Corresponding author: Marko Prous (
Academic editor: Hannes Baur
The
Prous M, Vikberg V, Liston A, Kramp K (2016) North-Western Palaearctic species of the
The host plant associations, details of larval morphology, and bionomy of only a few species of the
Larvae are cryptically coloured, with a largely green body (
Specimens examined or mentioned are deposited in the following collections:
The
Collection of Erik Heibo, Lierskogen, Norway
Collection of Ole Lønnve, Oslo, Norway
Collection of Veli Vikberg, Turenki, Finland
Lunds universitet, Lund, Sweden
Museo Zoologico di Università degli Studi, Napoli, Italy
Naturhistoriska riksmuseet, Stockholm, Sweden
Naturhistorisches
Swedish Malaise Trap Project, Station Linné, Öland, Sweden
Tromsø
Russian
Names of the mentioned host plants follow The Plant List (
Collecting data of the examined specimens is included in an excel file available at Dryad Digital Repository:
To photograph penis valves and lancets (valvula 1 or ventral part of saw), genital capsules and ovipositors were separated from the specimen and macerated in KOH (10–15%) for 6–10 hours at room temperature or treated with proteinase during DNA extraction (see below). Temporary or permanent slide preparations were made of dissected lancets and penis valves. For temporary slides, glycerine was used. After photographing, the lancets and penis valves were glued on a piece of cardboard, which was pinned with the corresponding specimen. For permanent slides, Euparal or PVA-mounting medium (mostly) was used (these specimens are labelled as ‘PR.
Photos were taken with a digital camera attached to a microscope. Composite images with an extended depth of field were created from stacks of images using the software CombineZP (Alan Hadley;
Morphological terminology follows
DNA was extracted and purified with an EZNA Tissue DNA Kit (Omega Bio-tek) according to the manufacturer’s protocol and stored at -20 °C for later use. Typically, the middle right leg was used for DNA extraction, but for males the whole genital capsule was often additionally used to increase DNA yield and to free penis valves from muscles for photographing. One mitochondrial and one nuclear region were used in phylogenetic analyses. Primers used for amplification and sequencing are listed in Table cytochrome oxidase subunit I gene triose-phosphate isomerase
Primers used for PCR and sequencing, with information provided on respective gene fragment, primer name, direction (forward, F or reverse, R) and location (internal, i or external, o) according to each gene fragment, primer sequence, standard annealing temperature, utilization (PCR/ sequencing), and reference.
Gene Region | Primer name | F/R i/o | Primer sequence 5'–3' | Annealing temperature (°) | PCR/Sequencing | Reference |
---|---|---|---|---|---|---|
|
SymF1 | F o | TTTCAACWAATCATAAARAYATTGG | 47 | PCR, seq | This study |
|
SymF2 | F o | TTTCAACAAATCATAAARAYATTGG | 47 | PCR, seq | This study |
|
sym- C1-J1718 | F i/o | GGAGGATTTGGAAAYTGAYTAGTWCC | 49 | PCR, seq | ( |
|
symC1- J1751 | F i/o | GGAGCNCCTGATATAGCWTTYCC | 47 | PCR, seq | This study |
|
C1-N1760 | R i/o | GGTARAAATCARAATCTTATATTAT | 47 | PCR, seq | ( |
|
SymR1 | R i/o | TAAACTTCWGGRTGICCAAARAATC | 47 | PCR, seq | This study |
|
SymR2 | R i/o | TAAACTTCTGGRTGTCCAAARAATCA | 47 | PCR, seq | This study |
|
A2590 | R o | GCTCCTATTGATARWACATARTGRAAATG | 49 | PCR, seq | ( |
|
TPI_29Fi | F o | GYAAATTYTTYGTTGGNGGIAA | 52 | PCR, seq | This study |
|
F o | GGNAAYTGGAARATGAAYGG | 56 | PCR, seq | ( |
|
|
F i | AARGGHGCNTTYACYGGNGA | 56 | Seq | ( |
|
|
R i | TCNGARTGDCCHADRATNACCCA | 52 | Seq | ( |
|
|
TPI385Fi | F o | GTRATYGCNTGYATYGGIGARA | 52 | PCR, seq | This study |
|
R o | GCCCANACNGGYTCRTAIGC | 56 | PCR, seq | ( |
|
|
TPI706R | R o | ACNATYTGTACRAARTCWGGYTT | 52 | PCR, seq | This study |
PCR reactions were carried out in a total volume of 15–20 µl containing 1–2 µl of extracted DNA, 0.6–0.8 µl (3–4 pmol) of primers and 7.5–10 µl of 2x Multiplex PCR Plus Master mix (QIAGEN). The PCR protocol consisted of an initial DNA polymerase (HotStar Taq) activation step at 95 °C for 5 min, followed by 38–40 cycles of 30 s at 95 °C, 90 s at 47–56 °C depending on the primer set used, and 30–70 s (depending on the amplicon size) at 72 °C; the last cycle was followed by a final 30 min extension step at 68 °C. 3 µl of PCR product was visualised on a 1.4% agarose gel and then purified with FastAP and Exonuclease I (Thermo Scientific). 1.0–1.5 U of both enzymes were added to 12–17 µl of PCR solution and incubated for 15 min at 37 °C, followed by 15 min at 85 °C. Purified PCR products were sent to Macrogen (Netherlands) for sequencing. To obtain unequivocal sequences, both sense and antisense strands were sequenced, using the primers listed in Table
Sequences reported here have been deposited in the GenBank (NCBI) database (accession numbers
Sequence data were analysed using the maximum likelihood method nearest neighbor interchanges subtree pruning and regrafting approximate likelihood-ratio test
Some of the Canadian Centre for DNA Barcoding
Barcode distance calculations were based on p-distances (proportion of nucleotide differences) and were taken from the BOLD BIN (Barcode Index Number) database (
The electronic identification key for the species of
A traditional dichotomous key was constructed manually to emphasise the most reliable characters (usually penis valves or lancets).
