Colin R. Hughes

Microsatellites and kinship

Many evolutionary studies, particularly kinship studies, have been limited by the availability of segregating genetic marker loci. Microsatellites promise to alleviate these problems. Microsatellite loci are segments of DNA with very short sequence motifs repeated in tandem; their often numerous alleles differ in the number of these repeat units. They are very common in eukaryotic DNA and can be amplified by the polymerase chain reaction, which allows the use of minute or degraded DNA samples. The alleles can be scored consistently and compared unambiguously, even across different gels.

Detection of highly polymorphic microsatellite loci in a species with little allozyme polymorphism

Microsatellite loci are regions of DNA containing tandem repeats of a short sequence motif; they occur abundantly in all eukaryotic genomes and have been shown to be a rich source of highly polymorphic genetic markers in humans and other mammals. These loci are particularly suitable for population studies because they can be relatively easily scored using a combination of polymerase chain reaction (PCR) amplification of each locus followed by electrophoresis to separate alleles. This paper details a method for finding these loci in any species. This method demonstrates that trinucleotide microsatellite loci are abundant and highly polymorphic in the social wasp Polistes annularis, whereas allozyme electrophoresis reveals very little polymorphism. The first six loci examined were all polymorphic with a mean observed heterozygosity of 0.62; in comparison average heterozygosity of 33 allozymes was 0.035. We suggest that this method can be used to detect variation where other methods have failed, making it an ideal tool for population and conservation geneticists who must deal with populations lacking other types of genetic variability.

PHYLOGENETIC RELATIONSHIPS AMONG PAPER WASP SOCIAL PARASITES AND THEIR HOSTS (HYMENOPTERA: VESPIDAE; POLISTINAE)

Abstract— Cladistic analyses of data from allozyme polymorphisms in paper wasp social parasites and their hosts do not support the hypothesis that social parasites are most closely related to their hosts. Electrophoretic data are adduced for nine species of Polistes, including all three known species of social parasites (Sulcopolistes) and their hosts. Three different coding methods are investigated; in no case do the social parasites cluster most closely with their hosts. Rather, there is limited evidence that they form a monophyletic group. However, formal taxonomic recognition of Sulcopolistes is not justified, as it renders Polistes sensu stricto paraphyletic. Although the social parasites are not most closely related to their hosts, hosts and parasites belong in the same subgenus and share many characteristics that may have facilitated the exploitation and deception practised by the parasites on the hosts.

Genetic Relatedness in Colonies of Tropical Wasps with Multiple Queens

The evolution of worker behavior in the social insects is usually explained by kin selection: although workers do not produce offspring, they do reproduce their genes by aiding the reproduction of relatives. The most difficult case for kin selection theory would be species in which workers are fully capable of reproducing but instead opt to rear brood of low relatedness. These conditions are perhaps best fulfilled by the swarm-founding wasps because they have little caste differentiation and their colonies usually have multiple queens, which should lower relatedness. Estimates of within-colony relatedness for three species in this group confirm that it is sometimes (but not always) very low. Inbreeding is negligible in these species, so the hypothesis that inbreeding may raise relatedness is not supported. The maintenance of worker behavior in such species is a significant challenge for kin selection theory.

Relatedness and queen number in the Neotropical wasp, Parachartergus colobopterus

Abstract The maintenance of sociality is most difficult to explain under circumstances where non-reproducing helpers are physiologically capable of reproducing and distantly related to the brood they rear. The Neotropical swarm-founding wasps are likely to fulfil these conditions because most taxa lack morphological differences between workers and queens, and they have many queens per nest, which is expected to substantially lower worker to brood relatedness. No morphological differences between workers and queens in Parachartergus colobopterus were detected. Colonies contained an average of 27 queens. However, relatedness among nestmates in P. colobopterus was higher (r=0·31) than would be expected on the basis of queen number alone because the queens themselves are very closely related (r=0·67) and because of large variation in numbers of queens among colonies. This makes the harmonic mean of queen number (five queens), which is the appropriate measure for investigating the impact of queen number on relatedness, much lower than the arithmetic mean. Reproductive dominance of one or a few queens within colonies was not a factor that greatly increased relatedness among workers. Taken together, these results support the cyclical oligogyny hypothesis for the maintenance of sociality in Neotropical social wasps.

