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Critical Comment: What We Don’t Recognize Can Hurt Us: A Plea for Awareness About Cryptic Species

Gerardo Pérez-Ponce de León Universidad Nacional Autónoma de México, [email protected]

Steven A. Nadler University of California - Davis, [email protected]

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Pérez-Ponce de León, Gerardo and Nadler, Steven A., "Critical Comment: What We Don’t Recognize Can Hurt Us: A Plea for Awareness About Cryptic Species" (2010). Faculty Publications from the Harold W. Manter Laboratory of Parasitology. 718. https://digitalcommons.unl.edu/parasitologyfacpubs/718

This Article is brought to you for free and open access by the Parasitology, Harold W. Manter Laboratory of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications from the Harold W. Manter Laboratory of Parasitology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Pérez-Ponce de León & Nadler in Journal of Parasitology (2010) 96(2). Copyright 2010, American Society of Parasitologists. Used by permission.

CRITICAL COMMENT . . .

J. Parasitol., 96(2), 2010, pp. 453–464 F American Society of Parasitologists 2010

What We Don’t Recognize Can Hurt Us: A Plea for Awareness About Cryptic Species

Gerardo Pe´rez-Ponce de Leo´n and Steven A. Nadler*, Departamento de Zoologı´a, Instituto de Biologı´a, Universidad Nacional Auto´noma de Me´xico, Ap. Postal 70-153, C.P. 04510, Me´xico D.F.; *Department of Nematology, University of California, One Shields Avenue, Davis, California 95616. e-mail: [email protected]

ABSTRACT: We conducted an extensive literature review on studies that issue of, ‘‘How similar do species need to be (if they are diagnosable based have used DNA sequences to detect cryptic species of parasites during the on morphology) to qualify as cryptic?’’ In the strictest interpretation of last decade. Each literature citation that included the term ‘‘cryptic’’ or cryptic species, once these species can be diagnosed based on morphology, ‘‘sibling’’ species was analyzed to determine the approach used by the they are no longer cryptic; however, from a purely practical standpoint, author(s). Reports were carefully filtered to retain only those that they may remain ‘‘hidden’’ during routine examination. Thus, it is useful recognized the existence of cryptic species centered on the use of DNA to distinguish between cryptic species sensu stricto (no morphological sequences. Based on analysis of these papers, we comment on the different differences are known) versus a functional definition of cryptic species (for ways that parasite cryptic species are discovered in studies focusing on which the application of the term is defined by the systematist). different aspects of the host–parasite relationship, or disciplines, within A bibliographic search for a 10-yr period (from January 1999 to parasitology. We found a lack of methodological and theoretical November 2009) in the ISI Web of Knowledge (http://apps.isiknowledge. uniformity in the discipline for finding and delimiting cryptic species, com) yielded 3,913 records (or ‘‘cryptic species reports’’) with the search and we draw attention to the need for standardizing these approaches. We term ‘‘cryptic species.’’ If the search term ‘‘sibling species’’ is used, the suggest that cryptic species, in the strict sense, are always provisionally number of records for the same period is 2,313. Interpretation of such cryptic, in that the possibility does exist that new morphological studiesor results requires caution, however, because these search terms also recover techniques will reveal previously unknown diagnostic structural differ- records on other ‘‘cryptic’’ biological phenomena involving species, e.g., ences which will permit rapid and practical morphological diagnosis. To cryptic behavior, cryptic coloration, and cryptic host-specificity. In avoid future taxonomic confusion, we recommend that parasitologists addition, papers on cryptic species are not exclusively based on molecular describe (and formally name) cryptic species following standard taxo- data. For example, Deunff et al. (2004) recently described a cryptic species nomic practice. of spinturnicid mite, a parasite of chiropterans, by associating biologic, biometric, and morphologic criteria together with the host’s ecoethology. Nevertheless, the number of such bibliographic records has increased Parasitologists discover and describe new species of parasites with every year since 1999, when 132 reports were published; in 2008, there regularity, and DNA-based taxonomic methods are increasingly used to were 513 records, and from just January to November 2009, 576 had been complement these descriptions. Undoubtedly, the routine discovery of new published. parasite species reinforces the belief that parasitism is one of the more When the same 10-yr period was searched in the ISI Web of Knowledge successful and common modes of life on earth, a belief which is also (ISI), using the terms ‘‘cryptic species’’ and ‘‘parasites’’ (and also ‘‘sibling illuminated by our increased understanding of parasite ecology, evolution, species’’ and ‘‘parasites,’’ as both terms have been used for the same and biogeography (Brooks and McLennan, 1993). The discovery of phenomenon in our discipline), with a focus on the major groups of cryptic species in nature (morphologically indistinguishable, but geneti- parasitic organisms (protists, helminths, and arthropods), 464 records cally distinct, species) has attracted the attention of systematists, were recovered (‘‘cryptic species reports’’). Although the number of ecologists, and evolutionary biologists. Implicit in their discovery are published studies reporting on cryptic species of parasites is increasing, it potential methods for detecting and delimiting cryptic species, and is not increasing at the same rate as in free-living taxa (Tables I, II). emphasis herein is given to the difference between cryptic species Careful filtering of these 464 records obtained from the ISI (plus manual prospecting (methods to detect putative cryptic species) and delimitation searches of the major parasitological journals to retrieve additional (testing that we have cryptic species). records not recovered during the ISI search) yielded 68 reports where Cryptic species have significant implications for evolutionary theory, cryptic species of parasites (or sibling species) also reference DNA biogeography, and conservation planning (e.g., Beheregaray and Caccone, sequences. Some papers refer to the potential presence of cryptic species, 2007; Bickford et al., 2007; Pfenninger and Schwenk, 2007; Trontelj and but no sequence data were presented, and these papers were not Fisˇer, 2009). The term ‘‘cryptic species’’ does not have a uniform meaning considered in Table III, e.g., Bolek et al. (2009). These 68 ‘‘filtered’’ as applied by different investigators either within, or outside of, papers include the recognition (or at least the potential presence) of cryptic parasitology. In the strictest sense, species are cryptic when no species among protists (apicomplexans, diplomonadids, trypanosomatids, morphological differences (qualitative structural, meristic, or morpho- and trichomonads), helminths (monogeneans, digeneans, cestodes, nema- metric) are known with which to diagnose them. Such species are most todes, and acanthocephalans), and arthropods (ticks, lice, and crusta- often recognized based on analysis of genetic evidence that falsifies the ceans) and also include different host groups including humans, livestock, hypothesis of a single species. However, many investigators use a much- and wildlife. Interestingly, only 17 of the 68 papers reported that voucher less strict concept with respect to morphological uniformity. For example, specimens had been archived in a parasite collection. During the decade of some will apply the term ‘‘cryptic species’’ to taxa that can be diagnosed the 1990s, several papers were published revealing the presence of cryptic based on morphology, but not easily so, such that the species have a high parasite species, although most of these studies were based on interpreta- degree of morphological similarity; this may lead to misidentification. This tion of multilocus protein electrophoretic data with a focus on assessing view reflects aspects of a definition provided by Bickford et al. (2007), levels of genetic divergence between taxa and, in some cases, distinguishing ‘‘. . .two or more distinct species that are erroneously classified (and among species (and discovering cryptic species) based on genetic distance hidden) under one species name,’’ in that such cryptic species are often criteria (e.g., Chilton et al., 1992; Pozio et al., 1992; Beveridge et al., 1993; discovered within what was formerly recognized as a single species Nascetti et al., 1993). (typically discovered using genetic data), but then a posteriori analysisof This review of the literature published during the last decade was the delimited species may reveal diagnostic morphological differences that conducted to identify reports that detected parasite cryptic species by were previously unrecognized. In the strictest sense, species that once were using sequence data from 1, or more, genes. The review was then used to not recognized as distinct, based on morphological evidence, may become assess the extent to which such species are found among parasitic so. However, application of the term ‘‘cryptic species’’ is often continued organisms. Meta-analyses of publication records for cryptic species have based on either taxonomic history or, more justifiably, because the species concluded that the discovery of cryptic species is likely to be non-random cannot be readily or practically diagnosed based on morphology due to with regard to taxon and biome (Bickford et al., 2007), but they have not their similarity and nature of the diagnostic characters. This raises the established if cryptic species are more common in particular habitats, latitudes, or taxonomic groups. If cryptic species are not randomly DOI: 10.1645/GE-2260.1 distributed, but are influenced by ecologic, historic, and abiotic factors,

