Tettigettalna josei (Boulard, 1982) (Hemiptera: Cicadoidea): first record in Spain, with notes on the distribution, genetic variation and behaviour of the species

Abstract The small cicada Tettigettalna josei (Boulard, 1982) was until recently only known from southern Portugal and was considered endemic to this country. Fieldwork in 2013 led to the first record of the species in Spain, expanding its known eastern range to Andalusia. The northern limits remain poorly defined but it appears that the distribution of Tettigettalna josei is restricted to the south Atlantic coastline in the Iberian Peninsula, with the highest densities found in Algarve. Some notes on behaviour and genetic variation of Tettigettalna josei are also given.


Introduction
Cicadas are well known for the acoustic signals produced by males during the mating season to attract females. Male calling songs are species-specific and easily distinguished by trained human ears. The acoustic signals are taxonomically valuable, being effective to distinguish between close cicada species and are especially useful to identify sibling species when they occur in the same geographical area (Boulard 1982).
The biodiversity of cicadas (Hemiptera: Cicadoidea) worldwide is remarkable but many taxa remain poorly known or even undescribed. Recent work in the Iberian Peninsula led to the revision and description of nine species under the genus Tettigettalna (Puissant and Sueur 2010). All species comprising the genus are small-sized (body length < 25 mm) and seven species are considered to be endemic to southern Iberia (Puissant and Sueur 2010, Simões et al. 2013, Sueur et al. 2004. The distribution boundaries of these species remain poorly defined because they are based on sporadic records and extensive field surveys have yet to be undertaken. Three Tettigettalna species (T. mariae, T. josei and T. estrellae) were described from Portugal and were thought to be endemic to this country, although their distributions in other parts of the Iberian Peninsula had never been investigated. Recent field surveys showed that the distribution of T. mariae actually extends to the neighbouring country, Spain, and this species is now considered as an Iberian endemism (Simões et al. 2013). As an outcome from intensive fieldwork for species of genus Tettigettalna in both southern Portugal and Spain during the summers of 2011-2013, we report here an update on the distribution range of T. josei, with the discovery of its presence in Andalusia in the summer of 2013.

Materials and methods
Several field surveys were conducted in the southern Iberian Peninsula (regions of Algarve and Andalusia) from the end of June until mid-August during the summers of 2011-2013. The fieldwork took place from 10:00 am to 07:00 pm with sunny weather and with temperatures ranging from 24 to 39°C degrees. Initial searches in 2011 were conducted by driving a car at low speed to allow the detection of the calling males. In 2012 and 2013, searches were targeted to areas of potentially suitable habitats. Geographical coordinates were determined with a GPS (Garmin, Oregon series 550t) for each site where male songs of Tettigettalna josei were heard or where specimens were collected.
Species were located through their calling song and their songs recorded in the field, followed by capture with a sweeping net. Acoustic signals were recorded using a Marantz PMD 661 Portable SD recorder (20 Hz -24 kHz) connected to a Telinga Pro 7 Dat-mic microphone (Twin Science) following the procedures given in Quartau et al. (1999). Captured specimens were conserved dry ( Fig. 1) at the general data bank on insect data at the Department of Animal Biology in the Faculty of Sciences, University of Lisbon (FCUL). A front leg was removed from each specimen and preserved in absolute ethanol for DNA isolation.
Time and frequency analysis of sound recordings from males were conducted with software Avisoft SASLab Pro (Specht 2012) as in previous analyses (e.g. Quartau et al. 1999, Simões et al. 2000. Acoustic recordings were analysed with a sampling rate of 44.1 kHz and a resolution of 16 bits. Spectra were computed using FFT with a resolution of 512 points and a Hamming Window. For each male, recordings of about one minute were analysed. Song terminology follows that of Gogala and Trilar (1999), Gogala and Trilar (2000).
Seven morphologic measures were taken from collected males in Spain as in Boulard (1982), namely, total length (TL), body length (BL), wingspan (WS), right hindwing length (aWL), head width (HW), mesonotum width (MW) and width at the level of the auditory capsules (ACD).
Whole-genome DNA was isolated with the E.Z.N.A.® Tissue DNA Isolation kit (Omega Bio-Tek). Sequences of 581 base pairs from the 5' region of the cytochrome c oxidase I (COI) mitochondrial gene were obtained from 15 males of T. josei. Sequences were obtained as described in Nunes et al. (2014) and deposited in GenBank (accession numbers KF977491-KF977505). A minimum spanning network was constructed with the medianjoining method (Bandelt et al. 1999) in NETWORK 4.6.1.1 (http://www.fluxusengineering.com). The input file was converted from FASTA to NEXUS format with CONCATENATOR 1.1.0 (Pina-Martins and Paulo 2008, http://cobig2.com/software).