Phylogenetic analyses of mitochondrial
Maximum likelihood tree of cytochrome oxidase subunit I approximate likelihood-ratio test , Austria , Canada , China , Germany , Spain , Estonia , Finland , France , United Kingdom , Italy , Morocco , Norway , Portugal , Sweden , United States of America , possible nuclear mitochondrial pseudogenes
Maximum likelihood tree of triose-phosphate isomerase approximate likelihood-ratio test
Lancets of
Lancets of
Lancets of
Lancets of
Lancets of
Lancets of
Lancets of
Lancets of
Lancets of
Lancets of
Penis valves of
Penis valves of
Penis valves of
Genetic data reveal five well separated subgroups within the
Because of the high similarity of the species in
Characters of the lancet that can be used for species identification are the shape of the tangium and serrulae, number of ctenidia, and the presence or absence of small spiny pectines. The tangium can have a distinct lobe (Figs
The clearest differences between species in the
1 | a Mesopostnotum smooth (Fig. |
|
b Claws without subapical tooth (Fig. |
||
c Mesepisternum smooth (Fig. |
||
d Antenna |
|
|
– | aa Mesopostnotum matt (Fig. |
|
bb Claws with subapical tooth (Figs |
||
cc Mesepisternum smooth or matt (Figs |
||
dd Antenna uniformly black or ventrally paler than dorsally (Figs |
|
|
2(1) | a Metafemur pale in most part (Figs |
|
– | aa Metafemur black in most part (Fig. |
|
3(2) | a Claws with large subapical tooth (Fig. |
|
b Antenna ventrally paler than dorsally (Figs |
||
c Metafemur whitish (Fig. |
||
– | aa Claws with small subapical tooth (Fig. |
|
bb Antenna uniformly black (Fig. |
||
cc Metafemur yellowish (Fig. |
||
4(2) | a Claws with long subapical tooth close to apical one (bifid) (Fig. |
|
– | aa Claws with small or large subapical tooth clearly separated from apical one (Figs |
|
5(4) | a Hind trochanters, trochantelli, and tibia partly pale | |
b Antenna (usually?) ventrally at least slightly paler than dorsally (Figs |
||
c In males, antennae with numerous and clearly visible stout black setae among finer paler ones (Fig. |
||
d Apical serrulae of lancet short and protruding, and tangium long and narrow (Fig. |
||
e Penis valve without membranous fold near tip of ventro-apical spine and pseudoceps with distinct dorsal depression in middle or basal part (Fig. |
|
|
– | aa Hind trochanters, trochantelli, and tibia uniformly black or brown | |
bb Antenna uniformly black (Fig. |
||
cc In males, antennae with only some barely visible stout black setae among finer paler ones (Fig. |
||
dd Apical serrulae of lancet long and flat, and tangium short and broad (Fig. |
||
ee Penis valve with membranous fold near tip of ventro-apical spine and pseudoceps without dorsal depression in middle or basal part (Fig. |
|
|
6(4) | a ♀ |
|
– | aa ♂ |
|
7(6) | a Tangium of lancet with distinct lobe (Figs |
|
b Mesepisternum smooth (Fig. |
||
c Claws with small subapical tooth (rarely with large) (Fig. |
|
|
– | aa Tangium of lancet without distinct lobe (Figs |
|
bb Mesepisternum smooth or matt (Figs |
||
cc Claws with small or large subapical tooth (Figs |
|
|
8(7) | a Antenna ventrally distinctly paler than dorsally (Fig. |
|
– | aa Antenna |
|
9(7) | a Inner surface of lancet with small spiny pectines (or dentes semicirculares) that reach sclerora (Figs |
|
– | aa Inner surface of lancet without small spiny pectines (Figs |
|
10(9) | a Mesepisternum smooth (Fig. |
|
b Lancet with numerous ctenidia (Figs |
||
c Apical serrulae of lancet short (Figs |
||
d Pterostigma basally dark brown and apically brown (Fig. |
|
|
– | aa Mesepisternum at least slightly matt (Figs |
|
bb Lancet with numerous or few ctenidia (Figs |
||
cc Apical serrulae of lancet short or long (Figs |
||
dd Pterostigma uniformly yellow or brown (Fig. |
|
|
11(10) | a Lancet with numerous ctenidia (Fig. |
|
b Apical serrulae of lancet long (Fig. |
|
|
– | aa Lancet with few ctenidia (Figs |
|
bb Apical serrulae of lancet short (Figs |
|
|
12(9) | a Lancet with few ctenidia (Fig. |
|
