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UDK 595.768.1
TAXONOMIC POSITION OF ANASTRANGALIA REYI
AND A. SEQUENSI COLEOPTERA, CERAMBYCIDAE BASED
ON MOLECULAR AND MORPHOLOGICAL DATA
A. M. Zamoroka1, D. V. Semaniuk1, V. Yu. Shparyk1, T.V. Mykytyn1, S. V. Skrypnyk2
1Vasyl Stefanyk Precarpathian National University,
vul. T. Shevchenka, 57, Ivano-Frankivsk, 76018 Ukraine
E-mail: andrii.zamoroka@pu.if.ua
2Khmelnytskyi National University,
vul. Instytutska, 11, Khmelnytskyi, 29000 Ukraine
Taxonomic Position of Anastrangalia reyi and A. sequensi (Coleoptera, Cerambycidae) Based
on Molecular and Morphological Data. Zamoroka, A. M., Semaniuk, D. V., Shparyk, V. Yu.,
Mykytyn, T. V., Skrypnyk, S. V.— Anastrangalia reyi (Heyden, 1889) and Anastrangalia sequensi
(Reitter, 1898) are morphologically similar species described in late of XIX century. e recent barcoding
revealed that A. reyi is almost identical to another species, Anastrangalia dubia (Scopoli, 1763), by the
sequence of nucleotides in cytochrome C oxidase subunit I (COI). Consequently, the taxonomic position
of these species is unclear. We have conducted a comprehensive meta-analysis of available data of COI
sequences combined with a study of morphological characters of the male genitalia of A.reyi, A. sequensi
and A. dubia. Based on 87 sequenced samples we built well-resolved phylogenetic maximum likelihood
tree. We found the clades of A. dubia, A. reyi and A. sequensi to be closely related and arranged in the dense
cluster. Despite this, numerous cases of introgressive hybridization of A. reyi and A. dubia were identi ed,
indicating an inadequate reproductive barrier between them. e study of morphological features of male
genitalia of A. reyi, A. sequensi and A. dubia shows minor di erences between them. Based on these
facts and the results of the phylogenetic analysis we propose to consider A. reyi and A. sequensi to be
subspecies of A. dubia.
Key words: Cerambycidae, taxonomy, molecular phylogeny.
Introduction
Anastrangalia reyi (Heyden, 1889) and Anastrangalia sequensi (Reitter, 1898) are morphologically
indistinguishable species, which initially were described as varieties of Anastrangalia dubia (Scopoli, 1763).
A. reyi was formally described by Claudius Rey under the name Leptura dubia race ochracea as having black
strip on the margin of the elytra (Rey, 1885). Heyden found this name to be preoccupied by Leptura scutellata
Vestnik Zoologii, 53(3): 209–226, 2019
DOI: 10.2478/vzoo-2019-0021
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210 A. M. Zamoroka, D. V. Semaniuk, V. Yu. Shparyk, T. V. Mykytyn S. V. Skrypnyk
var. ochracea Faust, 1879 and proposed a new replacement name Leptura reyi for the junior homonym, “Ray’s
Leptura” (Heyden, 1889). However, he did not indicate any reason for erecting of a new species or providing
its description. Decade later Edmund Reitter described Leptura sequensi as a species distinct from L. dubia. He
also found three forms of L. sequensi: var. rufopaca, var. pulchrina, var. tristina from East Siberia. According to
him, L. sequensi di ers from L.dubia by smaller temples (Reitter, 1898). is feature is typical also for A. reyi.
Both species have the pronotum subcylindrical with sparse erecting and dense decumbent pubescence. Despite
the absence of morphological di erences A. reyi and A. sequensi were recognized until now as distinct species
based mainly on geographical separation.
During the last decade due to intensive barcoding of European Coleoptera it was revealed that A. reyi does
not di er from A. dubia by the molecular signs of cytochrome c oxidase I (COI) (Rougerie et al., 2015; Hendrich
et al., 2015; Wu et al., 2016). Moreover, Hendrich et al. (2015) noted that all sequenced specimens of both
species were not grouped in separated clades on their phylogenetic trees; instead, they are completely mixed.
Rougerie et al. (2015) assumed possibility of the past or current hybridization between A. reyi and A. dubia.
ey also supposed continuing speciation with low divergence of both species. Wu et al. (2016) in their study
of the longhorn beetle larvae imported to the USA with solid wood packaging material found that A. reyi and
A. dubia are indistinguishable by molecular methods. us, they indicated both species as Anastrangalia sp.
in their study. e published data show some di culties in molecular identi cation of A. reyi and A. dubia
connected with low resolution of COI markers. us, the taxonomic position of A. reyi and A. dubia is unclear
as well as A. sequensi, which is morphologically similar to A. reyi.
In the current study we have conducted the comprehensive a meta-analysis of available data of COI
sequences, including analysis of A. reyi, A. sequensi and A. dubia morphological features of the male genitalia.
We have found evidence that di erence between all these species is considerably low and that they are
conspeci c. We proposed to consider A. reyi and A. sequensi to be subspecies of A. dubia.
Material and methods
e publicly available assembly data on DNA sequences of mitochondrial gene of COI of Anastrangalia
reyi (20 samples), Anastrangalia sequensi (4 samples) and Anastrangalia dubia (23 samples) were obtained
from GenBank as FASTA les. Additionally, we included 6 samples of Anastrangalia sp. that mentioned by Wu
et al. (2016) as doubtful identi cation of Anastrangalia dubia/reyi to the phylogenetic analysis. For evaluating
of the nesting of A. reyi and A. sequensi within Anastrangalia genera we added to phylogenetic analysis available
sequences of Anastrangalia sanguinea (1 sample) and Anastrangalia sanguinolenta (33 samples). All imported
nucleotide sequences were speci cally labeled for their identi cations on phylogenetic tree (see annex1). In
total 87 sequences of 5 species of Anastrangalia were included to phylogenetic analysis. Additionally, Cerambyx
cerdo (KM285966) and Cerambyx scopoli (KU917190) COI sequences were used as outgroup. Both species
belong to the type genus of family Cerambycidae.
