Notostraca Research Articles

Discriminating between the effects of founding events and reproductive mode on the genetic structure of Triops populations (Branchiopoda: Notostraca).

Crustaceans that initially colonize a freshwater temporary pond can strongly bias the subsequent genetic composition of the population, causing nearby populations to be genetically distinct. In addition, these crustaceans have various reproductive modes that can influence genetic differentiation and diversity within and between populations. We report on two species of tadpole shrimp, Triops newberryi and Triops longicaudatus “short”, with different reproductive modes. Reproduction in the tadpole shrimp can occur clonally (parthenogenesis), with self fertilization (hermaphroditism), or through outcrossing of hermaphrodites with males (androdioecy). For all these reproductive modes, population genetic theory predicts decreased genetic diversity and increased population differentiation. Here we use mitochondrial control region (mtCR) sequences and nuclear microsatellite loci to determine if the difference in reproductive mode affects the high genetic structure typical of persistent founder effects. Previous authors indicated that T. newberryi is androdioecious because populations are composed of hermaphrodites and males, and T. longicaudatus “short” is hermaphroditic or parthenogenetic because males are absent. In our data, T. newberryi and T. longicaudatus “short” populations were highly structured genetically over short geographic distances for mtCR sequences and microsatellite loci (T. newberryi: ΦST = 0.644, F ST = 0.252, respectively; T. l. “short”: invariant mtCR sequences, F ST = 0.600). Differences between the two Triops species in a number of diversity measures were generally consistent with expectations from population genetic theory regarding reproductive mode; however, three of four comparisons were not statistically significant. We conclude the high genetic differentiation between populations is likely due to founder effects and results suggest both species are composed of selfing hermaphrodites with some level of outcrossing; the presence of males in T. newberryi does not appreciably reduce inbreeding. We cannot exclude the possibility that males in T. newberryi are non-reproductive individuals and the two species have the same mating system.

Current knowledge of the South East Asian large branchiopod Crustacea (Anostraca, Notostraca, Laevicaudata, Spinicaudata, Cyclestherida)

The large branchiopod crustaceans (fairy shrimp, tadpole shrimp and clam shrimp) of South East (SE) Asia have only recently been examined with scientific vigor. Although more than 70 species have been reported for Asia and Indonesia, only six native taxa and one introduced species are known from SE Asia. The majority of records are from Thailand, with fewer than three records each from Laos, Myanmar, Malaysia, Cambodia, Vietnam, and Singapore. Eleven genera reported from adjacent regions may potentially occur in SE Asia. Spinicaudatan clam shrimp have been collected from Thailand and Cambodia, but have not been identified beyond genus. Four of the five native fairy shrimp species are endemic to SE Asia. The limited distributions of these few taxa suggest that this region has a distinct branchiopod fauna. In addition, we present new species and records of clam shrimp from Thailand. Future survey efforts will undoubtedly reveal additional taxa.

Uncovering Ultrastructural Defences in Daphnia magna – An Interdisciplinary Approach to Assess the Predator-Induced Fortification of the Carapace

The development of structural defences, such as the fortification of shells or exoskeletons, is a widespread strategy to reduce predator attack efficiency. In unpredictable environments these defences may be more pronounced in the presence of a predator. The cladoceran Daphnia magna (Crustacea: Branchiopoda: Cladocera) has been shown to develop a bulky morphotype as an effective inducible morphological defence against the predatory tadpole shrimp Triopscancriformis (Crustacea: Branchiopoda: Notostraca). Mediated by kairomones, the daphnids express an increased body length, width and an elongated tail spine. Here we examined whether these large scale morphological defences are accompanied by additional ultrastructural defences, i.e. a fortification of the exoskeleton. We employed atomic force microscopy (AFM) based nanoindentation experiments to assess the cuticle hardness along with tapping mode AFM imaging to visualise the surface morphology for predator exposed and non-predator exposed daphnids. We used semi-thin sections of the carapace to measure the cuticle thickness, and finally, we used fluorescence microscopy to analyse the diameter of the pillars connecting the two carapace layers. We found that D . magna indeed expresses ultrastructural defences against Triops predation. The cuticle in predator exposed individuals is approximately five times harder and two times thicker than in control daphnids. Moreover, the pillar diameter is significantly increased in predator exposed daphnids. These predator-cue induced changes in the carapace architecture should provide effective protection against being crushed by the predator’s mouthparts and may add to the protective effect of bulkiness. This study highlights the potential of interdisciplinary studies to uncover new and relevant aspects even in extensively studied fields of research.

