Marco Antonio Del Lama

Genetic structure of honeybee populations from southern Brazil and Uruguay

Apis mellifera scutellata was introduced to Brazil in 1956 and Africanized honeybee populations have now spread from Argentina to the southwestern United States. Temperate climatic restrictions seem to be a natural limit to Africanized honeybee expansion around parallels 35° to 40° SL. We used allozyme loci (Mdh-1 and Hk-1) and mtDNA haplotypes to characterize honeybee populations in southern Brazil and Uruguay and define a possible transition area between Africanized and European bees. Samples of 194 bee colonies were collected from ten localities between 30°-35° SL and 52°-59° WL. The mtDNA restriction patterns of these colonies were obtained through digestion of the mitochondrial genome by Eco RI, or by digestion by Bgl II and Xba I of the cytochrome B locus and the COI-COII intergenic region, respectively. The distribution limit of African bee colonies, i.e., those populations with only the African mtDNA haplotype and with a high proportion of African genes as shown by allozyme analysis, is located in northern Uruguay, with a hybridization zone located farther south in Uruguay. A gradual cline from north to south was observed, confirmed by mtDNA, racial admixture, and genetic distance analyses. No evidence of either gametic disequilibrium between nuclear markers or cytonuclear disequilibrium among the nuclear and mtDNA genotypes was detected, suggesting that the hybridization process has been completed.

Nectar-foraging behavior of Euglossine bees (Hymenoptera: Apidae) in urban areas

Euglossine bees have been described as long-distance pollinators because of their great flight capacities although flight capacity is not necessarily correlated to home range. Here we report the nectar-foraging behavior of two euglossine species (Euglossa cordata and Eulaema nigrita) in urban areas and the predictive power of wing wear as an age estimator of these bees, using mark-recapture techniques at Thevetia peruviana trees. A total of 870 bees were marked. Recapture rates were 33% (± 19.2) for E. cordata and 25% (± 2.5) for E. nigrita. Only 7 bees were sighted at a different site from where they were first captured. More than 75% of the individuals showed site-constancy at trees for at least 30 days. Wing wear accumulation rate was variable among individuals and it was a poor predictor of age for E. cordata. Our data show that euglossine bees may have small foraging ranges in urban areas, indicating that home ranges greatly differ from their flight capacity and homing ability.ZusammenfassungEuglossine Bienen (Prachtbienen) werden im allgemeinen als Weitdistanzbestäuber eingestuft, die grosse Sammelgebiete abdecken können. Sie sind dazu in der Lage aufgrund ihrer grossen Flugkapazität und dies obwohl Flugkapazität nicht notwendigerweise mit dem Heimatradius korreliert sein muss. In dieser Arbeit untersuchten wir das Nektarsammelverhalten von zwei Prachtbienenarten in Stadtgebieten, und wir bewerteten die Aussagekraft des Flügelabnutzungsgrads für die Altersabschätzung. Wir beschreiben zudem eine effiziente Methode zur Markierung dieser Bienen, die Etiketten leicht durch ihr intensives Putzverhalten verlieren können. Weibchen und Männchen von Euglossa cordata (nw = 596; nm = 53) und von Eulaema nigrita (nw = 106; nm = 115) (Tab. I) wurden markiert und später auf blühenden Bäumen der Art Thevetia peruviana (Apocynaceae) in einem Umkreis von 0,24–5,45 km im Stadtgebiet von São Carlos (SP, Brasilien) wiedergefangen. Die mittlere Wiederfangrate für Eg. cordata lag bei 33 % (± 19,2) und die für El. nigrita bei 25 % (± 2,5). Von den 213 wiedergefangenen Bienen wurden jedoch nur sieben an einem anderen Ort wiedergefangen als dem, an dem sie zuerst gefangen und markiert worden waren (Tab. II). Über 75 % der Bienen beider Arten zeigten ausserdem eine Ortskonstanz im Sammelverhalten an den Bäumen über mindestens 30 Tage hinweg. Bei Männchen war der Zeitraum allerdings kürzer als bei Weibchen (Abb. 3 und 4). Diese Ergebnisse weisen daraufhin, dass Prachtbienen bevorzugt über kurze Distanzen hinweg Nektar sammeln, dass sie ihre Sammelterritorien jedoch nach einiger Zeit wechseln können. Der Flügelabnutzungsgrad erwies sich als individuell stark variabel und schien bei Eg. cordata kein guter Altersindikator zu sein (Abb. 5). Unsere Daten zeigen, dass Euglossinen in Stadtgebieten aufgrund der Verteilung von Blüten relativ kleine Sammelgebiete haben können. Dies bedeutet, dass sich Heimatradien stark von den Faktoren Flugkapazität und Heimfindungsvermögen unterscheiden können.