Multiple alignments were generated using the Muscle so ware in the environment of SeaView 4 (Gouy
et al., 2010). Alignments were provided with unlimited iterations, and were edited manually to correct regions
containing missing data and to exclude unalignable positions. Phylogenetic trees were constructed using
maximum-likelihood (ML) and Bayesian methods with PhyML (Guindon, Gascuel, 2003). Analyses were
performed following a general time-reversible (GTR) model of sequence evolution using 4 categories of rates
variation with a gamma correction. We performed an approximate likelihood-ratio test (aLRT) for branch
support based on the log ratio between the likelihood value of the current tree and that of the best alternative
(Anisimova, Gascuel, 2006; Guindon et al., 2010). e optimal trees structure was estimated using the nearest-
neighbor interchange (NNI) algorithm for 5-branch trees. We also used neighbor-joining algorithm (BioNJ)
optimaizing trees topology for estimation of branch distance from COI sequences (Gascuel, 1997).
We also dissected 16 males of A. reyi, A. sequensi, A. dubia and A. sanguinolenta for the comparative analysis of
the genitalia morphology due to the classical practice in the taxonomical entomology (Simmons, 2014). Genitalia were
mounted and studied under the stereomicroscope Nikon SMZ-1 at 40× zoom. Photos were taken and processed by
USB camera DLT-Cam PRO 5 MP using DLTCamViewer x86, 3.7.7892 so ware package.
Measurements are given in the following format: min–max (M = mean). e following abbreviations are
used in the diagnoses: AW/L = aedeagus width to length ratio, PW/L = paramere lobes width to length ratio.
Results
Haplotypes and haplogroups. Detail comparative analysis of COI sequences
of A. rei, A.sequensi and A. dubia demonstrated presence of 20 nucleotide substitutions,
which distinguishing these taxa. Since, COI sequences are 658 nucleotide lengths all of them
ordered from 1st to 658th. us, nucleotide substitutions are nested in positions 22–206–
247–250–271–316–331–370–379–451–463–496–523–548–550–565–619–625–631–658.
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211Taxonomic Position of Anastrangalia reyi and A. sequensi (Coleoptera, Cerambycidae)…
Annex 1. e list of Anastrangalia COI sequences used in the study
GenBank
access code Abbreviation Country
of origin Coordinates
Anastrangalia reyi
KM449706 A_reyi_AU1 Austria 46.794 N 12.417 E
KM448004 A_reyi_AU2 Austria 46.708 N 12.587 E
KM446871 A_reyi_AU3 Austria 46.794 N 12.417 E
KM444576 A_reyi_AU4 Austria 46.796 N 12.409 E
KM444049 A_reyi_AU5 Austria 47.182 N 12.822 E
KM439252 A_reyi_AU6 Austria 46.796 N 12.409 E
KU918103 A_reyi_AU7 Austria 46.794 N 12.417 E
KU917280 A_reyi_AU8 Austria 46.708 N 12.587 E
KU916023 A_reyi_AU9 Austria 46.796 N 12.409 E
KU906588 A_reyi_AU10 Austria 46.796 N 12.409 E
KM451282 A_reyi_AU11 Austria 47.182 N 12.822 E
KM450755 A_reyi_AU12 Austria 46.794 N 12.417 E
KJ966266 A_reyi_FI1 Finland 60.238 N 25.14 E
KJ965629 A_reyi_FI2 Finland 60.675 N 27.005 E
KJ964792 A_reyi_FI3 Finland 66.288 N 29.646 E
KJ964213 A_reyi_FI4 Finland 65.116 N 25.829 E
KJ962569 A_reyi_FI5 Finland 65.116 N 25.829 E
KM286353 A_reyi_FR France 44.774 N 6.955 E
KU918610 A_reyi_IT1 Italy 46.720 N 12.301 E
KU908627 A_reyi_IT2 Italy 46.720 N 12.301 E
KY683642 A_sequensi_RU1 Russia 43.17 N 132.79 E
AF332939 A_sequensi_KO Korea not available
NC_038090 A_sequensi_CN China not available
KY773687 A_sequensi_CN1 China not available
KM447238 A_dubia_AU1 Austria 46.796 N 12.409 E
KM444513 A_dubia_AU2 Austria 46.804 N 12.453 E
KM451116 A_dubia_DE1 Germany 49.095 N 13.247 E
KM447620 A_dubia_DE2 Germany 47.491 N 11.095 E
KM444190 A_dubia_DE3 Germany 48.946 N 13.362 E
KM441763 A_dubia_DE4 Germany 49.095 N 13.247 E
KM439943 A_dubia_DE5 Germany 47.705 N 12.42 E
KM439442 A_dubia_DE6 Germany 47.705 N 12.420 E
KU910266 A_dubia_DE7 Germany 47.705 N 12.419 E
KU910193 A_dubia_DE8 Germany 47.705 N 12.420 E
KU908901 A_dubia_DE9 Germany 47.705 N 12.420 E
KM285974 A_dubia_FR1 France 42.739 N 2.200 E
KM286386 A_dubia_FR2 France 45.382 N 2.494 E
KM286142 A_dubia_FR3 France 46.332 N 6.063 E
KU918227 A_dubia_IT1 Italy 46.777 N 11.228 E
KM447713 A_dubia_IT2 Italy 46.778 N 11.242 E
KM444452 A_dubia_IT3 Italy 46.819 N 11.236 E
KM443724 A_dubia_IT4 Italy 46.778 N 11.242 E
KU913421 A_dubia_IT5 Italy 46.732 N 12.332 E
KM439224 A_dubia_IT6 Italy 46.733 N 12.332 E
KU906345 A_dubia_IT7 Italy 46.720 N 12.301 E
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212 A. M. Zamoroka, D. V. Semaniuk, V. Yu. Shparyk, T. V. Mykytyn S. V. Skrypnyk
KM440821 A_dubia_SL Slovenia 45.917 N 14.033 E
KY357754 A_dubia_USA e United States not available
Anastrangalia sp.
