Janine N. Caira

A DNA Sequence–Based Approach To the Identification of Shark and Ray Species and Its Implications for Global Elasmobranch Diversity and Parasitology

Abstract In an effort to provide a framework for the accurate identification of elasmobranchs, driven in large part by the needs of parasitological studies, a comprehensive survey of DNA sequences derived from the mitochondrial NADH2 gene was conducted for elasmobranchs collected from around the world. Analysis was based on sequences derived from 4283 specimens representing an estimated 574 (of ∼1221) species (305 sharks, 269 batoids), each represented by 1 to 176 specimens, in 157 (of 193 described) elasmobranch genera in 56 (of 57 described) families of elasmobranchs (only Hypnidae was not represented). A total of 1921 (44.9%) of the samples were represented by vouchers and/or images available in an online host specimen database (http://elasmobranchs.tapewormdb.uconn.edu). A representative sequence for each of the 574 species identified in this survey, as well as an additional 11 sequences for problematic complexes, has been deposited in GenBank. Neighbor-joining analysis of the data revealed a substantial amount of previously undocumented genetic diversity in elasmobranchs, suggesting 79 potentially new taxa (38 sharks, 41 batoids). Within-species p-distance variation in NADH2-percent sequence divergence ranged from 0 to 2.12 with a mean of 0.27; within-genus p-distance variation ranged from 0.03 to 27.01, with a mean of 10.16. These values are roughly consistent with estimates from prior studies based on barcode COI sequences for elasmobranchs and fishes. While biogeographic influences have likely shaped the diversification of the entire group, the traces left by older influences tend to be overprinted by newer ones. As a result, the most clearly interpretable influences are those associated with recently diverged taxa. Among closely related elasmobranchs, four regions appear to be of particular importance: (1) the Atlantic Ocean, (2) Arabian Sea, Persian Gulf, and Red Sea, (3) Southeast Asia, and (4) Australia. Each of these regions has a substantial proportion of taxa that are genetically distinct from their closest relatives in other regions. These results suggest that great care should be taken in establishing the identities of elasmobranch hosts in parasitological studies. Furthermore, it is likely that many existing host records require confirmation.

On the phylogenetic relationships among tetraphyllidean, lecanicephalidean and diphyllidean tapeworm genera.

This study had two main objectives: (1) to construct an extensive, explicit list of characters and character states that might serve as a starting point, and perhaps even a model, for the compilation of a more complete list of characters for all cestode taxa; and (2) to use this character list to generate a hypothesis of the phylogenetic relationships among species representing most of the tetraphyllidean, lecanicephalidean and diphyllidean genera. Specimens of one species in each of 48 genera of tetraphyllideans, eight genera of lecanicephalideans, the three genera of diphyllideans, two genera of proteocephalideans and two genera of trypanorhynchs, were examined as whole-mounts and sections, with light and scanning electron microscopy. A list of 120 morphological characters was compiled. Four phylogenetic analyses were conducted using PAUP* and/or NONA. The first was a comprehensive analysis with the 56 tetraphyllidean and lecanicephalidean species as ingroups and the remaining seven species as outgroups. The second was an analysis of the three diphyllidean species as ingroups and the two proteocephalidean and the two trypanorhynch species as outgroups. The third was an analysis of the eight lecanicephalidean species and the “tetraphyllideans” Echeneibothrium sp. and Pseudanthobothrium n. sp. as ingroups and an outgroup consisting of the seven species used as outgroups in the first analysis. In the fourth analysis, the ingroup consisted of the 14 hooked tetraphyllideans (onchobothriids), and the outgroup consisted of the seven species used as outgroups in the first analysis. The results of these analyses support the following phylogenetic hypotheses: The diphyllideans are monophyletic and Echinobothrium n. sp. and Macrobothridium sp. are more closely related to one another than either is to Ditrachybothridium macrocephalum. The tetraphyllideans, lecanicephalideans and proteocephalideans are more closely related to each other than they are to the diphyllideans or the trypanorhynchs. The ordinal status of the lecanicephalideans is dubious. The lecanicephalidean species are more closely related to some of the tetraphyllidean taxa than these tetraphyllidean taxa are to the remainder of the tetraphyllidean taxa. The proteocephalideans appear to belong within the tetraphyllidean clade. The “tetraphyllidean” species Echeneibothrium sp. and Pseudanthobothrium n. sp. are members of the lecanicephalidean clade. The position of “Discobothrium” n. sp. within the lecanicephalideans is dubious. Within the tetraphyllideans, the non-acetabulate species Litobothrium daileyi, Disculiceps galapagoensis and Cathetocephalus sp. are the most basal members of the group. The family Onchobothriidae is monophyletic, as it is currently defined. Within the onchobothriids, the uniloculate species are basal to the multiloculate species; the species with unipronged hooks are basal to the species with multipronged hooks. Although relationships among the phyllobothriids, as they are currently defined, remain poorly resolved, the family Phyllobothriidae is not monophyletic. These results suggest that some aspects of the classification of the lecanicephalidean and tetraphyllidean taxa require revision. However, such revision should be based on further analyses including a broader representation of the genera and species in these groups.

