New distribution records of subterranean crustaceans from cenotes in Yucatan (Mexico)

Abstract New records of 14 stygobiont crustacean species pertaining to six Malacostraca orders from 32 cenotes are presented, with their associated caves of the state of Yucatan, Mexico, together with an individual account for each species. Species composition of most of the investigated cenotes is examined for the first time. A thermosbaenacean and two amphipod species were not formally recorded to the cenote ecosystems of the state of Yucatan prior to our research. Distribution data of a cirolanid isopod previously known only from its type locality is also provided. Barcodes of mitochondrial cytochrome c oxidase subunit I for the reported peracarid species previously lacking this information have been included in present study as tools for species identification and a baseline of further molecular genetic analyses.


Introduction
'Cenotes' (the local name for water-filled sinkholes) are typical karst features of the Yucatan Peninsula in Mexico. In many cases, far-reaching networks of submerged subterranean cave passages extend from them (Mercado-Salas et al. 2013). Due to the mixing of fresh and saline water, a distinct stratification can be observed inside these anchialine systems (Bishop et al. 2015). Intrusion of saline water is found deeper as the distance from the coastline increases (Bauer-Gottwein et al. 2011). Therefore, most inland cenotes within the state of Yucatan are exclusively freshwater systems, except for a few rather deep ones with haloclines below 50 m in depth, and those located near the northern coastline of the Peninsula (Álvarez et al. 2005;Angyal et al. 2018).
Anchialine ecosystems in Yucatan contain a crustacean-dominated fauna that is adapted to hypogene conditions, such as the lack of sunlight and the low food resource availability . Stygobiont species are restricted to aquatic subterranean habitats (Botosaneanu 1986), and often exhibit conspicuous morphological adaptations to hypogene life, known as troglomorphisms. Such adaptations include structural reductions (e.g., loss of visual organs and pigmentation) or extensions (e.g., lengthening of appendages and complexity of sense organs) (Mejía-Ortíz et al. 2006;González et al. 2018) and physiological modifications (e.g., reduced metabolic rates and starvation resistance) (Hervant et al. 1999(Hervant et al. , 2001Bishop and Iliffe 2009). In 2016, prior to our systematic sampling, 47 stygobiotic crustacean species had been reported from anchialine ecosystems of the Mexican federal states of the Yucatan Peninsula, of which 22 were known from cenotes and submerged caves of the state of Yucatan (e.g., Holsinger 1977;Kallmeyer and Carpenter 1996;Álvarez et al. 2005;Suárez-Morales et al. 2006). Fourteen percent of these species belong to the subclass Copepoda (9 spp.), while the remainder belong to the orders Mysida (1 sp.), Stygiomysida (2 spp.), Amphipoda (1 sp.), Isopoda (5 spp.), and Decapoda (4 spp.).
According to the database of the Secretaría de Desarrollo Sustentable (SDS Yucatan), there are more than 3,000 registered cenotes and caves within this state. Current efforts are being directed to complete the descriptions of all registered cenotes, despite that only a small fraction of them have been biologically investigated to date. Ongoing research and explorations are necessary to describe the true biodiversity of these subterranean habitats, their geographical patterns, and changes in time. Thus, our aim was to improve our knowledge on the distribution and ecology of the stygobiotic crustacean fauna of the cenotes and their associated cave passages in the state of Yucatan. We aimed to provide data from cenotes that had never been investigated from a zoological point of view in order to extend the geographical range of crustacean species distribution and contribute to a precise biodiversity mapping of stygofauna in Yucatan. Additionally, we intended to collect samples for molecular and morphological studies so as to gain and make available to the public mitochondrial cytochrome c oxidase subunit I sequences (COI) of species that were lacking barcode information, setting the standard for studies and tools for species identification.