KY357756 Anastrangalia_sp_USA1 e United States not available
KY357755 Anastrangalia_sp_USA2 e United States not available
KY357753 Anastrangalia_sp_USA3 e United States not available
KY357752 Anastrangalia_sp_USA4 e United States not available
KY357751 Anastrangalia_sp_USA5 e United States not available
KY357750 Anastrangalia_sp_USA6 e United States not available
Anastrangalia sanguinea
KM849144 A_sanguinea_CA Canada 51.126 N 115.726 W
Anastrangalia sanguinolenta
KU909168 A_sanguinolenta_AU1 Austria 46.708 N 12.587 E
KM445964 A_sanguinolenta_AU2 Austria 46.794 N 12.417 E
KM444617 A_sanguinolenta_AU3 Austria 47.013 N 11.306 E
KU919418 A_sanguinolenta_DE1 Germany 53.358 N 12.891 E
KU912934 A_sanguinolenta_DE2 Germany 50.778 N 10.921 E
KU912144 A_sanguinolenta_DE3 Germany 50.709 N 11.270 E
KU911644 A_sanguinolenta_DE4 Germany 50.908 N 11.611 E
KU909503 A_sanguinolenta_DE5 Germany 53.404 N 12.866 E
KU907670 A_sanguinolenta_DE6 Germany 53.404 N 12.866 E
KU907549 A_sanguinolenta_DE7 Germany 50.772 N 10.656 E
KU907417 A_sanguinolenta_DE8 Germany 53.4047 N 12.866 E
KM450185 A_sanguinolenta_DE9 Germany 49.276 N 8.0899 E
KM448291 A_sanguinolenta_DE10 Germany 47.675 N 12.014 E
KM444762 A_sanguinolenta_DE11 Germany 50.004 N 7.805 E
KM443756 A_sanguinolenta_DE12 Germany 49.070 N 13.131 E
KU915111 A_sanguinolenta_DE13 Germany 50.326 N 12.236 E
KU914828 A_sanguinolenta_DE14 Germany 53.404 N 12.866 E
KU914539 A_sanguinolenta_DE15 Germany 53.404 N 12.866 E
KU914346 A_sanguinolenta_DE16 Germany 53.404 N 12.866 E
KU913708 A_sanguinolenta_DE17 Germany 53.404 N 12.866 E
KU913364 A_sanguinolenta_DE18 Germany 47.705 N 12.419 E
KU915601 A_sanguinolenta_DE19 Germany 53.358 N 12.891 E
KJ964113 A_sanguinolenta_FI1 Finland 61.042 N 28.712 E
KJ963700 A_sanguinolenta_FI2 Finland 65.132 N 25.944 E
KJ963360 A_sanguinolenta_FI3 Finland 61.924 N 25.731 E
KJ963259 A_sanguinolenta_FI4 Finland 65.116 N 25.829 E
KJ962892 A_sanguinolenta_FI5 Finland 61.454 N 27.404 E
KJ962498 A_sanguinolenta_FI6 Finland 61.924 N 25.731 E
KM286319 A_sanguinolenta_FR France 42.739 N 2.2 E
KU910516 A_sanguinolenta_IT1 Italy 46.732 N 12.332 E
KU908371 A_sanguinolenta_IT2 Italy 45.821 N 7.619 E
KU919382 A_sanguinolenta_IT3 Italy 46.628 N 12.229 E
KM452054 A_sanguinolenta_IT4 Italy 46.777 N 11.228 E
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213Taxonomic Position of Anastrangalia reyi and A. sequensi (Coleoptera, Cerambycidae)…
Fig. 1. Distribution of nucleotide substitutions within haplogroups: A — A. reyi (ArEu) (KJ964792); B — A. sequensi (AsFe) (KY683642); C — A. dubia (AdAl) (KM439943);
D — A. dubia (AdPy) (KM285974). e unic nucleotide substitutions are red circled; the common substitutions are blue marked.
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214 A. M. Zamoroka, D. V. Semaniuk, V. Yu. Shparyk, T. V. Mykytyn S. V. Skrypnyk
However, only 7 substitutions are crucial for distinguishing of A. rei and A. dubia. ese
found in positions 22–271–370–496–565–619–658. A. sequensi comprise 17 important
substitutions in the following positions 22–206–247–250–304–316–331–379–451–463–
496–523–548–550–619–625–631. e distribution of common and unic substitutions
within COI haplogroups of A. rei, A. sequensi and A. dubia is shown on g. 1.
We found a very low variatin of A. reyi COI sequences within all of Europe, which
forming the homogenous haplogroup named us European (hereina er: ArEu — A. reyi
European). e haplogroup ArEu contains next substitutions in positions: 22— a, 206— c,
247— c, 250— t, 271— g, 316— g, 331— a, 370— c, 379— c, 451— g, 463— t, 496— g,
523— t, 548— c, 550— a, 565— a, 619— g, 625— g, 631— a, 658— c. e haplogroup
ArEu consists two haplotypes (positions 22–271–370–496–565–619–658): ArEu-1 g–g–c–
g–a–g–c occupying Europe from the Alps to Lapland; ArEu-2 (g–a–c–g–a–g–c) presented
in Southern Finland.
All COI sequences of A. sequensi were origined from Paci c Coast of North Asia and
belong to one haplogroup. is haplogroupe was named Far East (hereina er: AsFe) and
contains substitutions in positions: 22— a, 206— c, 247— t, 250— c, 271— a, 316— a,
331— g, 370— t, 379— t, 451— a, 463— c, 496— g, 523— c, 548— t, 550— g, 565—
a, 619— a, 625— a, 631— g, 658— t. e haplogroup AsFe comprises two haplotypes
(positions 22–206–247–250–304–316–331–379–451–463–496–523–548–550–619–625–
631): AsFe-1 a–c–t–c–t–a–g–t–a–c–g–c–t–g–a–a–g distributed in North China and East
Siberia (Russia); AsFe-2 a–c–t–c–c–a–g–t–a–c–g–c–t–g–a–a–g found in Korean Peninsula.