Multiple global radiations in tadpole shrimps challenge the concept of 'living fossils'.

‘Living fossils’, a phrase first coined by Darwin, are defined as species with limited recent diversification and high morphological stasis over long periods of evolutionary time. Morphological stasis, however, can potentially lead to diversification rates being underestimated. Notostraca, or tadpole shrimps, is an ancient, globally distributed order of branchiopod crustaceans regarded as ‘living fossils’ because their rich fossil record dates back to the early Devonian and their morphology is highly conserved. Recent phylogenetic reconstructions have shown a strong biogeographic signal, suggesting diversification due to continental breakup, and widespread cryptic speciation. However, morphological conservatism makes it difficult to place fossil taxa in a phylogenetic context. Here we reveal for the first time the timing and tempo of tadpole shrimp diversification by inferring a robust multilocus phylogeny of Branchiopoda and applying Bayesian divergence dating techniques using reliable fossil calibrations external to Notostraca. Our results suggest at least two bouts of global radiation in Notostraca, one of them recent, so questioning the validity of the ‘living fossils’ concept in groups where cryptic speciation is widespread.

Variability in the functional role of Arctic charr Salvelinus alpinus as it relates to lake ecosystem characteristics

This study investigated how dietary habits vary with lake characteristics in a fish species that exhibits extensive morphological and ecological variability, the Arctic charr Salvelinus alpinus. Iceland is a hotspot of geological activity, so its freshwater ecosystems vary greatly in physical and chemical attributes. Associations of dietary items within guts of charr were used to form prey categories that reflect habitat-specific feeding behavior. Six prey categories were defined and dominated by snails (Radix peregra), fish (Gasterosteus aculeatus), tadpole shrimp (Lepidurus arcticus), chironomid pupae, pea clam (Pisidium spp.), and the cladoceran Bosmina sp.. These reflected different combinations of feeding in littoral stone, offshore benthic, and limnetic habitats. Certain habitat-specific feeding strategies consistently occurred alongside each other within lakes. For example, zooplanktivory occurred in the same lakes as consumption from offshore habitats; piscivory occurred in the same lakes as consumption from littoral benthic habitats. Redundancy analyses (RDA) were used to investigate how lake environment was related to consumption of different prey categories. The RDA indicated that piscivory exhibited by Arctic charr was reduced where brown trout were abundant and lakes were shallow, greater zooplanktivory occurred at lower latitudes and under decreased nutrient but higher silicon dioxide concentrations, and benthic resource consumption was associated with shallower lakes and higher altitudes. This study showed that trends previously observed across fish species were supported at the intraspecific level, indicating that a single species with flexible dietary habits can fill functional roles expected of multiple species in more diverse food webs.

High lability of sexual system over 250 million years of evolution in morphologically conservative tadpole shrimps.

BackgroundSexual system is a key factor affecting the genetic diversity, population structure, genome structure and the evolutionary potential of species. The sexual system androdioecy – where males and hermaphrodites coexist in populations – is extremely rare, yet is found in three crustacean groups, barnacles, a genus of clam shrimps Eulimnadia, and in the order Notostraca, the tadpole shrimps. In the ancient crustacean order Notostraca, high morphological conservatism contrasts with a wide diversity of sexual systems, including androdioecy. An understanding of the evolution of sexual systems in this group has been hampered by poor phylogenetic resolution and confounded by the widespread occurrence of cryptic species. Here we use a multigene supermatrix for 30 taxa to produce a comprehensive phylogenetic reconstruction of Notostraca. Based on this phylogenetic reconstruction we use character mapping techniques to investigate the evolution of sexual systems. We also tested the hypothesis that reproductive assurance has driven the evolution of androdioecy in Notostraca.ResultsCharacter mapping analysis showed that sexual system is an extremely flexible trait within Notostraca, with repeated shifts between gonochorism and androdioecy, the latter having evolved a minimum of five times. In agreement with the reproductive assurance hypothesis androdioecious notostracans are found at significantly higher latitudes than gonochoric ones indicating that post glacial re-colonisation may have selected for the higher colonisation ability conferred by androdioecy.ConclusionsIn contrast to their conserved morphology, sexual system in Notostraca is highly labile and the rare reproductive mode androdioecy has evolved repeatedly within the order. Furthermore, we conclude that this lability of sexual system has been maintained for at least 250 million years and may have contributed to the long term evolutionary persistence of Notostraca. Our results further our understanding of the evolution of androdioecy and indicate that reproductive assurance is a recurrent theme involved in the evolution of this sexual system.

