This redescription of Nidularia balachowskii is based on type material (see Material examined below), plus specimens from Israel collected by Bodenheimer and fresh material collected in Israel by us. Populations of Nidularia balachowskii from Quercus ithaburensis trees were studied and specimens were collected between 2010 and 2012 from the following nature reserves in northern Israel: Yehudiya Nature Reserve, Golan Heights (32°56'19"N, 35°39'56"E); Horshat Tal Nature Reserve, Upper Galilee (33°13'13.74"N, 35°37'45.65"E); Alonei Abba Nature Reserve, Lower Galilee (32°43'46.2"N, 35°10'18.47"E). Trees at each reserve were surveyed at least once a month and 150–200 branches (20–25 cm in length) were removed at each visit. The branches were taken back to the laboratory in large plastic bags and examined individually under a stereomicroscope for scale insects. Relevant specimens were slide-mounted for microscope examination using the protocol in Ben-Dov, Hodgson (1997). Specimens of Nidularia pulvinata (adult females and first-instar nymphs) became available from MNHN. Material of Nidularia japonica was not available and comparisons with this species were based on the original description by Kuwana (1918) as well on the redescription by Liu et al. (1997). Dry and mounted material of Nidularia balachowskii from Israel, are deposited in the ICVI, BMNH and MNHN.

Illustrations of the adult female and the first-instar nymph of Nidularia balachowskii are generalizations of several specimens, showing the dorsum on the left and the venter on the right, with enlargements of important structures arranged around the main drawing. The enlarged structures are not drawn to the same scale. Terms for morphological features follow chiefly those of Bullington and Kosztarab (1985), Baer and Kosztarab (1985) and Hodgson (1994). Measurements of specimens and of morphological structures were made using an ocular micrometer on an Olympus BX51 phase contrast microscope. Measurements of structures are given in microns (µm) and millimeters (mm). Body length was measured from the farthest points of the head to the posterior end of the body, and body width was the greatest width. Setae lengths were measured from the tip of its base (excluding the setal socket) to the apical tip of the setae. The frequency of each structure is given for the entire body. The range is taken from twenty specimens.

Abbreviations of specimen depositories are as follows: BMNH -The Natural History Museum, London, U.K.; ICVI - Coccoidea Collection, Department of Entomology, Agricultural Research Organization, Bet Dagan, Israel; MNHN - Museum National d’ Histoire Naturelle, Paris, France; and TAU - Tel Aviv University Insect Collection, Israel.

Turkey: Lectotype female (ICVI), here designated, and paralectotype female (MNHN), 21 km at road from Mardin to Diyarbakir, on branches and twigs of Quercus sp. (Fagaceae), 13.ii.1939, F.S. Bodenheimer. Bodenheimer (1941) did not select a holotype, and we regard the above-mentioned specimens as the original material studied by him as indicated on the slide labels.

Additional non-type material from Turkey as follows: Van-Koçet Road (alt.1625 m) on Quercus sp., 19.vii.2005, B. Kaydan (Yuzuncu Yil University, Turkey 2056); Hakkari -Üzümcü Road (alt. 956 m) on Quercus sp., 15.ix.2005, B. Kaydan (Yuzuncu Yil Universty, Turkey 2343); Van-Hakkari Road (alt. 1266 m) on Quercus sp., 16.ix.2005, B. Kaydan (Yuzuncu Yil Universty, Turkey 2370); Hakkari-Doğan (alt. 1032 m) on Quercus sp., 22.v.2005, B. Kaydan (Yuzuncu Yil Universty, Turkey 2688); Bitlis River (alt. 797 m) on Quercus sp., 23.vi.2006, B. Kaydan (Yuzuncu Yil Universty, Turkey 3036); Bitlis-Kavakbaşı (alt. 1365) on Quercus sp. 30.v.2007, B. Kaydan (Yuzuncu Yil Universty, Turkey 3419).

Israel: adult female, Daphne Oaks (= current name Horshat Tal Nature Reserve), on Quercus sp., 1.v.1939, F.S. Bodenheimer, (ICVI C:4805). This was the first record of this species from Israel. Additional females and first-instar nymphs, all collected off Quercus ithaburensis by M. Spodek: Yehudiya Nature Reserve, 10.x.2010, 7.xi.2010, 11.i.2011, 6.ii.2011, 16.x.2011, 6.xi.2011, 3.vi.2012, (ICVI C:4891, C:4912, C:4945, C:4970, MC:587, C:4970, MC:690); Horshat Tal Nature Reserve, 30.v.2010, 14.ii.2012, 27.ii.2011, 13.iii. 2012, (ICVI MC:228, MC:614, MC:430, C:5131); Alonei Abba Nature Reserve, 11.1.2011, (ICVI MC:385). First-instar nymphs; Yehudiya Nature Reserve, 6.iii.2010, 20.iii.2011, 24.iii.2012, (ICVI MC:140, MC:460, MC:637); Horshat Tal Nature Reserve, 13.iii.2012, 15.iii.2012, (ICVI MC:635, MC:636).

