Ecological and molecular diversity of Eulachnini aphids (Hemiptera: Aphididae: Lachninae) on coniferous plants in Lithuania

Based on research carried out from 2016 to 2018 there are twenty-six species of aphids of the tribe Eulachnini in Lithuania. Seventeen are members of the subgenus Cinara of the genus Cinara, three of the subgenus Cupressobium and two of the subgenus Schizolachnus. In addition, there are four species of the genus Eulachnus. Summarized information on the distribution and host specifi city of Eulachnini in Lithuania is presented. Nine species were in all climatic regions of Lithuania: C. (Cinara) brauni, C. (Cinara) hyperophila, C. (Cinara) neubergi, C. (Cinara) piceicola, C. (Cinara) pinea, C. (Cinara) pini, C. (Cinara) pruinosa, C. (Cupressobium) cupressi and C. (Cupressobium) juniperi. Five species of Lithuanian Eulachnini inhabit host plants of the genus Picea, three live on Larix, thirteen on Pinus, one on Abies, one on Thuja and three on Juniperus. Cinara (Cinara) piniphila was recorded on Pinus mugo and C. (Cinara) nuda on Pinus heldreichii for the fi rst time. Partial COI and EF-1α sequences of C. (Cinara) hyperophila, C. (Cinara) pilosa and C. (Cinara) piceicola were obtained for the fi rst time. Partial sequences of mitochondrial (COI) and nuclear (EF-1α) DNA of Lithuanian samples were used to explore molecular diversity using NJ trees, Automatic Barcode Gap Discovery (ABGD), General Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP). Species delimitation using GMYC (both on COI and EF-1α sequences), PTP (BI on COI) and ABGD (EF-1α) were the most consistent with traditional classifi cations. Pairwise between-species sample divergences (COI and EF-1α fragments) of the samples of the species complex C. (Cinara) pinea – C. (Cinara) piniphila indicate it is composed of a single species. Of the species of aphids that live on conifers, there are seven species of the tribe Eulachnini that are likely to shortly colonize Lithuania.

mainly of fragmentary faunistic records reporting the presence of 19 species of this tribe (for details, see Rakauskas et al., 1992Rakauskas et al., , 2008. Our pilot study in 2017 revealed the presence of 27 species of the tribe Eulachnini in Lithuania (Danilov et al., 2019). In the surrounding countries there are 34 species in Poland, 19 in Latvia, 21 in Belarus and 9 in the Kaliningrad region of Russia (Osiadacz & Hałaj, 2009;Nieto Nafria et al., 2013;Hałaj & Osiadacz, 2015;Wojciechowski et al., 2015). Forty species of Eulachnini are recorded in the Czech Republic, Slovakia and Austria (Holman & Pintera, 1977;Holman, 2009;Nieto Nafria et al., 2013;Wojciechowski et al., 2016;Kanturski et al., 2017), which are in the southernmost part of the Central European fl oristic province according to Frey & Lösch (2010). Lithuania is in the northernmost part of this fl oristic province. Due to global climate change, the Lithuanian fauna is expected to be enriched by species of Eulachnini from the South.
For the successful exploration of regional species diversity it is inevitable that DNA-based methods will have to be used in addition to ecological and morphological ones. These methods have proved to be a powerful tool INTRODUCTION Aphids of the tribe Eulachnini Baker, 1920 (Hemiptera: Aphididae: Lachninae) are phloem feeding insects inhabiting plants of the families Pinaceae and Cupressaceae. They are adapted to feed on particular parts of their host plants.
Of the species of Eulachnini, those of the genus Eulachnus del Guercio, 1909 andsubgenus Schizolachnus Mordvilko, 1909 of the genus Cinara Curtis, 1835, prefer the needles of their host plants. Other species of the genus Cinara inhabit the bark of roots, trunks, branches, twigs and shoots (Binazzi & Scheurer, 2009;Chen et al., 2016b;Albrecht, 2017;Blackman & Eastop, 2019). Aphids of the tribe Eulachnini can cause serious damage in tree nurseries, parks, forests and cultivated areas (Watson et al., 1999;Hopmans & Elms, 2013;Dara et al., 2019). The honeydew of these aphids, however, is the raw material for the so called "forest honey", which is an important commercial product in Europe (Binazzi & Scheurer, 2009).