We identi ed two COI haplogroups for A. dubia, which we named “Alpine”
(hereina er: AdAl) and “Pyrenean” (hereina er: AdPy). e AdAl haplogroup contains
the next substitutions in positions: 22— g, 206— t, 247— c, 250— t, 271— a, 316— g,
331— a, 370— t, 379— c, 451— g, 463— t, 496— a, 523— t, 548— c, 550— a, 565— a,
619— g, 625— g, 631— a, 658— t. e AdPy haplogroup contains following substitutions
in positions: 22— a, 206— t, 247— c, 250— t, 271— a, 316— g, 331— a, 370— c, 379—
c, 451— g, 463— t, 496— a, 523— t, 548— c, 550— a, 565— g, 619— a, 625— g, 631— a,
658— t.
e AdAl haplogroup comprises four haplotypes (positions 22–271–370–496–565–
619–658): AdAl-1 g–a–t–a–a–g–t distributed in the Alps and the North-West Balkans;
AdAl-2 g–g–t–a–a–g–t widespread among North foothills of Alps; AdAl-3 g–g–t–a–a–g–t
with additional sunstituions in positions 625— a and 646— c occupied territory from the
Northern Alps to the Ore Mountains and the Western Carpathians; AdAl-4 g–g–c–a–a–
g–t, which is known by one sequence obtained from the wooden pacadge imported to US
(Wu et al., 2016), unfortunately the territory of its origin is unknown.
e AdPy haplogroup includes the only haplotype (positions 22–271–370–496–565–
619–658) AdPy-1 a–a–c–a–g–a–t occurring from the Pyrenees to the South-Western Alps.
Table 1. e percentage (%) variation of di erence among haplotypes of A. reyi, A. sequensi and A. dubia
(A. sanguinolenta is given for comparison)
Haplotype ArEu-1 ArEu-2 AsFe-1 AsFe-2 AdAl-1 AdAl-2 AdAl-3 AdAl-4 AdPy-1
ArEu-1 0.00 0.15 2.74 2.43 0.61 0.46 0.76 0.46 0.91
ArEu-2 0.00 2.59 2.28 0.46 0.61 0.91 0.61 0.76
AsFe-1 0.00 0.15 2.43 2.58 2.59 2.89 2.43
AsFe-2 0.00 2.28 2.43 2.28 2.58 2.43
AdAl-1 0.00 0.15 0.46 0.46 0.61
AdAl-2 0.00 0.30 0.30 0.76
AdAl-3 0.00 0.61 1.10
AdAl-4 0.00 0.76
AdPy-1 0.00
A. sanguinolenta 12.16 12.01 12.46 12.61 12.01 12.16 12.16 12.31 12.01
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215Taxonomic Position of Anastrangalia reyi and A. sequensi (Coleoptera, Cerambycidae)…
Fig. 2. Phylogenetic tree of the genus Anastrangalia (with C. cerdo and C. scopolii as outgrouped).
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216 A. M. Zamoroka, D. V. Semaniuk, V. Yu. Shparyk, T. V. Mykytyn S. V. Skrypnyk
Fig. 3. Detailed phylogenetic subtree for the dubia group (hybrids of A. reyi and A. dubia are indicated by
arrows).
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217Taxonomic Position of Anastrangalia reyi and A. sequensi (Coleoptera, Cerambycidae)…
e level of di erence among COI haplotypes of A. reyi, A. sequensi and A. dubia is
shown in table 2.
Surprisingly, the di erence among A. dubia haplotypes is higher by an average of
0.2 % than between haplotypes of A. dubia and A. reyi. e COI sequences of A. sequensi
di ers from A. reyi by an average of 2.36 % and by 2.34 % from A. dubia. Comparison
these species with A.sanguinolenta showed at least 12 % di erence in the COI sequences.
us, the divergence between of A. reyi, A. sequensi and A. dubia is a very weak comparing
with the well separated species A. sanguinolenta.
Phylogenetic analysis. Based on 87 sequenced samples of the genus Anastrangalia
and 2 sequences of outgroups we have built a well-resolved phylogenetic maximum
likelihood tree ( g. 2). Nearly all branches are strongly supported based on an approximate
likelihood-ratio test (aLRT). e Anastrangalia maximum likelihood tree consists of two
strongly supported (1.00 aLRT) clades. e rst clade includes the dubia group of species
(e. g., A. dubia, A. reyi, A. sequensi). e sanguinolenta group (e. g., A. sanguinolenta
and A. sanguinea) constitutes the second clade. We found that the dubia-group species
grouped in a dense cluster, where the branches length do not exceed 0.01. is indicates
that A. dubia, A. reyi and A. sequensi are poorly di erentiated and their taxonomic status
should be revised. In the subsequent analysis, we recognize A. dubia, A. reyi and A.sequensi
as belonging to di erent evolutionary lineages (e. g., dubia lineage, reyi lineage and sequensi
lineage) of the same species.
e detailed phylogenetic subtree of dubia-group is presented on the g. 3. It consists
of two weakly separated clades, which di er by 2.5 %. e lesser clade presents sequensi-
lineage with two haplotypes AsFe-1 and AsFe-2. e bigger clade is an amount of the
successive sister branches of the dubia-lineage and the reyi-lineage. We found that the AdPy
haplogroup is well separated from the rest crown of the bigger clade. e AdPy branch
includes a sample that is phenotypically A. reyi, however, the COI sequence indicates that it
is A. dubia. e similarly we found several samples of phenotypically A. reyi in branches of
AdAl haplogroup, which in fact contain A. dubia mitochondrion genome. e reyi-lineage
(ArEu haplogrop) occupies top of the dubia-group successive tree and closely related to the
AdAl haplogroup. e ArEu haplogroup contains a sample of phenotypically A. dubia with
A. reyi COI sequence.
Discussion
Hybridization. e results of our phylogenetic analysis generally agree with
the ndings of other researchers that A. reyi and A. dubia are very similar in their COI
sequences (Rougerie et al., 2015; Hendrich et al., 2015; Wu et al., 2016). Hendrich and
colleagues (2015) noted that phenotypic A. reyi and A. dubia are mixed on the phylogenetic
trees based on COI sequences. ey indicate that molecular data are not consistent with
morphological features. Rougerie et al. (2015) and Wu et al. (2016) have reached very
similar conclusions on the impossibility of A. reyi and A. dubia discrimination based
on the molecular data. On the contrary to them, we found crucial molecular markers
for identi cation of A. reyi and A. dubia, which are described in the results (see above).