453 454 THE JOURNAL OF PARASITOLOGY, VOL. 96, NO. 2, APRIL 2010

TABLE I. Comparison of the total number of published papers (or ‘‘cryptic TABLE II. Comparison of the total number of published papers (or species reports’’) referencing cryptic species, to the total number of ‘‘cryptic species reports’’) referring to cryptic species in parasites (protists, published papers specific to parasites (protists, helminths, arthropods), in helminths, arthropods), with respect to other groups of organisms, from the general literature from 1999 to 2009.* 1999 to 2009.*

Publication ‘‘Cryptic species’’ + Number of published papers year ‘‘Cryptic species’’ ‘‘parasites’’({) Monogenea 7 1999 158 13 (2) Digenea 10 2000 180 11 (5) Cestoda 15 2001 201 16 (2) Parasitic Nematoda 17 2002 224 19 (7) Acanthocephala 2 2003 273 11 (3) Parasitic Arthropoda{ 7 2004 299 25 (5) Parasitic protists 10 2005 368 31 (10) Bryozoa 20 2006 453 26 (3) Insecta 126 2007 521 42 (9) Crustacea 81 2008 660 33 (12) Annelida 25 2009{ 576 25 (10) Mollusca 38 Equinodermata 20 * Source: ISI Web of Knowledge. http://apps.isiknowledge.com/WOS_GeneralSearch. Fungi 95 Data reported from January 1999 until November 2009. { Reports wherein DNA sequences specifically refer to the existence of cryptic Fish 197 species. Amphibia (‘‘frogs’’ + ‘‘caudata’’) 77 Reptilia (‘‘snakes’’ + ‘‘lizards’’) 60 then the frequency of cryptic species should differ among parasites due to Aves 18 their inherent ecological and life history variation. Parasite cryptic species Mammalia 76 have been recognized using molecular tools since the 1990s (Nadler, 1990), but the extent to which this phenomenon occurs has not yet been * Source: ISI Web of Knowledge. http://apps.isiknowledge.com/WOS_GeneralSearch. Data reported from January 1999 until November 2009. determined. The existence of cryptic species has obvious implications for { Includes crustaceans, ticks, and insects. obtaining an accurate inventory of extant biodiversity. Furthermore, the failure to recognize cryptic species in medically, economically, or ecologically important parasites can have serious negative consequences relatively common among parasitic organisms (Table III). These reports for the development of biological control measures, the monitoring and show that approximately 128 cryptic species of parasites were discovered control of human diseases and potential zoonoses, the management of in the last decade, although not all of these were characterized as such in agricultural and aquaculture pathogens, and for detecting the presence of the original publications and very few were formally described (and invasive species (see Leung et al., 2009 and references therein). Beyond named). These cryptic species contribute a small proportion of the these more-practical implications, the existence of undetected cryptic relatively large number of new species descriptions published annually in species and the radiation of cryptic species complexes have important parasitological and general zoology journals. Most descriptions of new potential impacts on understanding and developing parasite evolutionary parasite species are still based solely on morphological characters. theory, historical biogeography, and ecology (Hoberg and Brooks, 2008). However, to provide for the needed integration of morphological and Here, we evaluate the extent to which cryptic species have been reported molecular data in parasite systematics, it is strongly advisable to collect among parasites, and we also analyze the approaches by which these and preserve specimens for both morphological and molecular character- species have been discovered. This evaluation has also revealed a lack of ization. This recognizes the importance of genetic data, which should be uniformity in what parasitologists mean by ‘‘cryptic species,’’ coupled with less affected by host-induced phenotypic variation, for parasite species delimitation. The modern, integrated approach should assist efforts to an absence of consistency regarding the theoretical and methodological solve taxonomic problems, even though relatively few formal descriptions underpinnings by which these species are discovered. For instance, most of new parasite species incorporate molecular data. Modern taxonomic authors apply this terminology when they are incapable of distinguishing practices also require the preservation and deposition of voucher species that are morphologically very similar, i.e., ‘‘hidden.’’ Some have specimens in established parasite collections. used ‘‘cryptic’’ in other contexts, including ‘‘cryptic variation,’’ ‘‘cryptic host-specificity,’’ ‘‘cryptic host-associated divergence,’’ or for the hypoth- esis that parasites may reveal the ‘‘cryptic phylogeographic history of their POTENTIAL PROBLEMS IN RECOGNIZING hosts’’ (Hoberg, 1995), an idea subsequently adopted for applications of CRYPTIC SPECIES molecular data (Nieberding et al., 2004). The definition of ‘‘cryptic species’’ usually includes 2 elements, i.e., From the literature review, we identified some problems inherent with species that are morphologically indistinguishable, or practically so, and the recognition of cryptic species of parasites. Before addressing the genetically distinct lineages that are considered to represent separate different approaches to identify these species, we first discuss some of these species. Parasitologists have recognized cryptic species in different parasite problems. One difficulty is that there has been no agreement on what groups following this definition, with the first molecular approaches constitutes appropriate discovery methods, or analytical approaches, to employing protein electrophoretic data and testing whether or not genetic test hypotheses of such species. It is noteworthy that some authors are (allelic) population data were consistent with expectations of single, clearly discovering cryptic species, but the term cryptic species (or even panmictic species (e.g., Bullini et al., 1978; Nascetti et al., 1979; Baverstock sibling species) is not used in their papers (see, for example, Feliu et al., et al., 1985). During the last decade, nucleotide sequence-based methods 2000; Iwagami et al., 2000). Another problem regarding the lack of have replaced earlier molecular approaches, e.g., native proteins, RFLPs, uniformity in the way parasite cryptic species are found is related to the and RAPDs, and a common practice has been to obtain gene sequences to observation that their discovery is often peripheral to the main focus of the characterize levels of genetic variation over geographic space; however, investigation. Only occasionally has an article title included referenceto these same data can also be used to recognize the existence of cryptic cryptic parasite species (see, for example, Macnish et al., 2002; Miura et species. From published papers, it is often difficult to discern if al., 2005; Miller and Cribb, 2007; Razo-Mendivil et al., 2010). More investigators were deliberately prospecting for cryptic species (sensu frequently, the presence of cryptic species is a collateral finding resulting Blouin, 2002; Criscione et al., 2005; Vilas et al., 2005), or if their discovery from genetic analyses of other aspects of parasite evolution such as was accidental. Regardless, evidence suggests that cryptic species are phylogeography, population genetic structure, or phylogenetic analysis CRITICAL COMMENT 455 that includes samples of many individuals (Criscione and Blouin, 2004; similarity, the genetic species concept by genetic distance, and the Johnson et al., 2007; Bouzid et al., 2008). It is likely that, in many such phylogenetic species concept by monophyly. They reported that, with 1 cases, insufficient effort (and expertise) has been applied to assessing the exception, morphological species that were identified from multiple birds morphological diversity of the ‘‘cryptic’’ taxa. Another complication in had identical sequences for all infections (or differed only by a few parasitology is that the concept of cryptic species has been applied to synonymous substitutions) and were monophyletic by all tree reconstruc- larval forms when larvae are morphologically indistinguishable, but show tion methods (maximum parsimony, maximum likelihood, and Bayesian high genetic differentiation (e.g., Donald et al., 2004, 2007; Miura et al., inference). Only 1 species did not follow this pattern, representing instead 2005; Palm et al., 2008). However, there are many instances where larvae 2, or more, genetically distinct clades that were inferred to be cryptic or juveniles from different species are morphologically indistinguishable, species. It should be noted that the phylogenetic species concept is as occurs in many nematodes, yet their respective adult stages are applicable to an analysis of