Locations

This study was carried out in three tropical greenhouses located at Rotterdam Zoo: Amazonica, Victoria Serre and the Kwekerij.

Amazonica (51.9280°N, 4.4473°E, -1 m alt., Fig. 1) is a large circular greenhouse designed as a butterfly garden, opened in 2013. It has a diameter of 60 m and its highest point is 12 m. A total of 318 plant species are growing in open soil in Amazonica, mostly of South American origin. The average temperature is 25–28°C during daytime and goes down to 18–23°C at night. During warm summer days, the average temperature can be as high as 32–34°C.

The Victoria Serre (51.9260°N, 4.4529°E, -1 m alt., Fig. 2) is a large aviary built in 1945. It consists of three distinct but connected buildings, each hosting a different array of Asian bird species. In the first and second part, the vegetation is grown in patches of soil alongside a footpath. In the third part, the vegetation grows around a 10 m diameter pool containing giant water lilies. The diameter of the circular-shaped third part is 24 m and its highest point is circa 8 m. A total of 61 plant species are growing in open soil. The average temperature is 25°C during daytime and 20°C at night.

The Kwekerij (‘nursery') (51.9243°N, 4.4544°E, -1 m alt., Fig. 3) is a complex consisting of thirteen small greenhouses built in 1993, which function as nurseries for tropical plants. In contrast to the other greenhouses, it is not open to the public. The arthropod survey was carried out in four greenhouses with heights ranging from 9.8 to 11.6 m. The majority of the planting consists of a mixture of potted plants that can also be found in Amazonica and Victoria Serre. In one of the sampled greenhouses, several plants are (permanently) grown in an elongated patch of soil. Most of the greenhouses have the same temperature fluctuations as in Amazonica.

Sampling methods

The arachnids were surveyed in two separate periods: 4. Jun. – 10. Jul. (week 23–28) and 27. Aug. – 19. Sep. (week 35–38) 2018. Samples were collected each week using five methods. (i) Pitfall traps, composed of a 500 ml yoghurt can filled with circa 50 ml ethylene glycol solution. A total of five pitfall traps were placed per greenhouse, in the most dissimilar habitats possible. The traps were protected by a plastic lid supported by four clout nails. The pitfalls were emptied once a week. (ii) Banana baits, which were obviously primarily used to attract arthropods other than Arachnida, e.g. Drosophilidae and certain Coleoptera. A half peeled ripening banana was placed inside a plastic container on the ground, with 10 mm mesh covering the entrance, at two sites per greenhouse complex. The bananas were sampled once a week. (iii) Leaf litter samples, which consisted of sieving the top soil layer (i.e. 2 cm) within an area of 50 × 39 cm. The samples were sifted through a beetle sieve with a mesh size of 10 mm. The samples were taken weekly at different locations, once per greenhouse complex. In the Kwekerij, the samples were always taken at the only location with permanent vegetation. (iv) Hand searching, which included manually looking for arachnids and moving objects such as rocks, wood and litter. (v) Beating, consisted of shaking branches by hand over a sweep net. A few web-building spider species (i.e. Parasteatoda tepidariorum (C. L. Koch, 1841), Pholcus phalangioides (Fuesslin, 1775) and Uloborus plumipes Lucas, 1846) were counted instead of sampled, because of their unmistakable identification and common occurrence. At the start of the inventory, specimens of these species were collected to verify the identification. The exact sampling locations of hand searching and beating were randomised, with the aim of ultimately surveying as many locations as possible. Hand searching and beating were performed once a week, within a fixed period of time (i.e. 60 minutes). All samples and traps were evenly spread across each greenhouse.

Identification and COI-barcoding

All specimens were collected by transferring them into vials containing 96% ethanol. Identification of spiders was primarily done following Nentwig et al. (2019). The pseudoscorpions were identified with Legg & Farr-Cox (2016). Some specimens were photographed using a Zeiss Axioskop microscope equipped with an AxioCamMRc5 digital camera, creating stacked images with a great depth of field. Male specimens of Belisana ambengan were sent to Bernhard Huber (Zoologisches Forschungsmuseum Alexander Koenig, Bonn, Germany) for identification.

