João Stenghel Morgante

A Genetic–Morphological Characterization of Two Cryptic Species of the Anastrepha fraterculus Complex (Diptera: Tephritidae)

Abstract Biological characteristics of two entities of the Anastrepha fraterculus complex (Diptera: Tephritidae), referred to in previous publications as A. sp.1 aff. fraterculus and A. sp.2 aff. fraterculus, were further studied by a combined analysis of isozymes, karyotypes, morphometry, and crossings, in samples from 10 Brazilian populations. A survey of 16 enzymatic systems comprising 19 loci showed significant differences in the allele frequencies at four loci, FUM, ME, HEX, and LDH, allowing the recognition of two population clusters. These clusters also differ in their karyotypes, especially in the length of the sex chromosomes and in the size and location of heterochromatic regions. A morphometric analysis of wings and the aculeus in samples from five populations clearly showed a distinction between the two clusters but not between populations within each cluster. A phenetic analysis based on the Mahalanobis distance matrix also arranged the populations into the same two clusters. Crosses between populations of the same cluster showed no significant differences in egg hatching and in the adult sex ratio. However, a significant decrease in egg hatching was observed in the intercluster crosses. In crosses of cluster 1 males to cluster 2 females, a significant deviation in the sex ratio was observed according to Haldane’s rule, but not in the reciprocal crosses, indicating that a certain degree of reproductive isolation occurs between populations of cluster 1 and cluster 2. The results indicate that the two population clusters actually represent two cryptic species of the nominal species Anastrepha fraterculus, corroborating previous studies on this complex. We propose that the denominations A. sp.1 aff. fraterculus and A. sp.2 aff. fraterculus should be maintained until an appropriate taxonomic revision is made.

Phylogeography of the common vampire bat (Desmodus rotundus): Marked population structure, Neotropical Pleistocene vicariance and incongruence between nuclear and mtDNA markers

BackgroundThe common vampire bat Desmodus rotundus is an excellent model organism for studying ecological vicariance in the Neotropics due to its broad geographic range and its preference for forested areas as roosting sites. With the objective of testing for Pleistocene ecological vicariance, we sequenced a mitocondrial DNA (mtDNA) marker and two nuclear markers (RAG2 and DRB) to try to understand how Pleistocene glaciations affected the distribution of intraspecific lineages in this bat.ResultsFive reciprocally monophyletic clades were evident in the mitochondrial gene tree, and in most cases with high bootstrap support: Central America (CA), Amazon and Cerrado (AMC), Pantanal (PAN), Northern Atlantic Forest (NAF) and Southern Atlantic Forest (SAF). The Atlantic forest clades formed a monophyletic clade with high bootstrap support, creating an east/west division for this species in South America. On the one hand, all coalescent and non-coalescent estimates point to a Pleistocene time of divergence between the clades. On the other hand, the nuclear markers showed extensive sharing of haplotypes between distant localities, a result compatible with male-biased gene flow. In order to test if the disparity between the mitochondrial and nuclear markers was due to the difference in mutation rate and effective size, we performed a coalescent simulation to examine the feasibility that, given the time of separation between the observed lineages, even with a gene flow rate close to zero, there would not be reciprocal monophyly for a neutral nuclear marker. We used the observed values of theta and an estimated mutation rate for the nuclear marker gene to perform 1000 iterations of the simulation. The results of this simulation were inconclusive: the number of iterations with and without reciprocal monophyly of one or more clades are similar.ConclusionsWe therefore conclude that the pattern exhibited by the common vampire bat, with marked geographical structure for a mitochondrial marker and no phylogeographic structure for nuclear markers is compatible with a historical scenario of complete isolation of refuge-like populations during the Pleistocene. The results on demographic history on this species is compatible with the Carnaval-Moritz model of Pleistocene vicariance, with demographic expansions in the southern Atlantic forest.