all types of data (morphologic, molecular, morphologically distinct. Without linking these indistinct larval stages to combined evidence), and we do not necessarily advocate using different their adult stage, no complete morphological comparison is possible and, concepts for different types of data. therefore, there is no basis for describing as ‘‘cryptic’’ any species that are incompletely known or characterized. Without such a linkage, it would be possible to conflate larval genetic differentiation as evidence for one or APPROACHES FOR RECOGNIZING CRYPTIC SPECIES more new species when the distinct adult stages of the parasites may have The correct identification of parasite species, cryptic or not, is at the already been described and named. core of our understanding of biodiversity and the impact of parasitism in Another important, potential shortcoming is the absence of an nature. Cryptic species simply exacerbate the problem of correctly underlying species concept when researchers are delimiting species assessing biodiversity. In addition, research on the ecology or systematics (including cryptic species) in nature. The advantages and disadvantages of parasites may take different paths, depending entirely on the research of different species concepts are beyond the scope of this review, but there objectives of the investigator. Cryptic species of parasites are being are many recent discussions of this subject (e.g., Wheeler and Meier, 2000; discovered by taxonomists, especially those conducting molecular Brooks and McLennan, 2002). Although systematists may disagree on prospecting (sensu Blouin, 2002), but also by researchers specializing in what is the optimal species concept, they would probably agree that it is areas such as population genetics, life cycles, and veterinary or medical important to understand what you are looking for (have a species concept) parasitology. Cryptic species are of particular importance to humans in before trying to find or delimit species (Adams, 1998, 2001). Usually, it is cases where their discovery and diagnosis are important for diseases not explicit in parasite taxonomic papers what species concept is being involving agriculture (plant or animal health), companion animals, and followed (although see Nadler et al., 2000; Zietara and Lumme, 2003), and humans. Additionally, cryptic species are known to have major implica- clearly this should be communicated, and the choice of concept defended, tions for the implementation of effective control and surveillance because use of different concepts can lead to different decisions for the programs targeted for parasites of medical and veterinary importance, same data (Adams, 1998). Most papers recovered in the literature search or their vectors (e.g., Conn et al., 1997; Cepicka et al., 2005; Saijuntha et (Table III) used sequence divergence levels to assess conspecificity through al., 2007). application of a genetic yardstick, but this approach, although useful for A factor that has led to lack of uniformity in the way cryptic parasite species prospecting (Vilas et al., 2005), has been criticized as inappropriate species are recognized in nature is the expertise and focus of research for species delimitation for both practical and theoretical reasons (Nadler groups. Naturally, this leads to different starting points for research and et al., 2000; Nadler, 2002, 2005). Assessments of sequence divergence have different actions regarding recognition of cryptic species (Fig. 1). sometimes been accompanied by an evolutionary (phylogenetic) analysis Traditional alpha taxonomy mainly uses morphological attributes to to assess reciprocal monophyly, usually in the form of a neighbor-joining, distinguish species. In many groups, however, the identification of very maximum parsimony, maximum likelihood, or a Bayesian tree. These closely related species, with morphological data, may be difficult because approaches generally conform to the recommendation by Adams (1998), there may be a long time-lag between speciation and morphological which was that testing the hypothesis of lineage independence (species) in differentiation (Jousson et al., 2000), that is, morphological stasis. This is any particular case requires phylogenetic interpretation of data and the likely to be exacerbated in groups of organisms that have a paucity of potential for failure to recover such lineages. accessible morphological information (Zietara and Lumme, 2003). Other potential complications follow from how specimens are typically Molecular taxonomy makes use of 1, or more, molecular markers to collected and processed. Parasites are collected from their hosts during characterize organisms at various levels of the taxonomic hierarchy. Such fieldwork, and specimens are initially distinguished (sorted) based on a investigations might be used to estimate phylogenetic relationships among morphological species concept, i.e., a distinction is made among specimens previously identified species, or to test the hypothesis of conspecificity for and they are allocated to species level (morphospecies) based on existing morphospecies (or morphotypes) with independent genetic data, thus morphological diagnoses. In some cases, depending on the taxonomic potentially revealing the existence of previously unrecognized evolutionary complexity of the group, parasites are only readily separated as lineages (species) or, conversely, finding molecular data consistent with morphotypes, but not to morphospecies. For some parasite groups, conspecificity. Independent genetic evidence of conspecificity can also information on host-specificity and infection localization (tissue site) is provide a framework for documenting levels of intraspecific morpholo- also used as diagnostic evidence, when particular species of parasites are gical variability (including polymorphism among males, as in certain only known from a particular host species or from specific predilection Ostertagiinae) or, in the extreme, to demonstrate that 2 parasite sites within hosts. The pervasive influence of host species and traditional morphospecies (even those formerly assigned to different genera) represent morphological diagnostic methods on species status is evident from a the same species (Stevenson et al., 1996; Dallas et al., 2000; Desdevises et landmark study on reptile malaria. Perkins (2000) presented evidence that al., 2000; Bell and Sommerville, 2002; Li and Liao, 2003). Quite often, Plasmodium azurophilum, a parasite of lizards in the eastern Caribbean, parasitologists observe a parasite species with a broad host range, involves 2 cryptic species; she supported her conclusion with data for geographical distribution, or both, that is accompanied by morphological defining these species based on similarity, biologic, and phylogenetic variation (Hoberg et al., 1999). Typically, this variation is interpretedas species concepts. Sequence data from the mitochondrial cytochrome b occurring within the same species; for example, as normal population-level (Cyt b) gene showed that this lizard apicomplexan morphospecies was, in genetic variation, or perhaps as morphometric (size) differences correlated fact, 2 cryptic species (Perkins, 2000). These reproductively isolated species with the host species. Molecular tools offer the possibility to test the null are indistinguishable by light microscopy, but 1 species undergoes hypothesis that this would represent variation within a single species. schizogony only in erythrocytes and the other only in white blood cells. Cryptic species are found by different approaches that are based on the Bensch et al. (2004) suggested that avian haemosporideans have null hypothesis that researchers are dealing with a single species (Fig. 1). substantial potential for undiscovered cryptic species. This argument Often, parasitologists must deal with widespread species, defined in terms was used by Martinsen et al. (2006) to contrast morphologic versus of both host or geographic distribution range, or both, where morpho- molecular identification of these parasites, i.e., parasites were identified to logical characters do not provide evidence for separate morphospecies and species based on morphology and partial sequencing of the mitochondrial where morphological variability is low among members of different Cyt b gene. These data were analyzed by 3 species concepts (morphologic, geographic populations. A single parasite species might be found in genetic, and phylogenetic) and, as interpreted by these authors, the multiple host species that occur in sympatry, but also may be found morphological species concept requires grouping the parasites by associated with multiple host species in several localities. Given this wide 456 THE JOURNAL OF PARASITOLOGY, VOL. 96, NO. 2, APRIL 2010

