Anna K. Hundsdoerfer

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.

Sister species within the Triops cancriformis lineage (Crustacea, Notostraca)

We investigated the phylogenetic relationships among the three presently recognized subspecies of the tadpole shrimp, Triops cancriformis, using mitochondrial 16S and 12S rDNA sequences. Our results indicate that the taxon is divided into two distinct lineages. One lineage is formed of T. c. cancriformis populations and samples from northern Spain that had been classified as T. c. simplex in the most recent literature. The second lineage comprises all populations of T. c. mauritanicus and northern African populations of T. c. simplex. These two main lineages separated 2.3 to 8.9 million years ago, based on the range of inferred molecular clocks recognized for crustacean mtDNA sequence divergence. Percentages of divergence are in the range reported for recognized species in other notostracan lineages and we therefore propose to recognize them as two species, Triops cancriformis and Triops mauritanicus. The latter would comprise two subspecies in northern Africa, one consisting of the Moroccan populations of the former T. c. mauritanicus, the other comprising the African populations of the former T. c. simplex. It also includes three as‐yet unnamed lineages. A comparison of morphological characters with the molecular data revealed that the former T. c. simplex cannot be reliably separated from T. c. cancriformis, using morphological characters that have hitherto been used to distinguish among subspecies of T. cancriformis. Our investigation is the first to demonstrate the presence of T. c. cancriformis in Africa (Tunisia). The genetic haplotypes of these populations are identical with haplotypes also occurring in Central and Western Europe, as well as in Sicily. Therefore, we hypothesize that the African populations of T. c. cancriformis represent a result of repeated long‐distance dispersal across the Mediterranean Sea.

Molecular phylogeny of the Notostraca

We used a combined analysis of one nuclear (28S rDNA) and three mitochondrial markers (COI, 12S rDNA, 16S rDNA) to infer the molecular phylogeny of the Notostraca, represented by samples from the six continents that are inhabited by this group of branchiopod crustaceans. Our results confirm the monophyly of both extant notostracan genera Triops and Lepidurus with good support in model based and maximum parsimony analyses. We used branchiopod fossils as a calibration to infer divergence times among notostracan lineages and accounted for rate heterogeneity among lineages by applying relaxed-clock models. Our divergence date estimates indicate an initial diversification into the genera Triops and Lepidurus in the Mesozoic, most likely at a minimum age of 152.3-233.5 Ma, i.e., in the Triassic or Jurassic. Implications for the interpretation of fossils and the evolution of notostracan morphology are discussed. We further use the divergence date estimates to formulate a biogeographic hypothesis that explains distributions of extant lineages predominantly by overland dispersal routes. We identified an additional hitherto unrecognised highly diverged lineage within Lepidurus apus lubbocki and three additional previously unknown major lineages within Triops. Within T. granarius we found deep differentiation, with representatives distributed among three major phylogenetic lineages. One of these major lineages comprises T. cancriformis, the T. mauritanicus species group and two hitherto unrecognised T. granarius lineages. Samples that were morphologically identified as T. granarius diverged from the most basal nodes within this major lineage, and divergence dates suggested an approximate age of 23.7-49.6 Ma for T. cancriformis, indicating the need for a taxonomic revision of Triassic and Permian fossils that are currently attributed to the extant T. cancriformis. We thus elevate T. cancriformis minor to full species status as Triops minorTrusheim, 1938 and include in this species the additional Upper Triassic samples that were attributed to T. cancriformis. We further elevate T. cancriformis permiensis to full species status as Triops permiensisGand et al., 1997.

A revised molecular phylogeny of the globally distributed hawkmoth genus Hyles (Lepidoptera: Sphingidae), based on mitochondrial and nuclear DNA sequences.

The hawkmoth genus Hyles comprises some 29 species with a global distribution. In this study, we augment the previous taxon sampling with more species and add sequences from a nuclear gene to produce a refined phylogenetic hypothesis. A total evidence reconstruction based on Bayesian analysis of the combined mitochondrial (COI, t-RNA-Leu, COII; 2284 bp) and nuclear (EF1alpha; 773 bp) sequences is discussed and compared with the results from separate analyses of the two genes. The total evidence phylogeny corroborates many of the phylogenetic relationships previously postulated within the genus. In addition, the hitherto unsampled enigmatic species Hyles biguttata from Madagascar appears as sister group to Hyles livornicoides from Australia, although support for the relationship is relatively weak. The high level of differentiation of Hyles perkinsi from H. calida (both Hawaii), and the status of these two as sister species, is corroborated by both sources of sequence data. However, their phylogenetic position when mt DNA sequences alone are considered differs markedly from that under total evidence. The previously postulated relationships within the Hyles euphorbiae complex (HEC) s.s. are largely corroborated, but H. dahlii is now more closely related and the HEC s.l. is redefined to include H. zygophylli and H. stroehlei (two species that had not been studied previously using molecular data) and to exclude H. siehei and H. hippophaes. The nuclear sequences alone are insufficiently variable to fully resolve all lineages and the phylogeny suggests that nuclear gene swapping and incomplete lineage sorting have occurred implying recent divergence. The results from the total evidence analysis provide a phylogenetic hypothesis that both corroborates and complements the previous biogeographic scenario, and provides new insights into the origins of several of the included taxa.