Microsatellites for North American species of Triops (Branchiopoda: Notostraca)

We used 454 pyrosequencing to discover more than 3000 candidate microsatellite markers for three putative species of tadpole shrimp in the genus Triops. We selected 42 of these candidates, consisting of 12 systems identified in Triops longicaudatus “long,” 16 identified in Triops longicaudatus “short,” and 14 identified in Triops newberryi. Out of these potential loci we identified between five and eight polymorphic microsatellite systems per “species.” By implementing an additional cross amplification experiment with the microsatellite markers mentioned above and further testing 15 microsatellite markers initially identified in the European Triops cancriformis, we successfully transferred 17 microsatellite markers within the three North American Triops and found three new microsatellites, one polymorphic, that will amplify in T. cancriformis. The reported genetic methods are powerful molecular tools to acquire new detailed information at the interface of ecology, evolution and development, which will facilitate understanding phenotypic plasticity, breeding systems, and adaptation in this taxonomically-confusing group of “living fossils.”

Range-wide and local drivers of genetic structure in an endangered California vernal pool endemic crustacean

Phylogeographic and landscape genetic structure were studied for the endangered vernal pool tadpole shrimp (Lepidurus packardi) with the goal of identifying genetic units below the species level and understanding local patterns of gene flow. A portion of the mitochondrial cytochrome c oxidase I (COI) gene was sequenced from 208 individuals from throughout the range of the species (26 sites). The COI gene indicated the presence of five distinct clades or lineages within L. packardi. A majority of these clades were found in the southern portion of the species’ range. Sequence divergence between these clades was exceptionally high and historical demographic analysis did not indicate any changes in female effective population sizes. The divergent clades represent a pattern that is observed in other notostracan species. The presence of a single clade with a wide geographic distribution (northern portion of the study) suggests this area has experienced historical/recent gene flow, and is in contrast to the southern portion of the species range where divergent clades lie in close geographic proximity to one another. Amplified fragment length polymorphisms (AFLPs) and COI sequence data of 125 individuals samples from 14 pools revealed a pattern of isolation by distance across a 17 km span. The significant isolation by distance pattern suggests equilibrium processes override non-equilibrium processes (strong founder effects) at smaller geographic scales. These results suggest greater conservation attention to the southern portion of the species range (San Joaquin Valley) and the continuance of management practices that may assist in mitigating migration at local scales.

Review of a New Pest of Rice, Tadpole Shrimp (Notostraca: Triopsidae), in the Midsouthern United States and a Winter Scouting Method of Rice Fields for Preplanting Detection

Tadpole shrimp, Triops longicaudatus (LeConte), are a species of temporary, freshwater habitats. Tadpole shrimp are pests of rice production systems in California and have recently been found impacting Missouri rice fields and present in northeastern Arkansas rice fields. These impacts take the form of stand reduction by direct feeding on seedlings and uprooting seedlings during foraging. In addition, their foraging behavior causes water to become muddy, which reduces light penetration to submerged seedlings and consequently delays the development of the rice plant. Once rice is past the seedling stage, tadpole shrimp may be beneficial. This article provides information on the life cycle of tadpole shrimp, describes a new method for scouting for tadpole shrimp in rice fields, and provides scouting results and management implications.

Fighting parasites and predators: How to deal with multiple threats?

BackgroundAlthough inducible defences have been studied extensively, only little is known about how the presence of parasites might interfere with these anti-predator adaptations. Both parasites and predators are important factors shaping community structure and species composition of ecosystems. Here, we simultaneously exposed Daphnia magna to predator cues (released by the tadpole shrimp, Triops, or by a fish) and spores of the yeast parasite Metschnikowia sp. to determine how life history and morphological inducible defences against these two contrasting types of predators are affected by infection.ResultsThe parasite suppressed some Triops-induced defences: Daphnia lost the ability to produce a greater number of larger offspring, a life-history adaptation to Triops predation. In contrast, the parasite did not suppress inducible defences against fish: induction (resulting in smaller body length of the mothers as well as of their offspring) and infection acted additively on the measured traits. Thus, fish-induced defences may be less costly than inducible defences against small invertebrate predators like Triops; the latter defences could no longer be expressed when the host had already invested in fighting off the parasite.ConclusionsIn summary, our study suggests that as specific inducible defences differ in their costs, some might be suppressed if a target prey is additionally infected. Therefore, adding parasite pressure to predator–prey systems can help to elucidate the costs of inducible defences.

