Species delimitation and integrative taxonomy of the Reithrodontomys mexicanus (Rodentia: Cricetidae) cryptic complex.

Species boundaries are difficult to establish in groups with very similar morphology. As an alternative, it has been suggested to integrate multiple sources of data to clarify taxonomic problems in taxa where cryptic speciation processes have been reported. This is the case of the harvest mouse Reithrodontomys mexicanus, which has a problematic taxonomy history as it is considered a complex species. Here, we evaluate the cryptic diversity of R. mexicanus using an integrative taxonomy approach in order to detect candidate lineages at the species level. The molecular analysis used one mitochondrial (cytb) and two nuclear (Fgb-I7 and IRBP) genes. Species hypotheses were suggested based on three molecular delimitation methods (mPTP, bGMYC, and STACEY) and cytb genetic distance values. Skull and environmental space differences between the delimited species were also tested to complement the discrimination of candidate species. Based on the consensus across the delimitation methods and genetic distance values, four species were proposed, which were mostly supported by morphometric and ecological data: R. mexicanus clade I, R. mexicanus clade IIA, R. mexicanus clade IIIA, and R. mexicanus clade IIIB. In addition, the evolutionary relationships between the species that comprise the R. mexicanus group were discussed from a phylogenetic approach. Our findings present important taxonomic implications for Reithrodontomys, as the number of known species for this genus increases. Furthermore, we highlight the importance of the use of multiple sources of data in systematic studies to establish robust delimitations between species considered taxonomically complex.

Assessment of DNA Integrity From Trap-Captured Boll Weevil (Coleoptera: Curculionidae) for Use in a New PCR-Based Diagnostic Tool.

The boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), is a major pest of commercial cotton (Gossypium hirsutum) in the southern United States and throughout Central and South America. Efforts are underway to develop a PCR-based diagnostic tool that can be used to rapidly and accurately differentiate boll weevils from other weevil species that are commonly captured in pheromone traps. However, the quantity and integrity of weevil DNA must be sufficient for a successful PCR assay. Currently, active eradication programs service traps weekly, but post-eradication programs service traps at 2- or 3-wk intervals. Consequently, captured weevils may be dead, dismembered, and exposed to environmental conditions for prolonged periods which may adversely affect the quantity and quality of weevil DNA. We documented DNA quantity and integrity in boll weevils and weevil body parts aged in traps over a 3-wk period under field conditions. The quantity of DNA extracted from whole weevils, heads, abdomens, and legs generally remained sufficient (> 1 ng/µl) for successful PCR amplification throughout the 21-d period. The integrity (fragment length) of extracted DNA declined over time but generally was sufficient (> 700 bp) for successful amplification. PCR amplification of three marker genes validated that the quality and integrity of DNA extracted from dead weevils and individual weevil body parts aged in traps up to 21 d remained at sufficient levels for the PCR-based assay. However, our data also suggested that rain events may accelerate degradation of weevil DNA.

The role of Pleistocene climate change in the genetic variability, distribution and demography of Proechimys cuvieri and P. guyannensis (Rodentia: Echimyidae) in northeastern Amazonia.

The spiny rats, genus Proechimys, have the highest species richness within the Echimyidae family, as well as species with high genetic variability. The genus distribution includes tropical South America and Central America south to Honduras. In this study, we evaluate the phylogeographic histories of Proechimys guyannensis and P. cuvieri using cytochrome b, in a densely sampled area in northeastern Amazon where both species are found in sympatry in different environments. For each species, Bayesian and Maximum Likelihood phylogenetic analysis were congruent and recovered similar clades in the studied area. Bayesian phylogenetic analysis using a relaxed molecular clock showed that these clusters of haplotypes diversified during Pleistocene for both species. Apparently, the large rivers of the region did not act as barriers, as some clades include specimens collected from opposite banks of Oiapoque, Araguari and Jari rivers. Bayesian skyline plot analysis showed recent demographic expansion in both species. The Pleistocene climatic changes in concert with the geologic changes in the Amazon fan probably acted as drivers in the diversification that we detected in these two spiny rats. Proechimys cuvieri and P. guyannensis show genetic structure in the eastern part of the Guiana region. Greater genetic distances observed in P. guyannensis, associated with highly structured groups, suggest that more detailed studies of systematics and ecology should be directed to this species.

A new species of big-eared climbing rat, genus Ototylomys (Cricetidae: Tylomyinae), from Chiapas, Mexico.