Comparative material examined

Nidularia pulvinata: France: adult female and first-instar nymphs, Serignan, Vauctuse, on Quercus ilex, 18.v.1978, I. Foldi (ICVI C:4946); first-instar nymphs, Caumont (Avignon), on Quercus ilex, 11.iv.1978, D. Matile, J.P. Fabre, (MNHN 7337-4); Italy: adult femalePisa, on Quercus ilex, 5.iv.1988, D. Matile-Ferrero (MNHN10959, ICVI 5060)

Kermes quercus: Sweden: adult female, Skan, Near Lund on Quercus robur, 10.vi.2010, C.A Gertsson (ICVI C:4806); England: adult female , Wytham Wood, Berkshire on Quercus robur, 10.v.1965, S.C. Varley (BMNH 81-539); Poland; first-instar nymph, Warsaw on Quercus robur, 25.viii.1994, E. Podsiadlo (ICVI C:4798)

Kermes roboris: Hungary: adult female, Budapest, Plant Protection Institute-adjacent track on Quercus sp., 8.vi.1989, C.P. Malumphy (BMNH); England: adult female, Kent: Herne Bay on Quercus spp., 00.viii.1899, C.D Waterhouse (ICIV C:5071).

Specimens of the following adult female kermesidspecies, identified by YBD, were used in the molecular part of this study: Kermes nahalali Bodenheimer, Kermes echinatus Balachowsky, Kermes greeni Bodenheimer, Kermes quercus (Linnaeus), Kermes spatulatus Balachowskyand Nidularia balachowskii. Three specimens of each species were used as replicates except for Nidularia balachowskii, where six were used. Adult females, preserved in 96% ethanol, were examined under the stereomicroscope for the presence of hymenopteran parasitoid wasps prior to DNA extraction. Voucher specimens were slide-mounted using the cuticle of the actual specimens from which DNA was extracted. Slide mounting followed the protocol outlined in Ben-Dov, Hodgson (1997), and the voucher slides are deposited in the ICVI (Table 1). To provide some taxonomic context to our study, we included also DNA sequences of species belonging to other families within the Coccoidea: Asterolecaniidae, Coccidae, Diaspididae, Eriococcidae, Monophlebidae and Pseudococcidae. A species of aphid, Acyrthosiphon pisum (Hemiptera, Aphidoidea), was used as the outgroup species. These sequences were made available from GenBank (Table 1). The sequences of Kermesidae species obtained in this study are deposited in the GenBank under the accession numbers JX436113 - JX436154.

DNA was extracted from parasitoid-free adult females using the Cetyl trimethylammonium bromide (CTAB) method (Murray and Thompson 1980). Polymerase chain reaction (PCR) products were generated from the mitochondrial Cytochrome Oxidase I (COI) gene, and a fragment of the D2 and D3 regions of the 28S ribosomal DNA gene. PCR reaction was performed in a total volume of 25 µL containing 1 unit of dream Taq polymerase (Fermentas, USA), 2.5 µL of enzyme buffer supplemented with MgCl2, 0.2 µL of 25 mM dNTPs, 0.3 µL of 20 pmole for each primer, and 2 µL of DNA template. A 900 bp fragment of the 28S ribosomal RNA gene and a 400 bp fragment of the COI gene were amplified and sequenced. Primers for both genes were 28S forward 5’-GAC CCG TCT TGA AAC ACG GA-3’ and 28S reverse 5’-TCG GAA GGA ACC AGC TAC TA-3’ (Gullan et al. 2010). COI forward 5’-CAA CAT TTA TTT TGA TTT TTT GG-3’ (C1-J-2183 aka Jerry) and COI reverse 5’-GCW ACW ACR TAT AKG TAT CAT G-3’ (C1-N-2568 aka Ben) (Gullan et al. 2010). The COI barcode region (Herbert et al. 2003) was not used because it has failed to amplify in most scale insects tried to date (Schroer et al. 2008).

