Skip to main content

Molecular identification of Trichocera maculipennis, an invasive fly species in the Maritime Antarctic


Trichocera maculipennis, an invasive Diptera, was described for the first time in Antarctica in 2006 in a sewage system of one of the scientific stations on King George Island, South Shetland Islands, and started to increase its distribution within the island. To date, only taxonomical description of this species, based on morphological data has been available, as there were no molecular data recorded. In the present study, we present two methods of molecular identification of this species—based on partial cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S) genes. An appropriate and easy-to-use assay for proper and fast identification of invasive species is a key requirement for further management decisions, especially in such a fragile environment as found in terrestrial Antarctica.


Terrestrial habitats in Antarctica are limited to ice–free areas, consisting 0.2% of the continent’s surface [1]. Most of these regions are located on the coast of the continent, especially the Antarctic Peninsula and associated archipelagos, and a number of oases in East Antarctica [2]. Severe conditions, such as extreme low temperature, limited primary production, and very strong wind, make Antarctica inhospitable for terrestrial organisms. Therefore, the region’s biodiversity is very low, with very simple ecological structure [3]. Flora is limited to cryptogams, such as lichens and bryophytes, and two native flowering species, Deschampsia antarctica and Colobanthus quitensis. Animal species are represented mostly by micro-invertebrates, and only two species of macro-arthropods, both chironomiid flies, Parochlus steinenii and Belgica antarctica [4,5,6]. However, the recent increase in human activity, together with climate change, observed in Maritime Antarctica in last 30 years [7], have led the region to become more exposed to expansion of non-native species, introduced unintentionally by national operators of scientific stations or tourists visiting the region. Most of these species are not adapted to survive in the Antarctic environment, but some have already shown good adaptation to harsh conditions and have now become invasive. Known examples are: a grass, Poa annua [8], a chironomiid midge Eretmoptera murphyi [3], and a tipulomorph fly, Trichocera maculipennis [9, 10]. Invasive species may cause serious threats for ecosystem structure and function [3].

Trichocera (Saltrichocera) maculipennis, described for the first time in Antarctica in 2006 in the sewage system of one of the scientific stations on King George Island, South Shetlands Islands [10], started to increase its distribution within the island [9]. To date, only taxonomical description of this species, based on morphological data, is available. There are few taxonomic specialists on the family and the genus Trichocera, and none are known to participate in Antarctic expeditions. For most introductions of non-native species to Antarctica it is difficult to obtain rapid specialist identification. This was identified as an important constraint in the expedited management of Antarctic invasions by Hughes and Convey [11] Therefore, a rapid and accurate method of identification to species level is sought in order to facilitate necessary and appropriate control and eradication measures. The aim of this report is to provide a simple but reliable approach to identification of this species based on molecular data. This uses two widely applied molecular barcoding techniques based on amplification and sequencing of COI and 16S mitochondrial rRNA (mtrRNA) gene fragments.

Several congeneric species were added to the analysis to enable species placement among the distant and close members of the genus, estimated as such on the basis of their morphological similarity. This is the first report of the two phylogenetically informative methods applied in Trichoceridae family.

Trichocera maculipennis Meigen, 1818, is a Holarctic species, known from Arctic to the southern regions of Mediterranean zone and Far East [12, 13]. In the Southern Hemisphere, this species has been described only from the Kerguelen Island [14], and recently from King George Island in Antarctica [9, 10]. The species was probably introduced to both southern locations by human agency [9, 15], and widely dispersed between scientific stations of King George Is. Recognition of potential introduction routes and the origin of the species is very important to prevent further invasions, and to enable the implementation of appropriate management strategies.

Material and methods


Specimens of Trichocera (Saltrichocera) maculipennis Meigen, 1818: female [indicated as Polar in the diagrams], Arctowski Polish Station, data, collected from sewage system as described in [9] (IBB PAS, coll. B. Matuszczak); female [CH]: Switzerland, cave, 2. Galerie Sieben Hengste-Hohgant, alt. 1486 m, 26.12.1986–29.12. 1987 (MNHN, now ISEZ; coll. A. Hof); female: Poland, cave [PL] Pod Sokolą, 19.02.2018 (ISEA; coll. J. Zalewska). Trichocera (Saltrichocera) regelationis (L.), 1758, female, Poland, Ojców National Park, Wąwóz [gorge] Skałbania, 11.04. 1999 (ISEA, coll. A. Palaczyk); Trichocera (Saltrichocera) nordica Krzemińska & Gorzka, 2014 [16]: female, Finland, Oulanka Research Station, 5.09.2011; Trichocera (Saltrichocera) parva Meigen, 1804: female, Finland, Oulanka Research Station, 10.09.2011; Trichocera (Trichocera) major Meigen, 1818: female, Poland, Ojców National Park, Wąwóz [gorge] Korytania, 19.11.1992 (coll. E. Krzemińska). All specimens are housed in ISEA if not otherwise stated.