Conservation genetics of neotropical pollinators revisited: microsatellite analysis suggests that diploid males are rare in orchid bees.

Allozyme analyses have suggested that Neotropical orchid bee (Euglossini) pollinators are vulnerable because of putative high frequencies of diploid males, a result of loss of sex allele diversity in small hymenopteran populations with single locus complementary sex determination. Our analysis of 1010 males from 27 species of euglossine bees sampled across the Neotropics at 2–11 polymorphic microsatellite loci revealed only five diploid males at an overall frequency of 0.005 (95% CIs 0.002–0.010); errors through genetic nondetection of diploid males were likely small. In contrast to allozyme‐based studies, we detected very weak or insignificant population genetic structure, even for a pair of populations >500 km apart, possibly accounting for low diploid male frequencies. Technical flaws in previous allozyme‐based analyses have probably led to considerable overestimation of diploid male production in orchid bees. Other factors may have a more immediate impact on population persistence than the genetic load imposed by diploid males on these important Neotropical pollinators.

A REANALYSIS OF DIPLOID MALE FREQUENCIES IN EUGLOSSINE BEES (HYMENOPTERA: APIDAE)

NAMI CRISTINA TAKAHASHI,1 RUI CARLOS PERUQUETTI,1 MARCO ANTONIO DEL LAMA,1,2 AND LÚCIO ANTONIO DE OLIVEIRA CAMPOS3 1Laboratório de Genética Bioquı́mica, Departamento de Genética e Evolução, Universidade Federal de São Carlos, C.P. 676, CEP 13565-905, São Carlos, São Paulo, Brazil 2E-mail: dmdl@power.ufscar.br 3Departamento de Biologia Geral, Universidade Federal de Viçosa, CEP 36571-000, Viçosa, Minas Gerais, Brazil E-mail: lcampos@mail.ufv.br

Genetic differentiation of urban populations of Euglossa cordata from the state of São Paulo, Brazil

Males and females of Euglossa cordata collected inside flowers of Thevetia peruviana in urban areas of eleven cities of the state of São Paulo were analysed using allozymes and nine microsatellite loci. The analyses revealed that these populations have a high genetic diversity and are under genetic equilibrium, showing low population structuring and rare diploid males; consequently, high gene flow and effective population size (Ne) are inferred. These findings corroborate previous biological observations and phylogeographic evidence that report dispersion over long distances of Euglossini species in South America.