An allopatric population of big-eared climbing rats (Ototylomys) from the Northern Highlands of Chiapas, Mexico, is described as a new species. The new taxon is part of a unique montane rainforest community that includes several other endemic species in the limited geographic range between the Río Grijalva and the Central Depression of Chiapas. Several cranial, external, and molecular characters distinguish this new species of big-eared climbing rat from its more widely distributed congener, Ototylomys phyllotis. We performed principal component and discriminate function analyses of cranial measurements, and found that specimens of the new species consistently could be distinguished from other Ototylomys with strong statistical support. Compared with exemplars of Ototylomys from elsewhere in their range, the new species possesses a karyotype that differs by 3 additional biarmed chromosome pairs, is fixed or nearly fixed for distinct electromorphs at 12 allozyme loci, and the mean genetic distance exceeds 14%, based on comparisons of the mitochondrial cytochrome b gene between the new species of Ototylomys and representatives of O. phyllotis. The restricted distribution in montane karst rainforest suggests that the species and its habitat may be a matter of conservation concern.

Una población alopátrica de rata orejuda trepadora (Ototylomys) de las Tierras Altas del Norte de Chiapas, México se describe como una nueva especie. El nuevo taxón es parte de una comunidad única de bosque lluvioso montano que incluye varias especies endémicas en el área de distribución geográfica limitada entre el Río Grijalva y la Depresión Central de Chiapas. Varios caracteres craneales, externos, y moleculares distinguen la nueva rata orejuda trepadora de su congénere más ampliamente distribuido, Ototylomys phyllotis. Se realizaron análisis de componentes principales y de función discriminante de los caracteres craneales, y se encontró que los especímenes de La Pera fueron consistentemente distinguidos de otros Ototylomys con un fuerte soporte estadístico. En comparación con ejemplares de Ototylomys del rango, la nueva especie posee un cariotipo que difiere por 3 pares adicionales de cromosomas biarmados, está fijo o casi fijo por distintos electromorfos en 12 loci alozímicos. Adicionalmente, la media de la distancia genética comparada del gen mitochondrial citocromo b entre la nueva especie de Ototylomys y representantes de O. phyllotis, excede el 14%. La distribución restringida en el bosque lluvioso montano kárstico sugiere que la especie y su hábitat pueden ser de importancia para la conservación.

In the wake of invasion: tracing the historical biogeography of the South American cricetid radiation (Rodentia, Sigmodontinae).

The Great American Biotic Interchange (GABI) was greatly influenced by the completion of the Isthmus of Panama and impacted the composition of modern faunal assemblages in the Americas. However, the contribution of preceding events has been comparatively less explored, even though early immigrants in the fossil records are evidence for waif dispersals. The cricetid rodents of the subfamily Sigmodontinae are a classic example of a species-rich South American radiation resulting from an early episode of North American invasion. Here, we provide a temporal and spatial framework to address key aspects of the historical biogeography and diversification of this diverse mammal group by using mitochondrial and nuclear DNA datasets coupled with methods of divergence time estimation, ancestral area reconstruction and comparative phylogenetics. Relaxed-clock time estimates indicate that divergence of the Sigmodontinae began in the middle-late Miocene (ca. 12-9 Ma). Dispersal-vicariance analyses point to the arrival of a single lineage of northern invaders with a widespread ancestral distribution and imply that the initial differentiation between Central and South America gave rise to the most basal groups within the subfamily. These two major clades diversified in the late Miocene followed by the radiation of main tribes until the early Pliocene. Within the Oryzomyalia, tribes diverged initially in eastern South America whereas multiple dispersals into the Andes promoted further diversification of the majority of modern genera. A comparatively uniform background tempo of diversification explains the species richness of sigmodontines across most nodes, except for two akodontine genera with recent increases in diversification rates. The bridging of the Central American seaway and episodes of low sea levels likely facilitated the invasion of South America long before the onset of the post-Isthmian phase of the GABI.

Molecular phylogenetics and phylogeographic structure of Sumichrast's harvest mouse (Reithrodontomys sumichrasti: Cricetidae) based on mitochondrial and nuclear DNA sequences.

Sumichrast's harvest mouse (Reithrodontomys sumichrasti) is a montane rodent species widely distributed through the Mesoamerican highlands. We used sequence data from one mitochondrial (cytochrome b) and two nuclear (ß-fibrinogen and acid phosphatase type V) genes for a total of 1962 base pairs to estimate genealogical relationships and assess population genetic structure across the range of this taxon. Maximum likelihood and Bayesian approaches using cytochrome b resolved several major clades, revealing considerably more genetic diversity than observed in previous studies. The basal split in the tree topologies corresponded to the geographical separation among samples on either side of the Isthmus of Tehuantepec in México. We estimated an early Pleistocene or late Pliocene divergence between these two groups. We also recovered a well-supported clade south of the Nicaraguan Depression in Central America that we consider a separate biological species. The 12 networks generated using statistical parsimony (TCS) for cytochrome b sequence data were largely concordant with the phylogenetic analyses and we document the co-occurrence of two of these networks in central Veracurz. Phylogenies derived from ß-fibrinogen and acid phosphatase type V gene segments revealed less phylogenetic signal and did not separate samples of R. sumichrasti east and west of the Isthmus of Tehuantepec. The phylogeny estimated by combining the mitochondrial and nuclear sequence data was essentially identical to the cytochrome b gene tree.