Forests cover approximately 30% of Lithuania. Most consist of pine and spruce stands (https://www.forestgen. mi.lt). Nevertheless, information about the species of Eulachnini inhabiting coniferous plants in Lithuania consists eastern highlands and spruce stands scattered mostly in the Middle Lithuanian and Samogitian regions (https://www. forestgen.mi.lt). In addition to natural stands of conifers, commercial forests, decorative plantings and gardens, also ensure a high host plant diversity. In this paper, we summarize information on the diversity, ecological and molecular specifi city of aphids of the Eulachnini in Lithuania, present some taxonomic remarks and discuss the possible future development of the conifer-inhabiting aphid fauna in Lithuania.
(1) Needles 201 Havelka et al., Eur. J. Entomol. 117: 199-209, 2020doi: 10.14411/eje.2020.021 (1995, Binazzi & Scheurer (2009), Albrecht (2017) and Kanturski et al. (2017) were used. NIKON ECLIPSE E200 microscope with INFINITY ANALYSE 6.1 software was used for the microscopic analysis. Aphid material is deposited in the Life Sciences Centre of Vilnius University (Lithuania). For the molecular analysis, 161 samples of species of Eulachnini from many host plants and four climatic regions were used. A single aphid from a sample was considered to be a unique sample. A non-destructive extraction method was used so that each aphid could be mounted on a slide and used to confi rm its identity when the morphological and DNA identifi cation appeared to differ. Total genomic DNA was extracted from a single aphid using DNeasy Blood & Tissue kit (Qiagen). For the amplifi cation of COI fragments the primers LCO-1490 and HCO-2198(Folmer et al., 1994 were used. For the amplifi cation of EF-1α fragments the primers Ef3 and Ef6 (Jousselin et al., 2013) were used. PCR amplifi cation was carried out in a thermal cycler (Eppendorf) in 50 μl volumes containing 2 μl genomic DNA, 2.5 μl of each primer (0.5 μM), 25 μl of DreamTaq PCR master mix (Thermo Scientifi c) and 18 μl of nuclease free water (Thermo Scientifi c). The cycling parameters were as follows: denaturizing at 95°C for 5 min (1 cycle), denaturizing at 95°C for 30 s, annealing at 47°C (COI) or 50°C (EF-1α) for 30 s, extension at 72°C for 30-90 s (34 cycles in total) and fi nal extension at 72°C for 5 min (1 cycle). PCR products were purifi ed using Gene Jet PCR purifi cation kit (Thermo Scientifi c) and then sequenced at Macrogen Europe (Amsterdam, the Netherlands) and the Institute of Biotechnology (Life Sciences Centre, Vilnius University). The amplifi cation primers were also used as sequencing primers. DNA sequences for each specimen were confi rmed using both sense and anti-sense strands and aligned in the BioEdit Sequence Alignment Editor (Hall, 1999). Partial COI sequences were tested for stop codons and none were found. The GenBank Accession numbers are MH396414-MH396434, MK829820-MK829825, MN178356-MN178483 and MN192192-MN192351.
To evaluate within-species sequence diversity uncorrected p-distances were calculated for the COI and EF-1α fragments. Both exons and introns of EF-1α were included in the analyses. Sequences were also collapsed into haplotypes using FaBox 1.5 (Villesen, 2007). Neighbour-Joining (NJ) trees for each of analysed fragments were constructed based on within-and between-species distance matrices. MEGA 7 (Kumar et al., 2016) was used for calculating the distances and tree construction.