Comparative Phylogeography of the Atlantic Forest Endemic Sloth (Bradypus torquatus) and the Widespread Three-toed Sloth (Bradypus variegatus) (Bradypodidae, Xenarthra)

The comparative phylogeographic study of the maned sloth (Bradypus torquatus) and the three-toed sloth (Bradypus variegatus) was performed using a segment of mitochondrial DNA (mtDNA) control region. We examined 19 B. torquatus from two regions and 47 B. variegatus from three distant regions of Atlantic forest. This first characterization of molecular diversity indicates a great diversity (B. torquatus: h = 0.901 ± 0.039 and π = 0.012 ± 0.007; B. variegatus: h = 0.699 ± 0.039 and π = 0.010 ± 0.006) and very divergent mitochondrial lineages within each sloth species. The different sampled regions carry distinct and non-overlapping sets of mtDNA haplotypes and are genetically divergent. This phylogeographic pattern may be characteristic of sloth species. In addition, we infer that two main phylogeographic groups exist in the Atlantic forest representing a north and south distinct divergence.

Haldane's rule and other aspects of reproductive isolation observed in the Anastrepha fraterculus complex (Diptera: Tephritidae)

Some aspects of reproductive isolation between allopatric populations of two closely related species of the Anastrepha fraterculus complex (A. fraterculus sp. 1 and sp. 2) were evaluated in laboratory conditions. Most of the crosses were fertile in each species as well as between sp. 2 females and sp. 1 males. In the reciprocal cross only 41.7% of the matings yielded viable progeny. Egg hatching occurred at similar rates within the two species, but was significantly lower in the crosses between the species. Adult emergence did not differ significantly among crosses. The sex ratio of adult progeny within each species, as well as in the hybrid progeny derived from sp. 1 females crossed to sp. 2 males, did not differ from the expected 1:1 ratio. However, in the crosses between sp. 2 females to sp. 1 males, a significant deviation in the sex ratio in favor of females was observed, according to the Haldane’s rule. The results reinforce previous data which indicated that A. fraterculus sp. 1 and A. fraterculus sp. 2 are distinct biological entities.

Mating Behavior of Wild Anastrepha fraterculus (Diptera: Tephritidae) on a Caged Host Tree

The mating behavior of the South American fruit fly, Anastrepha fraterculus (Wiedemann) is described, following release of wild virgin flies onto a field-caged host tree (guava). Sexual encounters and attempted copulations occurred almost exclusively from 0700-0900 hours and were confined to leaf nodes or the bottom surface of leaves, where males stationed themselves, often forming leks, and appeared to emit a sex pheromone which was attractive to virgin females. There were no male visitations or sexual encounters on green guava fruit, even though females, after mating, frequently oviposited there.

OVIPOSITION DETERRING PHEROMONE INAnastrepha fraterculus FLIES

Anastrepha fraterculus (Wiedemann) females were found to deposit a water-soluble, durable, oviposition-deterring pheromone during ovipositor dragging on fruit after egg-laying. We present evidence that the occurrence of pheromone deposition after egg-laying, the amount deposited, and departure from the fruit without additional egg-laying after pheromone deposition are flexible traits inA. fraterculus, varying in expression according to fruit size and other factors. UnlikeRhagoletis, A. fraterculus males were not arrested by the pheromone.

Morphology, molecular phylogeny, and taxonomic inconsistencies in the study of Bradypus sloths (Pilosa: Bradypodidae)

Abstract This study focuses on morphological and molecular data analyses, misidentifications, and phylogenetic inconsistencies regarding Bradypus variegatus (the brown-throated sloth) and B. tridactylus (the pale-throated sloth). Misidentifications were recorded on 75 of 313 museum specimens of Bradypus. Almost 90% of the misidentified specimens were B. variegatus from north-central Brazil, erroneously attributed to B. tridactylus. These misidentified specimens are reported in taxonomic reviews as the southernmost records of B. tridactylus. A history of confusing nomenclature regarding sloth species exists, and these particular misidentifications could be attributable to the similarity in face and throat color between B. variegatus from north-central Brazil and B. tridactylus. The molecular phylogeny of morphologically confirmed sloth specimens exhibits 2 monophyletic lineages representing B. variegatus and B. tridactylus. The split time between these 2 lineages was estimated at 6 million years ago (mya), contradicting previous studies that estimated this divergence to be 0.4 mya. Taxonomic inconsistencies were detected when comparing the molecular phylogeny to previously published DNA sequences ascribed to B. tridactylus. Misidentification or introgression could underlie such phylogenetic incongruities. Regardless of their causes, these discrepancies lead to misstatements regarding geographic distribution, phylogeny, and taxonomy of B. variegatus and B. tridactylus.

