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.


Introduction
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 nonnative 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 1 3 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.

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 CaCl 2 , 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. 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).

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.

Results
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 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.

Discussion
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