Helminth communities of herons (Aves: Ardeidae) in southern Italy

The helminth communities of nine species of herons from southern Italy were studied and compared. Of 24 taxa found including seven digeneans, seven nematodes, six cestodes and four acanthocephalans, only five taxa were found in more than one heron species, and five of the 21 taxa that could be identified to species level were classified as 'heron specialists'. The total number of helminth species per heron species ranged from 1 in Botaurus stellaris to 9 in Ixobrychus minutus with infection levels generally low. A statistical comparison was carried out for herons with a sample size >. 5. At the infracommunity level, only I. minutus clearly differed from other heron species. Diversity parame…

Endoparasites of the blue whiting, Micromesistius poutassou from north-west Spain.

AbstractThe communities of metazoan endoparasites of blue whiting, Micromesistius poutassou, in waters of north-west Spain were analysed and a geographical comparison made with other localities. Four hundred blue whiting collected in July 1999 and September 2000 were examined for parasites, excluding the head and gills. Six species were found: Anisakis simplex s.l. (L3), A. physeteris (L3), Hysterothylacium aduncum (L2 and L3), Stephanostomum lophii (metacercaria), S. pristis (adult), and Prosorhynchus crucibulum (metacercaria). The latter is a new host record, and A. physeteris is reported for the first time in blue whiting from the north-east Atlantic. Host gender was not a significant pr…

Intestinal helminth fauna of striped dolphins (Stenella coeruleoalba) in the western Mediterranean: No effects of host body length, age and sex

The intestine of 52 (28 males and 24 females) striped dolphins (Stenella coeruleoalba) from western Mediterranean waters was examined for helminths. Animals were found stranded along the Spanish coasts during the morbillivirus epizootic in 1990. In accordance with observations from other pelagic tetrapods, including cetaceans from other geographical regions, the intestinal helminth community was depauperate. Four helminth species were found: three tetrabothriid cestodes, Tetrabothrius forsteri (prevalence: 96.2%; mean intensity [95% CI]: 47.4 [34.4–68.2]), Trigonocotyle globicephalae (9.6%; 1.8 [1.0–2.2]), and Strobilocephalus triangularis (23.1%; 5.3 [2.9–10.2]), and immature individuals o…

Host specificity ofOschmarinella rochebruniandBrachycladium atlanticum(Digenea: Brachycladiidae) in five cetacean species from western Mediterranean waters

AbstractWe investigated patterns of specificity of liver flukes (fam. Brachycladiidae) in a community of cetaceans from the western Mediterranean. The liver and pancreas of 103 striped dolphins,Stenella coeruleoalba, 18 Risso's dolphins,Grampus griseus, 14 bottlenose dolphins,Tursiops truncatus, 8 common dolphins,Delphinus delphis, and 5 long-finned pilot whales,Globicephala melas, were analysed for brachycladiid species. Two species were found:Oschmarinella rochebruniin striped dolphins (prevalence (P): 61.2%; mean intensity (MI) (95% CI): 34.2 (25.7–45.6)), andBrachycladium atlanticumin striped dolphins (P: 39.8%; MI: 7.1 (4.8–13.1)) and a single individual of common dolphin (P: 12.5%; in…

Long-Distance Travellers: Phylogeography of a Generalist Parasite, Pholeter gastrophilus, from Cetaceans

We studied the phylogeography and historical demography of the most generalist digenean from cetaceans, Pholeter gastrophilus, exploring the effects of isolation by distance, ecological barriers and hosts' dispersal ability on the population structure of this parasite. The ITS2 rDNA, and the mitochondrial COI and ND1 from 68 individual parasites were analysed. Worms were collected from seven oceanic and coastal cetacean species from the south western Atlantic (SWA), central eastern Atlantic, north eastern Atlantic (NEA), and Mediterranean Sea. Pholeter gastrophilus was considered a single lineage because reciprocal monophyly was not detected in the ML cladogram of all individuals, and seque…

Helminth communities of loggerhead turtles (Caretta caretta) from Central and Western Mediterranean Sea: the importance of host's ontogeny.

