First record of freshwater planarian Girardia sinensis Chen & Wang, 2015 (Platyhelminthes, Tricladida, Continenticola) in South Africa

The occurrence of the alien freshwater planarian Girardia sinensis Chen & Wang, 2015 is reported for the first time in sub-Saharan Africa. This species is a global invader of freshwater bodies as evidenced by its very recently reported discoveries in Europe, Asia and Australia. We used DNA barcoding and phylogenetic analysis to confirm species identity, as morphological identification alone is not accurate. Unintentional introduction via the aquarium trade through contamination is considered a possibility. Taken together with other reports of recent introductions of invasive freshwater species in South Africa, possibly through aquarium trade, this raises serious concerns about the ineffective control of aquarium trade.


Introduction
The first comprehensive review of aquatic, free-living flatworms in Africa was done by Young (1976), who listed 80 species in 29 genera.Other scholars (Sluys 2007;Schockaert et al. 2008) built upon this foundation and subsequently listed 85 species in 28 genera from the Afrotropical region.Recently, faunastic and taxonomic works on Afrotropical planarians have resulted in several new species descriptions and increased our understanding of their diversity and distribution in the region (De Vries 1988;Sluys 2007;Stocchino et al. 2002Stocchino et al. , 2012Stocchino et al. , 2014Stocchino et al. , 2017;;Harrath et al. 2019).Despite this, aquatic planarians are still poorly known in Southern Africa, and there are still no taxonomic keys to species or genera (Kawakatsu 1972;Appleton 2002).Lack of specialist knowledge and documentation results in alien species not being recognised (McGeoch et al. 2012).
Preliminary estimates of Young (1976) suggested a modest percentage of freshwater planarians in Africa as being non-native.However, recent estimates of freshwater planarian invasiveness are much higher (Sluys 2016;Stocchino et al. 2019;Fourcade et al. 2022).This high rate of invasiveness amongst free-living flatworms is surprising, as they tend to have a very limited dispersal ability (Lázaro and Riutort 2013;Sluys and Ruitort 2018).Although some taxa have the potential for natural long distance dispersal via migratory birds and wind, as they produce drought-resistant eggs (Young 1976), most free-living flatworms have instead been unwittingly transported by humans across their natural, historical boundaries (Sluys 2016).However, pin-pointing the exact mode of human-mediated introduction has been challenging, as there are several possible routes; including aquarium trade, ships' ballast waters, and soil or timber contamination (Lázaro and Riutort 2013;Sluys and Ruitort 2018;Stocchino et al. 2019).Once introduced, free-living flatworm species are often successful, as they can tolerate sub-optimal environmental conditions and reproduce via fissiparity, without the need for egg-laying (Sluys and Ruitort 2018).
Girardia Ball, 1974, is a genus originally native to South America, that spread to North America via natural dispersal routes but is now invasive in several other continents (Benítez-Álvarez et al. 2023).Invasive freshwater planarians exhibit puzzling dispersal routes (see, Solà et al. 2022) and Girardia is no exception.Girardia has since spread across the world, with a high probability of becoming invasive.The first dispersal of this genus into Europe occurred when Girardia tigrina (Girard, 1850) was recorded in the UK during the 1920s (Wright 1987).This species has subsequently been recorded across that continent (Ilić et al. 2018;Kanana and Ruitort 2019).Until recently, Girardia was not known from African waters.Benítez-Álvarez et al. (2022) reported the first record of this genus in Morocco, a country in the Palearctic region of Africa.However, there is currently no formal record of Girardia in sub-Saharan Africa.The current study reports for the firsttime the occurrence of a feral population of Girardia sinensis Chen & Wang, 2015 in South Africa.Originally described from China (Chen et al. 2015), where it may be non-native, G. sinensis is a native of North America, and has subsequently been introduced into many countries, including Australia, Morocco, Spain, Italy, France, Germany and Netherlands (Benítez-Álvarez et al. 2022(Benítez-Álvarez et al. , 2023)).Given the difficulties of morphological identification of fissiparous triclads lacking copulatory apparatus (Lázaro and Riutort 2013), the species level identification was confirmed using a fragment of the mitochondrial gene cytochrome oxidase I (COI).