Antibodies to Tacaribe serocomplex viruses (family Arenaviridae, genus Arenavirus) in cricetid rodents from New Mexico, Texas, and Mexico.

Blood samples from 4893 cricetid rodents were tested for antibody (immunoglobulin G) to Whitewater Arroyo virus and Amaparí virus to extend our knowledge of the natural host range and geographical distribution of Tacaribe serocomplex viruses in North America. Antibodies to arenaviruses were found in northern pygmy mice (Baiomys taylori), woodrats (Neotoma spp.), northern grasshopper mice (Onychomys leucogaster), oryzomys (Oryzomys spp.), deermice (Megadontomys nelsoni and Peromyscus spp.), harvest mice (Reithrodontomys spp.), and cotton rats (Sigmodon spp.) captured in New Mexico, Texas, or Mexico. Comparison of endpoint antibody titers to Whitewater Arroyo virus and Amaparí virus in individual blood samples indicated that the Tacaribe complex viruses enzootic in Texas and Mexico are antigenically diverse.

Monitoramento do ácido chiquímico no manejo de plantas daninhas com glifosato em pomar comercial de citros / Shikimic acid monitoring in weed control with glyphosate in a commercial citrus orchard

Com o objetivo de verificar o acúmulo de ácido chiquímico em plantas de laranja pêra (Citrus sinensis) num pomar comercial manejado com glifosato, um herbicida sistêmico de amplo espectro, foram coletadas amostras na Fazenda Jequitibá, tradicional no cultivo de citros, situada no Município de Santo Antônio de Posse, SP. O produtor aplicou de forma convencional Roundup® Original a 1.440 g.ha-1 de equivalente ácido (e.a.) do sal de isopropilamino de glifosato em 19/12/ 2006 na entrelinha de 15 plantas, deixando outras cinco como testemunha. A reaplicação de glifosato a 1.260 g.ha-1 de e.a. foi realizada em 2/4/2007. Em ambos os casos, imediatamente antes da aplicação e aos 3, 7, 10, 15, 20 e 35 dias após, foram coletadas 20 folhas de cada planta tanto da área tratada como da não tratada, analisando-se o teor de ácido chiquímico por cromatografia líquida de alta eficiência (CLAE) de forma isocrática após extração por micro-ondas. Os resultados mostraram não ocorrer acúmulo do ácido chiquímico nas plantas de laranja pêra, não havendo diferenças significativas nos teores deste composto entre o material proveniente da área tratada com glifosato e o daquela capinada manualmente.

In order to check the accumulation of shikimic acid in a traditional commercial grove of citrus "Pêra" cultivar (Citrus sinensis) managed for weed control with glyphosate, a systemic herbicide with wide spectrum, samples were collected at Fazenda Jequitibá, in Santo Antonio de Posse County, São Paulo State, Brazil. The producer applied the following treatments of Roundup Original® glyphosate at 1,440 g.ha-1 a.e. of the isopropylamine salt on 19 December 2006 between rows of 15 plants, leaving five others as control. The reapplication of glyphosate at 1,260 g ha-1 was done on 2 April 2007. In both cases, immediately before application and at 3, 7, 10, 15, 20 and 35 days thereafter, 20 leaves from each treated and untreated plants were collected for analysis of the content of shikimic acid by isocratic high performance liquid chromatography (HPLC) assisted with microwave. The results showed no significant differences in levels of shikimic acid between the material from the area treated with glyphosate and that weeded manually.

MONITORAMENTO DO ÁCIDO CHIQUÍMICO NO MANEJO DE PLANTAS DANINHAS COM GLIFOSATO EM POMAR COMERCIAL DE CITROS

ABSTRACT In order to check the accumulation of shikimic acid in a traditional commercial grove of citrus Pêra cultivar (Citrus sinensis) managed for weed control with glyphosate, a systemic herbicide with wide spectrum, samples were collected at Fazenda Jequitibá, in Santo Antonio de Posse County, São Paulo State, Brazil. The producer applied the following treatments of Roundup Original® glyphosate at 1,440 g.ha-1 a.e. of the isopropylamine salt on 19 December 2006 between rows of 15 plants, leaving five others as control. The reapplication of glyphosate at 1,260 g ha-1 was done on 2 April 2007. In both cases, immediately before application and at 3, 7, 10, 15, 20 and 35 days thereafter, 20 leaves from each treated and untreated plants were collected for analysis of the content of shikimic acid by isocratic high performance liquid chromatography (HPLC) assisted with microwave. The results showed no significant differences in levels of shikimic acid between the material from the area treated with glyphosate and that weeded manually.