We investigated the factors providing structure to the helminth communities of 182 loggerhead sea turtles, Caretta caretta, collected in 6 localities from Central and Western Mediterranean. Fifteen helminth taxa (10 digeneans, 4 nematodes and 1 acanthocephalan) were identified, of which 12 were specialist to marine turtles; very low numbers of immature individuals of 3 species typical from fish or cetaceans were also found. These observations confirm the hypothesis that phylogenetic factors restrict community composition to helminth species specific to marine turtles. There were significant community dissimilarities between turtles from different localities, the overall pattern being compat…

Parasites as fish population tags and pseudoreplication problems: the case of striped red mullet Mullus surmuletus in the Spanish Mediterranean

AbstractStudies of parasites as fish population tags often apply a single round of sampling to identify potential stocks or predict harvest localities. However, the lack of replication generates pseudoreplication, implicitly assuming that infection levels are more similar between samples from the same locality than between samples from different localities. We evaluated this assumption in the case of the striped red mullet Mullus surmuletus in three localities of the Spanish Mediterranean separated by c. 300 km. Samples of 25 fish of similar size were collected in each locality in the summer and autumn of two consecutive years. Prevalence and abundance of three long-lived parasite taxa diff…

Independent host switching events by digenean parasites of cetaceans inferred from ribosomal DNA

Cetaceans harbour a unique fauna of digeneans whose origin and relationships have sparked considerable debate during recent decades. Disparity in the species reported indicates that they do not share close affinities, but their unusual morphology has made their taxonomic identities and phylogenetic positions uncertain. Here we use sequence data to investigate the phylogenetic relationships of the main species of flukes infecting cetaceans. We sequenced the 18S, 28S and internal transcribed spacer 2 rDNA of digenean species representing all known families reported from cetaceans: Braunina cordiformis (Brauninidae), Ogmogaster antarcticus (Notocotylidae), Pholeter gastrophilus (Heterophyidae)…

Intestinal helminth fauna of the South American sea lion Otaria flavescens and fur seal Arctocephalus australis from northern Patagonia, Argentina

AbstractWe report on the intestinal helminth fauna of 56 South American sea lions, Otaria flavescens, and 5 South American fur seals, Arctocephalus australis, from northern Patagonia, Argentina. A total of 97,325 helminth specimens were collected from sea lions. Gravid individuals were represented by 6 species of parasites: 1 digenean (Ascocotyle (Ascocotyle) patagoniensis), 1 cestode (Diphyllobothrium spp.), 3 nematodes (Uncinaria hamiltoni, Contracaecum ogmorhini s.s., Pseudoterranova cattani) and 1 acanthocephalan (Corynosoma australe). In addition, third-stage larvae of 2 nematodes (Contracaecum sp. and Anisakis sp. type I) and 3 juvenile acanthocephalans (Andracantha sp., Profilicollis…

Presence of genital spines in a male Corynosoma cetaceum Johnston and Best, 1942 (Acanthocephala).

We collected 83 females and 80 males of Corynosoma cetaceum from 2 common dolphins, Delphinus delphis, collected in northern Patagonia (Argentina). Worms were most similar to specimens collected in other South American localities. However, 1 male had 2 spines adjacent to the genital pore and isolated from the rest of body spines. This finding confirms the recent reassignment of C. cetaceum to Corynosoma. Absence of genital spines is suggested to be avoided as the sole criterion to exclude specimens from Corynosoma or Andracantha.

A new species of Ascocotyle (Trematoda: Heterophyidae) from the South American sea lion, Otaria flavescens, off Patagonia, Argentina.