Materials and methods
Freshwater planarians were collected from the Korsman Bird Sanctuary (−26.195°;28.2911°) a natural depression pan that is 1627 m above sea level.The pan was 1.8 m deep with a surface area of 37 hectares at the time of collection.The pan (see Figure 1A) is fed mostly by storm water from an urban catchment of an upmarket residential area.The catchment receives most of its rainfall between November and March, with a long-term average of 744 mm and a record rainfall of 1242 mm in the 2022 hydrological year, measured locally.The pan has no natural outlet but during periods of high water level, water is pumped to Benoni Middle Lake in the Blesbokspruit river system.Korsman Bird Sanctuary water environmental parameter data (pH, electrical conductivity (EC), nitrates, ammonia (NH 4 ), sulphates (SO 4, phosphates (PO 4 ), chemical oxygen demand (COD) and faecal coliform counts) were sourced from monthly water quality data obtained from the City of Ekurhuleni.Water physico-chemistry recorded at the time of specimens collections were as follows: Temperature (°C) 14-19, pH 7.8-8.4,Conductivity (mS cm -2 ) 71-78, Nitrate (mg/l) 0.1, Ammonia (mg/l) 0.2-1.2,Sulphate (mg/l) 34-46, Phosphate (mg/l) 0.1, Sodium (mg/l) 61-77, COD (mg/l) 22-52, F coli (counts/100 ml) 12-37000.The eutrophic pan is subject to regular sewerage spills from an ageing sewer network and pumping stations within the perimeter of the Sanctuary.A sewer manhole within a few metres of the collection site overflows when the pump station is inoperative.
In May 2022, the lead author, a citizen scientist, found suspected Girardia sp.planarians in a water sample taken from flooded grassland in the vicinity of the sewer manhole.Three home-made planarian traps were placed in the vicinity of the find.The traps were made from small plastic containers drilled with 2 mm holes and provisioned with freshly killed Cornu aspersum (Müller, 1774) snails and buried in soft mud.The traps were removed after 36 hours and collectively contained 16 Girardia specimens.A previous attempt, provisioning the traps with pieces of cooked red meat, was unsuccessful in attracting planarians.However, on both attempts the traps attracted numerous leeches (Helobdella sp. and Alboglossiphonia sp.).The specimens were collected on the 6 th of August 2022.
The trapped specimens were not examined to check if they were asexual or not.However, one of the specimens collected earlier in May showed signs of recent fission.The specimens were measured before killing and the approximate extended body length ranged from 4 mm to 9 mm.Larger specimens were generally more highly coloured.Each specimen had a different spot pattern (Figure 1B).They were not starved before fixation and killed shortly after retrieval in 99% ethanol.11 specimens were submitted to Inqaba Biotech (Pretoria, South Africa), where the DNA extractions and sequencing was conducted, using standard procedures.An approximately 640-bp section of the COI gene was amplified using LCO1490 (50-GGTC AACAATCATAAAGATATTGG) and HCO2198 (50-TAAACTTCGGGT GACCAAAAAATCA) primers (Folmer et al. 1994), however the final length varied as ambiguous ends were visualized and trimmed using Chromas (Version 2.6.6).
In total, 54 COI sequences were used to infer the phylogenetic relationships of Girardia by using Maximum Likelihood and Bayesian Inference approaches.Four new sequences were obtained from the collected specimens (Table 1), with the remaining 50 sequences obtained from Genbank (Figure 2).The downloaded sequences were taken from Benítez-Álvarez et al (2022,2023), and represented six species of Girardia from across the world, including G. schubarti Marcus, 1946, G. sinensis, G. dorotocephala Woodworth, 1897, G. sanchezi Hyman, 1955, G. clandestina Sluys & Benítez-Álvarez, 2022, and G. tigrina.The sequences were aligned using the online version of MAFFT 7, using the default parameters (Katoh et al. 2019).The subsequent alignment was visualized and trimmed in AliView 1.27 (Larsson 2014).A phylogenetic tree was estimated using a Maximum Likelihood approach conducted in W-IQ-Tree (Trifinopoulos et al. 2016) with 1000 bootstrap replicates using the Ultrafast algorithm, using a partition scheme by codon (Table 2).Additionally, a Bayesian Inference approach was conducted in Mr Bayes 3.2.7a(Ronquist et al. 2012), using a partitioned scheme by codon and the models identified by the model selection tool in W-IQ-Tree (Table 2).The MrBayes test was conducted using the CIPRES science gateway (Miller et al. 2010; accessible at https://www.phylo.org/index.php/).The final alignment used for molecular analysis was trimmed to 827 base pairs, with 16% of the data ambiguous, or missing.