We used three methods of molecular species delimitation: distance-based Automatic Barcode Gap Discovery (ABGD) (Puillandre et al., 2012), tree-based General Mixed Yule Coalescent (GMYC) (Pons et al., 2006) and the Poisson Tree Processes (PTP) models (Zhang et al., 2013). Partial COI sequences and coding part of EF-1α fragment of Lithuanian samples were analysed using the graphic web version of the ABGD method (http:// wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html). Distances for each gene were calculated using MEGA 7 (Kumar et al., 2016) and a Kimura 2-parameter (K2P) model. The value of the relative gap width (X) was 1.00 for both fragments, with P min = 0.001 and P max = 0.02 for both fragments and other parameters by default. For tree-based methods, the following substitution models were used along with jmodeltest (Posada, 2008): HKY+I+G for COI and GTR+G for EF-1α fragment. One ultra metric tree for each fragment was constructed using the uncorrelated lognormal relaxed clock method implemented in BEAST v1.7.4 (Drummond & Rambaut, 2007), assuming a Yule tree prior. One run of 50 million generations with sampling every 5000 generations was performed. Convergence was checked for using Tracer 1.5 (Drummond & Rambaut, 2007). Sampled posterior trees were summarized using TreeAnnotator 1.7.4 (Drummond & Rambaut, 2007) to generate a maximum clade credibility (MCC) tree without the removal of burn-in. The GMYC method, as implemented in the R package SPLITS (http://www.rforge.r-project.org/projects/splits/) was then applied to the MCC tree and a lists of species derived from this phylogenetic tree. Contrary to GMYC, the PTP method does not require ultra metric trees as an input (Zhang et al., 2013). Bayesian inference trees for each fragment were built using MrBayes 3.2.1 (Ronquist & Huelsenbeck, 2003). One run of 1,000,000 MCMC generations with tree sampling every 1,000 generations was performed. Consensus trees with posterior probabilities were visualized using TreeView (Page, 1996). Trees were saved as newick format fi les and analysed using the bPTP web server (http://species.h-its.org/ptp/). All parameters used were those given by default, except for the number of generations (300,000).

Distribution, host plants and life cycles of species of Eulachnini in Lithuania
Our study revealed twenty-six aphid species of the tribe Eulachnini in Lithuania (Table 1). We failed to fi nd Cinara (Cinara) pinihabitans (Mordvilko, 1895), which was previously reported from the Curonian spit of Lithuania (Rakauskas et al., 2008). Most of the species (17) are members of the subgenus Cinara of the genus Cinara. There are 3 species of the subgenus Cupressobium and 2 of the subgenus Schizolachnus. The four climatic regions in Lithuania differ in terms of the diversity of Eulachnini (Figs 2-3): The highest species diversity was recorded in the Coastal region (23 species), whereas only 10 species were recorded in the Samogitian region. Species of the subgenus Cinara were placed in six ecological groups, each of which was associated with a different host plant. Species complex inhabiting host plants of the genus Pinus was the most common and numerous (8 aphid species). Of these species, fi ve were among the most common species of Eulachnini in all regions in Lithuania: C. (Cinara) brauni Börner, 1940, C. gi (Arnhart, 1930), C. (Cinara) pinea (Mordvilko, 1894) and C. (Cinara) pini (Linnaeus, 1758). Three of them (C. (Cinara) hyperophila, C. (Cinara) pinea and C. (Cinara) pini) mostly inhabited Pinus sylvestris Linnaeus 1753, which is the most common conifer in Lithuania. Two other widespread species of the subgenus Cinara, C. (Cinara) brauni and C. (Cinara) neubergi inhabited species of Pinus that are exotic to Lithuania: Pinus nigra J.F. Arnold 1785 group and P. mugo Turra 1765, respectively.