The PCR cycling conditions for 28S were 94°C for 4 min, followed by 35 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1.5 min, with a final extension at 72°C for 4 min. The PCR cycling protocol for COI was 95°C for 7 min, followed by 40 cycles of 95°C for 1 min, 45°C for 1 min, and 72°C for 1.5 min, with a final extension at 72°C for 5 min. Each reaction was examined by electrophoresis and bands were visualized with UV light. PCR products were excised from the gel and purified using the Zymoclean Gel Extraction Kit (Zymo Research, Irvine, CA). Purified PCR products were sequenced in both the forward and reverse directions at Hy-Labs (Rehovot, Israel).

Sequence alignments for both 28S and COI gene sequences were performed with MUSCLE 3.7 (Edgar 2004) and the results were adjusted manually where necessary to maximize alignment. The alignment data for each gene were used in maximum likelihood tree construction, using Kimura-2 parameter model (K2P) genetic distances (Kimura 1980). Both trees were generated using MEGA v.5 (Tamura et al. 2011) and branch support was estimated with 1000 bootstrap replicates.

We obtained a total of forty-two nucleotide sequences from the 28S and COI genes from Nidularia balachowskii (six individuals for each gene) and from five adult female Palaearctic Kermesidae species (three individuals for each species for each gene). 28S gene sequences (~700 bp) and COI sequences (~400 bp) from all species were recovered and aligned with sequences of Coccoidea species representing different families (obtained from GenBank). All species for which multiple specimens were sampled showed no interspecies variation. The maximum likelihood analysis of both genes resulted in tree typologies that show that Nidularia balachowskii is a distinct species within the monophyletic Kermesidae. Nidularia balachowskii is grouped together with other kermesid species and not with the other Coccoidea (Figures 5a+b). The bootstrap value that represents the separation between species of Kermesidae and species from other Coccoidea families is higher in the 28S tree typology, 74, compared to 60 obtained from the COI sequences.

Sequence divergence based on Kimura 2-parameter pairwise distance, between Nidularia balachowskii and the other five Kermesidae species ranged from 0.16–0.19 in the 28S gene region. This range is compared to the 0.2–0.3 sequence divergence range between Nidularia balachowskii and species from the four other Coccoidea families. In the COI gene region, the sequence divergence between Nidularia balachowskii and the six other Kermesidae species ranged from 0.06–0.13 and between Nidularia balachowskii and the four species from other Coccoidea families had a sequence divergence range of 0.8–1. Both trees show a strong relationship between Nidularia balachowskii and Kermes echinatus, indicating that they are closer to each other than to the other Kermesidae species examined.

Observations about the life history of Nidularia balachowskii were made in three nature reserves in northern Israel: Alonei Abba Nature Reserve, Horshat Tal Nature Reserve and Yehudiya Nature Reserve. The predominant oak species growing in these reserves is Quercus ithaburensis. In Alonei Abba Reserve, Quercus calliprinos trees are also present but they are less common. In Israel, Nidularia balachowskii has only been found on the trunks and branches of Quercus ithaburensis, where Nidularia balachowskii is an oviparous and univoltine species.

Gravid females were observed on branches and trunks of trees throughout March, during which time they oviposited 200 to 250 (range from 10 specimens) whitish eggs. Each egg was about 0.4 mm long and 0.2 mm wide. Once all of the eggs have been laid and the brood chamber full of eggs, the female dies and the dorsum becomes sclerotized. The sclerotized, convex body of the dead, post-reproductive female may remain on the host tree for a year or more after first-instar emergence.

Eclosion of first-instar nymphs occurs inside the brood chamber and nymphs emerge from the cavity under the dead female body. This takes place from end of March and throughout April. Crawlers settle in bark crevices on branches and on the trunks of the trees. Young teneral females are found on the branches from June to February. The females continue feeding and increase in body size throughout this period. Feeding was confirmed by observations of honeydew elimination. By late February, the dorsum of the female begins to expand greatly, increasing in convexity and sclerotization. The ventral surface of the abdomen becomes concave, forming the brood chamber into which the eggs are deposited. The ovipositing female secretes a woolly, white wax that surrounds its body margin. No injury has been observed to the oak hosts by Nidularia balachowskii in Israel.

We compared our observations of the host plant and development of Nidularia balachowskii in Israel to Bodenheimer’s 1941 records of this species. In Israel, this scale insect has only been found on the trunks and branches of Quercus ithaburensis trees, whereas Bodenheimer gives Quercus sp. as the host tree in Turkey (Bodenheimer 1941) and Iran (Bodenheimer 1944). The geographical distribution of Quercus ithaburensis is wide, extending also to Turkey and Iran, and so we may speculate that Bodenheimer’s Quercus sp. is probably Quercus ithaburensis ssp. macrolepis (Dufour-Dror and Ertas 2004).