b Serrulae of lancet flat (Fig. |
||
c Antenna (usually?) ventrally slightly paler than dorsally (Fig. |
|
|
– | aa Lancet with numerous ctenidia (Figs |
|
bb Serrulae of lancet flat or protruding (Figs |
||
cc Antenna uniformly black or ventrally paler than dorsally (Figs |
|
|
13(12) | a Mesepisternum (usually?) strongly matt (Fig. |
|
b Antenna uniformly black (Fig. |
||
c Pterostigma (usually?) uniformly yellow or brown (Fig. |
||
d Arctic habitats |
|
|
– | aa Mesepisternum (usually?) smooth or slightly matt (Figs |
|
bb Antenna uniformly black or ventrally paler than dorsally (Figs |
||
cc Pterostigma uniformly yellow to dark brown, or basally dark brown and apically brown (Figs |
||
dd |
|
|
14(13) | a Apical serrulae protruding (Figs |
|
b Antenna |
|
|
– | aa Apical serrulae flat (Figs |
|
bb Antenna uniformly black or ventrally paler than dorsally (Figs |
|
|
15(14) | a Pterostigma |
|
b Ctenidia of lancet more distinct (Figs |
|
|
– | aa Pterostigma |
|
bb Ctenidia of lancet less distinct (Figs |
||
16(14) | a Tangium of lancet without fold (Figs |
|
b Antenna uniformly black (Fig. |
||
c Pterostigma uniformly yellow (Fig. |
|
|
– | aa Tangium of lancet with fold (Figs |
|
bb Antenna ventrally slightly paler than dorsally (Fig. |
||
cc Pterostigma (usually?) basally dark brown and apically brown (Fig. |
|
|
17(6) | a Tergum 8 with apical projection (Fig. |
|
b Antennae ventrally distinctly paler than dorsally or uniformly yellow (Figs |
||
c Claws with large subapical tooth (Fig. |
||
d Mesepisternum smooth (Fig. |
||
– | aa Tergum 8 without apical projection (Fig. |
|
bb Antennae uniformly black to uniformly yellow (Figs |
||
cc Claws with small or large subapical tooth (Figs |
||
dd Mesepisternum smooth or matt (Figs |
|
|
18(17) | a Penis valve with membranous fold near or covering tip of ventro-apical spine (Figs |
|
b Claws with small subapical tooth (Fig. |
||
c Mesepisternum smooth (Fig. |
|
|
– | aa Penis valve without membranous fold (Figs |
|
bb Claws with small or large subapical tooth (Figs |
||
cc Mesepisternum smooth or matt (Figs |
|
|
19(18) | a Ventro-apical spine of penis valve less sharply bent (forming half circle) (Figs |
|
– | aa Ventro-apical spine of penis valve more sharply bent (being almost L-shaped) (Figs |
|
20(18) | a Pseudoceps of penis valve short and broad (Fig. |
|
b Mesepisternum smooth (Fig. |
||
c Antennae uniformly black (Fig. |
||
d Pterostigma (usually?) basally dark brown and apically brown (Fig. |
|
|
– | aa Pseudoceps of penis valve longer and narrower (Figs |
|
bb Mesepisternum smooth or matt (Figs |
||
cc Antennae uniformly black (Fig. |
||
dd Pterostigma uniformly yellow to uniformly dark brown (Figs |
|
|
21(20) | a Penis valve with weakly bent and broad ventro-apical spine, and with narrow pseudoceps without distinct dorsal depression in middle part (Figs |
|
– | aa Penis valve with different combination of characters (Figs |
|
22(21) | a Ventro-apical spine of penis valve narrow and with blunt tip (Figs |
|
b Antennae ventrally paler than dorsally (Fig. |
|
|
– | aa Ventro-apical spine of penis valve broad or narrow and with sharp tip (Figs |
|
bb Antennae uniformly black (Fig. |
|
|
23(22) | a Ventro-apical spine of penis valve narrow (Figs |
|
b Antennae uniformly black (Fig. |
|
|
– | aa Ventro-apical spine of penis valve broad (Figs |
|
bb Antennae uniformly black (Fig. |
|
|
24(23) | a Mesepisternum smooth to slightly matt (Figs |
|
b |
|
|
– | aa Mesepisternum |
|
bb Arctic habitats |
|
|
25(23) | a Pseudoceps of left and right penis valve without distinct dorsal depression in middle part and with weakly bent ventro-apical spine (Figs |
|
b Antennae uniformly black (Fig. |
|
|
– | aa Pseudoceps of left penis valve with distinct dorsal depression in middle part and with strongly bent ventro-apical spine (Fig. |
|
bb Antenna ventrally paler than dorsally (Fig. |
|
Externally, the most similar species are
Based on
Palaearctic. Specimens studied are from Estonia, Finland, Germany, Russia, and Sweden.