Six species of the subgenus Cinara coexist on native Pinus sylvestris. C. (Cinara) hyperophila usually feeds on shoots and twigs of mostly young pines and sometimes older trees. In autumn, it also inhabits needle-free parts of branches. Colonies are quite dense in spring, but become scattered later in the season. C. (Cinara) piniphila (Ratzeburg, 1844), C. (Cinara) pinea and C. (Cinara) pilosa (Zetterstedt, 1938) usually form small colonies or occur solitarily. C. (Cinara) piniphila occurred mostly on both fresh shoots and 1-2 year-old twigs throughout the entire season. Noticeably, we found this species on pines growing on poor sandy soil, mostly on dunes. C. (Cinara) pilosa and C. (Cinara) pinea occupy shoots and occasionally form mixed colonies on shoots of the lower branches of old trees. C. (Cinara) pilosa usually live on small shoots of old trees, but might also occur on mature (about 20 years old) pines. C. (Cinara) pinea occurs on both young and old trees. C. (Cinara) pini and C. (Cinara) nuda (Mordvilko, 1895) live in dense large colonies. C. (Cinara) pini commonly inhabits shoots and twigs both on young and old pines. Trunk and basal parts of old branches of immature trees are preferred by C. (Cinara) nuda. However, it can also be found on shoots and twigs in spring. Apart from P. sylvestris, we also found C. (Cinara) nuda on Pinus heldreichii Christ 1863 and C. (Cinara) hyperophila, C. (Cinara) pini, C. (Cinara) pinea and C. (Cinara) piniphila on P. mugo in Lithuania.
Five species of the subgenus Cinara inhabit Picea abies (Linnaeus) Karsten 1881, which is the second most common conifer in Lithuania. They coexist on the same host plant by utilizing specifi c microhabitats. The most common species were C. (Cinara) pruinosa (Hartig, 1841) and C. (Cinara) piceicola (Cholodkovsky, 1896). C. (Cinara) pruinosa occurred exclusively on Picea abies where it formed dense and often large colonies on old twigs, branches and the upper parts of the trunk in spring and autumn. They did not occur in the canopies of trees from mid-July to mid-August, probably due to migration to lower parts of the trunk and big roots as is reported by Blackman & Eastop (2019). C. (Cinara) piceicola inhabits twigs and young branches of Picea abies and sometimes the trunks and shoots. These two species sometimes form mixed colonies, although C. (Cinara) piceicola prefers the younger parts of trees. C. (Cinara) piceae (Panzer, 1800) was less common than the two above-mentioned species. The main reason is that this species may be overlooked due to its summer migration from branches and trunks to roots. These aphids were observed on trunks and branches of old and young trees in June-July and ovipositing oviparae on shoots and twigs in October. Apart from on Picea abies, C. (Cinara) piceae was also recorded on the exotic species Picea omorika (Pančić) Purkyne 1877 and P. alcoquiana (Veitch ex Lindley) Carrière 1867 in Lithuania. The rarest species of spruce-inhabiting aphid in Lithuania was C. (Cinara) costata (Zetterstedt, 1928) (Table 1), which lives in scarce colonies on small woody twigs on the lower branches in the crown and is covered in wax. The most common species on shoots and young twigs of Picea abies was C. (Cinara) pilicornis (Hartig, 1841).
Of the three species recorded on Larix in Lithuania, C. (Cinara) cuneomaculata (Del Guercio, 1909) was the most common. We recorded it living in small colonies on shoots and young woody twigs of Larix decidua Miller 1768, Larix kaempferi (Lambert) Carrière 1856 and on some unidentifi ed species of larch in the Costal Lowland, Middle Lithuanian Lowland and South eastern Highland regions. Dense colonies of C. (Cinara) laricis (Hartig, 1839) were found on young and old trees of Larix decidua where they occurred on the shoots in spring and branches and trunk later in the season. It was recorded at sites in the Costal Lowland and South eastern Highland regions. C. (Cinara) kochiana (Börner, 1939) forms dense and large colonies  on branches and trunk of young and old Larix decidua from midsummer until autumn. It was recorded only at one locality (Kapčiamiestis) in the southernmost part of the South eastern Highlands.
The only species of Eulachnini currently feeding on Abies in Lithuania is C. (Cinara) pectinatae (Nördlinger, 1880). The single fi nding of this aphid was recorded in the Kaunas botanical garden (Middle Lithuanian Lowland) on the exotic species Abies koreana E.H. Wilson 1920. Aphids were scattered individually on small branches among needles.