Evolutionary factors influencing the nature of parasite specificity.

This article considers how specificity patterns are shaped during the course of parasite evolution. Parasites are first and foremost specific to site, or microhabitat; host ranges are far more subject to change than is microhabitat. Specificity results from a number of convergent phenomena starting with habits (microhabitat and feeding styles) of free-living progenitors and the way in which the parasitic association arises (e.g., passive oral contamination as opposed to intrusive entry). These bias the types of interaction parasites have with the host, and, through this, the way specificity develops. Host ecology acts as an external factor affecting specificity and predominates in parasites that interact minimally with the hosts physiological and immune systems. Coevolutionary factors are more important in parasites that feed on host tissues or occur in extraintestinal sites. Here, parasites must present the right cues, and respond appropriately to the host defense system. The ability to generalize these cues and responses across host boundaries may act as a constraint on host range. The functional role of the host in the parasite life history also affects the degree of specificity; thus, parasites may act as host generalists in hosts that act as trophic channels to the final host. The role of competition in determining specificity is difficult to assess. However, competition has been reported to influence microhabitat and host distribution through interactive site selection and/or competitive seclusion.

Evolution of the Major Lineages of Tapeworms (Platyhelminthes: Cestoidea) Inferred from 18S Ribosomal DNA and Elongation Factor-1a

The interrelationships of the tapeworms (Platyhelminthes: Cestoidea) were inferred by analysis of complete gene sequences (approximately 2,200 bp) of 18S small subunit ribosomal DNA (18S) and partial gene sequences (approximately 900 bp) of elongation factor-1alpha (Ef-1alpha). New collections were made of 23 species representing each of the 14 currently recognized orders of tapeworms, including the Amphilinidea, Gyrocotylidea, and the 12 orders of the Eucestoda. Sequences were determined directly from polymerase chain reaction (PCR) products by either manual or automated methods. Nucleotide sequences of platyhelminth species outside of the Cestoidea were obtained for rooting the resulting trees. The 18S sequences were aligned with reference to the secondary structural features of the gene and the Ef-1alpha sequences were aligned with reference to their corresponding amino acid residues. Significant length variation among taxa was observed in the V2, V4, and V7 variable regions of the 18S gene. Such positions where sequences could not be aligned confidently were excluded from the analyses. Third codon positions of the Ef-1alpha gene were inferred to be saturated at an ordinal level of comparison. In addition, a short (approximately 35 bp) intron region of the Ef-1alpha gene was found to be shared only among the eucestode taxa, with the exception of Spathebothrium simplex (Spathebothriidea), which lacked the intron. Complete alignments showing structural features of the genes and sites excluded from analysis are provided as appendices. The sequence data were partitioned into 7 data sets in order to examine the effects of analyses on different subsets of the data. Analyses were conducted on the 2 genes independently, different codon positions of Ef-1alpha, amino acid sequences of Ef-1alpha, and combinations thereof. All subsets of the data were analyzed under the criterion of maximum parsimony as well as minimum evolution using both maximum-likelihood estimated, and LogDet-transformed distances. Results varied among the different data partitions and methods of analysis. Nodes with strong character support, however, were consistently recovered, and a general pattern of evolution was observed. Monophyly of the Cestoidea (Amphilinidea + Gyrocotylidea + Eucestoda) and Eucestoda and the traditionally accepted positions of the Amphilinidea and Gyrocotylidea as sister lineages to the Eucestoda were supported. Within the Eucestoda, the Spathebothriidea was found to be the sister of all other eucestodes. The remaining orders generally formed a diphyletic pattern of evolution consisting of separate difossate and tetrafossate lineages. This pattern was not universally observed among the analyses, primarily because the trypanorhynch and diphyllidean taxa showed instability in their phylogenetic position. Additional relationships that showed high levels of nodal support included a sister relationship between the Pseudophyllidea and Haplobothriidea and a clade uniting the Cyclophyllidea, Nippotaeniidea, and Tetrabothriidea. The Tetraphyllidea, as currently defined, was found to be paraphyletic without the inclusion of the orders Proteocephalidea and, possibly, Lecanicephalidea. Ordinal status of a monophyletic Litobothriidea, currently classified within the Tetraphyllidea, was found to be supported from a phylogenetic perspective.