Trinucleotide microsatellite loci for a social wasp, Polistes

that build open-faced paper nests in which offspring are reared. The lack of distinct morphological castes and great plasticity in social behaviour among females, combined with the ease of observing individually paintmarked adults, has made this a model genus for behavioural studies (Reeve 1991; Turillazzi & WestEberhard 1996). All species are eusocial, but at a relatively rudimentary level: colonies are relatively small (Reeve 1991), workers are not consistently different from queens (Haggard & Gamboa 1980), social relations are often characterized by strife (West-Eberhard 1969; Strassmann 1981a; Noonan 1981), and the queen in some species must act as the pacemaker, physically manipulating workers into working (Reeve & Gamboa 1983). Polistes has served as a model system for a wide variety of topics including dominance hierarchies (WestEberhard 1969), advantages of group living (Strassmann et al. 1988; Strassmann & Queller 1989; Strassmann 1991), kin selection (Noonan 1981; Strassmann 1981b; Queller & Strassmann 1988), kin recognition (Gamboa et al. 1987), usurpation (Klahn 1988), and social control by queens (Reeve & Gamboa 1983). To date, it has been difficult to combine detailed studies of behavioural interactions with studies of genetic relatedness because of the difficulty in obtaining precise estimates of relatedness for individual pairs of interactants with the available genetic markers (e.g. Strassmann et al. 1989). Clearly, a more polymorphic set of markers is needed. Microsatellite loci are highly polymorphic, codominant, and can be genotyped from many sources of DNA including alcohol-preserved tissues and sperm in a female’s spermatheca (Evans 1993; Queller et al. 1993; Peters et al. 1995). Here we present 18 new microsatellite loci derived from Polistes bellicosus and eight new loci from Polistes annularis. All of these loci contain trinucleotide repeat regions which are much easier to score unambiguously than are dinucleotide repeats. To obtain these loci we constructed very large partial genomic libraries in plasmids with inserts (not enriched for microsatellites) averaging 500 bp (Hughes & Queller 1993; see Strassmann et al. 1996, for the rationale of our approach and specific protocols). We separately probed replicate membranes with synthesized oligonucleotides containing 10 or 12 repeats of either AAT, AAC, AAG, CAT or TAG, five of the 10 possible trinucleotide repeats. We picked up hundreds of potential positives. Reprobing a Southern blot of the inserts indicated that most of these sequences contained repeats. We sequenced part or all of about 80 clones of P. bellicosus. Of these, 59 contained at least one repeat region. We designed polymerase chain reaction (PCR) primers for clones containing uninterrupted repeats, five or more repeats long for which we could read the flanking regions (Table 1). We did not design PCR primers around all repeats for reasons including the nature of the flanks, absence of sufficient flanking sequence, or because they were dinucleotide repeats, not the trinucleotides we were after. We evaluated these primers for heterozygosity on 3–24 unrelated individuals. Genomic DNA was prepared as described in Hughes & Queller (1993) or using protocol Strassmann.1 in Strassmann et al. (1996). PCR was carried out under oil in a 10 μL volume made up of 2 μL diluted genomic DNA (about a nanogram), 2 μL of primer mix (2.5 μM), 0.1 μL 10 mM dNTP mix, 1 μL 10× buffer (provided with Taq), 4.08 μL dH2O, 0.62 μl 25 mM MgCl2, 0.05 μL Taq polymerase (5 units/μL, Promega), 0.15 μL 35S dATP (12.5 μCi/μL). After an initial denaturing for 5 min at 95 °C, we carried out 30–35 cycles of 60-s denaturing at 92 °C, 60-s annealing (at a temperature optimized for the primers used: see Table 1) and 45-s extension at 72 °C. After that, 5 extra minutes at 72 °C allowed for the completion of the extension. PCR products were run on 6% denaturing acrylamide gels (Strassmann et al. 1996). All of the 18 microsatellite loci proved to be polymorphic in P. bellicosus though heterozygosity varied from 0.05 to 1 (Table 1). Seven of the eight loci from P. annularis proved to be polymorphic, with heterozygosities ranging from 0.17 to 1 (Table 1). We have published other microsatellites for Polistes annularis and for another social wasp, Parachartergus PRIMER NOTE

