New records of invasive hammerhead flatworms (Platyhelminthes, Geoplanidae, Bipaliinae) from Mexico using a citizen science platform, with an identification key to the species found in North America

We present new records of the invasive hammerhead flatworms Bipalium kewense Moseley, 1878 and Bipalium vagum Jones & Sterrer, 2005 (Platyhelminthes, Geoplanidae, Bipaliinae) from several states in Mexico based on iNaturalist and two vouchered specimens. This represents for Mexico the first review of distribution records of this group and highlights the importance of citizen science in monitoring the distribution of these ecologically important invasive predators. Methods for the collection and preservation of hammerhead flatworms, as well as an identification key, are proposed. invasive hammerhead flatworms Mexico a citizen science platform, with an identification key to the in North


Introduction
Among the many taxa that comprise the phylum Platyhelminthes (flatworms), only one family, Geoplanidae Stimpson, 1857, has been able to truly colonize terrestrial ecosystems ). This family of freeliving carnivores, commonly known as land planarians or terrestrial flatworms, is currently divided into five subfamilies, of which Bipaliinae Stimpson, 1857 is easily distinguished from the others by the presence of a greatly widened and conspicuous head (Fig. 1), earning them the common name of hammerhead flatworms (Álvarez-Presas et al. 2014).
Hammerhead flatworms are native to the island of Madagascar as well as Southeast Asia, from India to Japan and including Indonesia and the Philippines (Ogren and Kawakatsu 1987). However, several species have been accidentally introduced to several countries around the globe (Fig. 2), where they are considered invasive (Álvarez-Presas et al. 2014). Hammerhead flatworms are important predators of soft-bodied soil fauna, particularly earthworms and gastropods (Ducey et al. 1999(Ducey et al. , 2007, although they have been observed attempting predation upon tiny vertebrates such as Brahminy Blind Snake, Indotyphlops braminus (Daudin, 1803) (Serpentes, Typhlopidae) (Mizuno and Kojima 2017). They constitute a threat to the native soil fauna wherever they are introduced (Okochi et al. 2010;Justine et al. 2018).

Methods
Collection, recording and fixation of terrestrial flatworms. We followed the methodology described by Winsor (1998). Upon finding a specimen either resting under rocks, wood, or debris, or active at night   (Shorthouse 2010). The colored area only reflects the presence of the species but not its distribution within the countries. More detailed records for each country, including some of the records presented herein, are available on GBIF (B. kewense: https://www.gbif.org/species/2502938; B. vagum: https://www.gbif.org/species/6476069).
(especially during rainy and foggy weather), we placed it, individually, in a clean container (centrifuge tubes, test tubes, etc.) with some moist leaf litter or moss, and kept it in a cool, dark place. We then transported it to the workstation and placed it in a Petri dish filled to the brim with tap water and photographed it in a distended state. Following this, we drained the water and added a weak ethanol solution (~5%). This caused somewhat violent contractions in the specimen but anesthetized it in 10-20 min. After the specimen ceased movement and no longer reacted to stimuli, we carefully straightened it and photographed it (dorsally and ventrally) in this relaxed state.
Following the anesthetization process, we drained the weak ethanol solution and added "Winsor fixative" to the brim. This solution is hereby named after researcher Leigh Winsor, who proposed this modification of Tyler's fixative to fix terrestrial flatworms (Winsor 1998). It consists of 1-part commercial formaldehyde (37-40%) and 9-parts tap water, adding 20 g of anhydrous calcium chloride and 2 g of either cobalt chloride or cobalt nitrate for each liter of solution. If this solution is not available, carefully pouring boiling water on the living specimen and then placing it in commercial 70-96% ethanol or 8-10% commercial formaldehyde can be done instead. After the fixing media was added, the lid was placed on the Petri dish. The specimens are extremely fragile at this stage; therefore, care must be given in moving the Petri dish as little as possible, at least for the first 48 hours. The specimen is then left in the medium for at least a week, two if possible, refilling it when necessary. The specimens can be stored in Winsor's fixative at ambient temperature almost indefinitely but should not be stored in 8-10% commercial formaldehyde alone for more than 48 hours, as in our experience, it turns the specimens very hard and brittle. After fixation, the specimen was carefully washed in tap water and placed in 70% ethanol for long-term storage. Other histological techniques are explored in Winsor and Sluys (2018).
Correctly labeling of specimens is critical and must be done on water-resistant paper, with the data written with a pencil or solvent-resistant ink. It is important to add georeferenced locality data, date of collection, the collector's name, the fixing media, and codes for the photographs taken. The label must be inside the vial with the specimen, never on the outside. After specimens are properly prepared, photographed both dorsally and ventrally, and labeled, they should be deposited in museums or university collections, preferably those with collections of soil invertebrates or that have experts working on terrestrial flatworms.
The two physical vouchers were deposited in the Entomology Laboratory of the Facultad de Ciencias Forestales (FCF) of the Universidad Autónoma de Nuevo León (UANL) under the codes GEOPL001 and GEOPL002.
Records from iNaturalist. Records in this citizen science app were reviewed manually, searching for phylum Platyhelminthes, family Geoplanidae, subfamily Bipaliinae, as well as the genera Bipalium, Humbertium, Novibipalium, and Diversibipalium; the searches were limited to Mexico, including its islands. Those with obscured locality data were excluded from this study. A database was created using the 269 records found (190 for Bipalium kewense and 79 for Bipalium vagum). The determination of the species was corroborated using original and subsequent descriptions (Hyman 1943;Makino and Shirasawa 1983;Winsor 1983;Ogren 1987;Ball and Sluys 1990;Jones and Sterrer 2005;Justine et al. 2018;Wallace and Winsor 2020), from which an artificial identification key that employs external charac- ters was also created. The complete link (https://www. inaturalist.org/observations/) for each iNaturalist observation is abbreviated with the symbol # in the records list. The maps showing the records of the species were made using the website SimpleMappr (Shorthouse 2010 Identification. A very long species, up to 80 cm. Head laterally expanded into a semilunate headplate with recurved lappets. Dorsal ground color light brown; head with the same color of the dorsum or slightly darker. Dorsum with five longitudinal black to dark-gray or dark-brown stripes, median, paired lateral and paired marginal. Median stripe black, narrow, and well-marked; it starts behind neck and broadens over pharyngeal area. Lateral stripes lighter, broader, and with diffuse margins. Marginal stripes narrow, dark, and well-marked. Lateral and marginal stripes of each side unite behind neck to form an incomplete collar (Figs. 1B, 4). Ventral surface light-ochre, with white creeping sole distinct, ridged, and delineated by paired, narrow, diffuse grayish, longitudinal stripes which extend from the ventral portion of the collar to the posterior end; collar interrupted on each side of creeping sole (Winsor 1983;Justine et al. 2018   Identification. A small species, up to 5 cm long. Head laterally expanded but not recurved posteriorly, dark brown, sometimes with pigmentation concentrated into two black patches. Dorsal ground color pale brown to yellow-tan. Black collar at the neck, which extends ventrally and is interrupted at each side of the creeping sole. Dorsum with three longitudinal stripes that start at the collar: a broad median and sharply demarcated black stripe and two lateral broad, diffuse, dark-brown stripes (Figs. 1A, 5). Ventral side light, with white creeping sole distinct but not delineated by longitudinal stripes (Jones and Sterrer 2005;Ducey et al. 2007).

