Systematic position of Eulachnus cembrae Börner with description of hitherto unknown sexual morphs of E . pumilae Inouye ( Hemiptera , Aphididae , Lachninae )

The identity of Eulachnus cembrae Börner, 1950, stat. rev. from Europe, treated as a synonym of E. pumilae Inouye, 1939 from East Asia is clarified based on characters of sexual morphs. The oviparous female and alate male forms of E. pumilae are described and figured in detail for the first time and the poorly known sexual forms of E. cembrae are redescribed and figured in detail as well. Sexual morphs of the two similar species are compared, and significant differences clearly distinguishing those species are presented. A key to the identification of oviparous females and males of E. cembrae and E. pumilae as well as notes on host plants and distribution of these species are provided. The status of E. pumilae in the European aphid-fauna is clarified. Morphological characters of the sexual generation that may be useful for species identification are discussed.


Introduction
The Palaearctic genus Eulachnus Del Guercio, 1909 comprises about 13-18 species of small, narrow-bodied aphids, of which about 12 are known from Europe.They live often singly or in small colonies on the needles of Pinus spp., are hidden while feeding and become very active when disturbed (Blackman and Eastop 1994;Kanturski and Wieczorek 2014).Taxa from the genus Eulachnus are good examples for species and species-groups of unclear identity (Blackman and Eastop 2014).Such an example is the species pair E. pumilae Inouye, 1939 andE. cembrae Börner, 1950, stat. rev.In comparison to the other Eulachnus species they are characterized by the absence of dorsal sclerites and scleroites on the abdomen.
E. pumilae was described by Inouye (1939) from Hokkaido (Japan) from Pinus pumila, whereas Börner (1950) described E. cembrae from the Eastern Alps from P. cembra.Many authors treated those two species as synonyms (Inouye 1970;Ghosh 1982;Blackman and Eastop 1994).However,  Remaudière and Remaudière (1997) stated that E. cembrae should be treated as a separate species due to a difference in the number of accessory setae on the apical segment of the rostrum (2 setae in E. pumilae, no setae in E. cembrae), which is a difference also mentioned in Blackman and Eastop (2014).The problem of the identity of E. cembrae has not been resolved yet.The most recent papers of Mamontova (2011Mamontova ( , 2012) ) still treat these two species as synonyms.Moreover, in the Fauna Europaea E. pumilae is recorded as European species (Nieto Nafría et al. 2014), known only from Slovakia (Goffova and Wojciechowski 2013).
The descriptions of E. pumilae and E. cembrae were based on characters of the viviparous generation, although there were also sexual morphs in the type material of Börner (1950).Pintera (1968) briefly described the sexual generation of E. cembrae.Oviparae were characterized by numerous pseudosensoria; males were winged with numerous rhinaria on the antennae.Similar information was reported by Szelegiewicz (1978).The life cycle and sexual forms of E. pumilae were not described.Ghosh (1982) gave a description of sexual forms under the name E. pumilae, but this was in reality a description of sexual forms of E. cembrae.
The aim of this paper is to define the taxonomic status of these two species by morphological and biometric examination of their sexual morphs, especially sexual forms of E. cembrae from the type material.On the basis of the material deposited in the Natural History Museum, London (UK), a description of sexual forms of E. pumilae is provided as well as a redescription of the sexual generation of E. cembrae.Moreover, the role of the characters of the sexual generation is highlighted, especially the underestimated and rather rarely used features of the male genitalia.
Media with 1 fork.Hind legs long, covered by blunt and pointed setae, not longer than width of tibiae.Basal length of HT I 0.26-0.31times dorsal, 0.21-0.23 times ventral and 0.68-0.80times intersegmental length, with 2 dorsal and 14 ventral, pointed setae (Fig. 2k).HT II 0.35-0.38 times length of ant.segm.III and 0.72-0.83ant.segm.VI.Dorsal side of abdomen membranous with pointed setae, on abd.segm.I-VI 0.017-0.025mm long, on segm.VII-VIII 0.032-0.052mm long.Spinal setae arranged in two pairs on each segment arising from oval scleroites (Fig. 2l).Siphunculi very low with narrow cone-shaped base.Abd.segm.VI and VII sclerotised on whole surface.Cauda broadly rounded with numerous long, fine and pointed setae and very short spinules.Parameres present, located above basal part of phallus, clearly visible, basally fused.Their lobate parts, capitate in shape, arise into distinct, forceps-like projection toward base of phallus.Parameres dark pigmented, with numerous long setae on entire surface.Basal part of phallus club-shaped, brown, with few short setae in middle part.Sclerotized arms clearly visible, strongly sclerotized, dark pigmented.Proximal part robust and ends in triangular apex, distal part thinner.Sclerotized arms form upper half-circle-shaped structure that surrounds genital area (Fig. 3b, d).