is became possible a er we added to our analysis COI sequences of A. reyi obtained
by Pentinsaari et al. (2014) from Finland. Mentioned sequences are identical to the same
from the Alps obtained by Hendrich et al. (2015). We revealed that A. reyi grouped in the
dense monophyletic cluster ( g. 3), which are closely related to A. dubia. Despite that, we
identi ed numerous cases of the introgressive hybridization of A. reyi and A. dubia in zone
of overlapping of their areal in the Alps. is also has been noted by Rougerie et al. (2015).
We found that some phenotypic A. reyi contains mitochondrial COI as in A. dubia and vice
versa. ese are the cases of current unimpeded hybridization between both species as the
mitochondrial genome is non-recombinant and inherited only on the maternal line. e
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218 A. M. Zamoroka, D. V. Semaniuk, V. Yu. Shparyk, T. V. Mykytyn S. V. Skrypnyk
lack of reproductive barrier between A.reyi and A. dubia makes impossible to recognize
them as separated species. e distribution of A.reyi and A. dubia haplotypes and their
hybrids is presented in the table 3. By far, hybridization between A. reyi and A, sequensi is
unknown, however their introgression is possible in the zone of their areal overlapping in
the south of West Siberia.
Phylogeography. Generally, A. dubia, A. reyi and A. sequensi are vicariants,
which replace each other from Atlantic coast to Pacific coast throughout all Eurasia.
The altitudinal vicariance of A. dubia and A. reyi was observed by Brelih et al. (2006)
and Hellrigl et al. (2012). While the differences on COI sequences between A. reyi and
A. dubia is less than 1 % (table 1), the presence of numerous taxon-specific nucleotide
substitutions evidences a period of their isolation. The extremely low interpopulation
COI variation of A. reyi on the large territory from the Alps to Lapland indicates the
relatively recent and a very rapid expanding of its areal. A very similar level of the
interpopulation COI variation is typical for A. sequensi. On the contrary to them,
Table 2. Distribution of haplotypes within samples of A. dubia and A. reyi and their hybrids
Haplotype Positions of nucleotide substitutions
in COI sequence (from 1 to 658):
22–271–370–496–565–619–658
Samples
(see annex 1)
Anastrangalia dubia
AdPy-1 a–a–c–a–g–a–t A_dubia_FR1, A_dubia_FR2, A_
dubia_FR3, A_dubia_IT1
AdAl-1 g–a–t–a–a–g–t A_dubia_DE1, A_dubia_DE2,
A_dubia_AU2, A_dubia_DE3,
A_dubia_DE5, A_dubia_DE9,
A_dubia_IT2, A_dubia_IT3,
A_dubia_IT4, A_dubia_IT5,
A_dubia_IT7, A_dubia_SL, A_
dubia_US
AdAl-2 g–g–t–a–a–g–t A_dubia_AU1, A_dubia_DE6
AdAl-3 g–g–t–a–a–g–t
(additional substitutions in positions
625— a, 646— c)
A_dubia_DE4, A_dubia_DE7,
A_dubia_DE8, A_dubia_IT6
A. dubia (male) × A.reyi
(female) ArEu-1 g–g–c–g–a–g–c A_dubia_AU1
Anastrangalia reyi
ArEu-1 g–g–c–g–a–g–c A_reyi_AU2, A_reyi_AU3, A_
reyi_AU4, A_reyi_AU5, A_reyi_
AU7, A_reyi_AU8, A_reyi_AU10,
A_reyi_AU12, A_reyi_FI3, A_
reyi_FI4, A_reyi_FI5, A_reyi_IT1,
A_reyi_IT2,
ArEu-2 g–a–c–g–a–g–c A_reyi_FI1, A_reyi_FI2,
A.reyi (male) × A.dubia
(female) AdAl-1 g–a–t–a–a–g–t A_reyi_AU1, A_reyi_AU9, A_
reyi_AU11
A.reyi (male) × A.dubia
(female) AdAl-2 g–g–t–a–a–g–t A_reyi_AU6
A.reyi (male) × A.dubia
(female) AdPy-1 a–a–c–a–g–a–t A_reyi_FR
Anastrangalia sp.
ArEu-1 g–g–c–g–a–g–c Anastrangalia_sp_US5,
Anastrangalia_sp_US6
AdAl-1 g–a–t–a–a–g–t Anastrangalia_sp_US1,
Anastrangalia_sp_US2
AdAl-2 g–g–t–a–a–g–t Anastrangalia_sp_US3
AdAl-4 g–g–c–a–a–g–t Anastrangalia_sp_US4
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219Taxonomic Position of Anastrangalia reyi and A. sequensi (Coleoptera, Cerambycidae)…
the COI sequences of A. dubia vary highly on the territory of Europe constituting
at least 2 haplogroups and 5 haplotypes. Insufficient molecular data for A. dubia
from the Carpathians, the Balkans, Asia Minor and the Caucasus makes its complete
phylogeographic reconstruction impossible at the moment. Nevertheless, the high
interpopulation variation of A. dubia COI points to several isolated centres of its
formation. The time of the such centres existence refers to the Last Glacial Maximum
(LGM) 26.5–18.5 ka, when rapid reducing of forests distribution was occurred
(Terhurne-Berson, 2005; Svenning et al., 2008).