Institutional abbreviations.

IBB—Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.

ISEA—Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków. Poland.

MNHN—Museé national d’histoire naturelle, Neuchâtel, Switzerland.


DNA isolation from specimens

DNA isolation from insects was carried out as described by Gilbert et al. [17] with minor modifications as described below. The whole specimens were transferred into 1.5 ml microcentrifuge tubes and washed twice with 1 ml TE buffer (10 mM Tris–HCl, 1 mM EDTA pH 8.0) to remove residual ethanol from the sample, then 500 µl of lysis buffer (3 mM CaCl2, 2% (w/v) sodium dodecyl sulphate (SDS), 40 mM dithiotreitol (DTT), 50 µg/ml proteinase K, 100 mM Tris buffer pH 8 and 100 mM NaCl; final concentrations) were added. The homogenates were incubated overnight at 55 °C. Specimens were removed from the buffer, placed in 1 ml 95% (v/v) ethanol and replaced in their respective collections. The lysates were then extracted 3 times with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1; v/v/v) until the interface was clear. Nucleic acids were precipitated by addition of 0.7 volume of isopropanol. Three micro litres of glycogen (20 mg/ml) was added during the precipitation step to improve DNA yields. Samples were incubated at room temperature for 20 min and centrifuged using a MiniSpin Plus centrifuge (Eppendorf AG, Hamburg, Germany) at 14,000×g for 15 min. The supernatant was then removed and the DNA pellet washed twice in 500 µl room temperature 80% (v/v) ethanol, allowed to air-dry at 37 °C, and resuspended in 50 µl low-TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA). After isolation DNA quantity was measured using Qubit 3.0 fluorometer (Thermo Scientific, Waltham, USA) and High Sensitivity DNA quantification kit (Thermo Scientific, Waltham, USA). DNA concentration was normalized to final concentration of 5 ng/µl. All reagents used in the purification step were molecular biology grade and were purchased from Sigma.

PCR amplification and sequencing of mitochondrial barcodes

The standard cytochrome oxidase (COI) fragment was amplified using the primer pair described by Folmer et al. [18]:



16S mtrRNA fragment was amplified with following primer pair:



described by Simon et al. [19].

PCR products were amplified using KAPA Robust PCR kit (Roche, Basel, Switzerland). PCR reactions were carried out in 20 µl final volume consisting of: 4 µl of KAPA 2G A buffer, 0.4 µl of 10 mM dNTPs, 1U of KAPA Robust polymerase (5 U/µl), 0.5 µl of each primer (10 µM), 11.45 µl of PCR-grade water and 2 µl of DNA template (10 ng). Amplification reaction conditions for both sets of barcoding primers were as follows: 3 min of initial denaturation at 95 °C, followed by 38 cycles of 30 s at 95 °C, 20 s at 50 °C, 30 s at 72 °C, and final extension period of 2 min at 72 °C. The amplified products were visualized through agarose gel electrophoresis (1.5%, wt/v) and ethidium bromide staining. The amplicons were purified using EPPiC Fast kit (A&A Biotechnology, Gdańsk, Poland) and directly sequenced with the same primers used for PCR amplification. Sanger sequencing was done using BigDye Terminator v3.1 chemistry and ABI3730xl genetic analyzer (Thermo Scientific, Waltham, USA).

Data analysis

Sequence data were analyzed using FinchTV ver. 1.4.0 (Geospiza, Akron, USA). Consensus sequences were obtained with Seqman Pro ver. 9.1 software (DNAStar, Madison, USA).

Molecular phylogenetic analysis by maximum likelihood method

Phylogenetic tree of mitochondrial 16S mtr RNA and and COI gene fragments

The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model [20]. Evolutionary analyses were conducted in MEGA7 [21].

Nucleotide sequence submission and GenBank accession numbers

Accession numbers for each novel nucleotide sequence of COI and 16S mtrRNA genes of the Trichocera species are given in Table 1.

Table 1 List of species, collection sites, and GenBank accession numbers of COI and 16S mtrRNA sequences used in this study


Eight specimens were subjected to a manual DNA isolation procedure. PCR amplification of 16S mitochondrial rRNA fragment was successful for all eight of the tested samples. In the case of COI fragment PCR was successful only for six samples. In the case of the two remaining specimens, they did not produce a visible amplification product. Several attempts were made to analyze additional molecular barcodes such as nad6 or cytb using primer sets designed for Diptera [23] but only COI and 16S gene fragments were amplified successfully by PCR amplification. Obtained 16S mtrRNA and COI fragment consensus sequences were aligned against GenBank database using BLAST. The resulting sequence of the COI fragment was a 97% match to the Trichocera Barcode of Life Data system (BOLD) mitochondrial COI sequence (accession number KR386810.1) based on a BLAST comparison to the GenBank™ database. The 16S mtrRNA gene fragment showed 99% similarity to Trichocera bimacula mitochondrium sequence (accession number JN861750.1). Sequences obtained are listed in Table 1.