Fig. 1-3:

Impression of the three sampling locations at Rotterdam Zoo. 1. Amazonica; 2. Victoria Serre; 3. the Kwekerij

From the collected specimens, fourteen were subsampled for molecular analysis of the cytochrome c oxidase subunit I gene (COI) (Tab. 1). DNA extraction and amplification were performed at the Naturalis DNA Barcoding Facility. DNA primers LCO1490 and HCO2198 were used to amplify a 658-bp region of the mitochondrial COI gene. The resulting PCR products were sequenced at BaseClear using the Sanger sequencing method (for standardised protocols on extraction, amplification and sequencing, see Ivanova et al. 2009). The COI-sequences were matched against the BOLD database with the basic local alignment search tool (BLAST). Sequences with at least 99.0% similarity were declared a match (Hebert et al. 2003). The voucher specimens and parts of the collected material were deposited in the spider collection (RMNH.ARA) of Naturalis Biodiversity Center. Another part of the material is stored in the private collection of the first author.

Tab. 1:

List of arachnids recorded in the greenhouses at Rotterdam Zoo. Abbreviations: * – species recorded in the Netherlands for the first time; A – alien species (established) in the Netherlands; BB – banana baits; BE – beating; E – expansive alien species in the Netherlands; HS – hand searching; j – juvenile; LS – litter sieving; N – native species in the Netherlands; PT – pitfall traps; s – subadult

Data analysis

In order to evaluate the course of sampling, a growth curve illustrating the cumulative percentage of additional arachnid and arthropod species was used.To compare the differences in species richness between the sampling methods, a univariate analysis of variance was performed. Differences were considered to be significant at p<0.05 values.

Tab. 2:

List of the sequenced specimens that could be assigned to a Barcode Index Number (BIN) in the BOLD database, which verified the identification

Results

Twenty-two taxa were identified: twenty spider species, one mite taxon and one pseudoscorpion species (Tab. 1). Nine specimens could be identified by matching their COI-sequence against the BOLD database (Tab. 2). The majority of the species recorded in the greenhouses are alien to the Netherlands, the remaining ones are (semi)synanthropic.

Spiders (Araneae)

In total, twenty taxa (734 ex.) from twelve families were recorded (Tab. 1). Uloborus plumipes was the most dominant species overall, but completely absent from the Victoria Serre. Other very common species included Pholcus phalangioides, Hasarius adansoni (Audouin, 1826), Parasteatoda tepidariorum and Coleosoma floridanum Banks, 1900. Four species, Nesticella mogera, Heteroonops spinimanus, Triaeris stenaspis and Belisana ambengan, represent new records for the Netherlands. These and several other remarkable species are discussed in detail below.

Nesticidae

Fig. 4:

Habitus of adult male Nesticella mogera from the Amazonica greenhouse, 10.–18. Sep. 2018; body length: 2.1 mm

Oonopidae

Fig. 5:

Adult female Heteroonops spinimanus from the Amazonica greenhouse, 19. Sep. 2018; body length: 1.8 mm

Fig. 6:

Habitus of adult female Triaeris stenaspis from the Victoria Serre greenhouse, 27. Aug. – 3. Sep. 2018; body length: 1.7 mm

Pholcidae

Fig. 7:

Habitus of adult male Belisana ambengan from Wildlands Adventure Zoo Emmen, 2. Jul. 2019; body length: 1.3 mm

Fig. 8:

Right male palp in retrolateral view of Belisana ambengan from Wildlands Adventure Zoo Emmen, 2. Jul. 2019. Hairs not drawn and bulbal apophysis is bent inwards and not visible, see Huber (2005) for its morphology

Theridiidae

Uloboridae

Zodariidae

Mesostigmata

Pseudoscorpiones

Only one, native pseudoscorpion species (3 ex.) was found inside the greenhouses (Tab. 1).

Chthoniidae

General evaluation

The growth of the cumulative percentage of additional species slightly decreased over time in both arachnid and arthropod species (Fig. 9). Although the last survey week did not yield any additional arachnid species, the number of additional arthropod species (e.g. Diptera, Isopoda, Myriapoda) slightly increased. The arachnid species curve seems saturated, but the presumed asymptote is reached only in the last survey week. However, the arthropod species curve does not reach a clear saturation point.

