Systematics and biogeography of Zavrelia, Afrozavrelia and Stempellinella

(Diptera: Chironomidae)


Vitenskapsmuseet, NTNU  

Torbjørn Ekrem

  Seksjon for naturhistorie

Stempellinella tamaseptima (Photo by Eiso Inoue)

Project abstract

The project focuses on the taxonomy, systematics and zoogeography of the chironomid genera Zavrelia Kieffer Afrozavrelia Harrison and Stempellinella Brundin (Diptera). Through taxonomic revisions, and description of new species and immature stages, identification keys for all life stages will be erected, and morphological as well as molecular characters will be used to infer the phylogenetic relationship of the species in the two genera. The results will either support or refute the hypothesis that the genera Zavrelia, Afrozavrelia and Stempellinella should be synonymized, and provide a solid background for a revision of the current classification. Based on the resulting phylogeny, zoogeographical analyses will be performed and provide the most probable biogeographical history and area of origin for the two genera.

Background knowledge

The genera Zavrelia, Afrozavrelia and Stempellinella are members of the subtribe Zavreliina of the tribe Tanytarsini (e.g. Cranston et al. 1989, Ekrem & Sæther 2000, Sæther & Andersen 1998). Until present, adult males of the three genera have been separated solely on presence or absence of long microtrichiae between the ommatidia of their compound eyes (Cranston et al. 1989), the pupae by the extension of the armament on the abdominal tergite two (Pinder & Reiss 1986), and the larvae by differences in the pecten hypopharyngis and premandible (Pinder & Reiss 1983). Viewed separately, especially the adult male characters would normally be considered insufficient for generic separation in the Chironomidae, but in concert with the larval and pupal diagnostic characters they have been regarded to be appropriate. However, few species have had their immature stages described. Recently, I have obtained material from two different faunistic studies of groundwater springs: one in Berchtesgaden national park, Germany, and one in Luxemburg (project conducted by the National Museum of Natural History in Luxembourg). The material which was provided by I. Schrankel, E. Stur & S. Wiedenbrug contain a new species with intermediate character alternatives in the adult male. Moreover, specimens with associated larvae and pupae of an undescribed species from North America show adult male characteristics of one genus, and key to the other genus when relying on characters from the immature life stages. Hence, there are at least two species which indicate a possible synonymy of Zavrelia and Stempellinella.  

The genera include 17 described species world wide. There are eight Stempellinella and six Zavrelia species described from the Holarctic region (10 West Palaearctic, 5 East Palaearctic and 2 Nearctic), and one Afrozavrelia species from the Afrotropical region. In addition, there are two African species currently belonging to the genus Stempellina Kieffer (Thienemann & Bause in Bause, 1913) which probably should be transferred to Stempellinella (own observation), and perhaps as many as seven species from Europe, Asia, Australia, Brazil and North America which are new to science. Not only are the new species in need of description, but rather all of the species listed in table 1 are in need of revision and redescription to clarify their species boundaries. Moreover, their immature and adult female life stages are either insufficiently described in literature or not at all. Thus, a taxonomic revision of all species in the included genera is not only beneficial for freshwater ecologists, but a prerequisite to be able develop identification keys for all life stages, and to conduct proper phylogenetic and zoogeographical analyses.


Field work will be directed in particular to the sampling of Stempellinella, Zavrelia and Afrozavrelia species where freshly preserved alcohol material is scarce or absent in our collections. Also, material of potential sister group taxa will be collected, and a number of colleagues around the world has already been of great help in the supply of additional specimens from regions outside Europe. The fieldwork for 2004/2005 will mainly be directed to collect European species in which immature stages are unknown or inadequately described (e.g. Stempellinella sp. n. 7, Zavrelia atrofasciata and Zavrelia nigritula) and conducted in localities where the species are known to occur (German Alps, Luxemburg, Belgium). Moreover, it is highly desirable to obtain associated immature stages and fresh material of the African Stempellina and Afrozavrelia species, and a collecting trip to the Berg River in the Cape Province, South Africa where two of these species are known to occur (Freeman 1958) is planned for December 2004. 

For detailed examination, adults need to be dissected, cleared of musculature and mounted on microscope slides. Primarily, fresh, ethanol fixed specimens are used for DNA extraction to ease the amplification of long gene sequences. A tissue protocol using the enzyme proteinase-K will aid the extraction of DNA without destroying the exoskeleton, and replaces the usual clearing in KOH. Thus, all specimens used for sequencing can be slide mounted after DNA extraction, identified with certainty, and later used in reference collections.  

The right choice of genes is of importance for the success of the molecular systematic part of the project. Based on the experience of the molecular systematic group in Bergen, and the results of other studies, I plan to use the nuclear gene globin 2b, and the mitochondrial genes COI, COII and 16s. Primers are already developed for the gene COI for chironomids by Endre Willassen, and globin 2b has been used successfully sequenced from Chironomus by Martin et al. (2002).  