We describe a new heterophyid species, Ascocotyle (Ascocotyle) patagoniensis n. sp., based on specimens collected from the intestines of the South American sea lion Otaria flavescens from Patagonia (Argentina). Ascocotyle (A.) patagoniensis n. sp. is distinguished from the other species of the subgenus by the number of circumoral spines, which are arranged in 2 rows of 18 to 23. The new species also differs from the other species in having a gonotyl without papillae. The specimens exhibited the widest seminal receptacle described for a species of this subgenus. Species of the subgenus Ascocotyle usually infect fish-eating birds or mammals in freshwater or brackish habitats. Ascocotyle (A.) …

Helminth communities of owls (strigiformes) indicate strong biological and ecological differences from birds of prey (accipitriformes and falconiformes) in southern Italy.

We compared the helminth communities of 5 owl species from Calabria (Italy) and evaluated the effect of phylogenetic and ecological factors on community structure. Two host taxonomic scales were considered, i.e., owl species, and owls vs. birds of prey. The latter scale was dealt with by comparing the data here obtained with that of birds of prey from the same locality and with those published previously on owls and birds of prey from Galicia (Spain). A total of 19 helminth taxa were found in owls from Calabria. Statistical comparison showed only marginal differences between scops owls (Otus scops) and little owls (Athene noctua) and tawny owls (Strix aluco). It would indicate that all owl …

Description, microhabitat selection and infection patterns of sealworm larvae (Pseudoterranova decipiens species complex, nematoda: ascaridoidea) in fishes from Patagonia, Argentina

Third-stage larvae of the Pseudoterranova decipiens species complex (also known as sealworms) have been reported in at least 40 marine fish species belonging to 21 families and 10 orders along the South American coast. Sealworms are a cause for concern because they can infect humans who consume raw or undercooked fish. However, despite their economic and zoonotic importance, morphological and molecular characterization of species of Pseudoterranova in South America is still scarce. Methods: A total of 542 individual fish from 20 species from the Patagonian coast of Argentina were examined for sealworms. The body cavity, the muscles, internal organs, and the mesenteries were examined to dete…

Helminth fauna of the invasive American red-eared sliderTrachemys scriptain eastern Spain: potential implications for the conservation of native terrapins

AbstractIn this study we report on the helminth fauna of the invasive American red-eared slider Trachemys scripta in five localities from eastern Spain where this species co-occurs with two native, endangered freshwater turtles, i.e. Emys orbicularis and Mauremys leprosa. In total, 46 individuals of T. scripta were analysed for parasites. Adult individuals of three helminth species were found: the monogenean Neopolystoma orbiculare, the digenean Telorchis solivagus and the nematode Serpinema microcephalus. Telorchis solivagus and S. microcephalus are trophically transmitted parasites of native turtles that probably infected T. scripta through shared infected prey. Neopolystoma orbiculare in…

Life begins when the sea lion is ashore: microhabitat use by a louse living on a diving mammal host

AbstractAmong Anoplura, the family Echinophthiriidae includes species that infest pinnipeds and otters. Previous evidence obtained from pinnipeds infested by echinophthiriids, specifically from seals, indicates that flippers are the preferred infestation sites, while lice from fur seals select areas in the pelage. We studied habitat selection of Antarctophthirus microchir on South American sea lion pups (Otaria flavescens Shaw, 1800) from Patagonia, Argentina, during the austral summer of 2009. We found a clear pattern of habitat selection: eggs are laid on the dorsal surface; nymphs 1 hatch there and then migrate to the belly, where they develop into adults and copulate; and then ovigerous…

Population dynamics of Antarctophthirus microchir (Anoplura: Echinophthiriidae) in pups from South American sea lion, Otaria flavescens, in Northern Patagonia.

We analysed population dynamics of the louse Antarctophthirus microchir in pups of the South American sea lion, Otaria flavescens, at the Punta León rookery (Argentina) over a period of 2 years. A total of 136 pups were aged and marked at the beginning of the lactation period ashore, then sampled for lice at different times within 30 days. Sampling was restricted to the chest and belly, two sites where lice were especially abundant. This concentration on ventral areas might protect lice from thermal stress in the austral summer. Infestation patterns in pups ≤3 days old suggested that the potential for transmission increased from first nymphs to adults. Population trends of each instar with …

Feeding ecology of the loggerhead turtle Caretta caretta in the western Mediterranean