TABLE III. Published reports (data reported from January 1999 until November 2009) where DNA sequence data from nuclear or mitochondrial genes (or both) is used to reveal and diagnose, or to simply suggest the possibility of, cryptic species as a result of a diagnostic study, molecular prospecting, or phylogeographical analysis.

PY* Parasite group Molecular marker(s){ Reference{

1999 Teladorsagia boreoarcticus .NADH-4 .Hoberg et al. (1999) Cylicostephanus minutus .ITS1, ITS2 .Hung et al. (1999) 2000 Macvicaria/Monorchis .ITS1 .Jousson et al. (2000) Opecoelioides columbellae .ITS1 .Jousson and Bartoli (2000) Plasmodium azurophilum .Cyt b .Perkins (2000) Cryptosporidium parvum .HSP70 .Sulaiman et al. (2000) Contracaecum osculatum .ITS1, ITS2 .Zhu et al. (2000) 2001 Trypanosoma spp. .18S .Sehgal et al. (2001) Paranoplocephala spp. .ITS1 .Haukisalmi et al. (2001) 2002 Intestinal Nematoda in cattle (pairs of congeners) .ITS1, ITS2, NADH-4, COI .Blouin (2002) Gyrodactylus rugiensoides .ITS1, 5.8S, ITS2 .Huyse and Volckaert (2002) Columbicolla/Physconelloides .COI .Johnson et al. (2002) Hymenolepis nana .ITS1, COI .Macnish et al. (2002) Bolbophorus sp. .18S, ITS1, ITS2, 28S, COI .Overstreet et al. (2002) Ixodes holocyclus .ITS2 .Shaw et al. (2002) Pseudoterranova decipiens .ITS1, ITS2 .Zhu et al. (2002) Teladorsagia circumcincta .b-tubulin, ITS2, NADH-4 .Leignel et al. (2002) 2003 Gyrodactylus salaris/G. thymalli .COI .Hansen et al. (2003) Gyrodactylus spp. .18S, ITS1, 5.8S, ITS2, 28S .Zietara and Lumme (2003) Contracaecum ogmorphini .Cyt b .Mattiucci et al. (2003) 2004 Haemoproteus/Plasmodium .DHFR-TS .Bensch et al. (2004) Megathylacoides giganteum .28S .Rosas-Valdez et al. (2004) Derogenes aspina, Plagioporus shawi, .NADH-1, ITS1 .Criscione and Blouin (2004) Nanophyetus salmincola Larval digenea (Opecoelidae) .ITS2, 16S .Donald et al. (2004) Paranoplocephala omphalodes .COI .Haukisalmi et al. (2004) 2005 Progamotaenia exersi, P. macropodis, P. zschokkei .COI .Hu et al. (2005) Heterophydae/Philophtalmidae .COI, ITS1 .Miura et al. (2005) Pairs of closely related digeneans and cestodes .ITS1, ITS2, NADH-1 .Vilas et al. (2005) Pseudorhabdosynochus lantauensis .ITS1, 28S .Wu et al. (2005) Tetratrichomonas gallinarum .16S rRNA, ITS1, 5.8S, ITS2. .Cepicka et al. (2005) Echinococcus shiquicus .COI, NADH-1, atp6, Cyt b, rrnL, elp .Xiao et al. (2005) Contracaecum rudolphi .ITS1, ITS2 .Li et al. (2005) Plasmodium falciparum .Review paper .Gauthier and Tibayrenc (2005) 2006 Plasmodium, Haemoproteous, Leucocytozoon .Cyt b .Martinsen et al. (2006) Sanguinicolidae: Ankistromeces/Phthinomita .ITS2 .Nolan and Cribb (2006) Leucocytozoon toddi .Cyt b .Sehgal et al. (2006) 2007 Didymobothrium rudolphii .ITS2, 28S .Marques et al. (2007) Teladorsagia circumcincta .5 microsatellites .Grillo et al. (2007) Columbicola spp. .COI, 12S, EF-1a .Johnson et al. (2007) Soboliphyme baturini .NADH-4 .Koehler et al. (2007)} Hysterothylacium aduncum .ITS1, 5.8S, ITS2 .Klimpel et al. (2007)} Hovorkonema variegatum .ITS2 .Krone et al. (2007) Leptorhynchoides thecatus .ITS1, ITS2, COI .Steinauer et al. (2007) Anisakids, Raphidascarids .ITS1, 5.8S, ITS2 .Kellermanns et al. (2007)} Tentacularia coryphaenae .28S .Palm et al. (2007)} Geomydoecus spp. .COI, EF-1a .Light and Hafner (2007) 2008 Ligula intestinalis .ITS2, Cyt b, COI .Bouzid et al. (2008) Taenia polyacantha, T. taeniaeformis .COI, NADH-1 .Lavikainen et al. (2008) Pseudoleptobothrium sp. .Cyt b, EF-1a .Glennon et al. (2008) Gyrodactylus spp. .COI, ITS1, 5.8S, ITS2 .Kuusela et al. (2008) Ancylostoma caninum .COI .Miranda et al. (2008) Mesomermis flumenalis .COI .St-Onge et al. (2008) Eimeria sp. .ITS2 .Cantacessi et al. (2008) Contracaecum rudolphii .ITS1, 5.8S, ITS2 .Farjallah et al. (2008) Anisakis typica .ITS1, 5.8S, ITS2 .Palm et al. (2008) Anisakid nematodes .Review .Mattiucci and Nascetti (2008) Caligus elongatus .16S, COI .Oines and Schram (2008) Anoplocephaloides variabilis .COI .Haukisalmi et al. (2008) (Table III continued) CRITICAL COMMENT 457

TABLETableIII. III. Continued.Continued

PY* Parasite group Molecular marker(s){ Reference{

2009 Acanthoparyphium, Curcuteria .16S, ITS1 .Leung et al. (2009) Columbicolla spp. .COI .Malenke et al. (2009) Ligula intestinalis .15 microsatellites .Sˇtefka et al. (2009) Neoechynorhynchus golvani .ITS1, 5.8S, ITS2, 28S .Marı´nez-Aquino et al. (2009) Diphyllobothrium spp. .ITS1, 18S .Arizono et al. (2009) Dermanyssus spp. .COI, 16S, ITS1, 5.8S, ITS2, 18S .Roy et al. (2009) Trypanosoma congolense .7 microsatellites .Morrison et al. (2009) Anoplocephaloides dentata .COI, 28S .Haukisalmi et al. (2009) Contracaecum rudolphi .ITS1, ITS2 .Shamsi et al. (2009) Crassicutis cichlasomae .COI, ITS1 .Razo-Mendivil et al. (2010)

