Tânia M. Fernandes-Salomão

Phylogeography and historical demography of the neotropical stingless bee Melipona quadrifasciata (Hymenoptera, Apidae): incongruence between morphology and mitochondrial DNA

The stingless bees are among the most abundant and ecologically important social invertebrates in tropical communities. The Neotropical stingless bee Melipona quadrifasciata has two subspecies: M. quadrifasciata quadrifasciata and M. quadrifasciata anthidioides. The main difference between subspecies are the yellow metassomal stripes, which are continuous in M. q. quadrifasciata and discontinuous in M. q. anthidioides. Recently, two populations were described with continuous stripes and inhabiting clearly disjunct areas in relation to M. q. quadrifasciata. We sequenced 852 bp of the mtDNA COI gene from 145 colonies from 56 localities, and for the first time performed a detailed phylogeographic study of a neotropical stingless bee. Phylogenetic analyses revealed the existence of two clades exhibiting a south to north distribution: southern populations comprise the subspecies M. q. quadrifasciata, and northern populations are composed of M. q. anthidioides and two disjunct populations with continuous stripes. The divergence time of these two phylogroups was estimated between 0.233 and 0.840 million years ago in the Pleistocene, a period of climatic changes and geomorphological alterations in the Neotropical region. No evidence of genetic structure in relation to the tergal stripes was found, indicating that the morphological trait regarding the pattern of stripes on tergites is not an accurate diagnostic for the subspecies of M. quadrifasciata.ZusammenfassungStachellose Bienen gehören zu den häufigsten und ökologisch bedeutsamsten wirbellosen Tieren in tropischen Lebensgemeinschaften. Die neotropische stachellose Biene Melipona quadrifasciata hat zwei Unterarten: M. quadrifasciata quadrifasciata und M. quadrifasciata anthidioides. Das hauptsächliche Unterscheidungsmerkmal dieser beiden Unterarten sind gelbe metasomale Streifen, die bei M. q. quadrifasciata durchgehend, und bei M. q. anthidioides unterbrochen sind. Die Art ist entlang der Ostküste Brasiliens verbreitet, wobei M. q. quadrifasciata im Süden und M. q. anthidioides im Norden des Verbreitungsgebiets vorkommt.Vor kurzem wurden zwei Populationen mit zusammenhängenden Streifen beschrieben, die nördlich des von M. q. quadrifasciata besiedelten Gebietes in disjunkten Arealen vorkommen.In dieser Studie untersuchen wir die populationsgenetische Struktur und die historische Demographie von M. quadrifasciata mit Hilfe von Sequenzen der mitochondrialen DNA aus umfangreichem Probenmaterial (Abb. 1 und Tab. S1, online material). Dabei untersuchen wir zwei Hauptfragen: (i) korreliert die morphologische Variation (Verteilung des Streifenmusters auf dem Abdomen) mit der Variation der mitochondrialen DNA? Mit anderen Worten, stammen die Populationen mit zusammenhängenden Streifen aus derselben mütterlichen Linie ab? (ii) Welche demographischen Ereignisse können die beobachteten phylogeographischen Muster erklären?Zur Beantwortung dieser Fragen sequenzierten wir 852 bp der Untereinheit1 des Cytochromoxidase (CoI) Gens aus der mitochondrialen DNA von 145 Völken (1 Arbeiterin pro Volk), die entlang des Verbreitungsgebiets der Art gesammelt worden waren (Abb. 1). Die Ergebnisse von phylogeographischen Tests und Koaleszenzberechnungen machten deutlich, dass es zwei unterschiedliche Gruppen gibt: eine südliche Klade, die aus M. q. quadrifasciata besteht, und eine nördliche, die M. q. anthidioides sowie die beiden disjunkten Populationen mit durchgehenden Streifen beinhaltet. Interessanterweise sind also Formen, die durchgehende Streifen gemeinsam haben, nicht unbedingt monophyletisch; umgekehrt haben Individuen mit verschiedener Morphologie denselben mtDNA Haplotypen gemeinsam (H14 in Abb. 3). Wir schlagen vor, die durchgehenden tergalen Streifen in den diskjunkten Populationen als einen anzestralen Polymorphismus zu betrachten, da nach den Ergebnissen anderer Studien das Muster aus zusammenhängenden Streifen innerhalb der Untergattung Melipona (Melipona) ein plesiomorphes Merkmal zu sein scheint. Der Zeitpunkt der Divergenz der beiden Gruppen wurde auf die Zeit des Pleistozäns, zwischen 0,233 und 0,840 Millionen Jahren vor der Gegenwart bestimmt (online material, Abb. S2 und Tab. S1), die durch Klimaänderungen und geomorphologische Umbildungen in der neotropischen Region charakterisiert war. Ähnliche phylogeographische Muster wurden bei endemischen Wirbeltieren des Atlantischen Regenwaldes nachgewiesen, zum Beispiel bei Schlangen, Vögeln und Amphibien.