The current distribution of A. reyi coincides the spreading of Picea abies L. in
Europe; P.abies is the host plant for A. reyi as well as Pinus L. in general. The areal
of A. reyi (fig. 4) is disrupted into two parts: the eastern (Fennoscandia and the north
of Eastern Europe) and western (the Alps). Populations of the eastern part occupy
territories with dry and cold continental climate conditions reaching 65th parallel
north (Danilevski, 2014). The western populations inhabit the wet and cold climate of
the Alps and their spurs on the altitude over 1200 m a. s. l. (Brelih et al., 2006; Hellrigl
et al., 2012). The presence of A. reyi in the Pyreneans is doubtful because it has not
been mentioned in the detailed Cerambycidae Catalogue of the Iberian Peninsula
(Gonzalez Pena et al., 2007), and only generally marked “SP” [Spain] in the Catalogue
of Palearctic Coleoptera without any additional data (Löbl, Smetana, 2010). Since the
published papers that prove its presence in the Pyreneans are unknown for us, we
do not consider this area to the phylogeographyc analysis. As A. reyi lives in a cold
climate, we consider that it has evolved in the periglacial refugia of P.abies. There are
three main LGM refugia of P.abies known in Europe: The Massif Central (France),
the Pannonian Plain (Hungary) and the Dnister Valley (Moldova) (Terhurne-Berson,
2005). However, all these refugia were located far in the south, where introgressive
hybridization of A. reyi and A. dubia is highly probable. It should be noted that
hybrids of A. reyi and A. dubia are known only from the Alps and have not been
found in Fennoscandia. Thus, the LGM refugia of A. reyi are to be located in Eastern
Europe. The most possible periglacial refugia of A. reyi were isolated forests of P.abies
in the Don Valley (deposits age 30 ka) or surroundings of Plesheevo and Nero Lakes
(deposits age 18–15 ka) in Russia (Terhurne-Berson, 2005). The isolated microsites
of P. abies forests were scattered in the periglacial zone during LGM (Svenning et
al., 2008). The current extremely low variability of COI sequences in populations of
A. reyi indicates that it had to be emerged from a very small population restricted in
the such microsite and the founder effect had a place. The expanding of A. reyi areal is
believed to occur during rapid warming in Bølling-Allerød interstadial (11.7–10.7 ka).
During this period there was a rapid expansion of P.abies forests from the eastern
refugia to the west and north (Simakova & Puzachenko, 2005) and from the southern
refugia to the north and east (Terhurne-Berson, 2005). These well agree with the
molecular phylogeography of P.abies (Sperisen et al., 2001). At that time A. reyi was
widespread in Eastern and Central Europe, reached the Carpathians and the Alps but
not Britain. The areal had to be disrupted during the last cooling period in Younger
Drias (10.7–9.7 ka). The following warm and dry conditions in Boreal (9.7–7.5 ka) and
especially in Atlantic time (7.5–5 ka) caused extinction of A. reyi in the Carpathians
and its isolation on the Alps highlands.
Anastrangalia sequensi occupies the same areal (fig. 4) as its host plants the East
Siberian Picea obovata Ledeb. and the Far East Picea jezoensis (Siebold and Zuccarini)
Carriere. Along with Picea, Anastrangalia sequensi also infests Pinus, Abies, and Larix,
however their areals partly overlap. The lack of molecular data for A. sequensi from
the broad territories of Siberia does not allow us to reconstruct its phylogeography
completely and determinate of its LGM refugia. Nevertheless, the little data of available
COI sequences points to extremely high homogeneity of A. sequensi populations at
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220 A. M. Zamoroka, D. V. Semaniuk, V. Yu. Shparyk, T. V. Mykytyn S. V. Skrypnyk
Fig. 4. Current distribution of A. reyi (stars), A. sequensi (triangles) and A. dubia (squares) in Eurasia.
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221Taxonomic Position of Anastrangalia reyi and A. sequensi (Coleoptera, Cerambycidae)…
least from the Far East. Alike A. reyi, A. sequensi had to be spread from a restricted
LGM refugium to occupy current areal. Therefore, we indirectly identified possible
LGM refugium and ways of post-glacial migration of A. sequensi using the data on
the current and glacial vegetation cover of East Siberia and the Far East. At least three
LGM refugia of P. obovata are known in South Ural, Altai and Baykalia (Blyakharchuk,
2010). During the Bølling-Allerød interstadial, and later during Holocene P. obovata
had widespread within Siberia (Bezrukova, 2000; Blyakharchuk, 2010; Tollefsrud et
al., 2015). Picea obovata migrated from South Ural to West Siberia and the north of
Eastern Europe, from Altai it spread to the basin of Ob River and from Baikalia to
the basins of Yenisei River and Lena River and further to the Pacific coast of Asia
(Tollefsrud et al., 2015). We consider LGM refugia of A. sequensi to be located in
isolated P. obovata forests in the south of East Siberia. The most probable LGM
refugium for surviving of A. sequensi had to be Baykalia where P. obovata existed
at the time (Bezrukova, 2000). We rejected the possibility of South Ural and Altai as
A. sequensi LGM refugia for two reasons: firstly, A. sequensi is unknown from Ural
and West Siberia where A. reyi is widespread; secondly, Altai is the westernmost limit
of A. sequensi range (Danilevsky, 2014; Semaniuk, Zamoroka, 2018). We suggest that
spreading of A. sequensi to Altai is a Late Holocene event, occurred probably during
the Subboreal Time (5.7–2.5 ka). The assumption that there were also North Chinese
or Korean LGM refugia connected with P. jezoensis is unlikely. First of all, P. jezoensis
is widespread in the Far East including most of the Japanese Islands. Picea jezoensis is
known to have spread to Kyushu and Honshu Islands via the land bridge from Korean
Peninsula during LGM (Aizawa et al., 2007). However, A.sequensi, despite continental
Asia including Korean Peninsula, distributed also on the Northern Japanese Islands
(e. g. Sakhalin, Hokkaido, Kurile Islands) but it absents in the rest Japan (Semaniuk,
Zamoroka, 2018). Thus, spreading of A. sequensi in the Far East happened much later
after P. jezoensis migration. Nevertheless, the complete phylogeography of A. sequensi
will be explained only after wider molecular studies across all of its areal.