We studied the feeding ecology of juvenile loggerhead turtles Caretta caretta in the western Mediterranean based on the contents of the digestive tract of 54 turtles (range of CCL: 34–69 cm) seized in Barcelona (Spain) in 1991. Turtles had been captured in fishing trawls, but specific information about dates and localities is not available. Despite this limitation, we obtained interesting evidences about the foraging strategies of loggerheads, with potentially important conservation implications. We report 33 new taxa in the diet. Results indicated that western Mediterranean loggerheads feed in an opportunistic way. Numerically, fish made up the most important prey group, followed by pelagi…

Epibiotic barnacles of sea turtles as indicators of habitat use and fishery interactions: An analysis of juvenile loggerhead sea turtles, Caretta caretta, in the western Mediterranean

Abstract Sea turtles exploit a variety of habitats during their lifetime and are thus exposed to a number of anthropogenic threats, including interaction with fisheries. Mitigating this impact requires determining patterns of habitat use by turtles, which largely relies on data from marked individuals. We investigated the use of epibiotic barnacles as a cost-effective method to ascertain habitat use by sea turtles, choosing the juvenile loggerhead sea turtles (Caretta caretta) in the western Mediterranean as a model. A total of 200 turtles collected in the central Spanish Mediterranean between 1990 and 2017 were examined for epibionts: 25% were accidentally captured by pelagic longliners, 1…

Intestinal helminth fauna of the shortfin mako Isurus oxyrinchus (Elasmobranchii: Lamnidae) in the northeast Atlantic Ocean.

Large oceanic sharks represent a suitable model to investigate the influence of a host's oceanic conditions on the structure of its helminth communities. In this study, we describe the intestinal helminth fauna, and investigate determinants of infracommunity structure, in 39 specimens of shortfin mako Isurus oxyrinchus collected in the NE Atlantic. Six cestode species were found in the spiral valve of makos: 3 are typical from lamnid sharks, namely, gravid specimens of Clistobothrium montaukensis, Gymnorhynchus isuri and Ceratobothrium xanthocephalum, and 3 are immature specimens of cestode species common to several elasmobranchs, namely, Dinobothrium septaria, Nybelinia lingualis, and Phyl…

Spatial distribution and aggregation ofXenobalanus globicipitison the flukes of striped dolphins,Stenella coeruleoalba: An indicator of host hydrodynamics?

The role of lantern fish (Myctophidae) in the life-cycle of cetacean parasites from western Mediterranean waters

Myctophids (lantern fish) and cephalopods play a key role in trophic webs from the continental slope and oceanic waters linking the zooplankton to top predators. Many cetaceans feed on both lantern fish and cephalopods, and such prey would thus be expected to bridge the trophic gap in the life-cycles of helminths infecting cetaceans. However, information on the life-cycles of most of these helminths is extremely scanty. We examined the parasite fauna of myctophids and cephalopods in two areas from the western Mediterranean where at least 21 helminth taxa from cetaceans have been reported and both cetacean diversity and abundance is high. A total of 1012 individuals of 8 lantern fish species…

How students perceive the university's mission in a Spanish university: Liberal versus entrepreneurial education?

AbstractThis study explores the extent to which undergraduate students perceive a dichotomy between a liberal vs. an entrepreneurial model of university. This research is important in that it compares the stated aims of university education, particularly within the Bologna Process, with students' expectations regarding liberal education and employability. A Likert-type questionnaire containing 25 statements reflecting liberal and entrepreneurial features of university education was answered by 488 students from 10 disciplines at the University of Valencia, Spain, just before the implantation of the Bologna curriculum. An exploratory factor analysis revealed that students clearly perceive th…

