Research Article |
Corresponding author: Jelena Đuknić ( jelena.djuknic@ibiss.bg.ac.rs ) Academic editor: Art Borkent
© 2020 Jelena Đuknić, Vladimir M. Jovanović, Jelena Čanak Atlagić, Stefan Andjus, Momir Paunović, Ivana Živić, Nataša Popović.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Đuknić J, Jovanović VM, Čanak Atlagić J, Andjus S, Paunović M, Živić I, Popović N (2020) Simulium reptans (Linnaeus, 1758) and Simulium reptantoides Carlsson, 1962 from the Balkan Peninsula. ZooKeys 922: 141-155. https://doi.org/10.3897/zookeys.922.49306
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Simulium reptans (Linnaeus, 1758) and Simulium reptantoides Carlsson, 1962 are two species of the Simulium reptans group whose distribution is unclear because of their confusing taxonomy and systematics. Their genetic variability is well known for populations in northern and central Europe and shows that both species have two forms; however, the genetic variability of these species in southern and eastern Europe is unknown. To identify the status of these two species in southeast Europe, mtDNA was extracted from 19 individuals from 12 localities across the Balkan Peninsula. Phylogenetic analysis confirmed the existence of two species with 7.38–7.94% divergence. Each species was comprised of two clades, with 2.31% and 1.43% interclade divergence for S. reptans and S. reptantoides, respectively. This study revealed the presence of both species across the Balkans and that S. reptans occurs in this area in only one form (S. reptans B), while S. reptantoides is found in two genetic forms (A and B).
genetic variability, Simuliidae, southeast Europe
The systematics, population genetics, distribution and evolution of black flies (Diptera: Simuliidae) represent interesting research fields for scientists worldwide (e.g.,
Two species of this group, Simulium reptans (Linnaeus, 1758) and Simulium reptantoides Carlsson, 1962, have been extensively discussed in the literature. One of the problems with these species has been their frequent misidentification. Taxonomic features that distinguish them are given in only a few identification keys or scientific articles (
The second problem regarding these species has been their nomenclature, which is reflected by the high number of synonyms (
Both
According to the last inventory list (
Because of the work of
The aim of the present study was to fill in the knowledge gap in the distribution and genetic variation of these two species in southeastern Europe, i.e., to determine whether they are present in the Balkans or not and if so, in which molecular form(s).
From 2015 to 2017, samples of larvae and pupae of S. reptans and S. reptantoides were collected at 12 localities across the Balkan Peninsula as follows: Slovenia (SVN), Croatia (CRO), Bosnia and Herzegovina (BIH), Montenegro (MNE), Serbia (SRB), North Macedonia (MKD) and Bulgaria (BGR) (Table
Data for species S. reptans and S. reptantoides collected in the period 2015–2017. Species names are given according to the results of the study.
Accession Numbers | Species | Stage | River | Location | Country Alpha-3 code | Latitude /Longitude | Collection date | Collector |
---|---|---|---|---|---|---|---|---|
MK936587 | Simulium reptans | pupa | Sava River | near Čatež | SVN | 45.884078, 15.640831 | 03 Sep. 2015 | Paunović et al. |
MK936590 | pupa | Sava River | near Čatež | SVN | 45.884078, 15.640831 | 03 Sep. 2015 | Paunović et al. | |
MK936588 | pupa | Sava River | near Zagreb | CRO | 45.759639, 16.047861 | 04 Sep. 2015 | Paunović et al. | |
MK936589 | pupa | Humljani | Humljani | CRO | 45.578080, 17.798738 | 25 Sep. 2016 | Đuknić et al. | |
MK947040 | pupa | Strumica River | near Vasilevo | MKD | 41.497500, 22.643333 | 24 Jun. 2017 | Đuknić et al. | |
MK936596 | Simulium reptantoides | larva | Sava River | near Čatež | SVN | 45.884078, 15.640831 | 03 Sep. 2015 | Paunović et al. |
MK947041 | pupa | Neretva River | near Počitelj | BIH | 43.149052, 17.737837 | 31 Jul. 2016 | Đuknić et al. | |
