Andrea Gandolfi

Persistence of asexuality through mixed reproduction in Eucypris virens (Crustacea, Ostracoda).

The ostracod species Eucypris virens exhibits geographical parthenogenesis, with rare sexual populations in southern Europe and widespread asexual populations elsewhere. DNA sequence data from the nuclear ITS1 and mitochondrial COI regions have been used to estimate genetic variabilities and reconstruct phylogenies. The observed divergence was exceptionally high, with intraspecific maxima of 10.3% (ITS1) and 20.9% (COI) among European lineages, levels reported for interspecific comparisons of other taxa. Phylogenetic reconstructions reveal multiple origins of asexual clones from sexual populations. However, we argue that such data can only provide a lower limit on the number of origins of asexual reproduction, and an upper limit on the age of asexual lineages. Congruence between gene trees for different loci can provide support for the inference of long-term apomictic reproduction. Nuclear and mitochondrial data differ in their placement of some asexual clones, possibly indicating that genetic exchange has taken place between sexual and asexual lineages. Such intraspecific hybridization is one route to combine the benefits of both reproductive modes, and it might explain how asexuality managed to persist in E. virens even in long, evolutionary terms.

Intraindividual and intraspecies variability of ITS1 sequences in the ancient asexual Darwinula stevensoni (Crustacea: Ostracoda)

The lack of variability in ITS regions within individuals and within species has been explained as the result of concerted evolution. In fact, many examples of intraindividual variation in the ITS regions have been reported. Here we report evidence of within-individual variation of the ITS1 region in the obligate parthenogenetic species Darwinula stevensoni. We analysed 46 clones obtained from 12 individuals of D. stevensoni, from three Italian sites and one site in Luxembourg. Seven nucleotides out of 366 were variable. Most variability (80%) was found among clones within individuals, and the remainder of the variability was observed among individuals. No difference was found among populations or between habitats. The low intraspecific variability and the observation of recombinant molecules are evaluated in light of the relevant literature. The high percentage of variation within individuals and the occurrence of recombination without meiosis are discussed by considering the ancient asexual ‘status’ of the species.

Phylogenetic analysis of Macrobiotidae (Eutardigrada, Parachela): a combined morphological and molecular approach

Combined analyses of morphological and molecular data were used to resolve phylogenetic relationships within Macrobiotidae (Eutardigrada). Morphological data were analysed using a cladistic approach with a matrix comprising 15 taxa with 17 characters to obtain a phylogenetic reconstruction. Molecular data were obtained by sequencing the cytochrome c oxidase subunit I gene in seven species of Macrobiotidae and one of Eohypsibiidae (used as outgroup). The morphological character defining the family, symmetrical claw on each leg, turns out to be plesiomorphic. Moreover, neither morphological nor molecular analyses supports a monophyletic clade for the subfamily Macrobiotinae, whereas both support a well defined evolutionary line (Murrayinae) within the family. We propose elevating the latter to family level (Murrayidae) while temporarily retaining as valid the family Macrobiotidae (amending its diagnosis, including within it only Macrobiotinae). Murrayidae opens an interesting evolutionary prospective, because the entire line has differentiated without sexual reproduction, constituting an example of evolution of asexual lineages.

Egg diapause and clonal structure in parthenogenetic populations of Heterocypris incongruens (Ostracoda)

A study of the clonal structure of parthenogenetic populations of Heterocypris incongruens from rice-fields in Northern Italy carried out over two-year period is summarized. Significantly different levels of genetic polymorphism were found among populations. The coexistence of different electrophoretic clones and similar patterns of clonal seasonal succession have been observed in at least two different rice fields. The clone or group of clones present in fall, winter and early spring is substituted by other multilocus genotypes in late spring and summer. Different egg diapause induction mechanisms drive the clonal substitution in different clones. Here we report a new laboratory experiment designed to test the effect of temperature and photoperiod found in winter (12 °C 8:16 L:D), spring (24°C 12:12 L:D) and summer (28°C 16:8 L:D) conditions on the deposition and hatching of diapausal eggs in different multilocus genotypes. Clones respond in a way compatible with their sustained presence in the field in different seasons.

Heterocypris (Crustacea: Ostracoda) from the Isole Pelagie (Sicily, Italy): Hatching phenology of resting eggs

Abstract The hatching phenology of Heterocypris resting eggs was investigated in laboratory controlled conditions. Microcosms were set up using dry sediments from three freshwater temporary ponds (Cavallo Bianco, Aria Rossa and Vallone della Forbice) of the island of Lampedusa (Sicily) and kept at different temperatures and photoperiods. Hatching time was modulated by different environmental factors. Initial anoxia, induced by the decomposition of the sediments organic matter, delayed hatching in the microcosm set up with sediments from Vallone della Forbice which has a high organic matter content. Temperature and photoperiod in isolation did not seem to affect hatching significantly, while their interaction did. The coexistence of two female morphotypes with different reproductive modes could not be explained by a differential response to temperature and photoperiod. Coexistence was probably due to disturbance events, such as drying up of ephemeral pools. Some resting eggs (in different percentages in different microcosms) hatched only after a second hydration (after microcosms dried up). Hatching phenology is discussed in relation to genetic differences in egg responsiveness to hatching stimuli and to a bet‐hedging strategy in a year‐to‐year highly variable environment such as the ephemeral ponds of Lampedusa.