Anisakid larvae in the musculature of the Argentinean hake, Merluccius hubbsi

We report the infection levels of third-stage anisakid larva in the muscle of the Argentinean hake, Merluccius hubbsi, in relation to fish size and location in the musculature. The musculature of 42 hake was separated into hypaxial (ventral) and epiaxial (dorsal) parts and surveyed for nematode larvae. Two anisakid species were detected: Anisakis sp. (prevalence, 52.4%; mean +/- SD abundance, 1.2 +/- 1.7) and Pseudoterranova sp. (prevalence, 9.5%; mean +/- SD abundance, 0.2 +/- 0.7). Since the fish were gutted after capture, the occurrence of anisakids in the flesh indicates that the worms had migrated into the muscle before capture. The number of Anisakis sp. in muscle was not correlated w…

A new brachycladiid species (Digenea) from Gervais’ beaked whale Mesoplodon europaeus in north-western Atlantic waters

AbstractA new species of the digenean family Brachycladiidae Odhner, 1905 is described from the bile ducts of a Gervais’ beaked whale Mesoplodon europaeus Gervais (Ziphiidae) stranded on the North Atlantic coast of Florida. These parasites were assigned to Brachycladium Looss, 1899 and differed from other species of the genus in the relative size of the oral and ventral suckers, the form and size of the eggs and their extremely small body size. A canonical discriminant analysis was used to examine differences between these specimens and the smallest available individuals of B. atlanticum (Abril, Balbuena and Raga, 1991) Gibson, 2005, considered the morphologically closest species. The overa…

Parasite assemblages in the Western whip snakeHierophis viridiflavus carbonarius(Colubridae) from southern Italy

AbstractParasite assemblages of the Western whip snakeHierophis viridiflavus carbonariuswere investigated from the Calabria region in southern Italy. A total of 14 parasite taxa including 6 nematodes, 3 acanthocephalans, 2 cestodes, 2 digeneans and a single pentastomid was identified. Within the study area,H. v. carbonariusserves as the final host for seven species of helminths, of which only four (Hexametra quadricornis,Kalicephalus viperae,Paracapillaria sonsinoiandRenifer aniarum) can be considered as snake specialists, while one (Oswaldocruzia filiformis) is shared with other reptiles and amphibians, and two (Paradistomum mutabileandRhabdias fuscovenosa) with lizards. A large proportion…

Changes in epizoic crustacean infestations during cetacean die-offs: the mass mortality of Mediterranean striped dolphins Stenella coeruleoalba revisited.

In the summer and autumn of 1990, a cetacean morbillivirus caused a massive epizootic mortality of striped dolphins Stenella coeruleoalba in the western Mediterranean. Previous circum- stantial evidence suggested that the disease could also have increased host susceptibility to infesta- tions with epizoic crustaceans. In this study we provide strong evidence supporting this hypothesis. We examined striped dolphins stranded along the Mediterranean central coast of Spain from 1981 to 2004 (n = 136), and recorded data on prevalence, intensity of infestation, size and reproductive status of 2 sessile crustacean species specific to cetaceans, the phoront cirriped Xenobalanus globicipitis and the…

Transmission and Predictors of Burden of Lungworms of the Striped Dolphin (Stenella coeruleoalba) in the Western Mediterranean

Pseudaliid lungworms infect the lungs and sinuses of cetaceans. Information on the life cycle and epidemiology of pseudaliids is very scarce and mostly concerns species that infect coastal or inshore cetaceans. Available evidence indicates that some pseudaliids are vertically transmitted to the host, whereas others are acquired via infected prey. We documented pseudaliid infections in an oceanic cetacean, the striped dolphin (Stenella coeruleoalba) in the western Mediterranean, and investigated the possibilities of vertical vs. horizontal transmission and the potential influence of host body size, sex, and season on infection levels. We found two species of lungworm in 87 dolphins that stra…

Phylogenetic relationship among genera of Polymorphidae (Acanthocephala), inferred from nuclear and mitochondrial gene sequences.

