Introduction

Urban trees are vital for global warming resilience, community well-being, and city economies (Mullaney et al. 2015). City trees provide ecosystem services such as oxygen production, pollution reduction, temperature regulation, multitrophic interactions, containment of soil erosion, and water network conservation (Avella et al. 2016). However, woody perennials growing in urban landscapes are exposed to multiple abiotic stressors, including heat and drought stress, poor soil quality, air pollution and light disturbance (Czaja et al. 2020). Prolonged stress increases pathogen populations, herbivory, and insect transmission of pathogens, which can be critical for native and endemic tree species (Long et al. 2019). Phytoplasma diseases are an important economic and ecological problem for urban vegetation (Marcone et al. 2018). Typical symptoms such as leaf yellowing, phyllody, virescence, witches' broom and rosetting in shoot apices (Ermacora and Osler 2019) cause profound physiological, aesthetic and ecosystem service disturbances in phytoplasma-infected trees (Franco-Lara and Perilla-Henao 2019). Phytoplasmas affect trees in natural and urban environments around the world, includign alder (Alnus spp.) (Betulaceae), ash (Fraxinus spp.) (Oleaceae), conifer (Pinus spp.) (Pinaceae), elm (Ulmus spp.) (Ulmaceae), mulberry (Morus spp.) (Moraceae), paulownia (Paulownia spp.) (Paulowniaceae), poplar (Populus spp.) (Salicaceae), and sandalwood (Santalum album L.) (Santalaceae) (Marcone et al. 2018). In Colombia, in the Sabana de Bogotá, ‘Candidatus Phytoplasma asteris’ (group 16SrI) and ‘Candidatus Phytoplasma fraxini’ (group 16SrVII) infect at least eleven urban tree species: Acacia melanoxylon R.Br. (Fabaceae), Croton spp. (Euphorbiaceae), Fr. uhdei (Wenz.) Lingelsh. (Oleaceae), Eugenia neomyrtifolia Sobral (Myrtaceae), Liquidambar styraciflua L. (Altingiaceae), Magnolia grandiflora L. (Magnoliaceae), Pittosporum undulatum Vent. (Pittosporaceae), Po. nigra L. (Salicaceae), Quercus humboldtii Bonpl. (Fagaceae), Salix humboldtiana Willd. (Salicaceae) and Sambucus nigra L. (Adoxaceae) (Franco-Lara 2019).

Phytoplasmas are obligate plant pathogenic bacteria of the class Mollicutes, a member of the order Acholeplasmatales. Phytoplasmas have a reduced genome (530–1,350 kb), lack a cell wall and are transmitted by insect vectors, vegetative material, and rarely by seeds (Marcone et al. 1999; Bertaccini and Lee 2018). Hemipteran phloem-feeding insects such as leafhoppers (Cicadellidae), planthoppers (Cixiidae, Delphacidae, Derbidae, and Flatidae), and psyllids (Psylloidea) acquire phytoplasma from infected plants, which constitute a medium for their intracellular multiplication and dissemination in nature (Weintraub and Wilson 2010). During the acquisition period, nymphs and adults feed on the phloem sieve elements of a phytoplasma-infected plant (Alma et al. 2019); during the latency period, phytoplasmas multiply and colonize different organs of the insect by passing through the midgut (first barrier), reaching the hemolymph, multiplying and being transported to different organs of the insect’s body (Hogenhout et al. 2008). For an insect to be considered a vector, the phytoplasmas must cross the salivary gland cells (second barrier) and multiply inside this organ (Hogenhout et al. 2008). The inoculation period occurs when the phytoplasma-infected insect feeds on a healthy plant and inoculates the bacteria in the phloem by secreting its saliva (Alma et al. 2019).

Quercus humboldtii is an endemic tree species of Colombia and Panama, it is a dominant of Andean forest ecosystems and plays an essential ecological role as an umbrella species (Avella et al. 2016). Andean oak trees were planted in Bogotá to partially replace the invasive species Fr. uhdei affected by ‘Ca. P. asteris’ and ‘Ca. P. fraxini’ (Franco-Lara 2019) that from the decade of 1950 was planted in Bogotá. Quercus humbodltii was selected as an urban tree due to its tolerance to the climatic and edaphic conditions of the city, such as frost, high pollution, poor drainage, and slightly acidic soils with moderate to low fertility (Mahecha et al. 2010; Moreno et al. 2019). However, symptoms such as leaf yellowing, tufted foliage, general crown deformation, witches' broom, atypical elongation of apical shoots and deliquescent branching caused by phytoplasmas were reported in Andean oaks from Bogotá with a prevalence of 74% (n = 17) in a survey conducted in 2013 (Franco-Lara and Perilla-Henao 2014). A more extensive survey conducted in 2017 that included 238 randomly sampled trees in 17 zones of Bogotá, showed a disease prevalence (trees with at least one of the above symptoms) of 80% (n = 191/238). ‘Ca. P. fraxini’ was detected in 54.6% (n = 130), ‘Ca. P. asteris’ in 30.7% (n = 73) and mixed infections of both phytoplasmas in 8.8% (n = 21) of those trees (Lamilla et al. 2022).