The current areal of Anastrangalia dubia (fig. 4) coincides the spreading of Abies
Mill. species in Europe (Abies alba Mill., A. cephalonica Loudon and their hybrids),
Asia Minor and the Caucasus (Abies nordmanniana (Steven) and its hybrids, Abies
cilicica (Antoine & Kotschy), North Africa (Abies numidica de Lannoy ex Carriere).
Abies is the main food plant for A. dubia, but the larva also develops in decaying
wood of Picea, Pinus and other conifers. We consider A. dubia to survive during last
glaciation in A. alba LGM refugia in Europe. This explains the presence of A. dubia
in the Pyreneans, the Apennines, the Balkans, Asia Minor and the Caucasus, where
P. abies is completely absent at the present and during the last glaciation. The main
LGM refugia of A. alba located in the Southern Pyreneans, the South-Western Alps,
the Apennines and the Balkans (Terhurne-Berson, 2005). Locations of the LGM
refugia of Abies in North Africa, Asia Minor and the Caucasus are still unclear. The
post-glacial A. alba recolonization in Europe was started from the Pyreneans c. 10 ka
ago invading South France and the Western Alps (Terhurne-Berson, 2005). The AdPy
haplogroup of A. dubia spread from this LGM refugium. The different haplotypes of
AdAl haplogroup, apparently have colonized Central Europe independently. The most
probable LGM refugium of AdAl-2 and AdAl-3 situated in the Western Alps, where
A. alba forests were known at that time (Terhurne-Berson, 2005). Their migrations
occurred along the Northern Alps to the Ore Mountains and further to the Eastern
Carpathians (Terhurne-Berson, 2005). Migration has finished with A. alba in the
Eastern Carpathians at Subboreal Time (5.7–2.5 ka) (Kalinovych, 2003). The AdAl-1
haplotype is believed to migrate to the Alps from the Apennines LGM refugium. The
Southern Carpathian A. dubia had to migrate with A. alba forests from the Balkans,
the most probably from the Dinaric Mountains.
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222 A. M. Zamoroka, D. V. Semaniuk, V. Yu. Shparyk, T. V. Mykytyn S. V. Skrypnyk
Discrepancy between morphological and molecular data. We found a number
of the morphological and molecular incompatibilities between A. reyi, A. sequensi
and A. dubia. First of all, A. reyi and A. sequensi are completely identical in their
morphology. Second, the COI sequences of A. reyi and A. dubia are nearly identical.
Third, the morphology of A. dubia differs from both A.reyi and A. sequensi. Finally,
the COI sequences of A. sequensi are different from both A. reyi and A. dubia. Thus,
we conducted study of the male genitalia of A. reyi, A. sequensi and A.dubia, which is
a classical entomological practice for the identification of sibling species (Simmons,
2014). The aedeagus morphology of A. reyi, A. sequensi and A. dubia (fig. 5, A, B, C)
is nearly identical: the apical part of their aedeagus is slightly expanding and then
narrowing, sharply terminating by a small sclerotized tip. The main morphological
difference is the aedeagus width to length ratio: 0.179–0.185 (M= 0.182) in A. reyi,
0.207–0.227 (M= 0.217) in A. sequensi, and 0.160–0.173 (M = 0.167) in A. dubia.
Morphology of the male parameres is generally very similar for A. reyi, A. sequensi,
and A. dubia (fig. 5, E, F, G) differing by the lobes width to length ratio: 0.190–0.194
(M = 0.192) in A. reyi, 0.212–0.232 (M = 0.222) in A. sequensi, and 0.126–0.148
(M=0.137) in A. dubia. Consequently, the aedeagus and paramere lobes of A. dubia
are the longest and narrowest; A. sequensi — the shortest and widest; A. reyi—
Fig 5. Male aedeagi (A–D) and parameres (E–H) of A.reyi (A, E), A. sequensi (B, F), A. dubia (C, G) and
A. sanguinolenta (D, H).
A B C D
HGFE
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223Taxonomic Position of Anastrangalia reyi and A. sequensi (Coleoptera, Cerambycidae)…
intermediate between them. We also studied male genitalia of A. sanguinolenta
(fig. 5, D, H) and found them to have crucial differences from A. reyi, A. sequensi,
and A. dubia. This emphasizes close affinity of the specimens assigned to A. reyi,
A.sequensi and A. dubia.
The minor differences of the male genitalia of A. reyi, A. sequensi, and A. dubia as
well as numerous introgressive hybrids of A. reyi and A. dubia indicate insufficiency of
the reproductive barrier between them. While morphological and molecular features
of A. sequensi and A. dubia are the more or less differentiated, the positon of A. reyi
is unclear. Based on the facts of the intermediated morphology of the male genitalia,
the similarity of COI sequences and the results of the phylogenetic analysis shows
A. reyi is to be considered a hybridogenic lineage originated from hybridization of
A. sequensi and A. dubia in the past. We assume that A. sequensi was widespread
from East Europe to Siberia during the Riss-Würm interstadial (130–115 ka), where
it introgressed and hybridized with A. dubia. The climate changes during the Last
Glaciation (115–9 ka) induced disappearing of the forest ecosystems in North Eurasia
and extinction of A. sequensi in Europe, South Ural and West Siberia. However,
the surviving of the hybrid population of A. sequensi and A.dubia restricted in the
periglacial P. abies microsites gave rise of Holocene lineage of A. reyi. This is the
outstanding case of the founder effect.
Tax onomic summary. Consequently, the morphological and molecular di erences
between specimens assigned to A. reyi, A. sequensi and A. dubia are su cient for their
recognition as conspeci c rather than belonging to separate species, and as subspecies of
the same species. We therefore consider A. reyi syn. n. and A. sequensi syn. n. to be junior
synonyms of A. dubia and establish a subspecies rank for both of them: Anastrangalia dubia
reyi new rank and Anastrangalia dubia sequensi new rank. e position of the nominal
subspecies Anastrangalia dubia melanota (Faldennann, 1837) and Anastrangalia dubia
moreana (Pic, 1906) is unclear. Both of them possibly belong to Anastrangalia dubia dubia
(Scopoli, 1763).