Discussion
Aphids are a group of hemipterans whose classification is still controversial, as evidenced by uncertainties about the identity of many species in this group of insects (Blackman and Eastop 1994;Heie 1995).Many of these uncertainties at species level in aphid taxonomy might be resolved by studying morphs other than apterous and alate viviparous females, especially the sexual generation (i.e.oviparous females and males), which have strictly-established species characters and are likely to vary much less than the parthenogenetic forms (Hille Ris Lambers 1966;Wieczorek et al. 2013b).
The genus Eulachnus as a whole is an example for a group of aphids which needs revision, because many of the characters that have been used in species discrimination are subject to environmental influences (Blackman and Eastop 2014).This also applies to E. cembrae, E. pumilae and E. piniarmandifoliae Zhang from China, which form a separate group within the genus Eulachnus characterized by the absence of dorsal scleroites at the base of thoracic and abdominal setae.The type species of the discussed genus, E. agilis (Kaltenbach), as well as other European and Asiatic species, are identified by the presence of numerous scleroites with setae of various lengths and shapes on the dorsal side of the abdomen.On the generic level, this specific character occurs also in sexual morphs: oviparous females of E. pumilae and E. cembrae (sexual forms of E. piniarmandifoliae are unknown) can be easily recognized by the absence of dorsal sclerites and scleroites on the abdomen whereas in males only spinal scleroites with short setae are present on the abdomen.Those two species are similar with respect to the absence of the dorsal sclerotization of the thorax and the abdomen, but otherwise they significantly differ with respect to both morphological and biometric characters.In particular, sexual forms of E. pumilae possess two accessory setae on the ARS, as mentioned by Remaudière and Remaudière (1997), but also longer setae on the head, antennae and the abdomen.Oviparous females of E. pumilae differ from those of E. cembrae with respect to the ratios of body length to antennal length, and also with respect to individual ratios of HT I basal, dorsal, ventral and intersegmental length; measuring those ratios is always a good method to distinguish closely related species, especially in the tribe Eulachnini (Szelegiewicz 1978;Heie 1995).The oviparous females also differ by the number of pseudosensoria on the hind tibiae (Table 2), which is one of the most easily recognizable characters of parthenogenetic and sexual aphid females.The alate males of E. pumilae and E. cembrae differ significantly with respect to the number of secondary rhinaria on antennal segments III, IV and VI and the ratios of the ARS to the antennal segments VI or HT II (Table 3).As males are the rarest morphs of aphids, appearing only for a short period of time, the taxonomic value of the characters of their genitalia has not been fully exploited as yet.However, a comparative, systematic study of the male genitalia of the Aphididae has revealed a number of characters that may potentially be useful in discussions on the phylogenetic relationships, species identity and identification of these insects (Wieczorek et al. 2011(Wieczorek et al. , 2012(Wieczorek et al. , 2013a)).E. pumilae and E. cembrae, as most Lachninae, belong to a group of aphids with strongly modified genitalia, with parameres divided into lobate parts arising into projections, a well-developed basal part of the phallus and sclerotized arms forming the upper half-circle-shaped structure that surrounds the genital area (Wieczorek et al. 2012).On the species level, the shape of paramere projections (finger-like in E. pumilae, forceps-like in E. cembrae), the basal part of the phallus (crescent-shaped in E. pumilae, club-shaped in E. cembrae), and especially the structure of sclerotized arms (distal part robust, strongly flattened with a thorn-like process located on the inner edge in E. pumilae and thin in E. cembrae) are key characters in the identification of E. pumilae and E. cembrae.