Two insect vectors are known in the area. Amplicephalus funzaensis Linnavuori, 1968 and Exitianus atratus Linnavuori, 1959 (both Cicadellidae) were able to transmit ‘Ca. P. asteris’ and ‘Ca. P. fraxini’ experimentally to Phaseolus vulgaris L. (Fabaceae) (Perilla‐Henao et al. 2016) and E. atratus transmitted these phytoplasma to potatoes (Franco-Lara et al. 2023). Specimens of these leafhoppers, as well as other species of Cicadellidae and Psylloidea, have been captured on Andean oak trees (Lamilla et al. 2022), however, their role in the transmission of phytoplasmas to Q. humboldtii is unknown. Our aim was to identify Cicadellidae and Psylloidea taxa from the entomofauna collected on Andean oak and neighbouring trees in Bogotá, and to test for the presence of phytoplasmas on them. We hypothesized that since Q. humboldtii in Bogotá is frequently surrounded by kikuyu grass (Cenchrus clandestinus (Hochst. ex Chiov.) Morrone) (Poaceae) and other ornamental plants, where insect vectors feed (Silva-Castaño et al. 2019a, b) and complete their biological cycles (unpublished results), they may move between different plants dispersing phytoplasmas (Franco-Lara 2019).

Materials and methods

Study area

Bogotá, the capital of Colombia, is located in the high tropics at an altitude of 2,600 m It is one of the megacities in Latin America, with a total area of 1,587 km2, more than seven million inhabitants, and a populated urban area of 22.22% (Barrera 2010). Bogotá is divided into one rural and 19 urban boroughs (DANE 2020). In the urban area, more than one million trees have been planted, with a ratio of 0.18 per inhabitant, including 18,732 Andean oak trees (SIGAU 2022). The precipitation regime is bimodal, with a rainy season in April–May and October–November and a dry season in December-March and June–September, although in recent years, the seasons have blurred. There are no meteorological seasons in Colombia so the average temperature is similar during the year. However, temperatures change abruptly during the day; in 2019 when the sampling was conducted, the minimum annual average night temperature was 7.2 °C and maximum annual average day temperature was 25.5 °C (Ballesteros et al. 2020). The study was conducted in three contrasting urban areas with different degrees of anthropogenic influence, where insects were sampled on Andean oaks trees. The sampling site A was located at the Simón Bolívar Metropolitan Park (4°39´21″ N – 74°5′29″ W), a green lung of 40.5 hectares that belongs to the Teusaquillo borough, which has about 2,900 Andean oaks (SIGAU 2022). The Andean oaks sampled were surrounded by other species, such as Ficus americana Aubl. (Moraceae), Lafoensia acuminata (Ruiz & Pav.) DC. (Lythraceae), Podocarpus oleifolius D.Don (Podocarpaceae), Retrophyllum rospigliosii (Pilg.) C.N.Page (Podocarpaceae), Syzygium paniculatum Gaertn. (Myrtaceae), Tecoma stans (L.) Juss. ex Kunth (Bignoniaceae), Sa. humboldtiana, and Kikuyu grass (Fig. 1). Sampling site B was a pedestrian sidewalk (4°45′9.9″ N – 74°3′55.8″ W), located 11.2 km north of sampling site A in the Suba borough, where nearly 3,400 Andean oak trees are planted (SIGAU 2022). Trees such as Croton sp., Fi. americana, M. grandiflora, Prunus sp. (Rosaceae), and Sy. paniculatum were close to the sampled Andean oak trees (Fig. 1). Sampling site C was a wide median strip of a main road (4°39´20″ N – 74°6′18″ W). It was located 1.49 km west of sampling site A in the Fontibón borough in which 1,700 Andean oak trees grow (SIGAU 2022). Cenchrus clandestinus and other trees and shrubs, such as Erythrina rubrinervia Kunth (Fabaceae), Escallonia pendula (Ruiz & Pav.) Pers. (Escalloniaceae) and Fuchsia arborescens Sims (Onagraceae), were present at this site (Fig. 1).

Fig. 1
figure 1

Aerial map of Bogotá, Colombia, 2020 shows insect sampling sites on Andean oak trees and the general landscape structure of the site studies in Bogotá, Colombia—2019. Site A = A course of trees in Simón Bolívar Metropolitan Park (Teusaquillo borough); Site B = A pedestrian sidewalk next to an avenue (Suba borough); Site C = A wide median strip of a main roads (Fontibón borough). The blue circle indicates the location of the weather stations: 1) High Performance Centre (PM10 and PM2.5), 2) Botanical Garden (Precipitation and temperature), 3) Emmanuel D Alzon (Precipitation), 4) Suba (PM10 and PM2.5) and 5) UDCA (Temperature). N = north

Full size image

Insect sampling

At each selected site, insects were collected by direct and indirect sampling from four Andean oak trees with obvious phytoplasma symptoms (Fig. 2). Samplings were conducted once a month in April and October (rainy seasons), June and August (dry seasons) in 2019. Indirect sampling consisted of four 15 cm × 15 cm sticky traps placed on the lower branches, one for each cardinal point of each tree, left for one week. The cardboard traps had one side covered with yellow contact paper and a layer of Biotrapper© glue; the sticky side faced each cardinal point. Immediately after the coloured sticky traps were removed, the lower branches of each tree were shaken for 3 min and the insects that fell into a 0.6 m diameter beating tray were collected with a mouth aspirator. The direct samplings were conducted at 11:00 to 13:00 when the insects were more active. Additionally, in January 2020, leafhoppers and psyllids were collected from a 25 m linear transect from the vegetation surrounding the trees sampled at each study site. An entomological net (0.25 m diameter with 1.15 m handle length) was used to collect insects for grasses and a beating tray was used in trees Croton sp., E. pendula, Fi. americana, Fu. arborescens, L. acuminata, and M. grandiflora. All insect samples were preserved in 96% ethanol. Due to the COVID-19 pandemic, this sampling was not repeated.