MK936595 | larva | Zamna River | near Negotin | SRB | 44.297883, 22.354969 | 26 Apr. 2015 | Đuknić et al. | |
MK947046 | pupa | Urovica River | near Urovica | SRB | 44.399425, 22.407786 | 25 Apr. 2015 | Đuknić et al. | |
MK947048 | pupa | Urovica River | near Urovica | SRB | 44.399425, 22.407786 | 25 Apr. 2015 | Đuknić et al. | |
MK947045 | pupa | Ibar River | near Raška | SRB | 43.286957, 20.618514 | 11 Jun. 2017 | Đuknić et al. | |
MK947047 | pupa | Ibar River | near Raška | SRB | 43.286957, 20.618514 | 11 Jun. 2017 | Đuknić et al. | |
MK936591 | pupa | Rila River | Rila | BGR | 42.131866, 23.156651 | 17 Sep. 2017 | Đuknić et al. | |
MK947044 | larva | Neretva River | near Počitelj | BIH | 43.149052, 17.737837 | 31 Jul. 2016 | Đuknić et al. | |
MK947043 | pupa | Neretva River | near Počitelj | BIH | 43.149052, 17.737837 | 31 Jul. 2016 | Đuknić et al. | |
MK946294 | pupa | Tara River | near Kolašin | MNE | 42.863386, 19.527027 | 08 Aug. 2017 | Đuknić et al. | |
MK947042 | pupa | Cjevna River | near Podgorica | MNE | 42.382999, 19.278886 | 25 Mar. 2017 | Đuknić et al. | |
MK940493 | pupa | Lim River | near Prepolje | SRB | 43.393293, 19.642978 | 09 Aug. 2016 | Đuknić et al. | |
MK937592 | pupa | Rila River | Rila | BGR | 42.131866, 23.156651 | 17 Sep. 2017 | Đuknić et al. |
DNA extractions from larvae and pupae were performed in the Institute for Biological research “Siniša Stanković”. To avoid the risk of contamination by other DNA sources, the intestinal tracts of the larvae were removed. For the extractions we used the isolation kit “KAPA2G Express Extract Kit” (Kapa Biosystems, United States, Wilmington, Massachusetts). The quality of the DNA was checked by agarose gel (1%) electrophoresis. The barcoding region of the mitochondrial COI gene of two morphologically identified species, Simulium reptans (five individuals) and S. reptantoides (14 individuals), was amplified using the following primers: LCO1490 (5-GGTCAACAAATCATAAAGATATTGG-3) and HCO2198 (5-TAAACTTCAGGCTGACCAAAAAAT CA-3) (
In total, 90 sequences were analysed: five sequences of S. reptans and 14 of S. reptantoides collected from the Balkan Peninsula, 38 sequences of S. reptans and 33 of S. reptantoides downloaded from GenBank, and six sequences from the GenBank database were used as outgroups: two Simulium vernum Macquart, 1826, two Thaumalea testacea Ruthe, 1831 and two Culicoides brevitarsis Kieffer. The COI gene sequences for S. reptans and S. reptantoides that were downloaded from GenBank originated from Slovakia (19 sequences), Lithuania (8), Latvia (3), Sweden (12) and the UK (29), and are listed in Suppl. material
Maximum likelihood (ML) and maximum parsimony (MP) phylogenetic analyses were also carried out using MEGA6 software (
To assess branch support in the resulting ML and MP trees, 1,000 bootstrap replicates were performed. To calculate average genetic distances between clades and within each clade (bootstrap method: 1,000 replicates), the best-fitting model of base substitution was applied in MEGA6.
The best-fitting site evolution model priors within BEAST were selected according to a model selection run in MEGA6. We ran preliminary tests to examine the performance of strict versus uncorrelated log-normal relaxed clock priors. These preliminary analyses consisted of two independent runs, each for 6,000,000 generations, with sampling every 1,000 generation. We examined posterior density histograms in TRACER v1.6 (
DnaSP v6.10.01 was used (
Using the
All retrieved sequences had lengths ranging from 453 bp to 606 bp. The Tamura 3-parameter model with the gamma distribution of variation between the nucleotide positions (
Model | BIC | lnL | |
---|---|---|---|
T92+G | Tamura 3-parameter | 7364.018442 | -2643.969915 |
T92+G+I | Tamura 3-parameter | 7372.132269 | -2642.563464 |
HKY+G | Hasegawa-Kishino-Yano | 7373.999596 | -2638.033763 |
HKY+G+I | Hasegawa-Kishino-Yano | 7381.914365 | -2636.527782 |
TN93+G | Tamura-Nei | 7384.845828 | -2637.993514 |
The topology of the phylogenetic tree for S. reptans and S. reptantoides involves seven clades (Fig.