Cytogenetic and molecular analyses reveal a divergence between Acromyrmex striatus (Roger, 1863) and other congeneric species: taxonomic implications.

The leafcutter ants, which consist of Acromyrmex and Atta genera, are restricted to the New World and they are considered the main herbivores in the neotropics. Cytogenetic studies of leafcutter ants are available for five species of Atta and 14 species of Acromyrmex, both including subspecies. These two ant genera have a constant karyotype with a diploid number of 22 and 38 chromosomes, respectively. The most distinct Acromyrmex species from Brazil is A. striatus, which is restricted to the southern states of Santa Catarina and Rio Grande do Sul. Several cytogenetic and phylogenetic studies have been conducted with ants, but the karyotypic characterization and phylogenetic position of this species relative to leafcutter ants remains unknown. In this study, we report a diploid number of 22 chromosomes for A. striatus. The phylogenetic relationship between A. striatus and other leafcutter ants was estimated based on the four nuclear genes. A. striatus shared the same chromosome number as Atta species and the majority of metacentric chromosomes. Nuclear data generated a phylogenetic tree with a well-supported cluster, where A. striatus formed a different clade from other Acromyrmex spp. This combination of cytogenetic and molecular approaches provided interesting insights into the phylogenetic position of A. striatus among the leafcutter ants and the tribe Attini.

A RAPD marker associated with B chromosomes in Partamona helleri (Hymenoptera, Apidae)

The hymenopteran Partamona helleri is found in southwestern Brazil in the Mata Atlântica from the north of the state of Santa Catarina until the south of Bahia. This work shows that P. helleri can carry up to four B chromosomes per individual. In order to obtain more information about P. helleri B chromosomes, the RAPD technique was used to detect DNA fragments associated with these chromosomes. The results showed that the RAPD technique is useful to detect specific sequences associated with B chromosomes. One RAPD marker was identified, cloned and used as probe in a DNA blot analysis. This RAPD marker hybridized with sequences present only in individuals containing B chromosomes.

Honey physicochemical properties of three species of the brazilian Melipona

Physicochemical analyses were carried out to evaluate 27 samples of honeys from three species of the Brazilian genus Melipona (M. capixaba, M. rufiventris and M. mondury) from Espírito Santo and Minas Gerais States. The parameters water activity (Aw), percentage of soluble solids (Brix %), pH, acidity (meq/Kg) and moisture (%) were evaluated. The honey characteristics obtained from these samples were very similar to the ones from other Melipona species. However, regarding the honey from Apis (honey bee), only the pH values were similar. The low pH value and the high acidity detected in Melipona honey are potential factors for increasing the honey shelf life because they do not provide favorable conditions for the microbial development. On the other hand, the high level of water activity favors the growth of microorganisms, especially yeast, which demands a more careful handled and storage. The observed differences between Melipona and Apis honey reinforce the need for specific quality settings for stingless bee honey.

Pollen Sources for Melipona capixaba Moure & Camargo: An Endangered Brazilian Stingless Bee

Pollen samples were collected in three different periods from 11 Melipona capixaba Moure & Camargo hives and analyzed with melissopalynological methodology. A total of 33 pollen types were identified, of which 23 genera and 15 families were identified. The following families showed the highest pollen richness: Fabaceae (7), Myrtaceae (3), Solanaceae (3), Arecaceae (2), Asteraceae (2), Euphorbiaceae (2), Melastomataceae/Combretaceae (2), Rubiaceae (2), and Sapindaceae (2). The most frequent pollen types (>45%) were Eucalyptus, which generated great similarities between the samples, except one in which the Tibouchina was predominant. Although the majority of the pollen types showed low percentage values, the results demonstrated that M. capixaba has taken advantage of the polliniferous sources available in the Atlantic Rainforest as well as in the “Capoeira” (brushwood, secondary forest) and “ruderal” (field) plants, probably implying its importance as a pollinator of the native flora and of the exotic species.

Nuclear mitochondrial DNA: an Achilles’ heel of molecular systematics, phylogenetics, and phylogeographic studies of stingless bees

Mitochondrial-like DNA (numt) has been found in a variety of insect species. In this work, our objective was to create a phylogeographic hypothesis of Melipona capixaba based on the complete COI sequence. However, several inconsistencies were found, such as 1–2-bp-long indels and a stop codon within the putative amino acid sequences. This led us to infer that M. capixaba has numts. A phylogenetic analysis which included COI sequences of several species of Melipona Illiger, 1806 revealed that numts are also common among other species of the genus. Based on our results, we have proposed a checklist to help to identify the most conspicuous numts; however, it does not ensure that all numts will be identified, since not all numts present explicit signals. Therefore, we recommend taking the maximum care in phylogeographic and phylogenetic analysis within Melipona as well as Hymenoptera, since several evidences of numts were found within this order.

