Checklist of freshwater symbiotic temnocephalans (Platyhelminthes, Rhabditophora, Temnocephalida) from the Neotropics

Based on published records and original data derived from our research, we have generated a checklist of symbiotic temnocephalan fauna from 57 taxa of freshwater invertebrate and vertebrate hosts from 16 families included in four classes from the Neotropics. The checklist contains 38 nominal species from 3 genera belonging to the Temnocephalida families Diceratocephalidae, Didymorchidae and Temnocephalidae. All taxa (35) of the genus Temnocephala are endemic to the Neotropics and 14 (40%) are considered microendemic (i.e. only one record each from a single locality). While only one species and one variety of Didymorchis are known from the Neotropics; there are also two putative undescribed species of this genus. Only Diceratocephala boschmai (Diceratocephalidae) is reported as an introduced species from Uruguay. Host specificity to a particular group of invertebrates and vertebrates is the first evidence for the ecological and evolutionary associations that will be analyzed in future studies.


Introduction
Temnocephalida (Platyhelminthes, Rhabditophora) is the most diverse group of symbiotic turbellarians typically associated with crustaceans, with 122 valid species and 24 genera described in the world (Tyler et al. 2006(Tyler et al. -2012. Recently, Temnocephalida was confirmed as a monophyletic group included in Lymnotyphloplanida, which in turn makes up part of the Dalytyphloplanida clade, a major group of Rhabdocoela (Van Steenkiste et al. 2013). Within the Temnocephalida, the family Temnocephalidae Monticelli, 1899, is the most diverse, distributed in the Australian region with high species richness, but low host diversity, and in the Neotropics with an apparently lower number of temnocephalan species, but a greater diversity of host taxa Brusa 2009, Sewell 2013). In fact, in the Neotropics, 32 species belonging to the genus Temnocephala and four taxa belonging to Didymorchis, endemic to this region and associated with crustaceans, mollusks, insects and chelonians have been described Cannon 2001b, Garcés et al. 2013 and cited therein).
The inventory work of the temnocephalan fauna in the Neotropics began in the 18 th century, when the first species of Temnocephalida was described, Temnocephala chilensis (Moquin-Tandon 1846), associated with anomuran crabs, Aegla laevis (Latreille), from Chile (Damborenea and Cannon 2001a). Since then, more than 50 studies have been published regarding aspects of the temnocephalan fauna in the Neotropics, including descriptions of new species, analyses of temnocephalan symbiotic community structure of particular host species, and studies with phylogenetic and biogeographic inferences (e.g. Damborenea 1998, Volonterio 2007a, Garcés et al. 2013. However, in many cases, information about the reported biodiversity in particular geographical locations of these rabdocoel turbellarians is scattered among myriad bibliographic sources and difficult to access. Therefore, attempts to generate inventories and compile information are highly valuable for understanding the global diversity of freshwater flatworms (Schockaert et al. 2008). The main objectives of this paper are to compile all the available published accounts on the symbiotic freshwater temnocephalans from the Neotropics and to incorporate new data derived from our own work of the last few years to construct a checklist of symbiont-host associations.

Bibliographic search
All the published records on Neotropical temnocephalan species reported from Malacostraca (Decapoda), Gastropoda (Caenogastropoda), Insecta (Hemiptera, Megaloptera, Plecoptera and Trichoptera) and Reptilia (Testudines) strictly in freshwater systems were compiled. Databases such as Biological Abstracts, Biological and Agricultural Index Plus and Scopus, Google Scholar, Helminthological Abstracts, ISI Web of Knowledge, Turbellarian Taxonomic Database and Zoological Record were used to ensure that we retrieved all available information; the bibliographic search was undertaken up to June, 2014. We considered all the studies whose datasets provide taxonomic information regarding the Neotropical temnocephalan taxa, even those found in a single individual host. Papers containing compiled records of Neotropical temnocephalans that require taxonomic revisions due to problems were indicated (e.g. Vianna and Melo 2002). The host species names were used according with IUCN (2014), Tree of Life Web Project (Maddison and Schulz 2007), and with specific bibliography for Gastropoda (Cowie and Thiengo 2003) and Malacostraca (Melo 2003, De Grave andAshelb 2013). Furthermore, a species accumulative curve (Dove and Cribb 2006) was obtained to evaluate the extent of the Neotropical symbiotic temnocephalan inventory.