A nastrangalia dubia (Scopoli, 1763)
Leptura dubia Scopoli, 1763: 471, Leptura limbata Laicharting, 1784: 166*; Leptura notata Olivier, 1795:
11*; Leptura cincta Fabricius, 1801: 356*; Leptura chamomillae Fabricius, 1801: 359*; Marthaleptura
dubia, К. Ohbayashi, 196За: 9*; Anastrangalia dubia, Villiers, 1978: 177*; Anoplodera dubia, Silfverberg,
2004*; Corymbia dubia, Zeegers & Heijerman, 2008: 78*; Anastrangalia reyi syn. n.; Anastrangalia
sequensi syn. n.
Diagnosis: Body completely black. Elytra coloration vary. Male elytra fulvous with
black edging, apex and suture, rarely completely black. Female elytra red with black edging
and apex, frequently with big black stain on the disc or completely black. Pronotum longer
than wider (for males 1.6 times, for female 1.3 times) with median furrow, covered by the
dense decumbent hairs and the sparse standing hairs. e head shape varies due to temple
size (e. g. sharply angled or smoothed). Male aedeagus curved, subapical slightly expanded
then narrows sharply terminating by small sclerotized tip. Parameres long, narrow, touch
each other by the apex where covered by bunch of long hair.
Distribution: Europe (except Britain Island, Iceland), North Africa (North
Algeria), Asia Minor, the Caucasus, Siberia (except the most of the West Siberia), the
Far East.
1 Herea er, if not otherwise stated, for references with asterisk (*) see Löbl, Smetana (2010) and Dani-
levsky (2014).
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224 A. M. Zamoroka, D. V. Semaniuk, V. Yu. Shparyk, T. V. Mykytyn S. V. Skrypnyk
Anastrangalia dubia dubia (Scopoli, 1763)
Leptura dubia Scopoli, 1763: 47*; Leptura limbata Laicharting, 1784: 166*; Leptura notata Olivier, 1795: 11*;
Leptura cincta Fabricius, 1801: 356*; Leptura chamomillae Fabricius, 1801: 359*; Anastrangalia dubia dubia,
Slama et Slamova, 1996: 132*.
Type locality: “Carniolia” (= Krajina, Slovenia).
Diagnosis: Head: temple sharply angled. Aedeagus long and narrow, 0.175>AW/L
[0.160–0.173 (M = 0.167))]. Paramere long and narrow, 0.165 > PW/L [0.126–0.148
(M=0.137)]. Typical nucleotide substitutions of COI sequence in positions 22–271–370–
496–565–619–658: 1) a–a–c–a–g–a–t; 2) g–a–t–a–a–g–t; 3) g–g–t–a–a–g–t; 4) g–g–c–a–
a–g–t.
Distribution: North Africa (North Algeria), Mediterranean Basin, Central Europe,
Asia Minor, the Caucasus.
Anastrangalia dubia reyi new rank
Leptura reyi Heyden, 1889: 203; Leptura dubia race ochracea Rey, 1885: 277; Leptura inexspectata
Jansson & Sjöberg, 1928: 209, 212*; Marthaleptura inexpectata, К. Ohbayashi, 1963а: 9*; Anoplodera
reyi, Lundberg, 1986: 114*; Anastrangalia reyi, Villiers, 1978: 180*; Danilevsky et Smetana, 2010:
97*.
Type locality: Germany.
Diagnosis: Head: temple smoothed. Aedeagus relatively long and narrow,
0.175 < AW/L < 0.2 [0.179–0.185 (M = 0.182)]. Paramere relatively long and narrow,
0.165<PW/L<0.207 [0.190–0.194 (M=0.192)]. Typical nucleotide substitutions of COI
sequence in positions 22–271–370–496–565–619–658: 1) g–g–c–g–a–g–c; 2) g–a–c–g–a–
g–c.
Distribution: e Alps, Fennoscandia, Eastern Europe, the Southern Ural, Northern
Kazakhstan.
Coment: it is believed to be a hybridogenic lineage originated from A. sequensi and
A. dubia.
Anastrangalia dubia sequensi new rank
Leptura sequensi Reitter, 1898: 194, Leptura sequensi v. rufopaca Reitter, 1898: 194; Leptura
sequensi v. pulchrina Reitter, 1898: 194; Leptura sequensi v. tristina Reitter, 1898: 194; Leptura
(Leptura) sequensi var. baikalensis Pic, 1907: 6*; Leptura sequensi v. baicalica Pic, 1911: 4*; Leptura
(s. str.) sachalinensis Matsushita, 1933: 104*; Anoplodera (s. str.) sequensi, Gressitt, 1951 а: 89*;
Marthaleptura sequensi: Ohbayashi. 1963 а: 9*; Anastrangalia sequensi: Lobanov et al., 1981: 801;
Sama, Lоbl, 2010: 97*.
Type locality: “Ost-Sibirien: Quellgebiet des Irkut, Amur-Länder, Lena-Gebiet”.
Diagnosis: Head: temple smoothed. Aedeagus relatively short and wide,
AW/L > 0.2 [0.207–0.227 (M = 0.217)]. Paramere relatively short and wide;
PW/L >0.2 [0.212–0.232 (M=0.222)]. Typical nucleotide substitutions of COI
sequence in positions 22–206–247–250–304–316–331–379–451–463–496–523–
548–550–619–625–631: 1) a–c–t–c–t–a–g–t–a–c–g–c–t–g–a–a–g; 2) a–c–t–c–c–
a–g–t–a–c–g–c–t–g–a–a–g.
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225Taxonomic Position of Anastrangalia reyi and A. sequensi (Coleoptera, Cerambycidae)…
Distribution: Altai, Eastern Siberia, the Far East (except central and southern
Japanese Islands).
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Received 5 February 2018
Accepted 5 March 2019
Unauthenticated
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