Fig. 2
figure 2

Symptoms associated with phytoplasmas in the sampled Andean oak trees in Bogotá, Colombia – 2019. A Crown deformation, B abnormal elongation of internodes, C tufted foliage, D epicormic shoots, E witches’ brooms, F proliferation of axillary buds, G leaf yellowing

Full size image

In the laboratory, the insects were counted and grouped on Hexapoda’s orders under a Motic-DM143® stereomicroscope. Taxonomic identifications were based on external morphological characters for Cicadellidae (Young 1952; Linnavuori 1959; Dietrich 2005; Marques-Costa and Cavichioli 2012; Zahniser and Dietrich 2013; Catalano et al. 2014; Krishnankutty et al. 2016; Xue et al. 2020) and for Psylloidea (Rendón-Mera et al. 2017). The genitalia of some males were cleared with hot 10% KOH for a few minutes (Oman 1949) and observed under a Zeiss® microscope at 100x  and 400x. The specimens were photographed with a Nikon D5600® camera attached to a Zeiss Stemi 508® stereomicroscope (Fig. 3). To confirm the taxonomic identification of selected samples, molecular analyses of the mitochondrial DNA barcode region were performed by sequencing cytochrome c oxidase subunits I (COI) and II (COII) and of the nuclear 28S rRNA genes (unpublished results). One collected specimen per taxonomic unit of Cicadellidae and Psylloidea was deposited in the Entomological Collection of the Universidad Militar Nueva Granada (catalogue numbers UMNG-Ins 010545—UMNG-Ins 010556).

Fig. 3
figure 3

Psylloidea A-B and Cicadellidae C-H collected by direct sampling on Andean oak trees in Bogotá, Colombia, 2019. A Mastigimas sp. 1 (Calophyidae), B Acizzia uncatoides (Ferris Klyver, 1932) (Psyllidae), C Empoasca sp. (Typhlocybinae), D Dikrella (Readionia) sp. (Typhlocybinae), E Alebrini (Typhlocybinae), F Chiasmodolini (Eurymelinae), G Scaphytopius (Convelinus) sp. (Deltocephalinae), H Acocoelidia sp. (Neocoelidiinae). Bar = 1 mm

Full size image

Biodiversity analysis

The alpha diversity was estimated using the Margalef (DMg), Shannon‒Wiener equity (H'), and Simpson diversity (1-D) indexes for the total number of Cicadellidae and Psylloidea adults collected by direct and indirect sampling at each site. The beta diversity was estimated using the Jaccard index (Magurran 2004). Hill number series (N0, N1, and N2) were calculated to determine the effective number of species per site (Magurran 2004). All indexes were estimated using Past 4.05 software (Hammer et al. 2001). Comparisons of the species accumulation curves per study site with the nonparametric estimators Chao 2, Jackknife 1, Jackknife 2, and Bootstrap in EstimateS 9.1.0 software (Colwell 2013) were conducted to calculate whether the sampling effort allowed the capture of all the species representing the diversity (Magurran 2004).

To determine if quantitative variables (environmental abiotic variables: mean precipitation; maximum and minimum particular matter, PM10 and PM2.5; mean, maximum and minimum temperature) and qualitative variables (sampling site, sampling type, and sampling date) influenced the presence of Cicadellidae and Psylloidea adults in the studied sites, a factorial analysis of mixed data (FAMD) was performed using RStudio 2022.02 3 + 492 software with R version 4.2.1 (Kassambara 2017). Kruskal‒Wallis analysis (Conover and Iman 1981) followed by Dunn's multiple comparison analysis (Dunn 1964) was performed to determine the statistical significance of the abundance of Cicadellidae and Psylloidea adults in relation to environmental abiotic variables (significance level α = 0.05). Information on environmental abiotic variables was obtained from meteorological stations located near the sampling sites: High Performance Centre (PM10 and PM2.5) and Botanical Garden (precipitation and temperature) for site A and site C and Enmanuel D´Alzon (precipitation) and UDCA (temperature) for site B (Fig. 1).

Phytoplasma detection

DNA extraction

For Cicadellidae, total genomic DNA was extracted from the head, pronotum, scutellum, and first pair of legs of adults and the whole body of nymphs. For Psylloidea, DNA was extracted from the head, thorax, and wings. All specimens were processed with the Invisorb© Spin Tissue Mini Kit (INVITEK Berlin, Germany) following the manufacturer's instructions. The extracts were resuspended in 25 µl of elution buffer and stored at -20 °C.

Amplification of DNA by PCR

Phytoplasmas were detected in total DNA from a single specimen or in pools of two or three individuals of the same species by nested PCR of the 16S rRNA gene using the universal phytoplasmas primers P1A/P7A primers for the first round (Lee et al. 2004) following R16F2n (Gundersen and Lee 1996) and R16R2 (Lee et al. 1993) primers for the nested PCR. PCR reactions were conducted in a Thermal cycler Labcycler SensoQuest® in a final volume of 15 µl containing 0.05 U/µl Taq DNA polymerase (Bioneer, Korea) or genTaq® Taq polymerase (Laboratorio de Genética y Biología Molecular LTDA. Bogotá, Colombia), 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 µM of each primer and 2 µl of total DNA. PCR parameters corresponded to an initial denaturation cycle at 94 °C for 10 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min (P1A/P7A) or at 54 °C for 1 min (R16F2n/R16R2), extension at 72 °C for 2 min and a final extension cycle for 72 °C at 10 min. Plasmids carrying cloned DNA from ‘Ca. P. asteris’ and ‘Ca. P. fraxini’ were used as the positive controls, while Milli-Q water was used as a blank control. Amplicons were visualized by 1% agarose gel stained with ethidium bromide electrophoresis in TAE 0.5X (Tris–acetate-EDTA).