The most similar species is
Based on a
Palaearctic. Specimens studied are from Estonia, Finland, and Germany.
Smooth mesopostnotum (Fig.
Based on
Palaearctic, Nearctic. Specimens studied are from Austria, Canada, Estonia, Finland, Germany, Russia, and Sweden.
The most similar species is
Based on
Western Palaearctic. Specimens studied are from Finland, France, Germany, Italy, and Sweden.
Based on the external morphology, the most similar species are
Based on a
Western Palaearctic. Specimens studied are from Finland and Sweden.
Externally, perhaps the most similar species is
Based on
Western Palaearctic. Specimens studied are from Finland, Germany, Norway, and Sweden. According to the BOLD database, this species may also be present in North America. The identifications of North American specimens falling within BIN cluster
Based on the external morphology, the most similar species are
Based on
Western Palaearctic. Specimens studied are from Estonia, Finland, France, Germany, Sweden, and Switzerland.
Externally, perhaps the most similar species is
No data.
Unknown.
Western Palaearctic. Specimens studied are from Norway (Svalbard).
The most similar species to
Based on
Western Palaearctic. Specimens studied are from Austria, Finland, Germany, Great Britain, Russia, and Sweden.
The most similar species is
Based on
Western Palaearctic. Specimens studied are from Finland, France, Germany, Great Britain, Italy, Norway, Portugal, Spain, and Sweden.
The most similar species is
Based on
Holarctic. Specimens studied are from Canada, Estonia, Finland, France, Germany, Norway, and Sweden.
Based on the external morphology, the most similar species are
Based on
Unknown.
Western Palaearctic. Specimens studied are from Finland and Sweden.
Based on the external morphology, the most similar species are
Based on
Unknown.
Western Palaearctic. Specimens studied are from Finland, Norway, and Sweden.
The most similar species is
Based on
Western Palaearctic. Specimens studied are from Finland, Germany, Portugal, and Sweden.
Based on the external morphology, the most similar species are
Based on a
Western Palaearctic. Specimens studied are from Finland.
Based on the external morphology, the most similar species are
Based on
Western Palaearctic, Nearctic. Specimens studied are from Finland, France, Great Britain, Iceland, Norway, Sweden, and Switzerland. The species should be removed from the fauna of Denmark. Publications (e.g.
Based on the external morphology, the most similar species are
Based on
Western Palaearctic. Specimens studied are from Finland and Sweden.
Taxonomy of the species belonging to the
Even if most of the species treated here can be considered distinct, their identification unfortunately remains relatively difficult. For reliable results, lancets and penis valves should be studied. Nevertheless, we hope that the current revision removes most of the previous confusion about species identities, their names and the association of females and males, as well as enabling more reliable and confident identification of the species. One further issue that is worth following up is the identity of the species in North America, as barcoding has revealed close connections to Northern Europe (there are many identical or nearly identical barcodes between the continents; Fig.
Examination of most of the barcoded specimens from Europe revealed that most of the species within the
The study was supported by the Swedish Taxonomy Initiative (contract number dha 153/2011). For loans and gifts of material, as well as giving advice and valuable information, we would like to thank Ruth Ahlburg, John Grearson, Christer Hansson, Erik Heibo, Mikk Heidemaa, Iiro Kakko, Manfred Kraus, Jean Lacourt, Ole Lønnve, Pekka Malinen, Henri Savina, Olga Schmidt, Stefan Schmidt, Andreas Taeger, Hege Vårdal, and staff of the Swedish Malaise Trap Project (particularly Mattias Forshage, Kajsa Glemhorn, Dave Karlsson and Pelle Magnusson). Valuable suggestions made by reviewers Akihiko Shinohara, David Smith, and Villu Soon helped to improve the manuscript.