Abstract Acanthocephalans of the family Polymorphidae Meyer, 1931 are obligate endoparasites with complex life cycles. These worms use vertebrates (marine mammals, fish-eating birds and waterfowl) as definitive hosts and invertebrates (amphipods, decapods and euphausiids) as intermediate hosts to complete their life cycle. Polymorphidae has a wordwide distribution, containing 12 genera, with approximately 127 species. The family is diagnosed by having a spinose trunk, bulbose proboscis, double-walled proboscis receptacle, and usually four to eight tubular cement glands. To conduct a phylogenetic analysis, in the current study sequences of the small (18S) and large-subunit (28S) ribosomal RN…

Molecular phylogeny of the families Campulidae and Nasitrematidae (Trematoda) based on mtDNA sequence comparison.

Abstract Historically, the systematic arrangement of the genera within the family Campulidae, and its relationship with its allied family Nasitrematidae have been rather confused, particularly because only adult morphology has been available to classical taxonomic analysis. In this paper we provide a partial phylogeny of the genera of these families based on mtDNA from five campulid species: Campula oblonga, Zalophotrema atlanticum, Hadwenius tursionis, Oschmarinella rochebruni and ; and one nasitrematid, Nasitremaglobicephalae . Fasciola hepatica and Dicrocoelium dendriticum were used as outgroups. Maximum parsimony and neighbour-joining methods were applied. Both methods produced similar …

Morphology, performance and attachment function in Corynosoma spp. (Acanthocephala)

Background: Functional inference on the attachment of acanthocephalans has generally been drawn directly from morphology. However, performance of structures is often non-intuitive and context-dependent, thus performance analysis should be included whenever possible to improve functional interpretation. In acanthocephalans, performance analysis of attachment is available only for Acanthocephalus ranae, a species that solely relies on the proboscis to attach. Here we compare body morphology and muscle arrangement in 13 species of Corynosoma, which use their spiny body as a fundamental holdfast. A basic performance analysis using live cystacanths of two representative species is also provided.…

Recent increase of ulcerative lesions caused by Anisakis spp. in cetaceans from the north-east Atlantic

Abstract Species of Anisakis typically infect the stomach of cetaceans worldwide, often causing ulcerative lesions that may compromise the host's health. These nematodes also cause anisakiasis or allergic reactions in humans. To assess the risks of this emerging zoonosis, data on long-term changes in Anisakis infections in cetaceans are necessary. Here, we compare the prevalence and severity of ulcerative lesions caused by Anisakis spp. in five cetacean species stranded along the north-west Spanish coast in 2017–2018 with published data from 1991–1996. Open ulcers were found in 32/43 short-beaked common dolphins, Delphinus delphis; 3/5 striped dolphins, Stenella coeruleoalba; 1/7 bottlenose…

Assessing host-parasite specificity through coprological analysis: a case study with species of Corynosoma (Acanthocephala: Polymorphidae) from marine mammals.

In this paper we report an investigation of the utility of coprological analysis as an alternative technique to study parasite specificity whenever host sampling is problematic; acanthocephalans from marine mammals were used as a model. A total of 252 scats from the South American sea lion, Otaria flavescens, and rectal faeces from 43 franciscanas, Pontoporia blainvillei, from Buenos Aires Province, were examined for acanthocephalans. Specimens of two species, i.e. Corynosoma australe and C. cetaceum, were collected from both host species. In sea lions, 78 out of 145 (37.9%) females of C. australe were gravid and the sex ratio was strongly female-biased. However, none of the 168 females of …

Parasites as indicators of social structure and stock identity of marine mammals

Abstract Introduction: the use of parasites as biological indicators of marine mammals has not yet received all the necessary attention. The aim of this review is to show the value of parasite data in studies of stock identity and social structure. Methods: the application of the technique to marine mammals is hampered by the lack of control over sampling conditions and the paucity of information about the biology of their parasites. Relevant criteria for, suitable parasite tags are discussed. Case studies: previous work on marine mammals is presented to illustrate the usefulness and limitations of parasite data. A study of pilot whales revealed that differences in helminth infections betwe…

To Swim or Not to Swim: Potential Transmission of Balaenophilus manatorum (Copepoda: Harpacticoida) in Marine Turtles