RFLP analysis

Phytoplasma groups were detected by restriction fragment length polymorphism (RFLP) analysis by digestion of PCR amplicons with KpnI enzyme (New England Biolabs, United Kingdom) by incubation at 37 °C overnight (Duduk et al. 2013). The digested DNA was visualised in 3% agarose gels electrophoresis in TBE 0.5X and compared with positive control patterns. Amplicons corresponding to an infection by a single group of phytoplasmas were bidirectionally sequenced by the Sanger method at Macrogen, Korea.

Landscape structure analysis

A landscape structure analysis (Fig. 1) was performed for each sampling site with ArcGIS 10.5 software, in which a square buffer was defined. Within one kilometre of each sampling site, the categories of buildings (buildings + roads), trees, and grass were delineated using the image classification tool in ArcGIS 10.5 on satellite images from SAS. Planet software (http://www.sasgis.org/). The area for each category was estimated in kilometres (Fig. 1) using ArcGIS 10.5. Shannon’s diversity index (SDI) and Shannon's evenness index (SEI) were calculated to characterize the diversity and evenness of land use at each sampling site (Olsen et al. 2019) using the Patch Analyst tool version 5.2.0.16 in ArcGIS 10.5 (Elkie et al. 1999).

Results

Direct sampling method

A total of 1,281 Hexapoda specimens (adults n = 988, immatures n = 293) were captured from 12 Andean oak trees from all the studied sites in 2019. Ten orders of Hexapoda were recorded; Hemiptera was the most abundant taxon (adults n = 507, 39.6%; nymphs n = 140, 10.9%), followed by Psocoptera (n = 213, 16.6%), Hymenoptera (n = 146, 11.4%), Coleoptera (n = 65, 5.1%), Collembola (n = 19, 1.5%), Neuroptera (n = 19, 1.5%) and Diptera (n = 10, 0.8%). Blattodea, Thysanoptera, and Lepidoptera were represented by only one, two, and six specimens, respectively (Table 1). Site A showed a higher diversity, with Psocoptera (n = 187), Hemiptera (adults n = 92, nymphs n = 48), and Hymenoptera (n = 127) being the most abundant. Site C also had a high diversity, but Hemiptera were the dominant. Notably, a higher number of immature insects were collected at site C (Table 1). Site B had the lowest diversity, with Hemiptera as the dominant taxon (Table 1). Hemiptera was more abundant in the dry season at sites A (n = 60) and C (n = 233), whereas similar numbers of specimens were sampled in the dry (n = 56) and rainy (n = 58) seasons at site B. Psocoptera (n = 125) and Hymenoptera (n = 84) were more abundant in the rainy season than in the dry season, n = 62 and n = 43, respectively (Table 1).

Table 1 Hexapoda collected in Andean oak trees in Bogotá, Colombia – 2019
Full size table

Using the direct sampling method, 365 Cicadellidae (adults n = 234, nymphs n = 131) members of six taxa were collected from all sampling sites: Acocoelidia sp., Alebrini, Chiasmodolini, Dikrella sp., Empoasca sp. and Scaphytopius sp. In addition, three adults of Psylloidea belonging to two species: Acizzia uncatoides (Ferris Klyver, 1932) and Mastigimas sp. 1 were captured from the sites A and B (Table 2). Site A had four Cicadellidae taxa, while sites B and C showed same Cicadellidae taxa richness (n = 3). Scaphytopius sp. and Empoasca sp. were collected at all sites and Psylloidea specimens were present only at sites A and B. Similar numbers of Dikrella sp. were collected at site B (adults n = 10) and site C (adults n = 6), while Empoasca sp. showed similar abundance at site A (adults n = 8) and site C (adults n = 7), but only one specimen was found at site B. Scaphytopius sp. was the most abundant species at site C (n = 176 adults, n = 89 nymphs). No adults of Scaphytopius sp., were sampled at site B (Table 2), but the immature identification was made by a molecular approach (unpublished results). This species was more abundant in the dry season (June adults n = 7 and nymphs n = 31, August adults n = 133 and nymphs n = 13) than in the rainy season (April adults n = 27 and nymphs n = 11, October adults n = 9 and nymphs n = 34) at site C; however, similar numbers of specimens were captured in the dry season (June adults n = 8 and nymphs n = 7, August adults n = 3 and nymphs n = 9) and the rainy season (April adults n = 4 and nymphs n = 7, October adults n = 7 and nymphs n = 13) at site A.