16S mtDNA variation in Apis mellifera detected by PCR-RFLP

Phylogeographic and morphometric evidence can be used to cluster Apis mellifera subspecies into evolutionary lineages or branches. Mitochondrial DNA sequence and restriction site analyses have shown similar clustering of subspecies groups. Thus, mtDNA variation can be used to infer honey bee evolutionary relationships. In this paper, we describe three 16S mtDNA PCR-RFLP patterns, each one completely associated with a previously determined A, M, or C Dra I restriction pattern of the COI-COII region. These results indicate that the COI-COII and the 16S genes have had a very closely linked evolutionary history. Although distinct patterns were obtained with Eco RI, Alu I, Hinc II and Taq I, the best differentiation among the three patterns was observed with Dra I and Vsp I enzymes. Nucleotide sequence analysis of the16S gene fragment displayed 10 sites of base substitution (1.35%) among the three patterns and two insertions in the A. m. scutellata pattern.ZusammenfassungÄhnlich wie phylogeographische und morphometrische Evidenzen können auch Daten zu Variationen in der mitochondrialen DNA (mtDNA) herangezogen werden, um Schlüsse hinsichtlich evolutiver Beziehungen zu ziehen. Der Verdau der mitochondrialen intergenen COI-COII Region mittels DraI (Garnery et al., 1993) stellt gegenwärtig die Methode mit der besten Auflösung für die Charakterisierung und Zuordnung von Subspezies zu mitochondialen Linien dar. Populationsanalysen mittels mtDNA-Polymorphismen können dementsprechend ein wertvolles Werkzeug darstellen, um Richtungen und Mechanismen in der mtDNA-Genomevolution zu untersuchen. Als Teil einer Suche nach neuer mtDNA Variabilität beschreibt diese Studie die Amplifikations- und Restriktionsmuster für eine weitere mitochondriale DNA Region (16S) in Proben, die zuvor hinsichtlich ihrer COI-COII-Muster als A, M oder C klassifiziert wurden. Insgesamt wurden Proben von 38 Völkern mit M-Muster, 89 mit dem C- und 232 mit dem A-Muster gewonnen. Die Orte der Probennahmen und die jeweilige Zahl der untersuchten Völkern sind in Tabelle I zusammengestellt. DNA wurde individuell mittels Phenol-Chloroform aus Thoraxstücken extrahiert. Die DNA-Fraktion wurde für die PCR-Amplifizierung des 16S Locus verwendet und anschliessend mittels der folgenden Enzyme verdaut: Hinc II, Vsp I, Alu I, Taq I, Eco RI und Dra I.Die amplifizierten Fragmente und die Produkte der Endonukleasenverdaue wurden in 8 %, bzw. 10 % Polyacrylamidgelen aufgetrennt. Restriktionsanalysen der mitochondrialen DNA der Cytochrom b (Bgl II), COI (Hinc II) und COI-COII (Dra I) Regionen wurden für eine vergleichende molekulare Charakterisierung der Honigbienenproben durchgeführt. Anhand der 16S Amplifizierung und der Restriktionsanalyse wurden drei Muster festgestellt, die entsprechend ihrer assoziierten COI-COII-Muster als A, M und C bezeichnet wurden (Abb. 2). Das amplifizierte 16S-Genfragment wies in den C- und M-Proben eine Grösse von 740 bp und in den A-Proben eine Grösse von 742 bp auf. Paarweise oder multiple Vergleiche der DNA-Sequenzen zeigten Nukleotidsubstitutionen an 10 Positionen (1,35 %) (Abb. 1). Das gemeinsame Vorkommen der M- und A-Muster in Spanien belegt die Ergebnisse früherer Studien und weist darauf hin, dass dieses Land eine sekundäre Kotaktzone zwischen diesen beiden Linien darstellt. In der vorliegenden Studie zeigten 17 von 44 Völkern aus Italien das M-Muster und eines das A-Muster. Dieses Ergebnis bestätigt frühere Analysen, die zeigten, dass italienische Honigbienenpopulationen aus den M- und C-Linien der mitochondrialen DNA zusammengesetzt sind. Obwohl mittels Eco RI, Alu I, Hinc II und Taq I-Verdau gut unterscheidbare Muster erzeugt werden konnten, war die klarste Differenzierung für die drei Haplotypen bei Dra I und VspI I-Verdauen zu beobachten. Die 16S mtDNA Variation kann nicht nur eine alternative Methode für die Identifizierung der maternalen DNA-Abstammung eines Volkes darstellen, vielmehr kann der in dieser Arbeit aufgezeigte Polymorphismus, zusammen mit anderen Polymorphismen, sich auch als nützlich erweisen, um Ereignisse der mtDNA-Evolution bei A. mellifera zu untersuchen

Alocação sexual e seleção sexo-dependente para tamanho de corpo em Trypoxylon rogenhoferi Kohl (Hymenoptera, Sphecidae)

