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FIRST RECORD OF TINOCALLIS ZELKOWAE (TAKAHASHI, 1919) (HEMIPTERA APHIDIDAE CALAPHIDINAE) IN THE IBERIAN PENINSULA

Aphididae Calaphidinae)

All Tinocallis viviparous females are alate and usually present paired spinal and marginal tubercular processes; some of them also have conspicuous black pigmentation on dorsal body and/or forewings (Quednau, 2003;Kanturski et al., 2018). However, the seasonal variation of winged forms in spring, as opposed to summer and autumn (more pigmented and with less sensoria) makes the definition of some species complex (Blackman and Eastop, 2021). The taxonomy of Tinocallis has been revised by Quednau (2003) establishing four subgenera besides the nominal (Eotinocallis Quednau, 2003, Orientinocallis Quednau, 2003and Sappocallis Matsumura, 1919. Recently, a molecular study of sequences of cytochrome oxidase I (COI) of Calaphidinae revealed the presence of cryptic species (Lee and Lee, 2017).
The study of a colony of Tinocallis recorded on ornamental trees of Zelkova serrata in a public garden in the city of Valencia (southeast of Spain) allows us to record the Asiatic aphid T. zelkowae for the first time in the Iberian Peninsula.

MATERIALS AND METHODS
Aphid samples were collected for the first time in the Viveros Gardens in the city of Valencia from early September to middle of December of 2017, when the fall of the leaves ended and subsequently, the colonies were monitored during the years 2018 and 2019. In these two years and with a monthly periodicity, hundreds of winged females and dozens of sexuales (male and oviparae) were collected and studied in three trees of Zelkova serrata (coord. 39.478854, -0.368260) by the last of the authors (NPH) and finally, for this work, 20 specimens of each morph were measured. Tinocallis takachihoensis samples (used in the construction of the phylogenetic tree) were collected from Ulmus minor Mill. in Alcalá de la Selva (Santuario de la Virgen de la Vega), Teruel (18 th July 2017). Samples were preserved in 70% and 100% ethanol for morphological and molecular studies, respectively. The aphids (sample references: 944 and 840) and the DNA material and sequences were deposited in the aphidological collection of the Institute for Integrative Systems Biology (Mixt Centre of University of Valencia and CSIC).
The microscopic preparations and measurements were taken following the methodology exposed by Nieto Nafría and Mier Durante (1998) using a Leica microscope and the illustrations using a clear camera adapted to it. Microphotographs were taken with a Leica DC digital camera with IM1000 version 1.10 software. Living specimen photos have been taken by Ángel Umaran with a Canon E0S 600D.
Total DNA was extracted separately from three individuals of a sample of T. zelkowae that had been preserved in 100% ethanol since the collection date, following the HotSHOT (Hot Sodium HidrOxide and Tris) method (Truett et al., 2000) using 60 µl of both alkaline lysis and neutralizing reagents. A 710 bp fragment of the 5' region of the mitochondrial gene coding the cytochrome c oxidase subunit 1 (COI) was amplified using primers LCO1490 and HCO2198 described by Folmer et al. (1994). PCR reaction and sequencing procedures are outlined in Pérez Hidalgo et al. (2012). As expected, a 710 bp DNA fragment containing a portion of the mitochondrial COI gene was amplified through PCR from the aphids analyzed. After removing sequences corresponding to primers used in the PCR reaction, the sequences obtained from each sample consisted of 658 nucleotides. Chromatograms were revised, primers trimmed and sequences corresponding to each individual aphid assembled using the Staden package v1.6.0 (Staden et al., 1998).

According to Lee & Lee
Oviparae (Fig. II, 3) measure 1.25-1.40 mm and in life are yellowish, with some areas of the antennae (Fig. II, 3; Fig. V, 3), a series of dorsal spots and part of the legs being black or smoked. Dorsal setae are claviform and very long. The antennae (Fig. IV, 3) are 0.54-0.56 times the body and the terminal process 0.54-0.78 times the base. The posterior tibiae are clearly swollen (the diameter of the tibia in the middle is 0.037-0.047 mm and have 18 to 34 pseudosensoria (Fig.  V, 4). For other measurements and ratios, see Table I.
Males (   head (with red eyes) and thorax are blackish, and abdominal pigmentation is more marked than on females (Fig.  II, 4; Fig. V, 2). The antennae (Fig. IV, 2) have secondary sensoria in all flagellum segments (26-30, 8-11, 5-10 and 2-4 in the segments III to VI, respectively); the terminal process is shorter (0.055-0.065mm) than in viviparous female and 0.44-0.54 times the base of the antennal segment VI. For other measurements and ratios, see Table I.