New source of genetic polymorphisms in lepidoptera

The variability level of the ISSR (inter-simple sequences repeat) primer (GACA)4 was examined in the three Lepidoptera families Pyralidae, Sphingidae and Pieridae. Our study shows that the tetra-repeat (GACA)n is evidently present in sufficient numbers in these butterflies to provide informative DNA fingerprints. The variability is mostly rather high, but within a comparable range to other ISSR studies. Although less polymorphisms may be encountered in some butterfly families, this study indicates that high variability of this marker may be a common characteristic of Lepidoptera genomes. An appeal for a minimal level of standardization of ISSR-PCR data analysis is formulated to enable an exact comparison between the groups of organisms studied with this fingerprint technique.

Sequestration of phorbol esters by aposematic larvae of Hyles euphorbiae (Lepidoptera: Sphingidae)?

Summary.The larvae of the hawkmoth species Hyles euphorbiae have a conspicuous aposematic colouration and show gregarious behaviour. It has thus been suggested that they sequester phorbol esters from their food plants which include different species of the genus Euphorbia (Euphorbiaceae) for chemical protection against predators. To test this hypothesis in more detail, we fed larvae an artificial diet with three doses of 12-tetradecanoyl-phorbol-13-acetate (TPA), then examined the faeces and the larval tissues, such as integument, haemolymph and gut of the caterpillars for the presence of TPA. In order to determine the ability of the larvae to detoxify phorbol esters, other larvae were directly injected with a TPA solution and analysed in the same manner. Our study indicates that the larvae of Hyles euphorbiae do not sequester phorbol esters. Upon oral application TPA was not found in the larval integument or the haemolymph. Instead, it was mostly metabolised (about 70–90%). Nevertheless, about 10-30% were retained and recovered in the faeces. The larvae were also able to metabolise and thus detoxify the phorbol ester when TPA was injected directly into the body. These hawkmoth caterpillars are relatively large and have a gut full of plant material, which they regurgitate into the direction of the predator when attacked in nature. Since phorbol esters are very potent toxins and irritants, we postulate that the gut content (and especially the plant slurry disgorged as regurgitant from the anterior gut) alone could be aversive for a potential predator, even if some metabolism has taken place. Thus, although H. euphorbiae caterpillars do not actively sequester phorbol esters, their aposematic colouration appears to be based on chemical defence through phorbol esters retained in the gut.

Taxonomy, phylogeography and climate relations of the Western Palaearctic spurge hawkmoth (Lepidoptera, Sphingidae, Macroglossinae)

Hundsdoerfer, AK., Mende, MB., Kitching, IJ. & Cordellier, M. (2011). Taxonomy, phylogeography and climate relations of the Western Palaearctic spurge hawkmoth (Lepidoptera, Sphingidae, Macroglossinae). —Zoologica Scripta, 40, 403–417.

Larval pattern morphotypes in the Western Palaearctic Hyles euphorbiae complex (Lepidoptera: Sphingidae: Macroglossinae).

External morphological variation in larval pattern elements in the Hyles euphorbiae complex is documented and described from independent samples of numerous populations across Europe and North Africa. Variability in the distribution of black cuticle and other colours was found to be much higher within populations than previously believed and it proved difficult to characterize any species, subspecies and even local population unequivocally using this feature alone. A representative sample of larvae from the Canary Islands, Madeira, North Africa, Iberia, Italy, Germany, Eastern Europe, Malta, Greece and Yemen, is illustrated, supplemented by observations from photographs of larvae from the Middle East and Western Asia. Taxonomy-independent definitions of larval morphotypes demonstrate significant overlap between species. We discuss the results in the context of Western Palaearctic biogeography, postulating several areas of hybridisation between the more northern Hyles euphorbiae and more southern H. tithymali that have led to the mosaic distribution patterns observed, and consider the potential of these moths for tracking the future eff ects of climate change in the Mediterranean Basin.

Eight New Microsatellite Loci of the Western Palearctic Hyles euphorbiae Complex (Lepidoptera, Sphingidae)

We describe eight new micro satellite loci for hawkmoths of the Hyles euphorbiae complex. They are polymorphic (except for one locus in one population) with 2–18 alleles per locus, an expected heterozygosity between 0.14 and 0.94, and an observed heterozygosity between 0.10 and 0.75. As typical for Lepidoptera, the yield of new loci was low due to the presence of microsatellite gene families and variable flanking regions. These microsatellites provide informative results in population studies of the West Palearctic H. euphorbiae complex since cross amplification for H. euphorbiae and H. tithymali was successful for all but one locus.

Molecular phylogeny, morphology and taxonomy of Moroccan Triops granarius (Lucas, 1864) (Crustacea: Notostraca), with the description of two new species

We used three molecular markers to investigate populations of Triops granarius from a study area in western Morocco that had a north-south span of approx. 434 km, the most distant populations situated at more than 470 km distance from each other. Previous studies had already investigated two Triops granarius populations from this region and revealed their affiliation to the major phylogenetic lineage that includes Triops cancriformis. By contrast, based on the geographic position of the type locality and the morphology of the type, Triops granarius s.s. likely belongs to a clade that forms the sister group to American and Australian Triops, i.e. including Triops longicaudatus and Triops australiensis. In the present study a second, hitherto unknown phylogenetic lineage was discovered among Moroccan populations of Triops granarius s.l. Our phylogenetic analyses show that both Moroccan lineages of Triops granarius s.l. represent a pair of genetically and morphologically well differentiated sister species that should be separated from Triops granarius. We therefore formally describe them as two new species, Triops maximus sp. nov. and Triops multifidus sp. nov. The early larval stages of both species show a peculiar morphology with 10 to 15 setae on the exopodite of the 2nd antenna. The number of these setae was generally thought to span five to seven in Notostraca. Despite the fact that the antennal setae form a central part of the main locomotory organ in early instars, we found their number to vary by up to two between body-sides of single individuals.