Molecular phylogenetic analysis by Maximum Likelihood method for both sequences are presented at Figs. 1 and 2.

Fig. 1
figure 1

Evolutionary relationships of Trichocera species based on a fragment of mitochondrial COI gene

Fig. 2
figure 2

Evolutionary relationships of Trichocera species based on fragment of mitochondrial 16S ribosomal RNA gene

Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value, using 1000 bootstrap. The highest log likelihood was (-2270,1219). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site (next to the branches). The analysis involved nine nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 526 positions in the final dataset.

Initial tree for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value, using 1000 bootstrap. The highest log likelihood was (-1218,9058). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site (next to the branches). The analysis involved 11 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 513 positions in the final dataset.


Identification of T. maculipennis gave a satisfactory output by each method used (Figs. 1, 2); the specimens of this species from various localities, including those from the Polish Antarctic Arctowski Station (polar), cluster together. The sister branch is occupied by Trichocera regelationis, a closely related species, as inferred from similarity of morphological features (inner genitalia of male and female; spotted wing) based on which they were allotted previously to the regelationis group of species [24].

The remaining portions of both trees are difficult to compare, due to the separate sets of specimens and species analyzed in each.

The inner outgroup species is Trichocera (Trichocera) major, a representative of a different subgenus [25]; its sistergroup position to remaining trichoceriids is therefore expected and satisfactory. A branch next to the maculipennis + regelationis complex comprises two North American species, T. (S.) bimacula and T. (S). brevicornis, both similar to the species of the regelationis group; the former by spotted wings, the latter by male and female genitalia [26]. The position of T. (S.) nordica in the cluster is not so well supported by morphology of the genitalia.

Based on morphological data the method based on 16S ribosomal RNA gene would appear to be more consistent. T. (S.) bimacula, morphologically closely related to the maculipennis + regelationis complex, is located far on the COI-gene based tree.

Results presented herein could provide an important reference for future studies on Trichocera specimens, including new species identification projects and assessments of molecular variation between different geographical locations.

There were unfortunately some limitations to this study. The number of specimens collected for analysis was relatively small and our findings were potentially related to the limited number of specimens in collections. More extensive investigations with a larger number of samples are required for the future studies and for definitive findings to be made.

The presented data is a response to the Committee for Environmental Protection (CEP) recommendations regarding development of a standardized monitoring program to effectively control the spread of the flies in Maritime Antarctica, and identify a practical and coordinated management response for fly eradication [27, 28]. In this regard, use of molecular tools for proper and rapid identification of invasive species are key to improved management decisions, especially in such fragile environment as terrestrial Antarctica.


  1. Burton-Johnson A, Black M, Fretwell PT, Kaluza-Gilbert J (2016) An automated methodology for differentiating rock from snow, clouds and sea in Antarctica from Landsat 8 imagery: a new rock outcrop map and area estimation for the entire Antarctic continent. The Cryosphere 10:1665–1677

    Article  Google Scholar 

  2. Levy J (2013) How big are the McMurdo Dry Valleys?—estimating icefree area using Landsat image data. Antarct Sci 25:119–120

    Article  Google Scholar 

  3. Hughes KA, Worland MR, Thorne MAS, Convey P (2013) The non-native chironomid Eretmoptera murphyi in Antarctica: erosion of the barriers to invasion. Biol Invasion 15:269–281

    Article  Google Scholar 

  4. Chown SL, Convey P (2016) Antarctic entomology. Annu Rev Entomol 61:119–137

    CAS  Article  PubMed  Google Scholar 

  5. Convey P (2007) Antarctic ecosystems. In: Levin SA (ed) Encyclopedia of biodiversity, 2nd edn. Elsevier, San Diego

    Google Scholar 

  6. Frenot Y, Chown SL, Whinam J, Selkirk PM, Convey P, Skotnicki M, Bergstorm DM (2005) Biological invasions in the Antarctic: extent, impacts and implications. Biol Rev Camb Philos Soc 80:45–72

    Article  PubMed  Google Scholar 

  7. Turner J, Bindschadler R, Convey P, diPrisco G, Fahrbach E, Gutt J, Hodgson DA, Mayewski PA, Summerhayes CP (2009) Antarctic climate change and the environment. Scientific Committee for Antarctic Research, Cambridge, p 554

