bioRxiv preprint doi: https://doi.org/10.1101/2021.01.28.428687; this version posted January 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 RESEARCH NOTE 2 3 Another mystery snail in the Adirondacks: DNA barcoding reveals the first record of 4 Sinotaia cf. quadrata (Caenogastropoda: Viviparidae) from North America 5 6 Running header: Sinotaia in the Adirondacks 7 8 Ethan O’Leary, Donovan Jojo, Andrew A. David* 9 10 Clarkson University, Biology Department, Potsdam, New York, USA 11 12 Email addresses: 13 Andrew David: [email protected] 14 Ethan O’Leary: [email protected] 15 Donovan Jojo: [email protected] 16 17 *corresponding author 18 19 20 21 22 23 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.28.428687; this version posted January 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 24 ABSTRACT 25 Alien molluscs pose a serious threat to global freshwater diversity and have been implicated in 26 many ecosystem-altering invasion events over the past few decades. Biomonitoring surveys are 27 therefore a key tool for ensuring biosecurity in diversity hotspots and vulnerable habitats. In this 28 study, we use DNA barcoding to provide the first record of the viviparid, Sinotaia cf. quadrata 29 from North America. Reciprocal monophyly and low genetic divergence (uncorrected p- 30 distance: 0.004) with a Bellamya quadrata individual from the type region (China) provides 31 strong support for this identification. The species was recovered as part of a routine 32 biomonitoring survey of the Adirondack region of northern New York. Only three adults were 33 recovered (no populations or juveniles) indicating that the discovery represents a very recent 34 arrival. Considering the proximity of the sampling site from the massive St. Lawrence River, it is 35 likely that S. cf. quadrata was introduced into the St. Lawrence, probably via the aquarium plant 36 trade, and was able to spread into smaller river system in northern New York and possibly other 37 border states. This record represents the fourth alien viviparid, the third of which is of Asian 38 origin, that have made its way to New York waters. Future biomonitoring efforts for the 39 upcoming summer period will involve targeted searches for S. cf. quadrata to determine the 40 extent of its spread in the region. 41 42 43 44 45 46 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.28.428687; this version posted January 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 47 Aquatic invasive species (AIS) pose a serious threat to biological communities in both marine 48 and freshwater systems (David and Janac 2018). In the freshwater realm, notable events such as 49 the zebra mussel invasion of the Great Lakes of North America (Griffiths et al. 1991), the rapid 50 spread of the New Zealand mud snail across the United States (Loo et al. 2007) and more 51 recently, the march of the marbled crayfish across Europe (Hossain et al. 2019), has garnered 52 significant attention both within and outside of the invasion science community. While the 53 majority of introduced species rarely become invasive, the few that do can severely, and in some 54 cases permanently disrupt community dynamics and ecosystem functioning, ultimately leading 55 to biodiversity loss (Molnar et al. 2008). Early detection of AIS via frequent biomonitoring of 56 rivers and lakes is therefore a key task that must be undertaken to help prevent future invasion 57 events (Hamelin and Roe 2019; Pederson et al. in press). 58 The Adirondack Park, situated in northern New York, is the largest protected forest 59 reserve in the contiguous United States, and with more than 100,000 hectares of lakes and 60 streams, it is the largest protected freshwater system in the country (Erickson 1998). More than 61 half of the rivers within and around the park drain into the Laurentian Great Lakes and the larger 62 St. Lawrence River system which allows for bidirectional dispersal of organisms among these 63 three regions (Shaker et al. 2017). During the summer months, the park is a popular destination 64 for hikers, boaters and recreational anglers from around the country, and while New York State 65 environmental regulators have implemented policies and rules aimed at minimizing the potential 66 for non-indigenous species (NIS) introductions, the sheer size of the Adirondack region 67 combined with a small workforce make enforcing such policies difficult. The lakes and rivers of 68 the Adirondack region are now home to several non-indigenous molluscs including the zebra and 69 quagga mussels (Dreisenna polymorpha and D. bugensis), New Zealand mud snail 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.28.428687; this version posted January 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 70 (Potamopyrgus antipodarum), and three viviparids: the Chinese and Japanese mystery snails 71 (Cipangopaludina chinensis and C. japonica), and the banded mystery snail (Viviparus 72 georgianus) (David et al. 2017; David and Cote 2019). In July 2020, three snails were collected 73 from a boat launch site just outside the Adirondack Park and less than 1 km from the St. 74 Lawrence River as part of a long-term biomonitoring survey for aquatic invasive species (Fig. 1). 75 Both specimens were initially identified as ‘new’ viviparid records based on conspicuous 76 conchological features which differed from the well-established invasive viviparids already 77 present in the region. Here, with the aid of DNA barcoding, we report the first record of Sinotaia 78 cf. quadrata from northern New York and as far as we know, this record is also the first report of 79 the species within North American waterways. In addition to providing genetic confirmation of 80 the species, we also briefly discuss possible pathways and vectors which may have been 81 responsible for translocating it to this region. 82 As part of a long-term monitoring survey for AIS in the rivers and streams of the northern 83 New York, snails were collected from five different sites in July 2020, all located at or within the 84 vicinity of a boat launch dock (Fig. 1). Using a rapid assessment protocol outlined by David and 85 Cote (2019), sampling was carried out at each site for exactly one hour in the shallows 86 (maximum depth – 1.2 m). All specimens were transported live to the David Lab at Clarkson 87 University and sorted by Family and Genus using conchological characteristics and the 88 identification key of Jokinen (1992). Shell morphometrics were determined using the methods of 89 Chiu et al. (2002). A Vernier caliper (error margin: 0.05 mm) was used to determine shell length 90 (SL), shell width (SW), aperture length (AL) and aperture width (AW) and photographs of the 91 apertural and subapertural view of each snail was taken. Forty four out of the 47 snails collected 92 belonged to either Cipangopaludina chinensis or Viviparus georgianus, both of which are 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.28.428687; this version posted January 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 93 established invasive gastropods in the region. Three snails from one of the five sites (Brandy 94 Brook Boat Launch, Waddington NY - 44°52’03.75” N, 75°11’41.90” W) exhibited a spire that 95 was considerably shorter than the aforementioned species, though broad similarities in 96 taxonomically informative shell traits indicated they were all viviparids This prompted a 97 molecular investigation into their identities. All three snails were fixed in 99% ethanol for 48 hrs. 98 for DNA barcoding. Shells of both snails were cracked and ~2 mg of tissue just above the 99 operculum was dissected, air-dried and digested in a Proteinase K and lysis buffer solution 100 (Qiagen, Hilden, Germany). Genomic DNA from each sample was extracted using the D’Neasy 101 Blood and Tissue kit (Qiagen, Hilden, Germany) to produce aliquots with DNA concentrations 102 of 85 and 149 ng/µl. A ~710 fragment of the mitochondrial gene, cytochrome c oxidase 1 (CO1) 103 was amplified for both individuals using the forward and reverse primer pairs from Folmer et al. 104 (1994): (HCO2198 and LCO 1490). Polymerase Chain Reaction was carried out in a 25 µl 105 reaction mixture using the following cycling parameters: 92°C for 2 min, 92°C for 40 s, 51°C for 106 1 min, 68°C for 1 min and 68°C for 7 min. PCR products were visualized in a 1% agarose gel 107 stained with ethidium bromide (EtBr).