Biology and distribution
Tinocallis zelkowae is a monoecious holocyclic species with sexual morphs (winged males and oviparae) present in October-November in Japan (Inouye, 1968). It lives on undersides of leaves of Z. serrata and related species (Z. acuminata, Z. formosana, Z. schneideriana), although it has been collected frequently on Betulaceae (Alnus japonica, Carpinus sp.), Corylaceae (Corylus sieboldiana) and Fabaceae (Robinia, Glycine max and 'cultivated beans' (Holman, 2009;Blackman and Eastop, 2021). According to Blackman and Eastop (2021) records on Ulmus japonica may be misidentifications of other species of Tinocallis or cryptic species (Lee and Lee, 2017). The colonies of the Iberian Peninsula are abundant on the three Zelkova serrata sampling and have sexed from the first of October until the fall of the leaves, although it should be noted that adult males appear before (about 15 days) than the adult oviparous females.
It is a species of Eastern-Asiatic origin. It is known from Japan, Korea and China (Takahashi, 1919;Holman, 2009;Blackman and Eastop, 2021); in addition, it has been recorded from California (USA) and England (Stroyan, 1979;Kono, 1983), so this is the first record, which seems stable year through year.

DISCUSSION
Lee & Lee (2017) have described a new species of Tinocallis living on Z. serrata var. latifolia according to the molecular differences in the COI sequences and to slight differences in body size (1.19-1.47 mm in T. latifoliae; 1.63-2.47 mm in T. zelkowae), antennae (0.86-1.13 times the body in T. latifoliae; 0.53-0.79 in T. zelkowae) and with spinal tubercles more pigmented than those of T. zelkowae. However, the dorsal tubercles of winged viviparous females captured in Valencia are always strongly pigmented (Fig. III, 1) at all times of the year in which the observations took place. For this reason, we consider this character must be used with due precautions in the identification keys.
On the other hand, the recent description of T. latifoliae based on molecular sequences and morphological characters (Lee and Lee, 2017) means that identifications and descriptions of T. zelkowae previously made on morphological characters can be doubtful, since they could be based on specimens of T. latifoliae or from mixed colonies or even hybrid individuals. Currently, it can be stated that the species described by Takahashi (1919) and by Higuchi (1972) correspond to T. zelkowae. A separate question is to determine if under the name of T. zelkowae in his Atlas of the Drepanosiphinae of the World Quednau (2003) has included different forms of various species of Tinocallis living in Zelkova species from different countries, so a review with current taxonomic criteria would be necessary. Because of this, we consider that it is necessary to know (and describe) the sexual morphs of T. latifoliae in order to establish the differences between both species and that the descriptions must be accompanied by molecular studies, since it is possible that "there are other species of Tinocallis still to be described from Zelkova" (Blackman and Eastop, 2021).
Tinocallis zelkowae has the ability to live in the southeast of the Iberian Peninsula on Z. serrata even if it is a deciduous plant because it develops sexuales. Furthermore, winters are mild in the abovementioned area. This implies that T. zelkowae may be living in other areas of Europe where its plant is present. It is interesting to highlight that the population in Valencia changed in density throughout the years, from apparently stable and very abundant colonies in 2017 to only few individuals recorded in 2019. This opens questions about the real identity of the species presence in Spain: T. zelkowae is considered non-native, but up to date it cannot be considered invasive, because it is not cause of concern. It will be interesting to follow the colony development in future years to confirm or deny its stability. Valencia presents hot summers, with maximal temperature peaks of more than 30°C. In other works, it is clear that another species of the same genus, T. kahawaluokalani, decrease in density with hot urban temperatures (Parsons et al. 2019). The apparent slow disappearance of the colony could be linked to the same phenomenon.
It should also be noted that their presence has not been detected in Zelkova carpinifolia, which is located at very close distance (1.6 km) from the populations of T. zelkowae studied. There are no records on Z. carpinifolia of T. zelkowae but there are of other species of Tinocallis (T. nevskyi, T. saltans, T. zelkovae) (Blackman and Eastop, 2021). We do not know if the populations of T. zelkowae of England have been able to survive, but considering the above, it would not be possible to be present in England or in any other area of Europe living on some Zelkova used as an ornamental plant in parks and gardens.
Another species of the genus, T. nevskyi, is widely distributed in Europe (Germany, Austria, Belgium, Czech Republic, Denmark, Finland, France, Holland, Hungary, Italy, Poland, United Kingdom, Switzerland and Sweden) on different species of Ulmus and its presence in the Iberian Peninsula should not be ruled out.

ACKNOWLEDGMENTS
The authors are very grateful to Doc. Francesca Maria Gatti (Université de Strasbourg, Master in Translation, specialized in English and Russian) for reviewing the article in English. The authors want to express their special thanks to the librarian of the IVIA (Instituto Valenciano de Investigaciones Agrarias) Josep V. Castelló and to Dr. Masakazu Sano from National Agriculture and Food Research Organization of Japan for their help in providing all the Japanese references. Authors are also grateful for the contributions of the two reviewers.
Alice Casiraghi benefited from an Erasmus+ scholarship during the year 2018-2019, to do her Master thesis at the University of Valencia. Work accomplished in the context of Project CGL2015-68188-P, funded by "Ministerio de Economia, Industria y Competitividad" of Spain (MIMECO).