Both molecular and morphological data will be included in the phylogenetic analyses. The planned analyses will encompass methods and programs that use maximum parsimony, maximum likelihood and Bayesian likelyhood (e.g. NONA, WinClada, PAUP* and MrBayes). The comparison tests will include Bootstrapping and Bremer support calculations (Bremer 1994) to assess branch stability, incongruence length difference test (ILD) (Mickevich & Farris 1981) and modifications of ILD to measure the incongruence in the data set. 

The zoogeographical analysis will take advantage of methods like brooks parsimony analysis (BPA, Brooks 1990) and Dispersal-Vicariance Analysis (DIVA, Ronquist 1997) to explore the zoogeographical patterns of the species in Stempellinella and Zavrelia.


 The overlaying objectives of the proposed project are: 

Three main hypotheses will be tested:

Hypothesis 1: Stempellinella and Zavrelia are not separate monophyletic groups and the genera should be listed as synonyms.

This will be tested using phylogenetic analyses including all Stempellinella and Zavrelia species and representatives of their most probable sister groups.

Hypothesis 2: The sister group of Stempellinella, Afrozavrelia and Zavrelia combined is Stempellina.

Recent phylogenetic analyses of the tribe Tanytarsini based on morphological characters has suggested Stempellina Thienemann & Bause, Neostempellina Reiss, Seppia Ekrem & Sæther or Friederia Sæther & Andersen as the most probable sister groups to the two genera treated in the present project (Roque & Sæther submitted manuscript). Their findings will be tested by including species of all genera of the subtribe Zavreliina in the analysis.

Hypothesis 3: The group containing Stempellinella, Afrozavrelia and Zavrelia originated in Laurasia, and presence on the southern continents is due to later dispersal through the Middle East, Indonesia and Latin America.

This scenario is not uncommon for many groups of non-biting midges (e.g. Ekrem 2003), and the fact that only one species in Stempellinella or Zavrelia is found in South America and Australia supports the idea of an origin in Laurasia. The hypothesis will be tested by conducting biogeographical analysis on the most probable phylogeny, and if molecular data is included, it will be possible to estimate the origin of the group by using a molecular clock model.


Bause, E. (1913). Die Metamorphose der Gattung Tanytarsus und einiger verwandter Tendipedidenarten. Ein Beitrag zur Systematik der Tendipediden. Archiv für Hydrobiologie Supplement 2: 1-139.

Bremer, K. (1994). Branch support and tree stability. Cladistics 10: 295-304.

Brooks, D. R. (1990). Parsimony analysis in historical biogeography and coevolution: Methodological and theoretical update. Systematic Zoology 39: 14-30.

Brundin, L. (1947). Zur Kenntnis der schwedischen Chironomiden. Arkiv för Zoologi 39 A: 1-95.

Cranston, P. S., Dillon, M. E., Pinder, L. C. V. & Reiss, F. (1989). The adult males of Chironominae (Diptera, Chironomidae) of the Holarctic region. Keys and diagnoses. Pp. 353-502 in Wiederholm, T.: Chironomidae of the Holarctic region. Keys and diagnoses. Part 3. Adult males. Entomologica scandinavica Supplement 34.

Ekrem, T. (2003). Towards a phylogeny of Tanytarsus van der Wulp. Is morphology alone sufficient to reconstruct the genealogical relationships between species in Chironomidae (Diptera)? Insect Systematics & Evolution 34: 199-219.

Ekrem, T. & Sæther, O. A. (2000). Seppia, a new Afrotropical tanytarsine genus (Diptera: Chironomidae). Pp. 79-87 in Hoffrichter, O.: Late 20th Century Research on Chironomidae: an Anthology from the 13th International Symposium on Chironomidae. Shaker Verlag, Aachen.

Martin, J., Guryev, V. & Blinov, A. (2002). Population variability in Chironomus (Camptochironomus) species (Diptera, Nematocera) with a Holarctic distribution: evidence of mitochondrial gene flow. Insect Molecular Biology 11: 387-397.

Mickevich, M. F. & Farris, J. S. (1981). The implications of congruence in Menidia. Systematic Zoology 30: 351-370.

Pinder, L. C. V. & Reiss, F. (1983). The larva of Chironominae (Diptera: Chironomidae) of the Holarctic region. Keys and diagnoses. Pp. 293-435 in Wiederholm, T.: Chironomidae of the Holarctic region. Keys and diagnoses. Part 1. Larvae. Entomologica scandinavica Supplement 19.

Pinder, L. C. V. & Reiss, F. (1986). The pupae of Chironominae (Diptera: Chironomidae) of the Holarctic region - Keys and diagnoses. Pp. 299-456 in Wiederholm, T.: Chironomidae of the Holarctic region. Keys and diagnoses. Part 2. Pupae. Entomologica scandinavica Supplement 28.

Ronquist, F. (1997). Dispersal-vicariance analysis: A new approach to the quantification of historical biogeography. Systematic Biology 46: 195-203.

Roque, F. O. & Sæther, O. A. (in press). New Neotropical species of Nandeva, with a phylogeny of the tribus Tanytarsini (Diptera: Chironomidae). Tijdschrift voor Entomologie.

Sæther, O. A. & Andersen, T. (1998). Friederia, a new Afrotropical tanytarsine genus (Diptera: Chironomidae). Entomologica Scandinavica 29: 29-37.