The mean species richness per sample was found to significantly differ between the sampling methods. Out of the five collecting methods, hand searching yielded the significantly highest mean number of arachnid species per sample, followed by both pitfall trapping and litter sieving, while both beating and the banana baits had the lowest mean species richness (Fig. 10). Hand searching yielded four species not caught by the other methods, while the pitfall traps and the litter sieving resulted both in two unique and beating in one unique species.

Fig. 9:

The growth of the cumulative percentage of additional species over time.

Fig. 10:

The mean arachnid species richness per sample per sampling method, including SE-bars and statistical significance markings (with α level set at 0.05)

Impact on the natural environment

A subset of the alien species might be able to thrive outdoors. Mermessus trilobatus (Emerton, 1882) established successful populations in a wide variety of open natural habitats in the Netherlands and other European countries (van Helsdingen 2009, Holec et al. 2012). Ostearius melanopygius (O. Pickard-Cambridge, 1880) is frequently found in manure heaps, and also in natural areas (Roberts 1998, Nentwig et al. 2019). Parasteatoda tepidariorum and Steatoda grossa (C. L. Koch, 1838) are regularly found on outer walls of artificial structures in the Netherlands (pers. obs.). Apparently, these species are able to survive outside of buildings in the European temperate zone. All other alien species from Tab. 1 remain very strongly associated with heated buildings in the current Dutch climate, although in 2005 one stray individual of Hasarius adansoni was found in debris in a riverine flood area (van Helsdingen 2009). Their impact on natural habitats is therefore now insignificant, but it remains to be seen which species will protrude to the outdoors due to the increasing warming of the climate.

Species to be expected

The sampling stopped at the point when no further species were collected (Fig. 9): the presumed asymptote. However, Gotelli & Colwell (2011) state that the asymptote of a growth curve can be reached only when at least 20 samples are taken. Moreover, future imports of plants and accompanying soil can introduce additional arachnid species to Rotterdam Zoo. Therefore, it is expected that a subsequent survey will confirm the presence of additional species.

The composition of species found at Rotterdam Zoo shows overlap with other surveys of arachnid fauna in greenhouses (Snazell & Smithers 2007, Kielhorn 2008, Pfliegler 2014, Šestáková et al. 2017, Hula & Pešan 2018). In most studies, the dominant species is either Parasteatoda tepidariorum or Pholcus phalangioides, while Uloborus plumipes is the most ubiquitous species at Rotterdam Zoo. However, the unexpected discovery of a new pholcid spider for Europe at two zoos in the Netherlands is remarkable. Another similar example is the recent discovery of a small pholcid in a Dutch tropical greenhouse, Spermophora kerinci Huber, 2005 (Noordijk 2020); this species is further only known from Indonesian rainforests and a tropical greenhouse in England and Germany (Snazell & Smithers 2007, Kielhorn 2009). It is therefore suggested that arachnid communities in European tropical greenhouses are interconnected, probably due to similar importing routes for the planting material or due to exchange of plants or other products. For this reason, it is expected that some of the additional alien species sampled at other tropical greenhouses in Europe will be recorded in Dutch tropical greenhouses in the near future as well, e.g. Nesticodes rufipes (Lucas, 1846), Theotima minutissima (Petrunkevitch, 1929), Eukoenenia florenciae (Rucker, 1903) and Pseudanapis aloha Forster, 1959 (Snazell & Smithers 2007, Kielhorn 2008, Šestáková et al. 2017, Hula & Pešan 2018).

Comparison of collecting methods

The results of the current study are similar to other studies of arachnofauna in greenhouses involving comparable methods, e.g. Šestáková (2017), where hand collection yielded the highest total number of species and unique species. These results were to be expected, since hand searching covers most strata of the vegetation. Except for the banana baits, each method however yielded unique species (Tab. 1), and they are therefore complementary. The most surprising find, i.e. Belisana ambengan, was collected only by beating.

In future arachnid surveys in greenhouses, it is advised to use at least a combination of methods that sample the vegetation (hand searching or beating) and that yield epigeic arachnids (pitfall trapping or litter sieving) (for similar comparisons in the field, see Churchill & Arthur 1999, Jiménez-Valverde & Lobo 2005, Cardoso et al. 2008). Although the number of species did not differ significantly, pitfall trapping is favoured above litter sieving due to its passive and continuous nature. In order to gain more knowledge about arachnid communities in greenhouses, and their potential source function for invasive alien species, it is recommended to carry out more arachnid surveys in this relatively understudied habitat.