Three species of the subgenus Cupressobium of the genus Cinara are present in Lithuania, and C. (Cupressobium) juniperi (De Geer, 1773) is the most common. We found it in all climatic regions, where it formed small colonies on young shoots of the native conifer Juniperus communis Linnaeus 1753. Another species, C. (Cupressobium) mordvilkoi that inhabits the same host plant, was much rarer (three samples from the southernmost part of the South eastern Highlands, Table 1). These aphids feed on branches and stems in spring, root collar in summer and thin twigs at the end of September. A third species of the subgenus, C. (Cupressobium) cupressi (Buckton, 1881) was rather common feeding on shoots and small twigs of the exotic conifers Thuja occidentalis Linnaeus 1753 and Juniperus virginiana Linnaeus 1753 in Lithuania.
Of the two species of the subgenus Schizolachnus of the genus Cinara, the most common was C. (Schizolachnus) pineti (Fabricius, 1781). We found it in all climatic regions in Lithuania, except Samogitian, feeding in dense rows along the mature needles of mostly Pinus sylvestris. Some samples were also collected from P. mugo and P. nigra. Another species, C. (Schizolachnus) obscura (Börner, 1940), was much rarer and recorded mostly from P. nigra in the Coastal Lowland region, with one sample from the Middle Lithuanian Lowland region.
Of the four species of the genus Eulachnus the most common were E. agilis (Kaltenbach, 1843) and E. brevipilosus Börner, 1940, which were mostly associated with Pinus sylvestris and feed solitary on old needles. Some samples of both species were also collected from Pinus mugo. Our samples of E. rileyi (Williams, 1911) were mostly from Pinus nigra, but also P. mugo and P. sylvestris. We found this species in the Coastal Lowland, Middle Lithuanian Lowland and South eastern Highland regions, the same as the two above-mentioned species of Eulachnus. The only sample of Eulachnus nigricola (Pašek, 1953) was collected in the Coastal Lowland region (Botanical garden at Klaipėda) from the needles of P. heldreichii. 204 Havelka et al., Eur. J. Entomol. 117: 199-209, 2020doi: 10.14411/eje.2020.021

Molecular diversity and taxonomy of species of Eulachnini in Lithuania
Average and range of within-species genetic diversity (p-distances) based on COI (668 bp) and EF-1α fragments (1017 bp) of 26 species of the tribe Eulachnini from Lithuania are given in Table 2. It is noteworthy, that this is the fi rst record of the partial COI and EF-1α sequences of C. (Cinara) hyperophila, C. (Cinara) pilosa and C. (Cinara) piceicola. The values of average intraspecifi c p-distances for the COI fragment were higher than for EF-1α fragment and ranged from 0% to 1.68% and 0% to 0.58%, respectively. The number of haplotypes varied from 1 to 7 (for EF-1α) or 9 (for COI) ( Table 2). Only one COI haplotype was detected in samples of C. (Schizolachnus) obscura. Identical EF-1α sequences were recorded in samples of C. (Cinara) cuneomaculata and C. (Cinara) costata. Samples of C. (Cupressobium) mordvilkoi and E. brevipilosus were represented by a single haplotype of each fragment. The proportion of differences (p-distances) between our Eulachnini samples are represented by the NJ trees based on both fragments . Average intrageneric pdistances were 4.73% (COI) and 2.58% (EF-1α) for the genus Eulachnus, and 7.69% (COI) and 5.48% (EF-1α) for the genus Cinara. Mean values of intergeneric p-distances were 11.71% for COI and 10.94% for EF-1α. Within each subgenus of the genus Cinara the average values of p-distances were: 7.00% (COI) and 3.62% (EF-1α) for the subgenus Cinara, 4.64% (COI) and 1.82% (EF-1α) for the subgenus Cupressobium and 0.51% (COI) and 0.34% (EF-1α) for the subgenus Schizolachnus. Mean values of p-distances were lowest between the subgenera Cinara and Schizolachnus and reached 7.40% for COI and 5.52% for EF-1α. Average p-distances between the subgenera Cinara and Cupressobium were 10.55% for COI and 10.95% for EF-1α. The values of mean p-distances between subgenera Cupressobium and Schizolachnus were 9.07% for COI and 11.43% for EF-1α.