Colony life history and demography of a swarm-founding social wasp

Abstract  Colonies of social insects are sometimes viewed as superorganisms. The birth, reproduction, and death of colonies can be studied with demographic measures analogous to those normally applied to individuals, but two additional questions arise. First, how do adaptive colony demographies arise from individual behaviors? Second, since these superorganisms are made up of genetically distinct individuals, do conflicts within the colony sometimes modify and upset optima for colonies? The interplay between individual and superindividual or colony interests appears to be particularly complex in neotropical, swarm-founding, epiponine wasps such as Parachartergus colobopterus. In a long-term study of this species, we censused 286 nests to study colony-level reproduction and survivorship and evaluated individual-level factors by assessing genetic relatedness and queen production. Colony survivorship followed a negative exponential curve very closely, indicating type II survivorship. This pattern is defined by constant mortality across ages and is more characteristic of birds and other vertebrates than of insects. Individual colonies are long-lived, lasting an average of 347 days, with a maximum of over 4.5 years. The low and constant levels of colony mortality arise in part from colony initiation by swarming, nesting on protected substrates, and an unusual expandable nest structure. The ability to requeen rapidly was also important; relatedness data suggest that colonies requeen on average once every 9–12 months. We studied whether colony optima with respect to the timing of reproduction could be upset by individual worker interests. In this species, colonies are normally polygynous but new queens are produced only after a colony reaches the monogynous state, a result which is in accord with the genetic interests of workers. Therefore colony worker interests might drive colonies to reproduce whenever queen number happens to cycled down to one rather than at the season that is otherwise optimal. However, we found reproduction to be heavily concentrated in the rainy season. The number of new colonies peaked in this season as did the percentages of males and queens. Relatedness among workers reached a seasonal low of 0.21–0.27, reflecting the higher numbers of laying queens. This seasonality was achieved in part by a modest degree of synchrony in the queen reduction cycle. Worker relatedness reached peaks of around 0.4 in the dry season, reflecting a decrease to a harmonic mean queen number of about 2.5. Thus, a significant number of colonies must be approaching monogyny entering the rainy season. Coupled with polygynous colonies rearing only males (split sex ratios), this makes it possible for a colony cycle driven by selfish worker interests to be consistent with concentrating colony reproduction during a favorable season.

Developing microsatellites when they are rare: trinucleotide repeat loci in the northern mockingbird Mimus polyglottos

The great value of microsatellite loci to population studies spurs their development. However, finding loci is difficult when microsatellites are uncommon in the genome. Because trinucleotide‐repeat loci are rare in northern mockingbirds Mimus polyglottos, we sought a new method for developing a suite of loci. Here we show that a bacteriophage cloning vector, Lambda Zap Express (Stratagene, La Jolla, CA, USA) has several features which make it suitable for this purpose. Using this vector, we made a library of 150 000 size‐selected clones and screened with an AAT10 probe; 97 positives were identified. From these, 12 pairs of PCR primers were developed, nine of which amplify polymorphic loci. Certain combinations of these primer pairs enable simultaneous amplification of up to three loci.

EVOLUTION OF COLONIALITY IN BIRDS: A TEST OF HYPOTHESES WITH THE RED-NECKED GREBE (PODICEPS GRISEGENA)

Abstract Group breeding is perplexing, because individuals incur density-dependent fitness costs to breed in groups, yet no universal benefit appears to explain its evolution. Coloniality in birds, defined as dense nesting within limited territories, is a striking example of group breeding. Adaptive explanations for coloniality are diverse, and several research approaches have emerged. Here, we test models for the evolution of coloniality with a population of Red-necked Grebes (Podiceps grisegena). We distinguish habitat models from conspecific models for explaining the adaptive evolution of coloniality. Habitat models propose that the fitness benefits of colonial breeding are gained through the characteristics or location of the colony site, whereas conspecific models posit that the benefits of colonial breeding are gained solely from nesting near conspecifics. Red-necked Grebes are waterbirds that typically nest in a dispersed fashion, only rarely nesting in colonies. Both dispersed and colonial nesting co-occurred in the study population, and aquatic nests were found in three distinct habitat types. We gathered detailed demographic data and used analyses of covariance to test whether variation in habitat type or conspecific aggregation best explained differences in breeding success across the population. We further used fitness, genetic, and spatial data to test whether kin selection, sexual selection, or conspecific reproductive-success cueing may drive colony formation. The data provided strong support for a habitat-based model and inconsistently supported conspecific models. Our results suggest that colonial individuals of this population gain selfish benefits from relatively rare, high-quality habitat rather than from proximity of conspecifics. Evolución de la Colonialidad en las Aves: Una Evaluación de las Hipótesis en Podiceps grisegena