*PY5 Publication year. { Molecular markers. Mitochondrial: 16S. COI 5 cytochrome c oxidase subunit I; Cyt b 5 cytochrome b; NDH4 5 nicotinamide adenine dinucleotide dehydrogenase subunit 4; NDH1 5 ibidem, subunit 1; atp6 5 ATPase subunit 6; rrnL 5 large subunit rRNA. Nuclear: ITS 5 internal transcribed spacers; 16S, 18S, and 28S rRNA; elp 5 ezrin-radixin-moesin (ERM)-like protein; EF-1a 5 elongation factor 1 alpha; DHFR-TS 5 dihydrofolate reductase-thimidylate synthase; HSP70 5 70-kDa heat shock protein. { Papers where sequencing data were used to corroborate morphologically based distinct species are not included, unless a direct statement regarding cryptic species is made. } Papers that found no genetic evidence for cryptic (or sibling) species.

array of possibilities, a parasite species may show within-species variation absence of any known morphological differences—in that diagnosing such for some morphological traits (sometimes even diagnostic traits as species depends upon molecular evidence. originally defined in the differential diagnosis), or only show within- Hypothesis testing for cryptic species requires acceptance or rejection of species variation in terms of body size. In some cases, diagnostic the null hypothesis. Testing the null hypothesis with molecular data has differences in parasite morphology have been shown to be a host-induced taken several different forms, including phylogenetic analysis for potential variation within a single species (Downes, 1990). However, some types of evolutionary structure (reciprocal monophyly of individuals falsifying the morphological variation may be difficult to discern during the normal null) and population genetic analyses, e.g., patterns inconsistent with course of specimen examination. In other cases, insufficient sampling can panmixia of individuals falsifying the null hypothesis. If the null proposal result in a failure to detect cryptic species in widespread parasite species, as is accepted, then from the morphological perspective, any observed discussed by Hoberg et al. (2003) and Cook et al. (2005) for cestode structural variation represents intraspecific differences among individuals (Arostrilepis) diversity in arvicoline rodents. and may be due to normal intraspecific genetic variation, or to phenotypic Tests of cryptic species require postulation of the null hypothesis (Ho), plasticity, including host-induced variability (e.g., Pe´rez-Ponce de Leo´n, which is for a single species (Fig. 1). The alternate hypothesis is that the 1995). When the null hypothesis is accepted based on molecular data, species, as currently conceived, is represented by 2, or more, species. In some researchers have characterized observed intraspecific variability some cases, researchers have presented this alternate hypothesis (existence (geographic, morphologic, ecologic, etc.) as strain variation (e.g., of a cryptic species complex) as the null hypothesis for a particular host– O’Mahony et al., 2004; Kawazoe et al., 2008). In some cases, the main parasite system (e.g., Hoberg et al., 1999). Either way, proper recognition impetus for cryptic species prospecting is that such species were previously (and delimitation) of cryptic species in nature involves scientific hypothesis discovered in close relatives, yet such findings are not necessarily good testing. In most instances, parasitologists begin from a molecular predictors for other species groups (Shaw et al., 2002; Klimpel et al., 2007; perspective, sequencing specimens from a wide host or geographic range Koehler et al., 2007; Palm et al., 2007). Testing hypotheses of cryptic (or even genetically characterizing many individuals from several species can be affected by sampling error. For example, using too few characters, or molecular markers with low rates of substitution relative to populations). When appropriate analyses lead to the recognition of the time scale of speciation, may fail to reject the null hypothesis when independent lineages (rejecting Ho), a more detailed morphological separate species are present (Nadler, 2002). This is one reason that, for investigation can serve as ‘‘reciprocal illumination’’ (Hennig, 1966), species delimitation (rather than prospecting), multiple loci should be perhaps providing structural evidence consistent with separate species used. The absence of genetic differences at a single locus may not falsify or, alternatively, revealing no apparent morphological differences, thus the null hypothesis, but data from other loci could do so. For both providing for provisional recognition of cryptic species, i.e., the species prospecting and delimitation, faster-evolving loci should provide more remain in ‘‘taxonomic crypsis’’ following discovery (Schlick-Steiner et al., appropriate data than would more-conservatively evolving ones (Blouin, 2007). This requirement for ‘‘reciprocal illumination,’’ or comparison 2002; Nadler, 2002). For phylogenetic analyses, corroborating the same between morphological and molecular evidence, is necessary if reliable pattern of reciprocal monophyly with multiple loci is particularly strong inferences about the ‘‘cryptic’’ status of species are to be made (Jenkins et evidence of separate species (Nadler, 2002, 2005). Similarly, population al., 2005; Kutz et al., 2007). For completeness, this extends to genetic analyses of genetic structure will benefit from examination of morphological examination of all life cycle stages (when the life cycle is multiple loci, because not every locus will reflect a pattern of variation known), particularly those that have the greatest likelihood of showing consistent with a neutral genetic marker (Nadler, 1995). In this sense, there morphological differences. For example, examination of nematode larvae is a parallel between morphological and molecular approaches, i.e., both might cause investigators to conclude that 2 species are indistinguishable, are provisional upon the available data and, in theory, both are improved when the adults are morphologically distinct. The research interests of the by additional sampling of independent data, e.g., morphological investigator shape the hypothesis-testing path (Fig. 1), and research characters and genetic loci. Clearly, investigations that are exclusively outcomes can differ as a result. For example, one possible outcome is molecular, or only morphological, both have limitations. The most recognizing the presence of cryptic species based on phylogenetic analysis thorough studies include both types of information, with morphological of molecular data, whereas a more desirable outcome for describing conclusions being tested by genetic data and with molecular evidence for biodiversity would be the formal description (and naming) of the species. separate species being examined by a detailed morphological study of The discovery and delimitation of species using molecular data does not specimens representing each independent evolutionary lineage. Indeed, by guarantee the discovery of diagnostic morphological features for these design, some investigators propose using both sources of information to same species, even if such morphological differences exist. This leads to test the null hypothesis (e.g., Marques et al., 2007). Similarly, while practical challenges involving the formal description of the species—if they describing a substantial radiation of sanguinicolid blood flukes from 3 are cryptic in the strict sense, e.g., providing a differential diagnosis in the families of marine fishes, Nolan and Cribb (2006) used a species-level 458 THE JOURNAL OF PARASITOLOGY, VOL. 96, NO. 2, APRIL 2010