Survey work
Original data from our own studies of the last few years were included. A total of 11 taxa of decapod crustaceans of five families was examined for Argentine and Mexican temnocephalans. Furthermore, two species of Chelonia and one of Gastropoda from Argentina also were examined. Decapod crustaceans were collected with seine nets in one locality of central Mexico (Table 1). The collected decapod crustaceans were kept alive and examined for temnocephalans no more than 4 h after their capture. Decapod crustaceans were sacrificed and immediately examined for temnocephalans; external (e.g. carapace and claw surface) and internal structures (e.g. branchial cavity) were analyzed separately in Petri dishes with 0.65% saline solution, under a stereomicroscope. Gills from each decapod were also obtained and placed in tap water to search for temnocephalans. In the case of mollusk hosts, their mantle cavity was opened after sacrificing. Temnocephalan collections from live turtles were carried out by the catch-andrelease method (e.g. FAO 2012); therefore, the live turtles were identified directly in the field (L. Alcalde, personal communication). Temnocephalans were fixed with hot (steaming) 4% formalin or hot (steaming) distilled water. In some cases, specimens from the same host and with the same external aspect were fixed in 100% ethanol in the field for future molecular studies. All temnocephalans were processed following standard procedures (Sewell 2013

Results
In total, 60 papers have been published establishing host and locality records of the freshwater temnocephalan fauna in the Neotropics. The analysis of all available information (bibliographic and new original data) allowed us to establish a list of 38 symbiotic temnocephalan taxa in invertebrates and vertebrates in the Neotropical region, which are contained in four groups of hosts. Malacostraca (Decapoda): 4 taxa of Didymorchis associated with 3 taxa of crabs, 17 species of Temnocephala associated with 32 taxa of decapod crustaceans and only one species of Diceratocephala associated with one species of decapod crustacean; Gastropoda (Caenogastropoda): 5 species of Temnocephala associated with 5 taxa of freshwater snail hosts; Insecta: 1, 2, 2 and 1 taxa of Temnocephala associated with 1, 5, 3 and 1 taxa hosts of Trichoptera, Hemiptera, Megaloptera and Plecoptera, respectively; Chelonia (Testudines): 4 taxa of Temnocephala associated with 7 species of freshwater turtle hosts.
The results of this study are presented in the Table 1 which shows the symbiont-host list, where temnocephalans are organized by taxonomic groups and ordered alphabetically by family name. Then species within each family are listed alphabetically followed by authority name and date. The next category is the host species in which the temnocephalids were found, followed by the locality, and the bibliographic reference from which the information was obtained, except for those records established in the present work. In the temnocephalan species found in more than one host species, the latter are listed alphabetically, and host  Dioni (1967b) Temnocephala kingsleyae Damborenea, 1994 Kingsleya ytupora Araguari River, Amapa, BR 1°15'0''N; 49°55'0''W* Damborenea (1994) Temnocephala lamothei Damborenea and Brusa, 2008 Pomella megastoma Yabotí-Miní Stream, Misiones, AR 26°57'39"S; 53°49'23"W Damborenea and Brusa (2008) Temnocephala lanei Pereira and Cuoccolo, 1941 Trichodactylus sp.  Temnocephala travassosfilhoi Pereira and Cuoccolo, 1941 Trichodactylus petropolitanus San Bernardo, São Pãulo, BR 22°00'S; 49°00'W Pereira and Cuocolo (1941) species for which more than one locality was recorded, are listed together. Furthermore, a host-symbiont list (See Appendix 1) is taxonomically and alphabetically organized.