Key to species of hammerhead flatworms in North
America based on external characters 1a Transverse dark band (collar) behind the head complete ( Fig. 1A) or incomplete (Fig. 1B); head crescentshaped (Figs. 1A, B); median stripe does not extend into the head region (Fig. 1A, B) (Fig. 1C, D); head rounded ( Fig. 1C) or, if it is crescent-shaped, the median stripe extends into the head region (Fig. 1D, E) 1B); body usually grayish brown or opaque yellow; with a very narrow median dark stripe and two sets of lateral stripes, the external ones being less conspicuous than the ones closer to the median one, which are very bold (Fig. 1B) (Fig. 1C); body yellow-brown, median stripe does not extend into the head region (Fig. 1C); body without lateral and ventral stripes (Fig. 1C) (Fig. 1D); lateral stripes very poorly defined (Fig. 1D); color pale brown (Fig.  1D) (Fig. 1E); lateral stripes bold, very conspicuous (Fig. 1E); color yellow dorsally and ventrally; (Fig. 1E); three ventral longitudinal stripes, one on each side of the creeping sole and a broader, more diffuse one on the creeping sole; often >10 cm .. Diversibipalium multilineatum

Discussion
Bipalium kewense has been previously reported from Mexico by Winsor (1983), but the locality in the country was unspecified. Bipalium vagum has never been formally recorded for Mexico, so the records presented here are the first for the country and for 17 states.
The distribution patterns of both species correspond to the predictions made by Fourcade et al. (2022), who also used iNaturalist records. No hammerhead flatworms were recorded from the states of Aguascalientes, Baja California Sur, Chihuahua, or Sonora.
The iNaturalist platform has been recognized as a valuable tool for contributing to the knowledge of species' distributions (Chandler et al. 2017;Alzate-Cano and Hurtado-Pimienta, 2021). Given that hammerhead flatworm species recorded in North America can be confidently identified by photographs alone (when diagnostic features are visible), this citizen science app is appropriate to monitor their distribution on the continent, as it has been done previously for invasive (Hiller and Haelewaters 2019;Werenkraut et al. 2020) and endangered (Roux et al. 2019) species. However, although citizen science can be a very helpful tool, it cannot replace museum collections, and for this reason we decided to describe our collection and fixation techniques here.
Finally, we emphasize the need to study the genital structure of mature specimens of Diversibipalium multilineatum to properly assign it to one of the three "natural" genera. Since many introduced land planarians reproduce only asexually outside of their place of origin or preferred climate, not developing sex organs, molecular studies can also help characterize a species and its phylogenetic position (Justine et al. 2018.