Description
Taxonomic identification: Specimens collected in Spain were identified based on acoustic, morphological and genetic analysis. Acoustic analysis of the calling song of three males (Fig. 2, Table 1, Suppl. material 1) confirmed that their song's profile is in agreement with previous descriptions of Tettigettalna josei acoustics (Fonseca 1991). The calling song is composed of the repetition of a long sequence of phrases. Each phrase includes two parts, Part I with a long sequence of echemes separated by very short intervals and Part II shorter than Part I, at the end of the phrase, with echemes produced continuously and ever decreasing inter-echeme interval duration. Tettigettalna josei specimens have a broad spectrum near 9 -22.5 kHz with maximum energy around 17 kHz. For time domain variables, results obtained for Spanish specimens indicated an echeme duration ranging from 0.002 to 0.009s, with an average value of 0.004s. For the echeme period we found a range of 0.002 to 0.058s with an average of 0.015s. Morphological measurements of specimens collected in Spain are presented in Table 2. We found an average of 19.21 mm for total body length and 16.22 mm for hindwing length. These values are in general agreement with the ones previously reported for Tettigettalna josei (Boulard 1982). Sequences of cytochrome c oxidase I (COI) obtained from the three specimens collected in Spain (GenBank: KF977503-KF977505) were identical to the ones reported in Nunes et al. (2014)   Morphometric values (in mm) for each Tettigettalna josei male captured in Cartaya (Huelva, Andalusia): TL -total length, BL -body length, WS -wingspan, aWL -right hindwing length, HW -head width, MW -mesonotum width and ACD -width at the level of the auditory capsules.

Genetic variation:
We combined COI sequences from Nunes et al. (2014) and from this study in a dataset composed by 23 male specimens of T. josei from several locations across its distribution (Table 4) and constructed a minimum spanning network (Fig. 5a). The dataset included eight variable sites, resulting in nine haplotypes that differ from each other by single mutations. All mutations result in synonymous changes in the protein. Haplotypes H1 and H6 were the ones found in higher frequencies, but they were detected only in a few sampled locations, indicating that the distribution of some haplotypes tends to be localized (Fig. 5b).   List of males of Tettigettalna josei sequenced for the mitochondrial gene cytochrome c oxidase I (COI).

Distribution
Calling songs from males of T. josei were consistently heard in the region of Algarve, from the west Atlantic coast until the surroundings of the easternmost town, Vila Real de S. António (Fig. 3). A set of new locations and georeferenced coordinates of occurrence of T. josei are listed in Table 3, but they should not be considered as an exhaustive list. Tettigettalna josei is quite widespread in Algarve, reaching high densities (> 10 singing males) in open habitats covered with low vegetation (small bushes and dry grass) and well exposed to sunlight (Fig. 4). Consequently, their numbers were low in cultivated tree groves where herbaceous vegetation was removed but were easily found in uncultivated fields or small patches of marginal vegetation by roads, in the periphery of villages and in secondary dunes and cliffs near the sea. Tettigettalna josei was found in sympatry with two other species belonging to the same genus, T. argentata and T. mariae (Table 3). Males of T. josei were also found singing on trees, but they usually sing at heights below three meters, unlike their congenerics T. argentata or T. mariae, which often sing perched on high pine trees.
In July 2013, T. josei was also found in small numbers in Cartaya (Huelva, Spain), which extends its known distribution to Spain. This same area was visited the year before (see Simões et al. 2013), but T. josei was not found then. We surveyed other provinces of Andalusia in July of 2012 and 2013, but T. josei was not found so far in any other areas besides Huelva.  Table 4. Circle size is proportional to the number of specimens analysed (large = 3, medium = 2 and small = 1).