Sampling sites and sampling
We collected stygobiotic macro-crustaceans from 32 cenotes between May 2016 and January 2018 in cenotes of the state of Yucatan (shorter form: Yucatan) ( Figure 1, Table 1). Most of the cenotes studied are several kilometers away from the coast and contain only freshwater. In contrast, some cenotes near the coast have a halocline that divides the cave into freshwater and saline water habitats. Some of the cenotes studied belong to the 'Ring of Cenotes', a fracture zone with high density of sinkholes identified as the outer rim of the crater where the famous asteroid impacted Chicxulub 66 million years ago (González-Herrera et al. 2002;Bauer-Gottwein et al. 2011) ( Figure  1). Macro-crustaceans were collected during scientific cave dives using 50 ml sample

Molecular studies (COI barcode sequences)
DNA extraction of the peracarids studied was performed using QIAamp DNA Microkit (QIAGEN), following the manufacturer's instructions. A few pereopods of each animal provided the necessary material to extract DNA. For PCR amplification of mitochondrial COI, we used the primer pair LCO 1490 and HCO 2198 (Folmer et al. 1994). PCR reactions (25 μl) contained 13.85 μl mQ water, 2.5 μl 10× PCR buffer, 2.5 μl dNTP mix (2mM), 1.5 μl of each primers (5μM), 0.15 μl Fermentas Dream Taq (5U/ μl), and 3 μl DNA extract. PCR tempera ture conditions were set as follows: initial denaturation for 3 min at 94 °C, denaturation for 45 sec at 94 °C, hybridization for 45 sec at 48 °C, and polymerization for 1 min at 72 °C. After thirty cycles, a final extension for 3 min at 72 °C was performed. PCR products were purified using Exo SAP-IT Express PCR Product Cleanup (Affymetrix) accord ing to the manufacturer's instructions. The fragments were sequenced in both directions using PCR amplification primers with an ABI 3130 sequencer. Contigs were assembled and sequences were edited using BioEdit 7.1.11 sequence alignment editor software (Hall 1999): chromatograms of complement reverse and forward strings were compared, gaps were eliminated, while indels and stop codons were checked. 605-651 bp COI barcode sequences have been uploaded to the NCBI GenBank database. Accession numbers and localities are listed in Table 2.
Remarks. Our records show that this species is distributed in cenotes of central Yucatan and along the Ring of Cenotes. Among the two Stygiomysis species of the region, S. cokei proved to be rarer than Stygiomysis cf. holthuisi. New occurrences were recoded between 12-32 m deep in freshwater. In cenotes San Elias, Dzonotila and Yax-Kis it cooccurred with S. cf. holthuisi. Previously the species had also been reported in brackish habitats (Álvarez and Iliffe 2008;Álvarez et al. 2015).  Previous distribution. Gordon 1958;Botosaneanu 1980;Bowman et al. 1984;Pesce and Iliffe 2002;Álvarez and Iliffe 2008, Álvarez et al. 2015, Benítez et al. 2019.
Type locality is Devil's Hole, St. Martin, Lesser Antilles (France). The species is known from the Bahamas, Anguilla, Puerto Rico, and the Yucatan Peninsula. In Quintana Roo S. cf. holthuisi was recorded from cenotes Mayan Blue, Casa Cenote, Na'ach Wennen Ha, Bang, Odyssey, Muknal, and Tabano. From Yucatan the species was previously known only from a single locality, Cenote Mucuyche.
Type locality is Grutas de Balankanche (Yucatan). Widely distributed in the central and northern parts of the Yucatan Peninsula, known from several wells, cenotes and caves of Quintana Roo and Yucatan.
Remarks. Antromysis cenotensis was present in all the cenotes studied, except for Cenote Cervera. Álvarez et al. (2015) mentions that A. cenotensis occurs mostly above or occasionally below the halocline up to a depth of 16 m. In the present study, the species was only observed in freshwater habitats, in some cases as deep as the scope of the survey. Our findings prove this species as a common representative of the stygofauna of Yucatan, as it was found in more than 95% of the visited sites. Antromysis cenotensis is listed as "threatened" in the Mexican Red List of Threatened Species (NOM-059 SEMARNAT 2010). Remarks. At present, collected material is available from eight localities and a small M. troglomorpha population was also observed in Cenote San Elias. All the individuals were found in freshwater habitats, both in cave and cavern sections, where water temperature was between 26 and 27 °C. In cenote Kankirixche, some individuals were observed below 45 meters in depth. As a species recently described by our research group, one of the outcomes of present expeditions. As M. troglomorpha was found in approximately 30% of the visited sites, it does not appear to be a rare freshwater stygobiotic element in the Yucatan cenotes.  Previous distribution. Holsinger 1977Holsinger , 1990Reddell 1981;Álvarez and Iliffe 2008, Álvarez et al. 2015, Angyal et al. 2018, Benítez et al. 2019.