Table 2 Cicadellidae and Psylloidea (Hemiptera) sampled in the Andean oak trees in Bogotá, Colombia, 2019
Full size table

The alpha diversity for Cicadellidae, based on the Margalef index, varied from 0.38 at site C to 0.86 at site A, whereas the Simpson diversity index values ​​varied from 0.13 at site C to 0.49 at site A. The equity was also low, as the maximum value of the Shannon‒Wiener index was 0.89 (Table 3). The effective Hill numbers were consistent with the natural richness, evenness, and dominance observed during the samplings, with Scaphytopius sp. as the dominant species (N2 = 1) (Table 3). The Jaccard's similarity values ​​indicate that the diversity at site C was similar to that at site B (0.5), as two of the three taxa Dikrella sp. and Empoasca sp. were recorded at both sites (Table 2). The estimated species accumulation curves for Cicadellidae did not reach the asymptote for sites A and B (Fig. 4), as sampling was insufficient to collect all the taxa present at these sites. The Chao 2 and the Jackknife 1 estimators reached the asymptote for site A as they estimated richness of four taxa, in contrast with the Jackknife 2 and the Bootstrap estimators with which five Cicadellidae taxa were expected. For site B, the Chao 2 and Bootstrap parameters estimated four Cicadellidae taxa, but the nonparametric estimators Jackknife 1 and Jackknife 2 estimated five and seven Cicadellidae taxa, respectively. All the estimators reached the asymptote for site C (n = 3) (Fig. 4). No diversity analysis was performed on Psylloidea due to the low number of specimens.

Table 3 Alpha diversity of Cicadellidae and Psylloidea (Hemiptera) adults collected from Andean oak trees in Bogotá, Colombia – 2019
Full size table
Fig. 4
figure 4

Accumulation curves of observed taxa (S) and nonparametric estimators Chao 2, Jack 1 = Jackknife 1, Jack 2 = Jackknife 2, and Bootstrap of Cicadellidae and Psylloidea (Hemiptera) sampled on Andean oak trees in Bogotá, Colombia, 2019. Site A = A course of trees in Simón Bolívar Metropolitan Park (Teusaquillo borough); Site B = A pedestrian sidewalk next to an avenue (Suba borough); Site C = A wide median strip of a main roads (Fontibón borough)

Full size image

Indirect sampling method

A total of 3,157 Hexapoda specimens (adults n = 3,099, immatures n = 7, unidentified n = 51) were captured with 192 yellow sticky traps (4 traps per tree × 3 sites × 4 trees per site × 4 months of sampling) placed in the lower branches of Andean oak trees at three sites in Bogotá, Colombia, in 2019. Nine orders of Hexapoda were collected, and Diptera (n = 1,021, 32.3%), Thysanoptera (n = 809, 25.6%), Hymenoptera (n = 693, 22%), Hemiptera (adults n = 298, 9.4%; nymphs n = 7, 0.2%) and Psocoptera (n = 218, 6.9%) were the most abundant (Table 1). Thysanoptera (n = 792), Hymenoptera (n = 491), Diptera (n = 441), Psocoptera (n = 202), and Hemiptera (adults n = 128, nymphs n = 4) were the most abundant orders at site A, while Diptera, Hymenoptera and Hemiptera were the most common at sites B and C (Table 1). Similar numbers of Diptera were collected in the rainy (n = 292 and n = 276) and dry (n = 149 and n = 187) seasons at sites A and C, respectively, unlike site B, where the greatest abundance of Diptera was in the dry season (n = 73). Thysanoptera and Hymenoptera were abundant in both seasons, particularly at site A, and less abundant at site B. A larger number of Hemiptera were collected with the indirect rather than with the direct sampling method at site A (n = 128), but at sites A and C, the tendency was the opposite (Table 1). No differences were observed (H = 0.38 – p > 0.98) in the number of Hexapoda collected with the yellow sticky traps facing different cardinal orientations.

Using the indirect sampling method, 145 Cicadellidae (adults n = 138, nymphs n = 7) members of eleven taxa (Alebrini, A. funzaensis, Cicadellidae T1, Chiasmodolini, Deltocephalinae, Dikrella sp., Proconiini, Scaphytopius sp., Typhlocybinae T1, Typhlocybinae T2 and Xestocephalus sp.), and 126 adults of Psylloidea belonging to seven taxa (A. uncatoides, Ctenarytaina eucalypti (Maskell, 1890), Mastigimas sp. 2, Psylloidea T1, Psylloidea T2, Syncoptozus mexicanus Hodkinson, 1990 and Synoza cornutiventris Enderlein, 1918) were collected. Acizzia uncatoides (Psylloidea: Psyllidae) (adults n = 109), Scaphytopius sp. (adults n = 46, nymphs n = 2) and Dikrella sp. (adults n = 16) were the most abundant Cicadellidae species. At site A, more than twice the richness (n = 9) was found with the indirect sampling method compared with the direct sampling method; only two taxa were collected with both methods. At site B, the richness (n = 3) was equal with both sampling methods; at this site, Alebrini (Typhlocybinae), A. funzaensis (Deltocephalinae) and Dikrella sp. (Typhlocybinae) were collected. At sites A and C, Scaphytopius sp. was collected with both sampling methods, although the indirect sampling was less efficient. The number of A. uncatoides was similar in the dry season at all study sites, regardless of the orientation of the sticky traps (north n = 32, south n = 26, west n = 23, east n = 28). Scaphytopius sp. was more abundant in the rainy seasons (April n = 10 and October n = 4) than in the dry seasons (June n = 1) at site A, while at site C, a similar number of specimens was collected in the dry seasons (June n = 1 and August n = 14) and the rainy season (April n = 16). However, a similar number of specimens were collected in sticky traps located in any direction (north n = 11, south n = 12, west n = 7, east n = 16).