Evolution of the trypanorhynch tapeworms: Parasite phylogeny supports independent lineages of sharks and rays

Trypanorhynch tapeworms (Platyhelminthes: Cestoda) are among the most diverse and abundant groups of metazoan parasites of elasmobranchs and are a ubiquitous part of the marine food webs that include these apex predators. Here we present a comprehensive analysis of their phylogeny, character evolution and host associations based on 10years of sampling effort, including representatives of 12 of 15 and 44 of 66 currently recognized trypanorhynch families and genera, respectively. Using a combination of ssrDNA and lsrDNA (Domains 1-3) for 79 and 80 taxa, respectively, we maintain one-to-one correspondence between molecules and morphology by scoring 45 characters from the same specimens used for sequencing, and provide museum vouchers for this material. Host associations are examined through likelihood-based ancestral character state reconstructions (ACSRs) and by estimating dates of divergence using strict and relaxed molecular clock models in a Bayesian context. Maximum parsimony and Bayesian inference analyses of rDNA produced well-resolved and strongly supported trees in which the trypanorhynchs formed two primary lineages and were monophyletic with respect to the diphyllidean outgroup taxa. These lineages showed marked differences in their rates of divergence which in turn resulted in differing support and stability characteristics within the lineages. Mapping of morphological characters onto the tree resulting from combined analysis of rDNA showed most traits to be highly plastic, including some previously considered of key taxonomic importance such as underlying symmetries in tentacular armature. The resulting tree was found to be congruent with the most recent morphologically based superfamily designations in the order, providing support for four proposed superfamilies, but not for the Tentacularioidea and Eutetrarhynchoidea. ACSRs based on the combined analysis of rDNA estimated the original hosts of the two primary parasite lineages to be alternatively rajiform batoids and carcharhiniform sharks. This fundamental split provides independent support for rejecting the notion that rays are derived sharks, and thus supports the most recent molecular phylogenies of the Neoselachii. Beyond the basal split between shark- and ray-inhabiting lineages, no pattern was found to suggest that the trypanorhynchs have closely tracked the evolutionary histories of these host lineages, but instead, it appears that host-switching has been common and that the subsequent evolution of the parasites has been ecologically driven primarily through overlap in the niches of their shark and ray hosts. Using a relaxed molecular clock model calibrated by means of host fossil data, the ray-inhabiting lineage is estimated to have diversified around the Jurassic-Cretaceous boundary, whereas the shark-inhabiting lineage is estimated to have diversified later, in the Middle Cretaceous. Although the large error associated with the estimated divergence dates prevents robust conclusions from being drawn, the dates are nevertheless found to be consistent in a relative sense with the origins of their major hosts groups. The erection and definition of the suborders Trypanobatoida and Trypanoselachoida, for the major clades of trypanorhynchs parasitizing primarily rays and sharks, respectively, is proposed for the two primary lineages recovered here.