Species delimitation based on COI and EF-1α sequences gave different numbers of candidate species. The Automatic Barcode Gap Discovery (ABGD) method generated 24 and 19 candidate species based on partial COI sequences and the coding part of the EF-1α fragment, respectively (Tables 3-4). General Mixed Yule Coalescent (GMYC) method resulted in 29 species based on COI fragment and   Table 3. Species delimitation based on COI fragment data using Automatic Barcode Gap Discovery (ABGD), General Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP). Species names are those of the morphospecies identifi ed. For more details, see Table  S2. Species names, where molecular species delimitation defi nitely contradict the morphospecies status, are in bold.

DISCUSSION AND CONCLUSIONS
The values of average intraspecifi c and interspecifi c pdistances for COI and EF-1α fragments recorded in this study coincide with previously published data (Kim & Lee, 2008;Bašilova & Rakauskas, 2012;Coeur d'Acier et al., 2014;Rakauskas et al., 2014;Chen et al., 2016a;Arnal et al., 2019). Consequently, the NJ trees based on partial sequences of both fragments coincide rather well with the morphospecies (Figs 4-5), as does the grouping of the COI sequences using Automatic Barcode Gap Discovery (ABGD) and General Mixed Yule Coalescent (GMYC) methods (Table 3). These analyses reveal the taxonomic similarity of C. (Cinara) piniphila and C. (Cinara) pinea. Their average between-species sequence divergences were 0.73% (0-1.88%) for COI and 0.44% (0-1.19%) for EF-1α, which is the lowest among the species pairs analysed in this study. Sequences of both species were intermixed forming a mutual clade on both NJ trees, despite the presence of introns in the partial sequences of EF-1α (Figs 4-5). Samples of C. (Cinara) pinea and C. (Cinara) piniphila were also intermixed or merged into one group by the PTP, GMYC and ABGD analyses (Tables 3-4). Such discrepancies between morphospecies and genospecies strongly supports the synonymy of both species and coincides with reference data (Chen et al., 2012;2016b;Arnal et al., 2019). Actually, minor differences and overlapping values of some characters are presented in the identifi cation keys for both species as well as the data on their morphology and feeding site, see Table 5. In addition, our data reveal that the number of hairs on antennal segment II is 10-13 for C. (Cinara) piniphila compared with 5-10 for C. (Cinara) pinea. Maximum length of the hair on the fi fth abdominal tergite in our material was 108-191 μm for C. (Cinara) pinea and 26-70 μm for C. (Cinara) piniphila. Reference data also indicate subtle differences in the host plant preference of both species. C. (Cinara) piniphila prefers young pines growing in costal dunes attended by Formica cinerea, whilst C. (Cinara) pinea prefers open habitats: forest margins, clearings, rocks and dry meadows (Albrecht, 2017). The samples collected and monitoring data also Young shoots and 1-2 year-old twigs. Bark of 1-or 2-year-old twigs, among needles.
indicates C. (Cinara) piniphila inhabits young pines (P. sylvestris, P. mugo) in costal and continental dunes, attended by Formica cinerea, whilst C. (Cinara) pinea inhabits the same species of pine, but older trees growing on more fertile soils, attended by other species of ants. Therefore, more data are needed, including a morphological analysis of all morphs based on clonal material originating from hypothesized species-specifi c environmental conditions: 1-2 years-old twigs of young pines growing on poor sandy soil (C. (Cinara) piniphila) versus shoots of mature plants (C. (Cinara) pinea). In particular, the morphological analysis of male genitalia might be of great importance in the case of Lachninae aphids due to previously unknown valuable peculiarities (Wieczorek et al., 2012).
Based on the samples from Lithuania, C. (Schizolachnus) obscura and C. (Schizolachnus) pineti seem to be problematic taxa. Both species formed separate well-supported clades on the NJ trees (Fig. 4), yet values of betweenspecies p-distances between samples were low (average 0.81%, range 0.65-0.98% for COI; average 0.55%, range 0.43-0.75% for EF-1α). Morphological identifi cation of the samples (based on Albrecht, 2017; Blackman & Eastop, 2019) coincide with the species delimitation results of GMYC (COI and EF-1α), PTP BI (COI) and ABGD (EF-1α) methods, but were mismatched by the others (Tables  3-4). Our data confi rm the differences in host specifi city of both species already reported by Albrecht (2017) and Blackman & Eastop (2019). Most of our samples of C.