RED-NECKED GREBES BECOME SEMICOLONIAL WHEN PRIME NESTING SUBSTRATE IS AVAILABLE

Abstract During 1995–1998 we studied the dynamics of nesting dispersion in a population of 160–200 Red-necked Grebes (Podiceps grisegena) on Lake Osakis, a large, windblown lake in central Minnesota. Breeding grebe pairs arriving on the lake were highly attracted to several large floating cattail (Typha angustifolia) mats that had broken off and drifted away from the mainland to become “islands” snagged within rooted emergent beds. These mats provided prime nesting habitat protected from waves and predators that was chosen by nearly 40% of the breeding population. Most pairs establishing nests on the islands averaged 5–10 simultaneously active neighbors within 50 m, compared to pairs that established territories along the shoreline, most of which had no neighbors within 50 m. The overall success of the two strategies varied widely from year to year, depending on the water levels, nesting habitat availability, timing of windstorms, and predator activity. During most years, however, pairs that established nests on the mats hatched earlier in the season and had larger clutches and broods. The dense mats appeared to provide protection from wind and wave action, the primary cause of early nest loss. Our blood analysis work showed no genetic structure of the population and no tendency for neighbors to be closely related. However, pairs often returned to the same area and type of nesting situation in subsequent years. The behavioral flexibility of Red-necked Grebe pairs to nest in groups within prime habitat areas may enable them to breed successfully within unpredictable emergent wetland habitats. Los Zambullidores Podiceps grisegena Nidifican Semi-Colonialmente Cuando hay Disponibilidad de Buen Sustrato de Nidificación Resumen. Entre 1995 y 1998, estudiamos la dinámica de dispersión de nidos en una población de 160–200 zambullidores Podiceps grisegena en Lake Osakis, un lago grande afectado por el viento ubicado en Minnesota central. Las parejas de zambullidores que arribaron al lago fueron muy atraidas a varios parches flotantes de Typha angustifolia que se habían separado de la costa convirtiéndose en islas. Estos parches representaron el mejor hábitat de nidificación (protegido de las olas y los depredadores), y fueron escogidos por cerca del 40% de la población reproductiva. La mayoría de las parejas que establecieron sus nidos en las islas tuvieron en promedio 5–10 vecinos activos simultáneamente a menos de 50 m, en comparación con las parejas que ubicaron sus nidos a lo largo de la costa, la mayoría de las cuales no tuvieron ningún vecino a menos de 50 m. El éxito general de las dos estrategias varió año a año, dependiendo de los niveles de agua, la disponibilidad de hábitat de nidificación, el momento de ocurrencia de tormentas de viento y la actividad de los depredadores. Sin embargo, durante la mayoría de los años, las parejas que ubicaron los nidos en los parches flotantes tuvieron nidadas que eclosionaron más temprano, nidadas más grandes y un mayor número de nidadas. Estos parches densos parecieron conferir protección ante la acción del viento y las olas, las causas principales de la pérdida temprana de nidos. Nuestros análisis de muestras de sangre no mostraron estructura genética en la población, ni una tendencia a que los individuos vecinos estuvieran estrechamente relacionados. Sin embargo, las parejas a menudo regresaron a la misma área y la misma situación de anidación en años subsecuentes. La flexibilidad comportamental de las parejas de P. grisegena para anidar en grupos en las áreas con mejor hábitat les podría permitir reproducirse exitosamente en hábitats de humedales emergentes e impredecibles.