Egg size, yolk mass extrusion and hatching behavior in two cryptic speciesof Anastrepha fraterculus (Wiedemann) (Diptera, Tephritidae)

Variations in egg length were observed for two populations of cryptic species of Anastrepha fraterculus (Wiedemann). The eggs of type I flies were smaller than those of type II individuals. For both types, in regard to yolk mass extrusion, four classes of embryos were detected. Class 1: embryos that extrude masses at both extremities; class 2: embryos in which extrusion occurs only at the anterior pole; class 3: embryos that eliminate mass only at the posterior pole, and class 4: embryos that do not extrude any mass. Embryo class frequencies were similar for populations belonging to the same type, but different between types. Individual females may produce eggs from different embryo classes, but for any given female the pattern remains constant during a long period of oviposition. Variation in size of the extruded masses was similar for both populations. Individual females produced embryos with a small range of mass diameters, and different females produced masses of different mean size. However, individual mass size remained constant during oviposition. The results suggest the existence of genetic components involved in the control of this unusual process. Larvae of both types presented, just before eclosion, similar unusual behaviors: they ingest the anterior extruded mass, rotate 180°, absorb the posterior mass and eclose near the posterior pole. Data show that cryptic A. fraterculus type I and type II differs in regard to egg size as well as to the phenomenon of yolk mass extrusion

Identifying management units in non-endangered species: the example of the sloth Bradypus variegatus Schinz, 1825.

In this study we propose the analysis of genetic diversity of the common three-toed sloth, Bradypus variegatus, in an attempt to understand population structure, identify divergent intraspecific units, and contribute to the knowledge of biodiversity in the neotropical forests. We analyzed a 387 bp segment of the mitochondrial DNA control region in 28 individuals distributed in different localities of both Atlantic and Amazon forests. Our results demonstrated that the genetic diversity of B. variegatus is distributed in six management units, MUs. The observed MUs encompass six phylogenetic lineages and represent respectively north and south regions of Atlantic forest, three regions within the Amazon forest, and a transition region between these two biomes. Considering the fact that these MUs are concordant with phylogroups and endemism areas already described for other vertebrate species, we can say that the study of B. variegatus, a widely distributed and not endangered species, can help to identify areas for conservation biology purposes in neotropical rain forests.

A simple protocol for the extraction and sequence analysis of DNA from study skin of museum collections

Museum collections have been widely used as sources of biological samples for molecular biology studies and there are several methodologies and techniques to obtain and analyze DNA from tissues archived in museums, but most of these protocols have been developed for a specific tissue or are commercial kits. We present a simple protocol for extracting and amplifying DNA segments from sloth museum specimens. With this simple protocol we analyzed DNA fragments from 64% of 64 skin samples from three-toed sloths (Bradypus variegatus and Bradypus tridactylus) archived in three different museums: 43 samples from the University of Sao Paulo Museum of Zoology (Museu de Zoologia da Universidade de Sao Paulo, MUZUSP) Sao Paulo, Sao Paulo, Brazil; 18 samples from the Emilio Goeldi Museum (Museu Paraense Emilio Goeldi, MPEG), Belem, Para, Brazil; and 3 samples from the Museum of Vertebrate Zoology (MVZ) University of California, Berkeley, USA. The specimens sampled ranged in age from 18 to 108 years old. Our methodology allowed the recovery of up to 700 bp of mitochondrial DNA and 400 bp of nuclear genes. Thereafter, it is useful for genetic diversity studies of three-toed sloths and could be applied to other animals.