Bayesian phylogenetic tree based on the COI gene of two species, S. reptans and S. reptantoides. Species S. vernum, Culicoides brevitarsis and Thaumalea testacea were used as outgroups. Numbers above the branches represent posterior BA probabilities followed by ML and MP > 50% bootstrap support. Sequences (tree leaves) are given as GenBank accession numbers. Sequences in bold type with asterisks at the end of the accession number were obtained in this study. The colours of the clades are given according to the species and forms.
The Bayesian phylogenetic tree (Fig.
Nucleotide diversity within the monophyletic clades ranged from 0.50% within S. reptantoides B to 0.70% within S. reptans A (Table
Nucleotide diversity calculations and tests of neutrality; n – number of sequences, h – number of haplotypes, S – number of segregating sites, Hd – haplotype diversity ± standard deviation, Pi – nucleotide diversity ± standard deviation, Tajima’s D test and Fu’s Fs test.
Clades | n | h | S | Hd | Pi | Tajima’s D | Fu’s Fs |
---|---|---|---|---|---|---|---|
S. reptans A | 18 | 9 | 20/453 | 0.797±0.090 | 0.00698±0.00252 | -1.88682* | -1.912 |
S. reptans B | 24 | 9 | 14/418 | 0.663±0.107 | 0.00667±0.00171 | -1.07936 | -1.485 |
S. reptantoides A | 20 | 14 | 19/487 | 0.889±0.068 | 0.00631±0.00112 | -1.72802 | -8.315 |
S. reptantoides B | 27 | 19 | 19/544 | 0.949±0.032 | 0.00500±0.00057 | -1.79156 | -16.054 |
The interclade divergence for the COI sequence of S. reptans and S. reptantoides ranged from 1.43% (S. reptantoides A vs. S. reptantoides B) to 7.94% (S. reptans A vs. S. reptantoides A) (Table
Evolutionary divergence between clades based on the pairwise analysis of COI sequences.
Clades | 1. | 2. | 3. | 4. |
---|---|---|---|---|
1. Simulium reptans A | ||||
2. Simulium reptans B | 0.0231 | |||
3. Simulium reptantoides A | 0.0794 | 0.0738 | ||
4. Simulium reptantoides B | 0.0775 | 0.0792 | 0.0143 |
A total of 18 haplotypes of S. reptans were recognised in DnaSP (Table
A total of 33 haplotypes of S. reptantoides was recognised in DnaSP (Table
Phylogenetic analyses of sequences from samples of the Simulium reptans group revealed the presence of two major branches with four well-distinguished clades. Two branches represent previously defined species, S. reptans and S. reptantoides (
The typology of trees using different methods (ML, MP and Bayesian) showed the same position of the main clades, with high bootstrap values. We explain above the Bayesian tree topology. The positions of some lineages within these clades differed among the ML, MP and Bayesian phylogenetic trees. However, these differences do not have high bootstrap support and need to be analysed further.
Each species consisted of two clades that represented different molecular forms, A and B. The existence of these forms was described by
According to the latest inventory list, S. reptans is present in some Balkan countries, including Bosnia and Herzegovina, Greece, North Macedonia, Montenegro and Serbia.
Simulium reptantoides was originally described by Carlsson from an unspecified European country; thus, its type locality is unknown (
With the use of molecular barcoding, this study confirmed the presence of S. reptans throughout the Balkans and revealed that S. reptantoides is more widely distributed and has a higher frequency of occurrence in the Balkans than S. reptans. Based on previous studies (
The presence of S. reptantoides on the Balkan Peninsula indicates that some previous findings were misidentified or synonymised. Further analyses are needed in order to precisely delimit the distribution of this species and to explain the high intraspecific variability.
We are grateful to our colleagues from surrounding Balkan countries: Valentina Slavevska-Stamenković (Macedonia), Svetoslav Cheshmedjiev (Bulgaria), Predrag Mitrović (Bosnia and Herzegovina) for being our contact persons. We are grateful to the reviews, by both Peter Adler end a second anonymous reviewer that improved this publication with their suggestions and comments. This work was funded by the European Union Seventh Framework Programme for Research, Technological Development and Demonstration, grant agreement no. 603629-ENV-2013-6.2.1-Globaqua, and by Ministry of Education, Science and Technological Development of the Republic of Serbia.
The COI gene sequences for S. reptans and S. reptantoides downloaded from GenBank
Data type: table
Explanation note: Sequence downloaded from GenBank, Author of the sequence and Country where the original material was collected.