Results and discussion
Both the Maximum Likelihood (Supplementary material Figure S1) and Bayesian Inference (Figure 2) methodologies recovered phylogenetic trees with similar topologies.Unfortunately, the Maximum Likelihood tree provided  extremely poor resolution, as it recovered several polytomies and generally low bootstrap values.Additionally, it recovered a polyphyletic G. clandestina, and relationships between species were not resolved.Despite this, it recovered a strongly supported (97%) monophyletic clade of G. sinensis, which included the newly collected specimens from South Africa.Comparatively, with the exception of G. dorotocephala, species level clades were monophyletic and well supported in the Bayesian Inference analysis (> 0.90), although relationships between the species were not resolved.As with the Maximum Likelihood approach, the newly collected South African species were clustered together within G. sinensis in a monophyletic clade.This provides strong support for the assignment of the South African species to G. sinensis.Thus, the current manuscript reports the second record of this cosmopolitan species in Africa (Benítez-Álvarez et al. 2022), and a first from Sub-Saharan Africa.The discovery of this feral population shows the power of citizen and formal science working together (Daniels et al. 2022), as we were alerted to the presence of this population by a citizen scientist.In the case of the discovery at Korsman Bird Sanctuary, circumstantial evidence supports the theory of Girardia being introduced into the pan via the sewer network, possibly originating onwards from the aquarium trade.Freshwater invertebrates have been shown to have a high propensity of being accidentally transported as contaminants in the aquarium trade (Patoka et al. 2017).The initial discovery and subsequent successful trapping occurred within a few metres of a frequently overflowing sewer manhole.A nearby residence, connected to this sewer system, makes regular use of an aquarium service company.The company owner recognised photographs of the Girardia specimens as an aquarium pest.They were unaware of Girardia's environmental tolerance and believed that they would not survive in sewerage.As they were found within an area which had been drenched by waste water, this could suggest tolerance of severely degraded water quality.
Recreational lakes in and around Johannesburg surburbia appear to be on the receiving end of invasive species, largely coming off aquarium and pet trade (van Wilgen et al. 2010;Martin and Coetzee 2011).For example, Miranda et al. (2022) recently reported discovery of a new invasive snail, Sinotaia cf.quadrata (Benson, 1842), at Zoo lake.This site is about 30 km from where are reporting the new record of G. sinensis in South Africa.The full extent of the distributional occurrence of this species is unknown, however given the high connectivity of urban waters, it is highly unlikely that it is restricted to the reported site.These new records of invasive species in these lakes are alarming, given the already long list of invasive freshwater invertebrates in the country, some with massive environmental and socioeconomic impacts (Weyl et al. 2020).The next iteration of the National Invasive Species Report (e.g.Zengeya and Wilson 2020) should include these new species.However, to assess the invasive status of these species and their potential impacts (Zengeya and Wilson 2020), more information on their distributional occurrence is needed.As such, large scale assessment of the occurrence and ecological impact of these species in waters around Johannesburg is warranted.

Figure 1 .
Figure 1.Photographs showing A) the study location at Korsman Bird Sanctuary, B) Girardia sinensis specimens found onsite.Photographs by Jane Trembath.

Figure 2 .
Figure 2. Bayesian Inference phylogeny of Girardia sinensis and related species constructed using cytochrome oxidase subunit 1 (COI).Numbers given on each branch represent posterior probabilities.

Table 1 .
Genbank Accession Numbers of all specimens used in the Maximum Likelihood and Bayesian Inference approaches.

Table 2 .
Results of jModelTest2, and the relevant primers selected for the Bayesian Inference Test of phylogeny conducted in MrBayes