Proposal for a new tapeworm order, Rhinebothriidea

The polyphyletic nature of the tapeworm order Tetraphyllidea Carus, 1863 is addressed in part with the establishment of the new order Rhinebothriidea for a subset of the taxa formerly comprising the phyllobothriid subfamily Rhinebothriinae (Platyhelminthes: Eucestoda). Support for the order comes from Bayesian, maximum likelihood, and parsimony analyses of complete ssrDNA and partial (D1-D3) lsrDNA sequence data for 58 cestode species. These data consisted of novel data generated for 40 species in 15 genera of candidate rhinebothriines and the cathetocephalidean species Sanguilevator yearsleyi as well as comparable data taken from GenBank for an additional 18 cestode species in 17 genera. In total, the species analyzed consisted of two Cathetocephalidea, two Litobothriidea, two Lecanicephalidea, three Proteocephalidea, and 49 Tetraphyllidea. The tetraphyllideans consisted of three Onchobothriidae, three Serendipidae, and 43 Phyllobothriidae (one Thysanocephalinae, one Echeneibothriinae, five Phyllobothriinae, 35 candidate Rhinebothriinae and the poorly known Spongiobothrium). This work suggests that some elements of current membership in the group are in need of revision. For example, while inclusion of the echeneibothriine genus Echeneibothrium and the phyllobothriine genera Rhodobothrium and Anthocephalum, and also Spongiobothrium, in the Rhinebothriidea is supported, inclusion of Duplicibothrium and Caulobothrium in the new order is not. Histological sections and scanning electron microscopy of selected members of the study group suggest that the presence of bothridial stalks may serve as an effective morphological feature to characterise the order. The group is restricted to elasmobranchs, and appears to have a particular affinity for Myliobatiformes. The new order includes at least 13 genera. Intraordinal relationships were determined to be insufficiently stable to justify the formal reorganization of rhinebothriidean families at this time.

An investigation of the co-evolutionary relationships between onchobothriid tapeworms and their elasmobranch hosts.

There is general consensus that the living elasmobranchs comprise a monophyletic taxon. There is evidence that, among tetraphyllidean tapeworms, the approximately 201 hooked species (Onchobothriidae) may also comprise a monophyletic group. Determinations of host specificity are contingent upon correct specific identifications. Since 1960, over 200 new elasmobranch species and over 100 new onchobothriid species have been described. Some confidence can be placed in host and parasite identifications of recent studies, but specific identifications provided in older literature in many cases are suspect. There is some consensus among published works on the phylogenetic relationships among elasmobranchs. Phylogenetic relationships among onchobothriids remain largely unresolved. Elasmobranchs have been poorly sampled for onchobothriids; records exist for approximately 20% of the 911 species and approximately 44% of the 170 elasmobranch genera. Onchobothriids are remarkably host specific, exhibiting essentially oioxenous specificity for their definitive hosts. Multiple onchobothriid species commonly parasitise the same host species; in some cases these are congeners, in other cases these are members of two different onchobothriid genera. There is substantial incongruence between available host and parasite phylogenies. For example, Acanthobothrium is by far the most ubiquitous onchobothriid genus, parasitising almost all orders of elasmobranchs known to host onchobothriids, yet, there is no evidence of major clades of Acanthobothrium corresponding to postulated major subgroupings of elasmobranchs (e.g. Galea and Squalea or sharks and rays). Potamotrygonocestus appears to be among the most basal onchobothriid groups, yet it parasitises one of the most derived elasmobranch groups (the freshwater stingray genus Potamotrygon). It appears that congeners parasitising the same host species are not necessarily each others closest relatives. At this point the preliminary and limited available data suggest that, at least in this system, strict host specificity is not necessarily indicative of strict co-evolution. This study was extremely limited by the lack of available robust phylogenies for onchobothriids and elasmobranchs.

FOUR NEW SPECIES OF ACANTHOBOTHRIUM (CESTODA: TETRAPHYLLIDEA) FROM THE WHIPTAIL STINGRAY DASYATIS BREVIS IN THE GULF OF CALIFORNIA, MEXICO