28S junctions and chimeric elements of the rDNA targeting non-LTR retrotransposon R2 in crustacean living fossils (Branchiopoda, Notostraca)

The 28S rRNA genes of several metazoans are interrupted by site-specific targeting non-LTR retrotransposons, such as R2. R2 elements have been deeply analyzed but aspects of their retrotransposition mechanism and the origin of the wide diversity observed are still debated. We characterized six new R2 lineages in four tadpole shrimp species (Notostraca), samples deriving from a parthenogenetic population of Triops cancriformis (R2Tc_it) and from bisexual Lepidurus populations of L. lubbocki (R2Ll), L. couesii (R2LcA, R2LcB, R2LcC) and L. arcticus (R2La). All elements fit the canonical R2 structure but R2Ll which turned out to be a chimera with an additional ORF originating from another R2. Consistently with data on LINEs, R2Ll could be the result of recombination due to reverse transcriptase template jump. The analysis of 28S/R2 5′ end junctions further suggests aberrant homologous recombination, as observed in RNA viruses.

Toward a global phylogeny of the "living fossil" crustacean order of the Notostraca.

Tadpole shrimp (Crustacea, Notostraca) are iconic inhabitants of temporary aquatic habitats worldwide. Often cited as prime examples of evolutionary stasis, surviving representatives closely resemble fossils older than 200 mya, suggestive of an ancient origin. Despite significant interest in the group as ‘living fossils’ the taxonomy of surviving taxa is still under debate and both the phylogenetic relationships among different lineages and the timing of diversification remain unclear. We constructed a molecular phylogeny of the Notostraca using model based phylogenetic methods. Our analyses supported the monophyly of the two genera Triops and Lepidurus, although for Triops support was weak. Results also revealed high levels of cryptic diversity as well as a peculiar biogeographic link between Australia and North America presumably mediated by historic long distance dispersal. We concluded that, although some present day tadpole shrimp species closely resemble fossil specimens as old as 250 mya, no molecular support was found for an ancient (pre) Mesozoic radiation. Instead, living tadpole shrimp are most likely the result of a relatively recent radiation in the Cenozoic era and close resemblances between recent and fossil taxa are probably the result of the highly conserved general morphology in this group and of homoplasy.

Morphologic and Genetic Variation in Triops (Branchiopoda: Notostraca) from Ephemeral Waters of the Northern Chihuahuan Desert of North America

Abstract Tadpole shrimp are known to be important animals in the ecology of ephemeral wetlands. In the northern Chihuahuan Desert of North America, the tadpole shrimp fauna is composed of possibly three species in the genus Triops, which have variously been referred to as species, subspecies, and intraspecific variation. Our results support the presence of three morphologically distinct forms, which will be referred to herein as T. newberryi, T. longicaudatus “short,” and T. longicaudatus “long.” We report analyses of Triops spp. sampled in summer 2008 from 14 natural playas and man-made flood retention ponds. Data were recorded on meristic counts and quantitative measurements of morphological features. We also sequenced portions of the mitochondrial COI and ND1 genes for 72 shrimp, including individuals from all three morphological forms and for multiple ponds for each form where possible. The three forms were morphologically distinct for multiple characters and molecular analyses showed large difference...

Growing large and bulky in the presence of the enemy: Daphnia magna gradually switches the mode of inducible morphological defences