A scientific note on the characterization of microsatellite loci for Melipona mondury (Hymenoptera: Apidae)

Microsatellites are a class of markers much usedin population, phylogenetic and parentage studiesbecause of their high degree of polymorphism, co-dominance and high reproducibility (De la Ruaet al., 2001; Oliveira et al., 2006). Microsatellite se-quences are amplified by PCR, using primers thatflank the repeat sequences of nuclear DNA. The de-velopment of these primers, however, is the lim-iting factor for the use of these markers. Becauseof this difficulty, there are microsatellite primersdeveloped for only three species of stingless bee,

Development of a SCAR marker for the analysis of B chromosome presence in Partamona helleri (Hymenoptera, Apidae)

Chromosomes in hymenopteran insects cannot currently be analysed in adult individuals. The only available cytogenetic techniques need to be performed in larvae. Here we develop and implement a SCAR (Sequence Characterized Amplified Region) marker, associated with B chromosomes in the bee Partamona helleri, which has proven to be very useful to reveal B chromosome presence in adults from natural populations. The marker was tested in ten different colonies simultaneously analysed by both molecular (ten adults per colony) and cytogenetic (20 larvae per colony) techniques. The presence of the SCAR marker always showed the same pattern as B chromosome presence: both were present or absent in all individuals from a same colony, or both were present in only part of the individuals from a same colony. This molecular marker is thus a useful tool for analysing new aspects of this B chromosome system such as B frequency and geographical distribution, B transmission, or B effects in adult individuals.

Possible Introgression of B Chromosomes between Bee Species (Genus Partamona)

The origin of supernumerary (B) chromosomes is still a debated topic, with intra- and interspecific origins being the most plausible options. In the bee Partamona helleri, a sequence-characterized amplified region (SCAR) marker being specific to B chromosomes suggested the possibility of interspecific origin. Here, we search for this marker in 3 close relative species and perform DNA sequence comparison between species. The SCAR sequence does not show homology with other sequences in the databases, but does contain an open reading frame with sequence homology with a reverse transcriptase. Dot-blot hybridization using the SCAR marker as a probe confirmed that it is present in B-carrying, but not B-lacking larvae of P. helleri, and indicated its presence in adult individuals of P. cupira and P. criptica. Additionally, PCR amplification of the SCAR marker was successful on genomic DNA obtained from P. helleri and P. rustica larvae carrying B chromosomes, and on genomic DNA obtained from adult individuals of P. cupira, P. criptica and P. rustica. Finally, a comparison of the DNA sequence of the SCAR markers amplified from these 4 species showed very few nucleotide differences between the species. The complete association between B chromosome and SCAR presence and the scarce divergence observed for this DNA sequence between the 4 species analyzed suggest the possibility that this B chromosome has recently been transferred between species through several episodes of interspecific hybridization.

Integrating Paleodistribution Models and Phylogeography in the Grass-Cutting Ant Acromyrmex striatus (Hymenoptera: Formicidae) in Southern Lowlands of South America

Past climate changes often have influenced the present distribution and intraspecific genetic diversity of organisms. The objective of this study was to investigate the phylogeography and historical demography of populations of Acromyrmex striatus (Roger, 1863), a leaf-cutting ant species restricted to the open plains of South America. Additionally, we modeled the distribution of this species to predict its contemporary and historic habitat. From the partial sequences of the mitochondrial gene cytochrome oxidase I of 128 A. striatus workers from 38 locations we estimated genetic diversity and inferred historical demography, divergence time, and population structure. The potential distribution areas of A. striatus for current and quaternary weather conditions were modeled using the maximum entropy algorithm. We identified a total of 58 haplotypes, divided into five main haplogroups. The analysis of molecular variance (AMOVA) revealed that the largest proportion of genetic variation is found among the groups of populations. Paleodistribution models suggest that the potential habitat of A. striatus may have decreased during the Last Interglacial Period (LIG) and expanded during the Last Maximum Glacial (LGM). Overall, the past potential distribution recovered by the model comprises the current potential distribution of the species. The general structuring pattern observed was consistent with isolation by distance, suggesting a balance between gene flow and drift. Analysis of historical demography showed that populations of A. striatus had remained constant throughout its evolutionary history. Although fluctuations in the area of their potential historic habitat occurred during quaternary climate changes, populations of A. striatus are strongly structured geographically. However, explicit barriers to gene flow have not been identified. These findings closely match those in Mycetophylax simplex, another ant species that in some areas occurs in sympatry with A. striatus. Ecophysiological traits of this species and isolation by distance may together have shaped the phylogeographic pattern.