    Google Scholar 

  8. Olech M, Chwedorzewska KJ (2011) The first appearance and establishement of an alien vascular plant in natural habitats on the forefield of retreating glacier in Antarctica. Antarct Sci 23:153–154

    Article  Google Scholar 

  9. Potocka M, Krzemińska E (2018) Trichocera maculipennis (Diptera)—an invasive species in Maritime Antarctica. PeerJ 6:e5408.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Volonterio O, de Leon RP, Convey P, Krzemińska E (2013) First record of Trichoceridae (Diptera) in the maritime Antarctic. Polar Biol 36:1125–1131

    Article  Google Scholar 

  11. Hughes KA, Convey P (2012) Determining the native/non-native status of newly discovered terrestrial and freshwater species in Antarctica—current knowledge, methodology and management action. J Environ Manag 93:52–66

    Article  Google Scholar 

  12. Dahl C, Krzemińska E, Baez M (2002) Trichoceridae. In: Hjorth Andersen MCT (coordinator) Catalogo de los Diptera de Espana, Portugal y Andorra (Insecta). Monografias SEA 8. P. 82. Zaragoza

  13. Dahl C, Krzemińska E (2015) Trichoceridea: winter gnats. In: Boher J, Kristensen NP, Pape T, Vilhelmsen L (eds) The Greenland entomofauna: an identification of manual insects, spiders and their allies. Brill Publishing, Boston, pp 402–408

    Google Scholar 

  14. Seguy E (1953) Insectes Mallophages, Anoplures et Dipteres recueillis par M. P. Paulian aux Iles Kerguelen. Mem. Inst. Sci. Madagascar ser. E. IV. (Tananarive) Paris

  15. Dahl C (1970) Diptera: Trichoceridae of Kerguelen Island. Pac Insects Monogr 23:274–275

    Google Scholar 

  16. Krzemińska E, Gorzka D (2014) Subgenus Trichocera (Saltrichocera) in Scandinavia: a new species and first records from Finland (Diptera, Trichoceridae). Acta Zool Crac 57:1–10

    Google Scholar 

  17. Gilbert M, Thomas P, Moore W, Melchior L, Worobey M (2007) DNA extraction from dry museum beetles without conferring external morphological damage. PLoS ONE 2(3):e272

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3(5):294–299

    CAS  PubMed  Google Scholar 

  19. Simon Ch, Frati F, Beckenbach A, Crespi B, Liu H, Flook P (1994) Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am 87(6):651–701.

    CAS  Article  Google Scholar 

  20. Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10:512–526

    CAS  PubMed  Google Scholar 

  21. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

    CAS  Article  PubMed  Google Scholar 

  22. Geiger MF, Moriniere J, Hausmann A, Haszprunar G, Wägele W, Hebert PD, Rulik B (2016) Testing the Global Malaise Trap Program-How well does the current barcode reference library identify flying insects in Germany? Biodivers Data J.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Simon C, Buckley TR, Frati F, Stewart JB, Beckenbach AT (2006) Incorporating molecular evolution into phylogenetic analysis, and a new compilation of conserved polymerase chain reaction primers for animal mitochondrial DNA. Annu Rev Ecol Evol Syst 37:545–579

    Article  Google Scholar 

  24. Krzemińska E (1999) Three species with clear wings of the regelationis group: Trichocera (Metatrichocera) annulata, T. (M.) rufescens and a new species from Poland (Diptera, Trichoceridae). Acta Zool Crac 42(2):251–258

    Google Scholar 

  25. Krzemińska E (2002) A new subgenus and two new species of the genus Trichocera Meigen, 1803 (Diptera: Trichoceridae). Ann Zool 52(2):156–159

    Google Scholar 

  26. Pratt HD, Pratt GK (1984) The winter crane flies of the eastern United States (Diptera: Trichoceridae). Proc Entomol Soc, Washington 86:249–265

    Google Scholar 

  27. CEP XX Report (2017) Final Report of the XL ATCM, Beijing, China 2017. Secretariat of the Antarctic Treaty Buenos Aires.

  28. Hughes KA, Pertierra LR (2016) Evaluation of non-native species policy development and implementation within the Antarctic Treaty area. Biol Conserv 200:149–159

    Article  Google Scholar 

Download references


We are grateful to Christophe Dufour (MNHN) for a donation of a specimen of Trichocera maculipennis from Switzerland. Authors would like to thank participants of 42. and 43. Polish Antarctic Expedition to Henryk Arctowski Station for their help with collecting samples.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Marta Potocka.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Potocka, M., Krzemińska, E., Gromadka, R. et al. Molecular identification of Trichocera maculipennis, an invasive fly species in the Maritime Antarctic. Mol Biol Rep 47, 6379–6384 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Invasive species
  • Antarctica
  • Trichocera
  • Non-native species
  • Molecular identification
  • Insects
  • Alien species