Two populations of the wasp Trypoxylon rogenhoferi Kohl, 1884 from Sao Carlos and Luis Antonio, State of Sao Paulo, Brazil, were observed and sampled from May 1999 to February 2001 using trap-nests. This mass-provisioning wasp was used to test some aspects of optimal sex allocation theory. Both populations fit all the predictions of the models of Green and Brockmann and Grafen. Maternal provisions determined the size of each offspring, and females allocated well-stocked brood cells to daughters, the sex that benefits most being large. This strategy resulted in a difference in size between the sexes. In Sao Carlos, female weight at emergence was 1.18 times that of males, in Luis Antonio this value was 1.13. The brood cell volume was correlated with both wing length and weight at emergence in both sexes, and the chance that a given brood cell contained a male offspring decreased with increased brood cell volume. In T. rogenhoferi female body size was related to fitness. Larger females were able to collect more mass of spiders per day, the spiders they captured were heavier, and they provisioned more brood cells per day. They also produced larger daughters. For males, no relationship between body size and fitness was found, but the data were scarce. Since the patterns of provisioning were variable among different females in both study sites, it is possible that the females not follow a unique strategy for sex allocation. The sex ratio and/or investment ratio in the Sao Carlos population was female-biased and in Luis Antonio, male-biased. In spite of the influence of trap-nests diameters on male production in Luis Antonio, there is some evidence that in Sao Carlos population the local availability of prey and/or lower rate of parasitism may be major forces in determining the observed sex ratio, but further studies are necessary to verify such hypothesis.

Gene Variation, Population Differentiation, and Sociogenetic Structure of Nests of Partamona seridoensis (Hymenoptera: Apidae, Meliponini)

Gene variation and the differentiation of two populations of Partamona seridoensis (Hymenoptera: Apidae: Meliponini) from the Caatinga biome, a semiarid ecosystem unique to Brazil, were estimated through allozymic and microsatellite analyses. These populations exhibited similar low degrees of enzyme gene variation. Observed genotype frequencies at the allozyme and microsatellite loci were in accordance with Hardy–Weinberg equilibrium in the two populations. Both markers demonstrated that the two populations are not genetically homogeneous and must be considered distinct populations. The occurrence of private alleles at the allozyme and microsatellite loci corroborates this differentiation, sustaining the hypothesis of a low level of interpopulation gene flow. The phenotypic segregations clearly demonstrated that the progeny inside each nest were the result of mating between the queen of the colony and only one male.

Differentiation of Melipona quadrifasciata L. (Hymenoptera, Apidae, Meliponini) subspecies using cytochrome b PCR-RFLP patterns

Melipona quadrifasciata quadrifasciata and M. quadrifasciata anthidioides are subspecies of M. quadrifasciata, a stingless bee species common in coastal Brazil. These subspecies are discriminated by the yellow stripe pattern of the abdominal tergites. We found Vsp I restriction patterns in the cytochrome b region closely associated to each subspecies in 155 M. quadrifasciata colonies of different geographical origin. This mitochondrial DNA molecular marker facilitates diagnosis of M. quadrifasciata subspecies matrilines and can be used to establish their natural distribution and identify hybrid colonies.

Milk protein polymorphisms in Brazilian Zebu cattle

Five bovine milk protein polymorphisms were studied in Zebuine cattle raised in Brazil, through horizontal electrophoresis on starch gel containing urea and 2-mercaptoethanol, using basic and acidic buffer systems. Allelic frequencies for a-La, b-Lg, aS1-Cn, b-Cn and k-Cn loci were estimated in six Gyr herds (N = 283), six Guzerat herds (N = 205), one Nelore herd (N = 17) and one Sindi herd (N = 22), all from Sao Paulo or Minas Gerais State, Brazil. Genotypic frequencies observed for each locus and breed studied are in accordance with the assumption of genetic equilibrium, demonstrating absence of high inbreeding levels for the breeds tested. The FST value found indicated significant genetic differentiation among breeds; however, the Gyr and Guzerat herds showed significantly different gene frequencies. Genetic distance estimates among zebuine breeds studied and the Holstein breed, taken as a reference for a taurine breed, showed strong differences between these two racial groups