Diversity of Cicadellidae and Psylloidea in Andean oak trees

Cicadellidae and Psylloidea showed low alpha diversity (Margalef index < 2.43) and low equity (Shannon‒Wiener index < 1.53) based on the indirect sampling results. The diversity was low for the three study sites (Simpson index < 0.66), and the effective number of species ​​(N2) indicated two dominant species for Cicadellidae and one for Psylloidea (Table 3). The species composition was different for each study site. Sites A and C were more similar (Jaccard's similarity = 0.3) than site B because they shared the Typhlocybinae T2, Dikrella sp. and Scaphytopius sp. (all Cicadellidae) taxa. Regarding Psylloidea, site A was similar to site B (Jaccard's similarity = 0.5) because they shared A. uncatoides, S. mexicanus and S. cornutiventris (all Psylloidea) (Table 2). The estimated species accumulation curves for Cicadellidae in the three sampling sites did not reach the asymptote; for example, for site A, the Chao 2 and Jackknife 2 estimators indicated that the taxa richness could be three and two times the observed value, respectively. According to the Bootstrap and Jackknife 1 estimators, the number of Cicadellidae taxa should be between 11 and 15 for site A, and using the Jackknife 2 estimator, seven (site B) and eight (site C) taxa were expected (Fig. 4). For Psylloidea at site B, the nonparametric estimators Chao 2 and Bootstrap estimated six taxa, but the estimated accumulation curves did not reach the asymptote. Similarly, the Jackknife 1 and Jackknife 2 estimators ranged between 8 and 10 Psylloidea taxa for this site. For sites A and C, Jackknife 2 estimated seven and six taxa, respectively, Chao 2 estimated the same richness of Psylloidea taxa (n = 4), and Jackknife 1 estimated six (site A) and five (site C) taxa. The Bootstrap estimator estimated five (site A) and four (site C) Psylloidea taxa. Nymphs of Cicadellidae collected by direct sampling and the Typhlocybinae (Cicadellidae) specimen captured by the indirect method were not included in the diversity analysis.

Taxonomic identification of insects and phytoplasma detection

Morphological characters, examination of the genitalia, and sequences of COI, COII, and 28S gene fragments were conducted for the taxonomic identification of Cicadellidae and Psylloidea (unpublished results). All the Cicadellidae and Psylloidea taxa collected by direct sampling were tested for phytoplasma infection by amplification of the 16S rRNA gene except those used for taxonomic identification. In total, sixty-nine Cicadellidae samples were tested for phytoplasma infection, three adults Scaphytopius sp., from site C and a pooled sample (three specimens) from site A were infected with groups 16SrI or 16SrVII (Table 4). One of four specimens of Dikrella sp. sampled at site C was infected with group 16SrVII. In one Empoasca sp. specimen from site A, 16SrI and 16SrVII groups were detected. The specimens of Dikrella sp., Empoasca sp. and Scaphytopius sp. used for phytoplasma detection were captured on Andean oak trees. Additionally, one specimen of Chiasmodolini captured on L. acuminata at site A was infected with group 16SrVII. Six Alebrini specimens captured on Croton sp. at site B were tested, but none of them were positive for phytoplasmas (Table 4). The only Acocoelidia sp. (Cicadellidae) specimen found was handed to the entomology collection and was not tested, nor was Psylloidea, whose DNA was not suitable for the detection of phytoplasma groups. The quality of the obtained sequences was too poor to conduct bioinformatic analyses.

Table 4 Phytoplasma groups detected by RFLP analysis in adult Cicadellidae sampled from Quercus humboldtii, Bogotá, Colombia
Full size table

Diversity of Cicadellidae and Psylloidea in plants neighbouring sampled Andean oak trees

Acocoelidia sp., Alebrini, Chiasmodolini, Empoasca sp., Scaphytopius sp. (all Cicadellidae), and A. uncatoides (Psylloidea), species that had been previously collected in Andean oaks, were also found in Fi. americana, L. acuminata, and C. clandestinus growing near the sampled Andean oak trees. Four Cicadellidae and three Psylloidea taxa were found in neighbouring plants, with S. cornutiventris (Psylloidea) (n = 60), Alebrini (Cicadellidae) (n = 52) and Chiasmodolini (Cicadellidae) (n = 31) being the most abundant. Higher numbers of Cicadellidae specimens were collected from nearby plants at sites A (n = 62) and B (n = 55). Regarding Psylloidea, (n = 31) and (n = 36) specimens were collected at sites A and B, respectively (Table 5).

Table 5 Diversity of Cicadellidae and Psylloidea in plants neighbouring sampled Andean oak trees in three sites of Bogotá, Colombia, 2020
Full size table

Landscape structure analysis and diversity

The factorial analysis of mixed data (FAMD) explained 18.3% and 10% of the variability in the first and second axes, respectively, for all comparisons (Fig. 5). The sampling site influenced the abundance of Cicadellidae and Psylloidea collected with direct sampling (H = 10.8—p < 0.01; site Ab, site Ba, and site Cb). Significant differences (H = 25.9—p < 0.0001) were also found regarding the number of Cicadellidae and Psylloidea and the type of sampling, which was not observed in the FAMD. No significant differences were observed for other tested variables, such as sampling dates (direct sampling: H = 0.7—p > 0.05; indirect sampling: H = 4.4—p > 0.05), sampling sites (indirect sampling: H = 1.9—p > 0.05) or environmental variables (p > 0.05).