(Schizolachnus) pineti were from Pinus sylvestris, whereas those of C. (Schizolachnus) obscura were from the P. nigra group. Therefore, the taxonomic status of these species awaits a proper analysis as was recently suggested by Kaszyca-Taszakowska et al. (2019).
From the ecological viewpoint, pine-dwelling species of the subgenus Cinara co-exist in Lithuania by exploiting different species of pine or different microhabitats on the same host plant. This is refl ected in our NJ trees based on COI and EF-1α fragments  and coincides with the Bayesian tree based on six gene fragments, including both aphid (COI, Cytb, Aph and EF-1α) and Buchnera aphidicola (GroEL and His) DNA sequences (Jousselin et al., 2013;Meseguer et al., 2015). The evidence that speciation of the Eulachnini was mostly host plant mediated is very strong. Nevertheless, one should not ignore the possible role of climatic niche, landscape history and geographic barriers (Jousselin et al., 2013;Meseguer et al., 2015;Arnal et al., 2019).
Our data provide information relevant to the discussion on the reliability of molecular markers and molecular species delimitation (Chen et al., 2012(Chen et al., , 2016aJousselin et al., 2013;Arnal et al., 2019). Namely, our data indicate that most species are distinct in their ecological and morphological characteristics. Accepting that the "traditional" species delimitation is correct, the most reliable molecular species delimitation was that based on the Automatic Barcode Gap Discovery (ABGD) method and COI sequence data set. It correctly delimited 24 of the 26 morphospecies used in the present study (Table 3). It also confi rmed the taxonomic uncertainty of the above-mentioned C. (Cinara) piniphila -C. (Cinara) pinea species complex. In addition, it confi rmed that C. (Schizolachnus) obscura and C. (Schizolachnus) pineti are closely related taxa (see above for more details). In summary, it correctly identifi ed all the material sampled. COI partial sequences were reliable also in combination with the General Mixed Yule Coalescent (GMYC) model (29 genospecies and 26 morphospecies, Table 3). In contrast, molecular species delimitation using EF-1α fragment analysis was least compatible with traditional taxonomy of all the methods used in this study (Table 4). Noticeably, the PTP method was most reliable when used with the EF-1α data matrix: it generated 20 (BI) and 21 (ML) candidate species against 26 morphospecies. In the case of the COI data it was 32 (ML) and 34 (BI) against 26 (Tables 3-4). In conclusion, niche analysis was the best way to delimit species. Morphological, molecular and other information is important, however, when diagnosing species already delimited on ecological grounds.
Currently the Lithuanian fauna of Eulachnini is rather poor compared with that of the southernmost Central European fl oristic province (27 species against 40 species in the Czech Republic, Slovakia and Austria). This is due to the poorer biodiversity of coniferous plants with only four conifers listed in the native fl ora of Lithuania: Pinus sylvestris, Picea abies, Juniperus communis and Taxus baccata Linnaeus 1753, the latter of which is a rare relict species (Ozolinčius et al., 2003). Currently, sixteen species of Eulachnini occur exclusively on native species of conifers in Lithuania (6 species) or mostly on native conifers (10 species, Table 1). Nine species of Eulachnini were recorded only on exotic species of conifers in Lithuania. Of the four samples of Eulachnus rileyi, three were collected from exotic species of pine and one from native P. sylvestris. Nevertheless, succession in plant communities due to global warming and increasing introductions of exotic plants (both natural and anthropogenic) might shortly enrich the Lithuanian fauna of Eulachnini. One of the possible exotic Lithuania newcomers is C. (Cinara) (Del Guercio, 1909), which is already in Poland and is reported producing sexual morphs important for spreading northwards. In addition, this spe-cies can overwinter on roots anholocyclically (Durak & Durak, 2015).