FIGURE 1. Chart depicting potential hypothesis-testing paths for discovering parasite cryptic species. taxonomy based on a combined molecular, biological, and morphological phylogenetic analysis of sequences; if the evolutionary lineages are approach. These authors recognized a priori that morphology alone may distinguished by morphological characters, the species are described be insufficient for the unequivocal identification of parasite species; they (and named) using both the sequence and morphological data (e.g., used molecular methods to augment the traditional morphological Curran et al., 2006; Pe´rez-Ponce de Leo´n et al., 2008). However, if only approach, concluding that cryptic species are an increasingly important genetic data distinguish the evolutionary lineages (i.e., no correlated consideration for accurately characterizing parasite biodiversity. morphological differences are discovered), then falsification of the null If data analysis leads to the rejection of the null hypothesis (Fig. 1), then hypothesis, based on molecular data in the absence of morphological several scenarios can result. From the morphology-based approach, differences, is indicative of cryptic species sensu stricto (see Overstreet et finding diagnostic characters can lead to the traditional description of the al., 2002; Rosas-Valdez et al., 2004; Chilton et al., 2007; the latter study new species, as is characteristic of comparative morphological analysis. was based on allozyme electrophoresis). Traditionally, the decision-making process of ‘‘rejecting the null hypoth- If the hypothesis-testing process leads to the rejection of the null esis’’ (presence of a multiple species) has been made through comparisons proposal, from the perspective of the DNA-based approach, the existence of a taxonomic expert referencing differential diagnoses (and physical of cryptic species sensu stricto means that the delimited species are either specimens) of other congeners. Most often, no explicit procedure is indistinguishable morphologically, or are so similar that they have not presented in concluding that a taxon merits recognition as a distinct been distinguished. If the null hypothesis has been rejected, research species. Species diagnosed in this way do not reflect hypotheses testing, in generally takes 1 of 2 directions (Fig. 1). One, or more, cryptic species may any formal sense, given that there is no explicit methodology for analyzing be discovered and delimited, but without formal scientific description (e.g., data that leads to falsification. Instead, this approach is an implicit appeal Hung et al., 1999; Macnish et al., 2002; Cepicka et al., 2005; Wu et al., to the experience and expertise of the taxonomist. Although many of these 2005; Miranda et al., 2008). Some authors, after recognizing the existence decisions may be correct, they do little to bolster confidence in the of cryptic species (but not necessarily delimiting them), discuss the objectivity and consistency of species delimitation as a science (Adams, implications of cryptic diversity or cryptic diversification (sometimes 1998; Nadler, 2002). erroneously referred to as ‘‘cryptic speciation’’) for the investigation, More recent systematic studies of parasites show that, for many which is typically focused on ecological and evolutionary questions rather taxonomic groups, molecular tools are very valuable and efficient than on systematics, per se (e.g., Jousson et al., 2000; Sehgal et al., 2006; resources, not only for initial ‘‘prospecting,’’ but for providing additional Grillo et al., 2007; Steinauer et al., 2007). In some cases, cryptic species characters useful for delimiting and diagnosing species. Most typically, have been recognized, but not described, with the rationale that there is no molecular sequences are obtained for samples of individuals from within, mechanism for the formal description of species based only on genetic or among, infrapopulations, and the null hypothesis is rejected because data (Andrews et al., 1998). However, there is no rule in the International reciprocal monophyly is revealed for the taxa (individuals) based on a Code of Zoological Nomenclature (ICZN, http://www.iczn.org/iczn/index. CRITICAL COMMENT 459 jsp) preventing such a description, although there is also no article devoted conducted a posteriori analysis, revealing that morphological differences to the description of species based solely on molecular data nor is there a were consistent with genetic differences, thus leading to the recognitionof provision specifically focused on description of cryptic species. cryptic species in the system. When morphological diagnosis of species is difficult, or species-level Investigators working with monogeneans of the genus Gyrodactylus, diversity is poorly understood, e.g., cryptic species complexes, for the where cryptic species seem to be very common, have proposed a seemingly accurate characterization of species-level diversity it is critically important novel approach for presenting species descriptions that involves both to obtain molecular and morphological data from the same, individual morphological and molecular data (Zietara and Lumme, 2003; Kuusela et specimens. More typically, different sub-samples of specimens are al., 2008). With this approach, the definitive species recognition is based prepared using different procedures, one optimized for morphology and on the nucleotide sequence of nuclear ribosomal DNA (internal another for DNA, but this is less than ideal, particularly when there are transcribed spacers) and mtDNA, on evaluating the number of nucleotide few specimens from a host or if the sample may contain several substitutions, the presence of indels, and on pair-wise divergence. A morphologically similar congeneric species. Even when strong evidence molecular diagnosis is presented, and morphometric and morphological is found to delimit 2, or more, species using molecular data, authors can be diagnoses are established for each new species, with illustrations of the reluctant to propose nomenclatural changes based on molecular data haptoral structures, along with all relevant biological and geographical alone, when morphological diagnosis is uncertain (see Nolan and Cribb, data and information on deposition of specimens and sequence data. In a 2006; Marques et al., 2007). Commonly, more detailed morphological similar way, Nolan and Cribb (2006) presented a remarks section in their study is suggested as a necessary step to investigate the possibility of subtle publication that included a molecular diagnosis immediately following the morphological differences consistent with genetically recognized species, morphological description of each sanguinicolid digenean species they or even to emphasize other biological data such as growth requirements, discovered in marine fishes. metabolism, host preference, or geographical distribution. However, when Other investigations that may lead to the recognition of cryptic species cryptic species of parasites are discovered using a molecular approach, are those of phylogeographic and population genetic analyses (Fig. 1). morphological re-examination of the specimens for diagnostic traits often These approaches usually begin without a taxonomic focus because their follows, sometimes complemented by a more thorough morphometric main goal is to investigate the population genetic structure, or the analysis or use of new data types, e.g., scanning electron microscopy. phylogeography, of a single species. Most typically, mitochondrial genes These and other approaches can lead to a formal description of cryptic are employed for phylogeography whereas microsatellites, amplified species. fragment length polymorphisms (AFLPs), or other nuclear gene markers Some examples are available that illustrate how research on cryptic are used for estimating population genetic structure (Grillo et al., 2007; species can successfully progress from discovery through description. For Sˇtefka et al., 2009). Population-level analyses are useful for inferring example, feather lice species of Columbicola have been the subject of which aspects of the host–parasite relationship and life history may have intensive investigation (e.g., Johnson et al., 2002; Johnson et al., 2003; shaped the genetic structure of the species, including geography, host- Johnson et al., 2007; Bush et al., 2009; Malenke et al., 2009); molecular specificity, and life cycles (e.g., Nadler, 1995; Criscione and Blouin, 2004; phylogenetic analysis of 49 of the 80 species within the genus (Johnson et Bouzid et al., 2008; Glennon et al., 2008). In studies of population genetic al., 2007) revealed a considerable divergence that was correlated with host structure, estimated parameters include measures such as F-statistics (and associations, and this was used as evidence to delimit more than 30 cryptic their haplotype equivalents), including FST and FIS, effective population species. In the initial paper describing their discovery (Johnson et al., size, nucleotide and haplotype diversity, parsimony networks, and 2007), it was indicated that additional morphological studies were required estimated migration rates. Characteristics of the genetic markers prior to taxonomic revision. This was followed by more detailed (dominant vs. codominant expression) also influence the types of analyses morphological evaluations and descriptions (Bush et al., 2009). Similarly, that can be conducted. For example, direct assessments of interbreeding the nematode Teladorsagia was found to represent a species complex in through the determination of heterozygote frequencies requires codomi- Holarctic ruminants, based on reciprocal monophyly inferred from nant markers. High levels of genetic structure, a reduction of hetero- mitochondrial NDH4 sequences and on estimates of sequence divergence zygotes relative to expected numbers, and parsimony networks with large and nucleotide diversity (Hoberg et al., 1999). These authors formally numbers of inferred substitutions separating individuals, might be described the new species Teladorsagia boreoarcticus as part of the original explained by the lack of genetic exchange that is characteristic of cryptic publication, reporting discovery of the cryptic species. species, wherein individuals are separated by reproductive isolation rather Researchers working with other groups of parasites have sometimes than by geographic barriers to gene flow. For instance, Bouzid et al. taken similar, integrated approaches to delimiting species and describing (2008) studied 2 factors (geography and host specificity) that affected the them. For example, Xiao et al., (2005) investigated taeniid cestodes of genetic structure of Ligula intestinalis, a widespread tapeworm with larvae Echinococcus under the premise that potential cryptic species should be infecting freshwater fishes. They found different evolutionary mechanisms considered in their taxonomy. These authors used a phylogenetic analysis at the local and global geographical scales, based on sequences of nuclear of 5 mitochondrial and 1 nuclear gene to describe a new Echinococcus and mitochondrial genes (ITS2, Cyt b, and cox1), for 109 tapeworms from species (Echinococcus shiquicus) from the Tibetan fox and plateau pika in 13 host species and 18 localities. These authors found genetically divergent China. An investigation of the molecular systematics of larval tapeworms and well-separated clusters in different geographic areas sampled globally, (Mesocestoides) from dogs and coyotes (Crosbie et al., 2000) showed at and reproductive isolation was apparent for clades distributed sympa- least 3 distinct monophyletic groups; this information was expanded upon trically and infecting the same definitive host, suggesting the likelihood of by Padgett et al. (2005), who used multiple genetic loci in combination separate (cryptic) biological species (labeled as clades A and B in their with a morphometric analysis and an hypothesis-testing framework to study to represent these 2 different species), although specific taxonomic demonstrate that these 3 clades within Mesocestoides were distinct species. recommendations were not made. One of these species was conspecific with