The decapods are the most species-rich host group with temnocephalans (27), followed by the insects (5 taxa) and snails (5 species). Of the 38 taxa of Temnocephalidae listed in this work, all appear to be specific to particular host groups, while at least only one species of the family Diceratocephalidae have successfully associated with hosts after their anthropogenic introduction, i.e. Diceratocephala boschmai. The most widely distributed species are T. axenos, T. chilensis and T. iheringi, which are present in 9 and 10 crab host species and 5 snail host species, along 20, 25 and 49 localities, respectively.
In terms of hosts, Hydromedusa tectifera (a turtle) is the host with the highest temnocephalan species richness with 4 taxa, followed by Aegla neuquensis, A. platensis, Dilocarcinus pagei (decapod crabs) and Pomacea canaliculata (snail), all with 3 species; meanwhile, 49 host taxa show only one record of temnocephalid taxa for one locality.
The species accumulation curve for Neotropic temnocephalans plotted against the total number of species (Figure 1) shows irregular growth over 15 decades of studies in Temnocephalida (each decade divided into two periods of five years). This graph shows that the asymptote has not been reached yet and, if the systematic studies of the group are continued, a significant increase in the number of species in the Neotropical region can be expected. This graphic also reflects two important periods of research. The first shows the initial prospecting for temnocephalid species in the Neotropical region, between 1890 and the beginnings of the 20th century. The second period, beginning around 1970, shows an increase in the research on temnocephalans from different host species, with some stationary periods.

Discussion
The genus Temnocephala is an endemic component of the Neotropical region (Damborenea and Cannon 2001a). At the moment, it includes 35 taxa, of which 14 (40%) are considered microendemic (only one record for locality) ( Table 1). In total, 57 host taxa are associated with one or more temnocephalan taxa, which belong to seven orders and 14 families within four classes. It is worth pointing out that each major group of hosts is characterized by a particular assemblage of temnocephalan species, with host specificity at family level. For example, 17 taxa of Temnocephala are associated with three families of freshwater crab hosts (Aeglidae, Pseudothelphusidae and Trichodactylidae), while five Temnocephala species are associated with 8 taxa of freshwater shrimps included in three families (Cambariade, Palaemonidae and Parastacidae). Information about the natural history of this endemic genus is key to understanding the role of different factors that shaped its diversification patterns across several hydrological basins in the Neotropics and the possible implications of codivergence with host groups (see below) (e.g. Thompson 2005, Martínez-Aquino et al. 2014b. In this inventory, only Diceratocephala boschmai was detected as an introduced species because of translocation together with their crustacean hosts, the invasive redclaw Cherax quadricarinatus in Uruguay (Volonterio 2009a), due to human activities such as aquaculture and breeding of ornamental species (Lodge et al. 2012, Saoud andGhanawi 2013). According to several authors, D. boschmai causes a detrimental economic impact because of an aesthetic effect of the eggs on the body surface of the C. quadricarinatus (Herbert 1987, Volonterio 2009a. However, it is more important to mention the detrimental biological and ecological impact of these introduced species (e.g. Ahyong andYeo 2007, Larson andOlden 2012). To date, the values of ecological infection parameters (e.g. prevalence and abundance; see Bush et al. 1997) are unknown not only locally but globally. These parameters are required to measure the effect of this symbiotic association -both introduced species, C. quadricarinatus and D. boschmai -to detect the extent of the spread of D. boschmai to other crustacean taxa, especially endemic crabs in their natural ecosystems (Jones and Lester 1993, Chivavaya 2013, du Preez and Smit 2013. Furthermore, the introduced populations of D. boschmai in natural hydrological systems in Uruguay represent a serious problem of displacement to the endemic populations of Neotropical Temnocephala species because of interspecific competition between symbiotic organisms (Gelder 1999, Sicard et al. 2006, Witte et al. 2008, Tsuchida et al. 2011, Ohtaka et al. 2012. In this context, the data generated in this checklist can be used to support conservation strategies for freshwater biodiversity (Cardoso et al. 2011a, b, Stendera et al. 2012, Collen et al. 2013).