RESUMO Com o objetivo de verificar o acúmulo de ácido chiquímico em plantas de laranja pêra (Citrus sinensis) num pomar comercial manejado com glifosato, um herbicida sistêmico de amplo espectro, foram coletadas amostras na Fazenda Jequitibá, tradicional no cultivo de citros, situada no Município de Santo Antônio de Posse, SP. O produtor aplicou de forma convencional Roundup® Original a 1.440 g.ha-1 de equivalente ácido (e.a.) do sal de isopropilamino de glifosato em 19/12/ 2006 na entrelinha de 15 plantas, deixando outras cinco como testemunha. A reaplicação de glifosato a 1.260 g.ha-1 de e.a. foi realizada em 2/4/2007. Em ambos os casos, imediatamente antes da aplicação e aos 3, 7, 10, 15, 20 e 35 dias após, foram coletadas 20 folhas de cada planta tanto da área tratada como da não tratada, analisando-se o teor de ácido chiquímico por cromatografia líquida de alta eficiência (CLAE) de forma isocrática após extração por micro-ondas. Os resultados mostraram não ocorrer acúmulo do ácido chiquímico nas plantas de laranja pêra, não havendo diferenças significativas nos teores deste composto entre o material proveniente da área tratada com glifosato e o daquela capinada manualmente.

Molecular systematics of Middle American harvest mice Reithrodontomys (Muridae), estimated from mitochondrial cytochrome b gene sequences.

We estimated phylogenetic relationships among 16 species of harvest mice using sequences from the mitochondrial cytochrome b (cyt b) gene. Gene phylogenies constructed using maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) optimality criteria were largely congruent and arranged taxa into two groups corresponding to the two recognized subgenera (Aporodon and Reithrodontomys). All analyses also recovered R. mexicanus and R. microdon as polyphyletic, although greater resolution was obtained using ML and BI approaches. Within R. mexicanus, three clades were identified with high nodal support (MP and ML bootstrap, Bremer decay and Bayesian posterior probabilities). One represented a subspecies of R. mexicanus from Costa Rica (R. m. cherrii) and a second was distributed in the Sierra Madre Oriental of Mexico. The third R. mexicanus clade consisted of mice from southern Mexico southward to South America. Polyphyly between the two moieties of R. microdon corresponded to the Isthmus of Tehuantepec in southern Mexico. Populations of R. microdon microdon to the east of the isthmus (Chiapas, Mexico) grouped with R. tenuirostris, whereas samples of R. m. albilabris to the west in Oaxaca, Mexico, formed a clade with R. bakeri. Within the subgenus Reithrodontomys, all analyses recovered R. montanus and R. raviventris as sister taxa, a finding consistent with earlier studies based on allozymes and cyt b data. There was also strong support (ML and BI criteria) for a clade consisting of ((R. megalotis, R. zacatecae) (R. sumichrasti)). In addition, cytb gene phylogenies (MP, ML, and BI) recovered R. fulvescens and R. hirsutus (ML and BI) as basal taxa within the subgenus Reithrodontomys. Constraint analyses demonstrated that tree topologies treating the two subgenera (Aporodon and Reithrodontomys) as monophyletic (ML criterion) was significantly better (p>0.036) and supported polyphyly of R. mexicanus (both ML and MP criteria - p>0.013) and R. microdon (MP criterion only for certain topologies; p>0.02). Although several species-level taxa were identified based on multiple, independent data sets, we recommended a conservative approach which will involve thorough analyses of museum specimens including material from type localities together with additional sampling and data from multiple, nuclear gene markers.

Hantavirus pulmonary syndrome in Central Bolivia: relationships between reservoir hosts, habitats, and viral genotypes.

In August 2002, two cases of hantavirus pulmonary syndrome (HPS) were confirmed in Mineros and Concepcion, within the Santa Cruz Department of Bolivia. Extensive alteration of the native ecosystem, from dense forest to pasture or sugarcane, had occurred in both regions. An ecologic assessment of reservoir species associated with the human disease identified a single hantavirus antibody-positive Oligoryzomys microtis from Mineros and three hantavirus antibody-positive Calomys callosus from Concepcion. In Mineros, the virus from the O. microtis was 90% similar to sequences published for Rio Mamore virus. Viral nucleotide sequences from two C. callosus were 87-88% similar to the sequence of Laguna Negra virus. The viral sequence from the C. callosus was 99% identical to viral sequences obtained from the HPS patient in this area, implicating C. callosus as the host and Laguna Negra virus as the agent responsible for the HPS case near Concepcion.