Fig. 5
figure 5

Factor analysis of mixed data of sampling method, sampling site and sampling date of Cicadellidae and Psylloidea collected from Andean oak trees in Bogotá, Colombia, 2019. Site A = A course of trees in the Simón Bolívar Metropolitan Park, Site B = A pedestrian sidewalk next to an avenue, Site C = A wide median strip of a main roads

Full size image

All Andean oak trees sampled (n = 12) showed symptoms associated with phytoplasma infection, such as leaf yellowing, crown deformation, abnormal elongation of internodes and witches’ broom. Some symptoms were present in some trees; proliferation of axillary buds was observed at site A (n = 2), site B (n = 3) and site C (n = 1); tufted foliage was observed at site C (n = 3 trees), and epicormic shoots were observed at site B (n = 1) and site C (n = 1). The Shannon diversity index (SDI) was 1.1 for site A, 0.99 for site B and 0.83 for site C, suggesting different land structures for each study site. However, Shannon’s evenness index (SEI) revealed an even urban structure, as the SEI was 0.79, 0.90, and 0.60 for sites A, B, and C, respectively.

Discussion

Urban Andean oak trees in Bogotá provide a habitat for an important diversity of insects with different ecological functions that may mediate multiple interactions above and below the plant (Del-Claro 2004; van Dam and Heil 2011). These include the ecological interactions that occur in pathosystems. In this work, we generated information about the role that Cicadellidae and Psylloidea may play in the transmission of phytoplasmas to and from Andean oaks in an urban environment, such as Bogotá.

In samplings conducted on twelve trees in 2019, we collected individuals of ten Hexapoda orders in comparison with a sampling conducted by Lamilla et al. in 2017 in which seven Insecta orders were collected from 102 trees of 17 locations in Bogotá by shaking the lower branches of Andean oak trees (Lamilla et al. 2022). In Lamilla´s et al. (2022) work, the sampled trees were in parks or street sidewalks and were sampled once, whereas in this work, three contrasting sites were sampled on four occasions. As far as we know, there are no other studies about the richness and diversity of phytoplasma insect vectors in urban environments, with which we could compare our data, so it is difficult to determine if our observations point out to a rich or a poor environment. In this study, the landscape structure of the three sampling sites differed and significantly influenced the diversity of insects collected (Fig. 5). The diversity and landscape structure determine mutualistic or competitive relationships between taxa and may explain the existence of certain dominant groups over others (Del-Claro 2004). Anthropogenic pressure, local dynamics of abiotic conditions (pollution, temperature, relative humidity, rainfall regime), vegetation type and cover, as well as tree age, development, and phytoplasma symptoms of the sampled Andean oaks, could regulate the hexapod diversity at each site. When the sampling sites were selected, the idea was to sample three contrasting places. However, the landscape structure analysis showed that they were not very different (Fig. 1) because in all of them, the building areas were predominant. In urban environments, biodiversity is linked to the size of patches and the presence of biological corridors that connect these patches (Beninde et al. 2015). The three sampling sites were highly fragmented, which might negatively influence insect diversity. Even so, ten orders of Hexapoda were captured, some of them in high abundance. Not necessarily a highly urbanized area can indicate that species richness and abundance decrease. In contrast, studies carried out on pollinators have shown high biodiversity in urban environments (Baldock et al. 2015). In addition to landscape structure, environmental variables and seasonality can shape the abundance of insects such as Cicadellidae (Pinedo-Escatel and Moya-Raygoza 2018).

Two sampling methods were used to capture as many taxa as possible. Considering the overall results, the most abundant orders were Diptera, Hymenoptera, Thysanoptera and Hemiptera. In the work of Lamilla et al. (2022), Hemiptera was the most abundant taxon, although the abundance and richness varied at different sampling sites. In this work, Hemiptera was dominant in the three sampling sites with direct sampling but was less abundant with indirect sampling. In contrast, Diptera, Hymenoptera and Thysanoptera were the dominant taxa by indirect sampling. The direct sampling method is a useful tool for capturing insects because it is fast and easy to implement. With this method, we captured almost the same orders of Hexapoda as with the indirect sampling method. Furthermore, immature Coleoptera, Hemiptera, Lepidoptera and Neuroptera were sampled, indicating that several taxa may be completing their life cycle on Andean oak trees.

Cicadellidae and Psylloidea contain several phytoplasma insect vector species (Weintraub and Beanland 2006). In the survey conducted by Lamilla et al. (2022), specimens of 21 Cicadellidae taxa were collected in 17 locations, in comparison with 13 taxa collected in this work in three locations, suggesting that the richness of Cicadellidae associated with Andean oak trees did not vary considerably in the different city boroughs. The common Cicadellidae taxa captured in both surveys were Acocoelidia sp., Alebrini, Chiasmodolini, Dikrella sp., Empoasca sp. and Scaphytopius sp. The species accumulation curves from our study suggest that up to 15 Cicadellidae taxa may be present; therefore, our sampling effort was not sufficient to collect the existing diversity. Longitudinal studies are needed to study the population dynamics of the species of interest. In the tropics, where climatic conditions such as day and night average temperatures and day length remain more or less constant throughout the year, the fluctuation of the insect populations is difficult to follow. In this and other works conducted by our team (Silva-Castaño et al. 2019a; unpublished results), we have not been able to describe the dynamic population pattern of the species under observation. For instance, the Cicadellidae taxon diversity of the sampling sites varied greatly in the same season, and our results indicate that environmental abiotic variables such as the mean precipitation, maximum and minimum particulate matter (PM10 and PM2.5), and mean, maximum and minimum temperatures did not explain the community structure in any case. Insect diversity and population dynamics seem to be greatly influenced by local microconditions rather than general environmental and climatic conditions.