Mayaweckelia cenoticola Holsinger, 1977
Type locality is Cenote Xtacabiha (Yucatan). From Yucatan the species was also known from Cueva de Orizaba, Cenote Nohchen, Grutas de Tzab-Nah and Grutas de Santa Maria. From Quintana Roo there were records from Cenote Actun Ha (Carwash), Cenote de las Ruinas, Cenote de San Martin, Cenote de Santo Domingo, Cueva de Tancah, Odyssey, Bang and Tabano. From the state of Campeche, the species was known from the Volcán de los Murciélagos cave.
Remarks. Mayaweckelia cenoticola proved to be rarer than M. troglomorpha, since it was recorded from only four cenotes. In Cenote Bebelchen we found some individuals in the roots of trees near the surface at the entrance region. Holsinger (1990) found that the species is associated mainly with freshwater habitats, with few populations occurring in weak brackish water. Individuals found in the Ox Bel Ha System (Quintana Roo) by Álvarez et al. (2015) and Benítez et al. (2019) also occurred in freshwater.  Previous distribution . Holsinger 1990;Álvarez and Iliffe 2008;Álvarez et al. 2015;Angyal et al. 2018;Benítez et al. 2019.
Remarks. Tuluweckelia cernua was both the most frequent and abundant stygobiotic amphipod in the present study. Additional observations were from cenotes Yaal Utsil, El Virgen, and Dzalbay. In contrast with previous reports (e.g. Holsinger 1990), T. cernua always occurred in freshwater habitats. Individuals were collected between depths of 5-50 m. The species co-occurred with M. troglomorpha in five cenotes. These are the first distributional records of T. cernua for the state of Yucatan. Known localities of this species have almost tripled, increasing its distribution range into the Yucatan inland area.

Yucatalana robustispina Botosaneanu & Iliffe, 1999
Remarks. Individuals of Y. robustispina were collected in a third of all localities visited, where it occurred in freshwater between 3 and 49 m in depth. In eight cenotes Y. robustispina co-occurred with the isopod C. anops. Agreeing with our observations, previous records referred specimens caught in freshwater between 5-50 m in depth (Botosaneanu and Iliffe 1999. Known localities of this species have been doubled.  Previous distribution. Iliffe 2000, 2006;Álvarez and Iliffe 2008;Rocha-Ramírez et al. 2009. Type locality is Cenote Sabak Ha (Yucatan). This species had only been collected from its type locality until our expeditions.
Remarks. We here provide the first records after the original description, which was based on a single specimen collected at 60 m in depth near the halocline at a salinity of 1.4 g/l Iliffe 2000, 2006). The three newly collected individuals were found in freshwater habitats, both in cavern and cave zones below 19 m in depth. The species was found in approximately 10% of the studied cenotes always as solitary individuals. Therefore, C. yunca seems to be a rare element of the Yucatan freshwater cenote ecosystems.  Type locality is Buya Uno, allotype was collected from Cenote Cervera and paratypes from Dzilamway, all cenotes in Dzilam de Bravo region (Yucatan north coast). This species was recently recorded at the Ox Bel Ha system south of Tulum (Benítez et al. 2019) and the Ponderosa system north of Tulum (Espinasa et al. 2019).

Typhlatya mitchelli Hobbs & Hobbs, 1976
Type locality is Cenote Kabahchen (Yucatan). The species occurs in numerous caves and cenotes throughout the peninsula in Quintana Roo and Yucatan.

Typhlatya pearsei Creaser, 1936
Type locality is 'Balam Canche Cave' (Grutas de Balankanche, Yucatan). The species is widely distributed within the northern part of the Yucatan Peninsula; it occurs in Quintana Roo, Yucatan, and Campeche.
Remarks. Despite previous studies stating that T. pearsei has the largest of Typhlatya's distribution range in the Yucatan Peninsula (Álvarez et al. 2015), we only collected individuals in a few localities, where it occurred in freshwater, both near the surface in open cenote pools and in deeper cave passages up to 26 m in depth. This species is listed as "least concern" in the IUCN Red List (De Grave et al. 2013b) and as "threatened" in the Mexican Red List of Threatened Species (NOM-059-SEMAR-NAT 2010).
Remarks. Reddell (1981) mentions the species as an "ever-present element of fauna of pools and lakes in caves in the Yucatan Peninsula". In addition to the above listed localities, we also observed the species in cenotes Yaal Utsil, Pol Box, San Elias, Dzonotila, Flor de Liz, X'baba, Chihuo Hol, and Yax-Kis. Specimens were recorded in both cave and cavern sections, up to 38 m in depth. Benítez et al. (2019) also found individuals around and below the halocline in cenotes belonging to the Ox Bel Ha system. Creaseria morleyi is listed as "threatened" in the Mexican Red List of Threatened Species (NOM-059-SEMARNAT 2010) and as "least concern" in the IUCN Red List (De Grave et al. 2013c).