Biology
Reproductive behaviour: During our fieldwork we observed the copulatory mating behaviour of T. josei. We had witnessed a few ongoing copulations in other Tettigettalna species (T. argentata and T. helianthemi) but had never seen how the process is initiated. One male of T. josei was first noticed in a branch tip of a small stone pine (Pinus pinea) while singing and its unusual behaviour caught our attention.
The male was moving frantically up and down or circling while singing. We noticed a female standing still in the same branch but on the opposite side of the male, thus out of sight of the male. The female was standing still and produced wing-flicks at regular and short intervals (see Boulard (2006) for a review on cicadas wing-flicking). After a few wing-flicks the male eventually moved into the females' direction, turned around the branch and made body contact with her. The male immediately mounted on the female's dorsum and initiated a sideway copulation (Fig. 6). The couple stood quietly and in silence for about three minutes, being the female's front legs firmly attached to the branch. When the couple become apart, the female remained in the branch while the male took off a minute later to a nearby branch and resumed his calling song. A female of this species was seen laying their eggs on a Fennel stem (Foeniculum vulgare) at about 1 m from the soil. Cicadas of genus Tettigettalna are particularly vulnerable during copulation and oviposition because they tend to resist fleeing away if threatened.

Taxon discussion
The present data clarify the currently known distribution range of Tettigettalna josei in the south of the Iberian Peninsula, encompassing the region of Algarve (Portugal) and the province of Huelva (Spain). Morphological, acoustic and genetic analyses confirmed the identity of specimens collected in Cartaya (Huelva) as belonging to T. josei.
Previous records on the distribution of this small cicada species were sparse and limited to Portugal (Sueur et al. 2004). Hence the species has been considered until now as endemic to Portugal. The new record of T. josei in Spain in July 2013 refutes this status and the species must now be added to the list of the Spanish cicadas and, therefore, should be considered as an Iberian endemism.
The region of Huelva was previously surveyed in August 2012 with the detection of T. mariae (Simões et al. 2013) but not of T. josei. This could be explained by the low densities of T. josei found in Spain, since only about five singing males were detected in the region in 2013. Additionally, we have noted that the emergence peak of T. josei might be slightly earlier (June-July) as compared to other Tettigettalna species ( T. argentata and T. mariae: mostly July). Consequently, the number of singing males of T. josei should decline earlier, in mid-August, justifying why we might have missed T. josei in Spain during our surveys in mid-August 2012.
Data obtained so far indicates that populations of T. josei are acoustic, morphological and genetically homogeneous throughout the distribution range of the species. The genetic analysis of COI gene showed no evidence of population structure. Haplotypes differ by single mutations from each other and form a star-like haplotype network. In spite of this, some differences in the distribution and frequency of each COI haplotype seem to reflect the expected trend for low dispersal in these cicadas. As demonstrated before in a species of genus Cicada (Simões and Quartau 2007), emerged specimens usually experience limited dispersal during the mating season, remaining near the emergence site, where the singing males may form choruses.
The distribution of T. josei overlaps with an area under severe human pressure. The coastline of Algarve has been intensively exploited for beach tourism and golf. However, land management associated with tourism facilities seem to have less impact on the persistence of T. josei than the perturbation caused by farming practices that deplete the shrub and grass cover of the soil (e.g. plowing, harvesting or intensive grazing). Fortunately for cicadas, these practices are not severely intense in the coastal region of Algarve and crops are usually small sized and patchy, allowing populations of T. josei to persist all over the region. In contrast, monocultures such as olive and stone pine woods in the Spanish region of Andalusia occupy extensive areas and are regularly maintained to restrain the growth of shrub-like vegetation under the trees. These differences in land management might help to explain the small effective numbers of T. josei detected so far in Spain, and favours instead the prevalence of Tettigettalna species that are frequently found on trees, such as T. mariae or T. aneabi.