Summary 1. Phenotypic plasticity in defensive traits has been proven to be effective in ecosystems with frequently changing predator regimes. However, if a single dominant predator exerts predation pressure on each ontogenetic stage, prey should adapt by developing defensive traits for each life stage within a cost-benefit framework. This may require a change of defensive mechanisms between juvenile and adult life stages. 2. In this study, we examined the morphological defences of the cladoceran Daphnia magna Strauss induced by the tadpole shrimp Triops cancriformis Bosc. We tested for the induction of morphological defences by directly exposing daphnids to the predator and conducted a life span experiment to determine if the expression of the induced morphological defences varies throughout the life span of D. magna. In addition, we studied the adaptive value, i.e. the effectiveness, of the Triops-induced morphological defences in D. magna by conducting predation trials. 3. We found that, in D. magna, the expression of an array of inducible morphological defences, which act synergistically to provide effective protection, changes during the daphnids lifetime in response to the tadpole shrimp T. cancriformis. This gradual switch in the protective function of single traits between juvenile and adult stages represents a novel functionality and complexity of inducible defences. Both direct contact with the predator and chemical cues (kairomones) released by T. cancriformis induce an increased body length, body width and an elongation of the tail spine in D. magna. This study is the first to show that kairomones released by a predator can induce ‘bulkiness’ as a defensive mechanism in Daphnia. Finally, we demonstrate the effectiveness of the Triops-induced morphological defences (i.e. an elongated tail spine and increased bulkiness) by conducting predation trials. 4. Our study provides rare evidence for morphological defences in D. magna, and in addition shows that prey species gradually switch between plastic traits to maintain effective defences throughout their entire lifetime. Hence, our results help to shed light on the mechanisms governing phenotypic plasticity within natural populations.

Ecosystem-level effects of bioturbation by the tadpole shrimp Lepidurus packardi in temporary pond mesocosms

An example of ecosystem engineering gaining attention in aquatic systems is bioturbation, the disruption of sediment at the water–sediment interface due to burrowing and foraging. One consequence of bioturbation can be increased turbidity from suspended sediment, which generally inhibits macrophyte growth and reduces ecosystem functioning. Conversely, bioturbation may promote invertebrate species richness by unearthing dormant cysts. Temporary-pond crustaceans are not widely regarded as agents of bioturbation, but on the basis of aquaria observations we hypothesized that certain taxa can disturb the sediment and create highly turbid water. We tested this hypothesis by removing crustaceans from mesocosms lined with vernal pool soil. Compared to this treatment group, mesocosms containing crustaceans had extremely high turbidity from suspended sediment, as well as reduced total macrophyte cover. We also found clear compositional differences in macrophyte communities between treatments, driven largely by differences in water physicochemistry, including turbidity. Regression analysis linked most of the bioturbation to the endangered notostracan Lepiduruspackardi Simon 1886, which was a strong predictor of turbidity in our mesocosms. We also found a trend toward increased crustacean species richness in our mesocosms in the presence of this taxon. An analysis of published data from King et al. (1996) suggests that this trend may extend to natural vernal pools. Overall, our results suggest that L. packardi may have large effects on vernal pool communities, likely mediated in part through its disturbing of the sediment.

Phylogeny, molecular ecology and taxonomy of southern Iberian lineages of Triops mauritanicus (Crustacea: Notostraca)

We investigated the phylogeography of the main lineages in the tadpole shrimp Triops mauritanicus Ghigi in the south-western Iberian Peninsula, using mitochondrial 12S and 16S rDNA sequences. Our results indicate that a fourth, hitherto unknown main phylogenetic lineage occurs in Iberia, so that in total, the species is divided into six distinct clades, comprising T. m. mauritanicus, T. m. simplex Ghigi, and four as yet unnamed lineages that appear to be endemic to Iberia. Percentages of sequence divergence among the main clades in T. mauritanicus reach the range reported for recognized species in other notostracan lineages. A thorough morphological investigation also revealed that the differentiation among these lineages is higher than previously thought, and that populations of three of the main clades within T. mauritanicus can be reliably separated from each other and from the remaining lineages based on the morphology of adult males. The remaining clades also show a significant level of morphological differentiation, but include a certain proportion of populations for which the additional application of molecular methods is needed for a reliable determination. The geographic distributions of 12S haplotypes are indicative of frequent dispersal events and gene flow among populations belonging to the same main lineage, but give no evidence of recent migration events among different main lineages, suggesting that there is no gene flow among the latter. Our data thus suggest that the six main lineages within T. mauritanicus represent distinct species. We therefore describe the Iberian lineages as T. baeticus Korn n. sp., T. emeritensis Korn & Pérez-Bote n. sp., T. gadensis Korn & García-de-Lomas n. sp., and T. vicentinus Korn, Machado, Cristo & Cancela da Fonseca n. sp., and reinstate T. simplex Ghigi to full species status. Our data confirm the general, previously recognized pattern of a lower dispersal probability in gonochoric Triops taxa. However, we found evidence that passive dispersal in Triops may be further complicated by a strong habitat dependence of dispersal probability, mediated by prevailing dispersal vectors.