According to Fauna Europaea (Nieto Nafría et al. 2014) E. pumilae is a European species, recorded from Slovakia.The checklist of Aphidomorpha from Slovakia (Goffova and Wojciechowski 2013) also reported this species.Goffova and Wojciechowski (2013) cited the paper of Pašek (1952), whereas in this work on the genus Eulachnus (Protolachnus in the original) only E. agilis, E. bluncki (= E. rileyi), E. nigricola and E. cembrae were listed.The record of E. pumilae in Slovakia given by Fauna Europaea cites probably Holman and Pintera (1977), where the authors treated E. cembrae as a synonym of E. pumilae.The latter record from Ukraine presented by Mamontova (2012) should also be treated as E. cembrae.In the description as well as in the figure the author presents the apterous viviparous female with ARS without accessory setae, which is the key character to distinguish these two species.Moreover, the characters of sexual morphs overlap with features of E. cembrae.Thus all records of E. pumilae in Europe are in fact records of E. cembrae, and E. pumilae does not occur in Europe.
Separateness of these two similar species is also reflected by their biology: E. cembrae is a European species, recorded mostly from locations in central European mountain ranges (the Alps, the Carpathians) (Börner 1950;Heinze 1962;Pašek 1952;Szelegiewicz 1968;Chumak 2004).It may also occur in other, submontane regions (e.g.artificial plantings of P. cembra in Zakopane, Poland (Szelegiewicz 1978) or in the Botanical Garden in Cracow (Kanturski and Wieczorek 2014)).E. pumilae, on the other hand, should be treated as an East Palaearctic species, recorded from Japan, Korea, India and East Siberia (Inouye 1939(Inouye , 1970;;Szelegiewicz 1974;Ghosh 1982;Pashchenko 1988;Lee et al. 1994).Host plants of both species mostly belong to the subsection Cembrae of the Pinus section Strobus.E. cembrae is always associated with the Swiss stone pine P. cembra and occasionally with P. strobus (Holman 2009), whereas E. pumilae is associated with P. koraiensis, P. parviflora, P. pentaphylla, P. pumila and P. strobus (Blackman & Eastop, 2014).Recent molecular studies have shown that P. cembra is clearly separated from the closely related P. koraiensis, P. parviflora and P. pumila which form a distinct clade (Liston et al. 1999;Wang et al. 1999;Gugerli et al. 2001).
Detailed morphological and biometric analysis of the sexual morphs of the studied species, including type material of E. cembrae designated by Börner, supported by biological data, definitely distinguish the studied species as separate taxa.
Key to oviparous females of E. cembrae and E. pumilae.

Figure 3 .
Figure 3. External male genitalia of Eulachnus pumilae (a, c) and E. cembrae (b, d): bp -basal part of phallus with sclerotized arms consists of short proximal (solid arrow), long distal (dotted arrow) part and upper half-circle-shaped structure that surrounds the genital area (arrow-head), P -parameres, C -cauda.

Table 2 .
Main morphological differences between oviparous females of E. pumilae and E. cembrae AL-antennae length, BLbody length, ANT IV-antennal segment IV length, ANT IIIant.segm.III length, HT I bL-first segment of hind tarsus basal length, HT I dL-HT I dorsal length, HT I iL-HT I intersegmental length, HT I vL-HT I ventral length ARS-apical segment of rostrum, HT II-second segment of hind tarsus length.
fused.Their lobate parts arise into distinct, finger-like projection toward base of phallus.Parameres dark pigmented, with numerous, long setae on entire surface.Basal part of phallus crescent-shaped, light brown, with numerous long setae.Sclerotized arms clearly visible, strongly sclerotized, dark pigmented.Proximal part robust, ends in triangular apex.Distal part also robust, strongly flattened with thorn-like process located in inner edge of arm.Sclerotized arms form upper half-circle-shaped structure that surrounds genital area (Fig.