Mesocestoides vogae and, Similarly, during early stages of their research, some authors interested although none was characterized as ‘‘cryptic,’’ nevertheless, these authors in characterizing the genetic structure of parasites in order to address illustrated the practical application of sequence data to test the hypothesis ecological and evolutionary questions uncover the presence of distinct of lineage independence and species status for cestodes. Similarly, research evolutionary lineages that leads to recognition of cryptic species. For on the ascaridoid nematode Toxocara revealed that original reports of example, as part of a study to examine the effects of life cycles on the Toxocara canis in Malaysian domestic cats were inaccurate; subsequent distribution of genetic variation within and among parasite populations molecular studies showed that this parasite represented a separate species with allogenic and autogenic life cycles, Criscione and Blouin (2004) from T. canis and Toxocara cati, the common ascaridoid of domesticated discovered 2 divergent mitochondrial ND1 lineages among the digenean cats (Zhu et al., 1998). Originally identified as a cryptic species (Toxocara Deropegus aspina. To test whether these 2 mtDNA haplogroups cf. canis), further taxonomic study led to its description as Toxocara represented cryptic species, they sequenced the internal transcribed spacer malaysiensis (Gibbons et al., 2001). Subsequent mtDNA genome 1 (ITS1) of the rDNA gene. There was a complete concordance between sequencing has provided additional molecular support for this distinction taxa delimited by mtDNA and ITS1, a result indicating no (or minimal) (Gasser et al., 2005; Jex et al., 2008). For trematodes, Miller and Cribb introgression between the 2 lineages of D. aspina and, therefore, they (2007) described cryptic species of digeneans in marine fishes from considered them to be genetically distinct species (D. aspina A, and D. Australia. To test their morphologically based taxonomic approach, they aspina B) and analyzed their genetic structures separately. A similar sequenced 3 nuclear ribosomal regions (partial 28S, ITS1, and ITS2) and situation was found by Glennon et al. (2008) while studying host 460 THE JOURNAL OF PARASITOLOGY, VOL. 96, NO. 2, APRIL 2010 specificity of monogeneans from rhinobatid rays in southern Australia. To processed for morphological examination, or even if such specimens have determine whether the 3 species of monogeneans found in the ray been properly preserved for that purpose. Properly archived specimens are Trygonorhyna fasciata were restricted to this host, these authors used Cyt essential for future comparative work on cryptic species, including b sequences and found that, not only were these monogeneans not strictly archival material for molecular work. Several recent parasite survey and host-specific, but that the genetic structure of Pseudoleptobothrium inventory projects (e.g., The Beringian Coevolution Project or the aptychotremae (found in 3 of the 5 rhinobatid species surveyed in freshwater fish helminth parasite fauna in Mexico) serve as exemplars Australian coastal waters) was genetically homogeneous across most for host and parasite sampling strategies, preservation for morphological localities. However, 1 population showed an unusually high Cyt b and molecular studies, archiving of specimens, and integrated molecular divergence level. Sequencing of the nuclear gene elongation factor 1-a and morphological investigations that have examined broader questions corroborated the deep mtDNA divergence, suggesting that this clade concerning the distribution of parasite biodiversity (Hoberg et al. 2003; represents a cryptic species. Cook et al., 2005; Pe´rez-Ponce de Leo´n and Choudhury, 2010). The future In these cases, where a taxonomic approach is not the major focus of the of parasite systematics will be substantially complicated, if reports of research, cryptic species are inferred, but typically no effort is made to re- putative cryptic species accrue without proper delimitation or description examine the specimens and to corroborate the molecular findings with (and a valid name), because scientists will be required to deal with detailed morphological studies. Without such an effort, the ‘‘cryptic organisms of uncertain status (real species or not?) as well as with their status’’ of such taxa remains enigmatic, and such species often remain ‘‘labels,’’ e.g., Species A, Species B, etc. Recently, Hoberg et al. (2009) undescribed and without a proper scientific name. In many cases, research explored the critical role of permanent and well-supported museums or results may only suggest that unknown species are present, with natural history collections as foundations for systematic research in the confirmation requiring sequencing of additional loci, more detailed traditional sense, but also for all aspects of our discipline that depend analyses for hypothesis testing, e.g., corroboration of lineage indepen- upon the meaningful reference to a parasite by scientific name. In dence, and suitable morphological studies to differentiate between cryptic addition, museum collections and their associated (curated) data create an species and those that are morphologically diagnosable (or even previously empirical record that promotes our understanding of the biosphere described). The desired outcome for newly discovered cryptic species is through time. For example, while studying a range of taxonomic that they are properly described and that the initial molecular studies that (nomenclatural) and biogeographic questions about pinworms and pikas led to their discovery are followed by detailed morphological studies that from the American west, Hoberg et al. (2009) demonstrated the critical have the potential to reveal unrecognized structural differences. Although importance of type and voucher specimens and the role of museum such cryptic species may be indistinguishable based on current morpho- repositories for documenting species diversity and changes in faunal logical practice, it remains possible that future developments in structure over time (see also Brooks and Hoberg, 2000; Hoberg, 2002). morphological tools, e.g., microscopy techniques, staining methods, etc., Research on cryptic species of parasites is still in its infancy, but it is may provide methods that permit their diagnosis based on structural very likely that these species are much more common than previously features. In this sense, the strict cryptic status of such species remains thought. Data available thus far (Table III) are insufficient to permit provisional, although the practical matter of diagnosing these species ona generalizations, as are those made by authors such as Bickford et al. routine basis may remain difficult—even following the discovery of (2007) and Pfenninger and Schwenk (2007). For instance, the observation structural differences between species. that more cryptic species have been discovered among parasitic nematodes This emphasis on integrative molecular and morphological approaches (Table II) has to be considered in relation to the comparatively large to parasite systematics raises the important issue of the lack of sufficient, number of research groups worldwide investigating the molecular traditionally trained taxonomists to undertake the scope of research that systematics of anisakids. Researchers in Australia, Italy, and the United results from molecular investigations. Classically trained taxonomists have States have demonstrated several instances of cryptic species among a wealth of information on parasite morphology and natural history, anisakid nematodes, and because of their comparatively large size, knowledge that is critical for the required comparative analyses of known molecular methods (e.g., allozyme electrophoresis) have long been versus potentially new species and for the formal description of these practical for ascaridoid nematodes (for a review see Mattiucci and species. As pointed out by Baldwin et al. (1999), addressing this issue Nascetti, 2008). Within other parasite groups, few comparable efforts have requires not only efforts to strengthen traditional taxonomic expertise and been made to discover cryptic species, so it is premature to search for infrastructure, but changing attitudes among scientists themselves so that trends among different taxonomic groups. It has been suggested in a few systematists are considered as critical to university research as are the cases that certain host–parasite associations might show a larger number faculty who study fundamental molecular processes using reductionist of cryptic species, such as anoplocephalid and hymenolepidid tapeworms approaches. in arvicolid rodents (Hoberg et al., 2003, but also see Haukisalmi et al., 2004, 2008, 2009), or in protostrongylid nematodes in ungulates (Cook et CONCLUSIONS AND FUTURE DIRECTIONS al., 2005). As discussed by Hoberg and Brooks (2008), it is the history and structure of hosts and their parasites that can promote such species-level The discovery of cryptic species has a direct impact on our assessment of diversity (including cryptic species complexes). The fact that parasites parasite biodiversity (Poulin and Morand, 2004). Increased precision of contribute a small fraction to the total of ‘‘cryptic species reports’’ (sensu cryptic species discovery and description will result if parasitologists Bickford et al., 2007; Pfenninger and Schwenk, 2007) does not establish follow a common, theoretical framework and methodology for finding that cryptic species are less common among parasites than are free-living and delimiting cryptic species in nature. It is clear that research on cryptic organisms. The literature search for general patterns of cryptic species,as species has 3 steps. The first step is the recognition of potential cryptic discussed by Bickford et al. (2007) and Pfenninger and Schwenk (2007), species, sometimes discovered through cryptic species prospecting. The should be interpreted cautiously because these reports were not second step is their delimitation through hypothesis testing (potential individually verified for content. However, this may also reflect that falsification of the null hypothesis of a single species), and the third step is cryptic species research has proceeded at a slower rate within our that of formal description (and naming). As previously noted, the discipline. recognition of parasite cryptic species is frequently achieved when We predict that there are very large numbers of parasite cryptic species taxonomy is not the major focus of the research. However, simply to be discovered and speculate that they may account for a substantial recognizing potential cryptic species, without actually delimiting and fraction of parasite biodiversity in some clades. Host–parasite systems are describing them, will lead to increased taxonomic uncertainty that is known to represent rich macroevolutionary mosaics, with empirical counterproductive to research progress and synthesis in parasite systema- studies indicating that host-switching and geographical dispersal of tics. This also holds true for other research areas such biogeography, parasites are more common phenomena than is strict (maximum) ecology, and evolutionary biology. Molecular research on potential cospeciation (Hoberg and Brooks, 2008). Predictions concerning parasite cryptic species should be applied in concert with comparative morpho- biodiversity, including cryptic species, should consider this broader array logical research, and voucher specimens should be preserved and of macroevolutionary possibilities. Research programs on biodiversity and deposited in established museum collections to facilitate future systematic cryptic species will be enhanced as more parasite taxonomists use study, perhaps with improved techniques. molecular approaches that include infrapopulation-level sampling of Unfortunately, it can be difficult to ascertain, from some published individuals and analysis of multiple genetic loci. Molecular prospecting papers involving potential cryptic species, if specimens have been studies will increase in frequency as molecular methodologies become CRITICAL COMMENT 461

