be identified by molecular markers and are therefore morphologically cryptic taxa.

Complex phylogeography in Rhinoclemmys melanosterna: conflicting mitochondrial and nuclear evidence suggests past hybridization (Testudines: Geoemydidae).

We examined differentiation within the Colombian wood turtle Rhinoclemmys melanosterna, and among R. melanosterna and the closely allied species R. diademata, R.funerea and R. punctularia, based on 1060 base pairs of the mitochondrial cyt b gene. We also assessed the phylogenetic relationships among these species using 2050 bp of mtDNA (partial cyt b, 12S and 16S genes) and 3620 bp of nuclear DNA (partial Rag 1, Rag 2, C-mos, R35 and ODC genes). There is considerable phylogeographic structuring within R. melanosterna, with seven distinct clades distributed across the species' range. These clades correlate to some extent with previously described differences in the dorsal pattern of head coloration. Individual and combined analyses of mitochondrial and nuclear DNA indicated contradictory relationships among R. melanosterna, R. diademata, R. funerea and R. punctularia. Mitochondrial DNA sequences revealed R. melanosterna to be non-monophyletic with respect to R. diademata, R. funerea and R. punctularia. In contrast, R. melanosterna constituted a well-supported monophyletic clade using nuclear DNA. This conflict between mitochondrial and nuclear data suggests past gene flow among the allopatrically and parapatrically distributed species R. melanosterna, R. diademata, R. funerea and R. punctularia. Compared to the other Rhinoclemmys species, the taxa under study are weakly differentiated. To assess their taxonomic status, further research is warranted using additional nuclear markers and additional samples of R. diademata, R. funerea and R. punctularia. For the time being, a continued classification of R. melanosterna, R. diademata, R. funerea and R. punctularia as distinct species is justified owing to their allopatric and parapatric distributions, and to conserve the established usage of names that is based on morphological and karyotypic differentiation.

Deep genealogical lineages in the widely distributed African helmeted terrapin: evidence from mitochondrial and nuclear DNA (Testudines: Pelomedusidae: Pelomedusa subrufa).

We investigated the phylogeographic differentiation of the widely distributed African helmeted terrapin Pelomedusa subrufa based on 1503 base pairs of mitochondrial DNA (partial cyt b and ND4 genes with adjacent tRNAs) and 1937 bp of nuclear DNA (partial Rag1, Rag2, R35 genes). Congruent among different analyses, nine strongly divergent mitochondrial clades were found, representing three major geographical groupings: (1) A northern group which includes clades I from Cameroon, II from Ghana and Ivory Coast, III from Benin, Burkina Faso and Niger, IV from the Central African Republic, and V from Kenya, (2) a northeastern group consisting of clades VI from Somalia, and VII from Saudi Arabia and Yemen, and (3) a southern group comprising clade VIII from Botswana, the Democratic Republic of Congo, Madagascar and Malawi, and clade IX from South Africa. Malagasy and continental African populations were not clearly differentiated, indicating very recent arrival or introduction of Pelomedusa in Madagascar. The southern group was in some phylogenetic analyses sister to Pelusios, rendering Pelomedusa paraphyletic with respect to that genus. However, using partitioned Bayesian analyses and sequence data of the three nuclear genes, Pelomedusa was monophyletic, suggesting that its mitochondrial paraphyly is due to either ancient introgressive hybridization or phylogenetic noise. Otherwise, nuclear sequence data recovered a lower level of divergence, but corroborated the general differentiation pattern of Pelomedusa as revealed by mtDNA. This, and the depth of the divergences between clades, indicates ancient differentiation. The divergences observed fall within, and in part exceed considerably, the differentiation typically occurring among chelonian species. To test whether Pelomedusa is best considered a single species composed of deep genealogical lineages, or a complex of up to nine distinct species, we suggest a future taxonomic revision that should (1) extend the geographical sampling of molecular data, specifically focusing on contact zones and the possible sympatric occurrence of lineages without admixture, and (2) evaluate the morphology of the various genealogical lineages using the type specimens or topotypical material of the numerous junior synonyms of P. subrufa.