Spectral sensitivity of light-induced hatching and expression of genes mediating photoreception in eggs of the Asian tadpole shrimp Triops granarius

Embryonic development of the Asian tadpole shrimp Triops granarius is arrested at an early stage of organogenesis under darkness, whereas exposure to light resumes its development and promotes hatching. To better understand the photoreception systems involved in the light-induced hatching of T. granarius eggs, we examined the spectral sensitivity of hatching and the expression of genes involved in photoreception (opsins) and phototransduction (arrestin 2). Hatching of eggs is induced by light of broad wavelengths, from ultraviolet (UV) (395nm) to red (660nm), suggesting that visual pigments are involved in photoreception. Next, we focused on opsins which are protein moieties of visual pigments and arrestin 2 which is involved in a phototransduction cascade. Transcripts of several opsin genes and the arrestin 2 gene were detected in T. granarius eggs. Thus, we present a possibility that T. granarius eggs use visual pigments for light perception in light-induced hatching.

Fairy, tadpole, and clam shrimps (Branchiopoda) in seasonally inundated clay pans in the western Mojave Desert and effect on primary producers

BackgroundFairy shrimps (Anostraca), tadpole shrimps (Notostraca), clam shrimps (Spinicaudata), algae (primarily filamentous blue-green algae [cyanobacteria]), and suspended organic particulates are dominant food web components of the seasonally inundated pans and playas of the western Mojave Desert in California. We examined the extent to which these branchiopods controlled algal abundance and species composition in clay pans between Rosamond and Rogers Dry Lakes. We surveyed branchiopods during the wet season to estimate abundances and then conducted a laboratory microcosm experiment, in which dried sediment containing cysts and the overlying algal crust were inundated and cultured. Microcosm trials were run with and without shrimps; each type of trial was run for two lengths of time: 30 and 60 days. We estimated the effect of shrimps on algae by measuring chlorophyll content and the relative abundance of algal species.ResultsWe found two species of fairy shrimps (Branchinecta mackini and B. gigas), one tadpole shrimp (Lepidurus lemmoni), and a clam shrimp (Cyzicus setosa) in our wet-season field survey. We collected Branchinecta lindahli in a pilot study, but not subsequently. The dominant taxa were C. setosa and B. mackini, but abundances and species composition varied greatly among playas. The same species found in field surveys also occurred in the microcosm experiment. There were no significant differences as a function of experimental treatments for either chlorophyll content or algal species composition (Microcoleus vaginatus dominated all treatments).ConclusionsThe results suggest that there was no direct effect of shrimps on algae. Although the pans harbored an apparently high abundance of branchiopods, these animals had little role in regulating primary producers in this environment.

Branchiopods (Anostraca, Notostraca) from Protected Areas of Western Montana

Fairy shrimp (order Anostraca) and tadpole shrimp (order Notostraca), members of the crustacean class Branchiopoda, are important components of many freshwater and saline wetlands (Dodson and Frey 2001, Brendonk et al. 2008). These animals are especially common in seasonal wetlands, where populations are maintained by banks of resting eggs that may remain dormant for decades or longer (Brendonck 1996). Both orders are distributed worldwide (Brendonk et al. 2008). Fairy shrimp are more prone to endemism, with several species listed as threatened or endangered, and new species are described frequently (e.g., Rogers 2006, Rogers et al. 2006). Most of the comparatively few species of tadpole shrimp are widespread, but Lepidurus packardi is endemic to central California and the recently-described L. cryptus is restricted to the Pacific Northwest (Rogers 2001).

Genetic variability and reproductive modalities in European populations of Triops cancriformis (Crustacea, Branchiopoda, Notostraca)

The genetic variability of the tadpole shrimp Triops cancriformis was studied through the analysis of 12 nuclear microsatellite loci. Seven of these loci were newly obtained from an enriched genomic library using a trinucleotide biotinylated probe (CAG)11. The study was conducted on six populations that previous genetic studies demonstrated to be poorly differentiated, notwithstanding their different reproductive strategies (gonochorism, parthenogenesis, androdioecy). The newly isolated microsatellite loci showed a higher genetic variability than those previously isolated, probably in relation with their different motif structure. Different patterns of variability were scored at both locus and population levels, the latter probably being caused by the different reproductive modalities. Wright's F‐statistics, AMOVA, and ISOLDE analyses showed that populations are well structured and isolated, again as a result of different reproductive modalities, but also of geographic distances and low migration flow.