We observed that Nidularia balachowskii is an oviparous, univoltine species in Israel, similar to Bodenheimer’s (1941) observations in Turkey. Earlier observation on the other two species of Nidularia indicatedthat both Nidularia pulvinata in Italy (Viggiani 1991) and Nidularia japonica in China (Liu et al. 1997) are univoltine. All three species of Nidularia have only been recorded so far on oak trees (Ben-Dov et al. 2012). Koteja (1980) redescribed Nidularia pulvinata and noted that young specimens were covered with a fragile layer of wax and that, during expansion of the dorsum, this layer breaks into pieces and the females then secrete a nest-like ovisac ventrally and laterally. Kuwana (1918) and Liu et al. (1997) both describe a nest-like ovisac for female Nidularia japonica. In Israel, the teneral adult female of Nidularia balachowskii also produces a thin layer of dorsal wax but gravid females do not produce a nest-like ovisac. The eggs of Nidularia balachowskii are deposited into the egg cavity beneath the venter of the female, as described by Bullington and Kosztarab (1985) and Podsiadlo (2005a) for other kermesid species.

Some morphological characters of adult female Nidularia balachowskii are compared with those of Nidularia pulvinata, Kermes quercus and Kermes roboris (type species of Kermes) in Table 2, in order to evaluate the generic and family placement of the former species. All four species possess the following synapomorphic traits: three-segmented labium, bilocular pores on the venter, simple pores on the dorsum, quinquelocular pores surrounding the spiracles, and tubular ducts on the venter.These characters are some of the synapomorphic characters of kermesid adult females that have been described by Ferris (1955), Koteja (1980), Bullington and Kosztarab (1985) and Hodgson (1997).

Within the genus Nidularia, adult female Nidularia balachowskii share with Nidularia pulvinata the following characters: (i) one-segmented antennae; (ii) absence of legs; (iii) absence of setae-pore clusters on venter; (iv) ventral position of anal ring, and (v) an anal ring with setae and cells, whereas the two Kermes species have: four, five or six-segmented antennae, and possess legs, setae-pore clusters on venter, anal ring placed on dorsum, and an anal ring without setae and cells. Comparing Nidularia balachowskii and Nidularia pulvinata, the most obvious distinguishing feature is the presence of quinquelocular pores on the spiracle peritreme of Nidularia pulvinata.

The morphological characters of first-instar nymphs of Nearctic kermesids were summarized by Baer and Kosztarab (1985). Kuwana (1931) and Hu (1986) outlined some distinctive characters for separating first-instar nymphs of Oriental species of kermesids, and Balachowsky (1950, 1953) reviewed the characters of first-instar nymphs of some Palaearctic kermesids.

The first-instar nymphs of Nidularia balachowskii share the following characters with other Kermesidae species: (i) six-segmented antennae; (ii) three-segmented labium; (iii) simple pores forming longitudinal lines on the dorsum (iv) dorsal setae; (v) anal ring with cells and setae; (vi) microspines in rows on abdominal segments, and (vii) bilocular pores on venter. This last character is sometimes overlooked.

Nidularia balachowskii can be distinguished from other Kermesidae species by the form of its marginal setae. Nidularia balachowskii has sharply spinose, apically pointed and slightly curved setae, each 9–15 µm long. This differs from Nidularia pulvinata which has hair-like setae (Koteja 1980) and Nidularia japonica which has setose setae that are somewhat conical at the base (Kuwana 1918, Liu et al. 1997).The first-instars of Palaearctic Kermes species that have been described possess conical, hair-like or spatulate marginal setae (Kuwana 1931, Balachowsky 1950, 1953, Sternlicht 1969, Hu 1986, Liu et al. 1997, Podsiadlo 2005b, Pellizzari et al. 2012). There is no unique set of characters that distinguishes the first-instar nymphs of the genus Nidularia from the Palaearctic Kermes examined.

The DNA-sequence data for Nidularia balachowskii, six other species of Palaearctic Kermesidae, and species representing six other Coccoidea families showed that gene fragments of both COI and 28S separated Nidularia balachowskii from other Coccoidea species, and clearly placed Nidularia balachowskii in the Kermesidae. This study confirms that Nidularia balachowskii is a distinct species, clearly distinguishable from other closely-related kermesid species.