Examination of the spiral intestines of 29 whiptail stingrays, Dasyatis brevis, from 6 sites in the Gulf of California, México in 1993 and 1996 resulted in the discovery of 4 new species of Acanthobothrium: Acanthobothrium bullardi, Acanthobothrium dasi, Acanthobothrium rajivi, and Acanthobothrium soberoni. This brings the total number of species of Acanthobothrium known from the eastern Pacific Ocean to 34. With 22 additional species reported from elasmobranchs from the western Atlantic Ocean, and over 100 species globally, the number of interspecific comparisons required to justify the designation of a new species is rather unwieldy. To facilitate these and future comparisons, and in the absence of a phylogenetic hypothesis for this genus, the 56 species of Acanthobothrium from these 2 geographic regions were categorized for 4 characters: total length (≤ or >15 mm), number of segments (≤ or >50 segments), number of testes (≤ or >80), and symmetry of poral and aporal ovarian lobes. These 56 taxa and their categories are presented in tabular form. Based on these characters, A. bullardi, A. dasi, and A. rajivi are category 2 species (they are relatively small, possess few segments, relatively few testes, and exhibit asymmetrical ovaries); A. soberoni is a category 6 species (it is a relatively longer worm with a larger number of segments, but with fewer testes and an asymmetrical ovary). All 4 species differ from 1 another and from species in other geographic regions in further subtleties of these 4 characters as well as hook size and relative length of hook prongs, cirrus sac size, genital pore position, number of columns of testes anterior to the cirrus sac, and number of postvaginal testes. Five specimens that appear to represent a new species of Acanthobothroides were also collected. This species is figured and some details of the morphology are described, but the material was considered to be insufficient to allow for formal description of this species at this time. This is the first record of either of these genera of onchobothriid tapeworms from the Gulf of California.

Morphological investigations into Floriceps minacanthus (Trypanorhyncha: Lacistorhynchidae) with analysis of the systematic utility of scolex microtriches

Specimens of Floriceps minacanthus Campbell & Beveridge, 1987 are reported from Carcharhinus melanopterus (Quoy & Gaimard, 1824) taken in the waters surrounding the island of Kirimati in the Republic of Kiribati in the Central Pacific Ocean. This represents a new host and a new locality record. The description of this species is expanded to include the presence of an ovary which is bilobed in cross section and a hermaphroditic duct. The surface features were examined by scanning electron microscopy. With the exception of the lateral margins of the bothridia and the apical region of the scolex, the scolex was found to be covered with palmate microtriches interspersed with filiform microtriches. The palmate microtriches varied in number of digitiform extensions depending on location. Elongate, bifid microtriches were present in the transition zone between the proximal and distal surfaces along the lateral margins of the bothridia. The apical region of the scolex was covered with filiform microtriches. The surface of the anterior margin of the strobila was covered with broad, flattened scale-like structures. The pattern of microthrix distribution was repeated for each of the bothridia of an individual, and this pattern was intraspecifically invariable. Comparison of our results with those from the seven other trypanorhynch species examined by previous authors with SEM indicates that there is much interspecific variability in both the morphology of microtriches present as well as their location on the scolex. This, coupled with the lack of intraspecific variability, confirms that these structures are systematically informative.

FIVE NEW SPECIES OF ACANTHOBOTHRIUM (TETRAPHYLLIDEA: ONCHOBOTHRIIDAE) FROM THE FRESHWATER STINGRAY HIMANTURA CHAOPHRAYA (BATOIDEA: DASYATIDAE) IN MALAYSIAN BORNEO

Five new species of Acanthobothrium (Tetraphyllidea: Onchobothriidae) are described from the spiral intestine of the Freshwater whipray, Himantura chaophraya, in the Kinabatangan River in Malaysian Borneo. Based on criteria set forth in a previous categorization scheme for species of Acanthobothrium, these consist of 3 Category 1 species, Acanthobothrium asnihae n. sp., Acanthobothrium saliki n. sp., and Acanthobothrium zainali n. sp.; a Category 8 species, Acanthobothrium etini n. sp.; and a Category 2 species, Acanthobothrium masnihae n. sp.. Acanthobothrium asnihae n. sp. differs from all Category 1 species in its possession of a horizontal band of weak musculature that divides the posterior loculus in half. Among Category 1 species, A. saliki n. sp. differs from all but Acanthobothrium southwelli in its possession of postovarian testes. It differs from A. southwelli in its possession of fewer testes and a greater number of proglottids. Acanthobothrium zainali n. sp. differs from the 25 other Category 1 species in a combination of overall size, muscular pad and hook shape, arrangement and number of testes, ovary configuration in cross section, position of ovarian isthmus, and genital pore position. Acanthobothrium etini n. sp. is distinguished from all 5 other Category 8 species in its lack of testes from the proglottid antiporal and postporal regions and in testis number. Acanthobothrium masnihae n. sp. differs from the 35 other Category 2 species in its possession of fewer testes, postporal testes, or a greater number of proglottids. A key to Acanthobothrium species parasitizing H. chayophraya is presented. This represents the first report of Acanthobothrium from freshwater stingrays belonging to a family other than the Potamotrygonidae.