Our results show that Scaphytopius sp. is a recurrent and abundant species in Andean oak trees in Bogotá, since adults and immatures were collected on them. Usually, insect females lay their eggs on host plants that offer enough resources for nymphs to develop (Lauzière and Sétamou 2009). Additionally, in the case of Cicadellidae, the immatures lack wings, so they cannot migrate easily to other plants; thus, we concluded that Andean oaks are the main plant host of Scaphytopius sp. and only four specimens out of 44 were positive for phytoplasmas in this study. The online global database of Hemiptera Phytoplasma Plant Interactions reports that seven Scaphytopius species are phytoplasma vectors in Canada, the United States, Brazil, and Colombia (Trivellone 2019). Scaphytopius fuliginosus (Osborn 1923) carries phytoplasmas of group 16SrI and has been associated with disease symptoms in soybean in Colombia (Granada 1979), and group 16SrIIIZ is associated with broccoli stunt disease in Brazil (Eckstein et al. 2014).

Due to our findings, we suggest that Scaphytopius sp. plays a role in phytoplasma dispersal between Andean oaks and so far, at least 14 species of the Scaphytopius sp. genus have been reported in Colombia (Freytag and Sharkey 2002; Pinzón 2007), but none of them corresponds with the Andean oak species. More studies are needed to determine its taxonomic identity and to describe its biology. Additionally, Chiasmodolini (Eurymelinae), Dikrella sp. and Empoasca sp. (both Typhlocybinae) specimens were positive for phytoplasmas in this study. Both subfamilies, Eurymelinae and Typhlocybinae, contain known phytoplasma vector species (Weintraub et al. 2019). These three taxa were present in low abundance, although Empoasca sp. was an abundant species in Lamilla et al. (2022) survey. It is likely that Empoasca sp. completes its life cycle in Andean oak trees since we collected abundant nymphs in previous samplings that were identified using DNA barcodes (unpublished results). Several species of the Empoasca genus have been associated with the transmission of phytoplasmas and other pathogens (Acosta et al. 2017; Salas-Muñoz et al. 2018; García-Cámara et al. 2019; Trivellone 2019). A few Empoasca sp. specimens were collected from neighbouring plants, such as Fi. americana, L. acuminata and Fu. arborescens. Chiasmodolini specimens were also captured in Fi. americana, L. acuminata and the grass C. clandestinus, while Alebrini was abundant in Croton sp., suggesting that they move between different trees. All DNA extractions conducted to test the presence of phytoplasma were performed from the insect heads, where the salivary glands lay. This supports the idea that they can transmit phytoplasmas because salivary gland infection is a requisite for phytoplasma transmission (Alma et al. 2019). However, to confirm their role as phytoplasma vectors, transmission tests must be conducted for these species (Weintraub and Beanland 2006).

Amplicephalus funzaensis and E. atratus (both Cicadellidae) are known phytoplasma vectors in the Sabana de Bogotá (Perilla-Henao et al. 2016; Franco-Lara et al. 2023). In this work, we captured one A. funzaensis specimen with a yellow sticky trap; Lamilla et al. (2022) captured 104 and 12 specimens of A. funzaensis and E. atratus, respectively, in the trees they sampled. Grass is the main host of these two species, so it is possible that they visit Andean oak trees occasionally when the grass is tall, or the trees have low branches. Our observations suggest that these species are not the main phytoplasma vectors of Andean oaks, but they may play a marginal role in transmitting phytoplasmas to and from the grass, as has been hypothesized previously (Lamilla et al. 2022).

Some species of Cicadellidae and Psylloidea were collected in Andean oak trees and neighbouring plants; therefore, we propose that in addition to the movement of insect vectors from grass to trees and vice versa (Lamilla et al. 2022), different species of Cicadellidae move to and from trees or shrubs dispersing phytoplasmas. Additionally, although the populations of the insect vectors may fluctuate in different locations, they seem to be present throughout the year, which may explain the widespread phytoplasmas in this ecosystem.

Regarding the Psylloidea species, we collected individuals of two species (A. uncatoides and Mastigimas sp. 1) with the direct sampling method, while Lamilla et al. (2022) collected one species. However, several specimens were captured in neighbouring plants. The fact that they were captured with yellow sticky traps suggests that these Psylloidea taxa are occasional visitors to Andean oak trees. The abundance of A. uncatoides in yellow sticky traps in the present study may be due to flight interference, as the species has also been recorded in the genera Acacia (Fabaceae), Ficus (Moraceae) and Magnolia (Magnoliaceae) (Rendón-Mera et al. 2017). Trees that are also part of the urban woodland in Bogota and specimens of these plant genera were present in the tested sites. However, despite our attempts, we were not able to determine if they carried phytoplasmas, and therefore, their role as insect vectors in Bogotá remains unclear. However, taxa within Psylloidea have been implicated as important in the transmission of phytoplasmas (Weintraub and Wilson 2010; Cruz et al. 2018). It is necessary to conduct additional studies to test more Psylloidea specimens.

Our results provide information about phytoplasma insect vectors in tropical urban areas and will allow future insect diversity comparisons. We propose that several Cicadellidae taxa may be involved in phytoplasma transmission in urban trees in Bogota. Although the population dynamics of these insects are unknown, they can be collected on trees throughout the year, with important implications for the epidemiology of the disease. Our study contributes to the knowledge of the entomofauna interactions with urban trees in Bogotá. This is relevant because some megacities propose climate change monitoring programmes using urban trees (Brune 2016) and leafhoppers as indicators (Santos et al., 2024).