One hundred sixty eight years have passed since the first description and record of a temnocephalan from the Neotropics (Damborenea and Cannon 2001a), and, currently, ±236 records of temnocephalans have been published. However, considering the number of described species and the time passed, it can be stated that most of the diversity of Temnocephala remains yet to be described. There is also a significant number of potential hosts that have not been studied with regards to symbiotic temnocephalans. On the other hand, Schoackaert et al. (2008) mentioned that the few species recorded in South America were mostly recorded up to about 1970. Based on the species accumulation curve (Figure 1), this study shows clearly the increase in knowledge about the biodiversity of the temnocephalan fauna in recent times, but based on all of the information compiled for Neotropic temnocephalans, we show the necessity to continue inventory work. The Neotropic temnocephalan fauna contains 31% of Temnocephalida taxa described at the moment, representing 37 taxa allocated to two genera. This checklist presents data on almost all the extant species of temnocephalans along their distributional ranges in 11 Neotropical countries, which represents 35% of the total political territories (i.e. countries) in the Neotropics ( Figure 2). Argentina, Brazil and Uruguay are the countries with the most records of temnocephalans and with the most endemic species of Temnocephala, which are represented by 6, 9 and 4 species, respectively, while Colombia, Costa Rica, Mexico and Peru hold 1, 2, 1 and 1 endemic species, respectively. The relatively high number of records in Argentina, Brazil and Uruguay can imply that in these countries there are more research groups working with turbellarians compared to other Neotropical countries (e.g. Damborenea and Brusa 2008, Volonterio 2010. Therefore, the values of endemism for these particular countries are subjective -a function of the research effort -and it is probable that the endemism may be increased/decreased in future studies from different Neotropical countries. With regards to its exclusively Neotropical distribution, morphological evidence (mosaic syncytial plates) (e.g. Joffe 2001, Damborena andCannon 2001b), plus the recorded host specificities shown in this study (Appendix 1), allow for the inference that the biological radiation of Temnocephala may be the result of a complex combination of ancestral allopatric speciation processes (as a result of the separation of South America and Australia), plus the diversification of their host groups (e.g. Parastacidae) in South (and subsequent radiation in Central) America. For example, the species of Temnocephala associated with mollusks appear to be a morphologically homogeneous group with a phylogenetic structure (Volonterio 2007a, Damborenea andBrusa 2008). On the other hand, the almost exclusive distribution in the Southern Hemisphere of the family to which Temnocephala belongs (Temnocephalidae) is noteworthy and alludes to a Gondwanian origin (Gelder 1999, Cannon andJoffe 2001). However, a reliable molecular clock of the Temnocephalida is required to support or reject this hypothesis. Future studies combining research programs in integrative taxonomy (Schlick-Steiner et al. 2010, Ceccarelli et al. 2012, Fujita et al. 2012) with approaches of historical association (e.g. genes, organism and areas; see Page and Charleston 1998) will decipher the evolutionary history of Temnocephala.
At least 60 papers have been published dealing with the records of Neotropic symbiotic temnocephalans; however, the scarcity of studies in many countries is clear, and needs to be rectified. For example, some countries comprising complex geographic areas (i.e. Mexican Transition Zone, South American Transition Zone) only have one record of these turbellarians, and the diversity of the four major hosts groups is also unknown (Martínez-Aquino et al. 2014a). Therefore, we contend that future survey work should be strategic, aimed at enhancing the biodiversity inventory, combining identification of the host spectrum with choice of appropriate drainages based on biogeographic, faunistic, and hydro-logic data and on lessons from other freshwater symbiotic Platyhelminthes (e.g. Pérez-Ponce de León and Choudhury 2010, Martínez-Aquino et al. 2014c).