Discussion
While there are more than 3,000 registered cenotes in the state of Yucatan (SDS Yucatan census), less than five percent have been zoologically investigated. Results herein confirm that the region deserves more attention and that the geographical, bathymet-ric, and fresh/salt water distribution of stygobiotic species is far from being fully understood. In order to contribute to the management of the vulnerable cenote ecosystems and their highly specialized endemic stygofauna, collecting as much information as possible about the biology of Yucatan aquifers would be paramount. This data should include reports on the species' distribution, density and rarity, taxonomy, ecology, as well as characteristics of their habitats related to their biology, such as the amount of epigean originated organic sources or the degree of anthropogenic pollution in cenotes.
Prior to this study, the amphipod T. cernua was only known from Quintana Roo, mostly associated with saltwater habitats in anchialine cenotes near the northeastern coastline of the Peninsula (Holsinger 1990;Rocha et al. 1998;Álvarez and Iliffe 2008;Álvarez et al. 2015). Contrary to previous findings, all individuals were found in freshwater habitats during our study (Angyal et al. 2018). Rocha et al. (1998) and Pesce and Iliffe (2002) mentioned observation records of 'thermosbaenaceans' from cenotes Yuncu, Mucuyche, Pabakal (Papakal), and Grutas de Tzab-Nah (all in Yucatan). However, these individuals had never been identified at the species level and it seems no voucher information of the potentially collected specimens is available. The present study confirms first records for T. cernua and T. unidens in the state of Yucatan. Together with the amphipod M. troglomorpha, which was discovered and described within the frame of herein presented expeditions (Angyal et al. 2018) and the new cave isopod Curassanthura yucatanensis Álvarez, Benítez, Iliffe & Villalobos, 2019, the list of stygobiotic crustaceans recorded for the state of Yucatan raised from 22 (in 2016) to 26. In addition, the cirolanid isopod C. yunca was only known from its type locality, but we now provide distribution data for this species in three other localities. Our results show that the stygiomysid S. cf. holthuisi has historically been unrecognized, unsampled or ignored. This specific contribution proves that inland cenotes have been understudied and distribution patterns of stygofauna are still unknown. Due to the previously lacking zoological information for the vast majority of the cenotes investigated in our study, most of the distribution records presented here are new.
A closer morphological and molecular analysis of the Typhlatya species in Yucatan is recommended in order to distinguish cryptic species that may be causing confounding biodiversity and ecological patterns in the Yucatan Peninsula.
Among the 14 crustacean species listed, prior to this study, cytochrome c oxidase subunit I sequences were publicly available only for the decapods T. mitchelli, T. pearsei, T. dzilamensis, and C. morleyi. The currently published COI barcode gene fragments can aid future molecular research on the peracarid fauna of Yucatan's cenote ecosystems by facilitating their identification, as well as in the recognition of cryptic species.
The mysid A. cenotensis, the atyid shrimps T. mitchelli and T. pearsei and the palaemonid shrimp C. morleyi are listed in the Mexican and IUCN red lists of threatened species (SEMARNAT 2010;De Grave et al. 2013a, b, c). These species are present in most cenotes throughout the Yucatan Peninsula and can be considered a selected group of species whose protection will act as an umbrella in protecting other less common ones. On the other hand, there are rare species with an extremely narrow distribution range, which are not yet under legal protection. This makes these species even more vulnerable to urbanization and environment deterioration. Therefore, we suggest the inclusion of narrow endemic species into the national and international protection lists, such as the isopod C. yunca or the atyid shrimp T. dzilamensis.
The number of new records provided in this work shows a historic lack of biodiversity surveys in underwater caves of inland cenotes of the state of Yucatan. Most of the biodiversity and its distribution patterns are currently biased towards large populations, easily accessible sites, and touristic attractions. Our efforts yield a greater understanding of the distribution patterns of stygofauna in Yucatan cenotes.