easier and less expensive. Standardization of the way we discover and BOUZID, W., J. Sˇ TEFKA,V.HYPSˇA,S.LEK,T.SCHOLZ,L.LEGAL,O. delimit cryptic species is vital for the development of more-robust KALTHOUM,B.HASSINE, AND G. LOOT. 2008. Geography and host estimates of their contribution to parasite biodiversity. This will lead to specificity: Two forces behind the genetic structure of the freshwater more accurate characterizations of parasite biota, but within the broader fish parasite Ligula intestinalis (Cestoda: Diphyllobothriidae). Inter- context of ‘‘integrative taxonomy,’’ wherein evidence is obtained from a national Journal for Parasitology 38: 1465–1479. wide range of sources, including morphology, molecular biology, host- BROOKS, D. R., AND E. P. HOBERG. 2000. Triage for the biosphere: The specificity, or biogeography, and is used not only to refine our estimates of need and rationale for taxonomic inventories and phylogenetic species numbers but to further understand the ecological, evolutionary, studies of parasites. Comparative Parasitology 67: 1–25. and biogeographical histories of host–parasite associations. ———, AND D. A. MCLENNAN. 1993. Parascript: Parasites and the language of evolution. Smithsonian Institution Press, Washington, ACKNOWLEDGMENTS D.C. 429 p. ———, AND ———. 2002. The nature of diversity: An evolutionary voyage This paper was written during the sabbatical leave of G.P.P.L. to the of discovery. Chicago University Press, Chicago, Illinois, 668 p. Department of Nematology, University of California–Davis. Thanks are BULLINI, L., G. NASCETTI,S.CIAFRE,F.RUMORE,E.BIOCCA,S. due to the program, UC MEXUS-CONACyT (University of California MONTALENTI, AND G. RITA. 1978. Ricerche cariologiche ed elettrofor- Institute for Mexico and the United States–Consejo Nacional de Ciencia y etiche su Parascaris univalens e Parascaris equorum. Atti della Tecnologı´a, Me´xico), for the fellowship awarded. We thank Andre´s Accademia Nazionale dei Lincei Rendiconti Classe di Scienze Fisiche Martı´nez-Aquino and 2 anonymous reviewers whose comments greatly Matematiche e Naturali 65: 151–156. improved this manuscript. This study was also partially supported by BUSH, S. E., R. D. PRICE, AND D. H. CLAYTON. 2009. Descriptions of eight grants from the Consejo Nacional de Ciencia y Tecnologı´a (CONACyT, new species of feather lice in the genus Columbicola (Phthiraptera: No. 83043) and the Programa de Apoyo a Proyectos de Investigacio´n e Philopteridae), with a comprehensive world checklist. Journal of Innovacio´n Tecnolo´gica (PAPIIT-UNAM IN 209608) to G.P.P.L.; Parasitology 95: 286–294. S.A.N. was supported by a NSF PEET award (DEB-0731516). CANTACESSI, C., S. 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