Salinity tolerance of Darwinula stevensoni (Crustacea, Ostracoda)

Abstract Relatively few laboratory tests of the effect of salinity on ostracod species exist. Here we report the results of acute and chronic bioassay tests to study the effects of six different salinities (deionised water, 22, 120, 922, 9600 μS‐cm‐1, and marine water) on survival and reproductive parameters (number of eggs and hatching). The test was carried out on individuals of Darwinula stevensoni from different localities characterised by different salinity (from 111 μS‐cm‐1 at Monate, Italy, to 3440 μS‐cm‐1 at Hollandersgatkreek, Belgium). Individuals belonging to three life cycle stages were used (pre‐adult, adult, ovigerous). Surprisingly low mortality was observed in all treatments and for all life stages, independently of population of provenance. Only in marine water was mortality noticeable although reproductive parameters were hardly affected. Given the high genetic homogeneity of the species, our results seem to support the hypothesis of high phenotypic plasticity for salinity tolerance in D. stevensoni.

Heterocypris (Crustacea, Ostracoda) from the Isole Pelagie (Sicily, Italy): Population genetics

Abstract We analysed the genetic structure of five populations of Heterocypris from small temporary ponds on the islands of Lampedusa and Linosa (Isole Pelagie, South of Sicily) where we have observed two different adult females morphotypes. The most genetically‐differentiated population, Cavallo Bianco, is made up by females only showing a morphology typical of H. incongruens. All other populations, from both islands, are bisexual and their adult females present a lobe‐like expansion of the selvage (lamella hyalina) on the posterior margin of the left valve. In one locality, Vallone della Forbice, both female morphotypes occur. The analyses of individuals raised in the laboratory microcosms confirmed the genetic differentiation between morphotypes and their different reproductive modes. The adult females without a lamella hyalina are apomictic, whereas females with this feature are am‐phimictic. Genetic differentiation of the two female morphotypes is compatible with the hypothesis that the two morphotypes represent different species of Heterocypris although, such a morphological differentiation is not unusual in populations of the same parthenogenetic species, and we found evidence of hybridisation between males and parthenogenetic females.

Re-evaluation of three related species of the genus Branchipus Schaeffer, 1766 (Branchiopoda: Anostraca) by morphological and genetic analyses

The systematics of the genus Branchipus Schaeffer, 1766 (Branchiopoda: Anostraca) has been based on morphological taxonomy only and thus remains poorly resolved or, at best, questionable. Among the seven species described in the genus, a detailed comparison of Branchipus schaefferi Fischer, 1834 with both B. pasai Cottarelli, 1969 and B. visnyai Kertesz, 1956 is necessary to clarify their status. The use of morphological characters and, in particular, the appearance of second antennae and antennal appendages in males still cause taxonomic confusion among these three species. Herein, we use two molecular markers (allozymes and mtDNA sequences) and scanning electron microscopy to determine consistent differentiation patterns and test for congruence with the shape of the genus diagnostic trait, the frontal shield. We analyzed specimens belonging to 11 populations and covering a wide geographic distribution: B. pasai from Italy, B. schaefferi from Spain and Morocco, and B. visnyai from Malta and Italy. The extent of genetic divergence among populations evaluated by allozyme variation at 13 enzymatic loci and a mtDNA marker suggests the existence of different taxa and the absence of gene flow between them. The molecular results, together with morphological observations, did not provide support for the existing subdivision in the three nominal species B. pasai , B. schaefferi , and B. visnyai and places the validity of previously proposed taxonomic groups in discussion.

Low genetic variability in the ancient asexual ostracod Darwinula stevensoni

Abstract The genetic variability and genotypic (clonal) structure of 34 populations of the ancient asexual Darwinula stevensoni was assayed using allozymes. In this study of 1964 individuals from nine countries in Europe, Israel and South Africa, little genetic variability was found. Out of nine enzyme systems only two revealed sufficient enzyme activity to produce consistent zymograms. Only one locus (Gpi 1) was polymorphic, and only in 14 populations: 83% of all screened individuals from all over Europe shared the same genotype. There was no significant deviation of Hardy—Weinberg equilibrium in 10 out of 14 polymorphic populations, a result unexpected in an asexual species. Among possible causes we discuss null alleles whose presence would make genotype frequencies discordant with Hardy‐Weinberg equilibrium. Allele and genotype frequencies were different in lakes and rivers (heterozygotes were dominant in rivers, homozygotes in lakes). Habitat choice might be driven by a different starvation tolerance by different genotypes.

DESCRIPTION OF SOME MENDELIAN-INHERITED RAPD MARKERS FOR HETEROCYPRIS INCONGRUENS (OSTRACODA)

Abstract The inheritance of 65 RAPD markers obtained from 15 arbitrary primers has been tested in two sexual populations of the ostracod Heterocypris incongruens. The markers were selected using the criteria of reproducibility and banding-pattern clarity from the amplification products yielded by 40 primers. The expected inheritance model was inferred from the progeny banding pattern and tested with the appropriate statistics. All 65 markers followed a Mendelian pattern of inheritance and indicated that the Spanish and the Italian populations studied are genetically different.