Species of Balaenophilus are the only harpacticoid copepods that exhibit a widespread, obligate association with vertebrates, i.e., B. unisetus with whales and B. manatorum with marine turtles and manatees. In the western Mediterranean, juveniles of the loggerhead sea turtle, Caretta caretta are the only available hosts for B. manatorum, which has been found occurring at high prevalence (>80%) on them. A key question is how these epibionts are transmitted from host to host. We investigated this issue based on experiments with live specimens of B. manatorum that were cultured with turtle skin. Specimens were obtained from head-started hatchlings of C. caretta from the western Mediterranean. …

Constructional morphology and mode of attachment of the trunk ofCorynosoma cetaceum (Acanthocephala: Polymorphidae)

Dead specimens of Corynosoma cetaceum were used to describe the trunk musculature of this species and to infer the use of the trunk as a secondary holdfast. Inferences were based on trunk muscle arrangement, changes in trunk shape, size and distribution of spines, and geometry of tegument thickness. The foretrunk of C. cetaceum is swollen and forms a spiny disk that is bent ventrally. The disk is flattened by several groups of muscles not described previously, which seem able to finely adjust the disk surface over the substratum. Disk attachment appears to be accomplished by two dorsal neck retractor muscles specialized in pulling the anchored proboscis into the foretrunk. This mechanism ha…

Ontogenetic Habitat Selection by Hadwenius pontoporiae (Digenea: Campulidae) in the Intestine of Franciscanas (Cetacea)

The linear habitat selection of 4 sequential maturity stages (1, 2, 3, and 4) of the trematode Hadwenius pontoporiae in the intestines of 26 South American dolphins Pontoporia blanvillei was investigated. The franciscana is a suitable host for H. pontoporiae because all 26 hosts were infected, the infrapopulations being composed mostly of gravid (stage 4) worms. Most trematodes were found in the first third of the intestine. The niches of the maturity stages decreased from stage 1 to 4. Gravid worms favored the most anterior part of the duodenum, whereas stages 1, 2, and 3 occurred more posteriorly in every host. The distributions of the maturity stages showed a narrow site fidelity and wer…

Corynosoma cetaceumin the Stomach of Franciscanas,Pontoporia blainvillei(Cetacea): an Exceptional Case of Habitat Selection by an Acanthocephalan

Adult acanthocephalans are typically found in the intestine of vertebrates, where they can readily absorb nutrients. However, Corynosoma cetaceum has been frequently reported in the stomach of cetaceans from the Southern Hemisphere. The ecological significance of this habitat was investigated by examining data on number, sex ratio, maturity status, biomass, and fecundity of C. cetaceum in different parts of the digestive tract of 44 franciscanas Pontoporia blainvillei. Individual C. cetaceum occurred in the pyloric stomach (PS) and, to lesser degrees, in the duodenal ampulla (DA) and the main stomach (MS). Females outnumbered males in all chambers, although the sex ratio was closer to 1:1 i…

Sex-specific impact of inbreeding on pathogen load in the striped dolphin.

The impact of inbreeding on fitness has been widely studied and provides consequential inference about adaptive potential and the impact on survival for reduced and fragmented natural populations. Correlations between heterozygosity and fitness are common in the literature, but they rarely inform about the likely mechanisms. Here, we investigate a pathology with a clear impact on health in striped dolphin hosts (a nematode infection that compromises lung function). Dolphins varied with respect to their parasite burden of this highly pathogenic lung nematode (Skrjabinalius guevarai). Genetic diversity revealed by high-resolution restriction-associated DNA (43 018 RADseq single nucleotide pol…

Living in a harsh habitat: epidemiology of the whale louse, Syncyamus aequus (Cyamidae), infecting striped dolphins in the Western Mediterranean

Whale lice (Cyamidae) are ectoparasitic amphipods exclusive to cetaceans. Data on their epidemiology usually come from species infecting baleen whales, which are large, slow-moving hosts. In this study, we provide data on infection parameters, population structure and microhabitat selection in a whale louse exclusive to delphinids, Syncyamus aequus, with the aim of comparing them with those from cyamid species from large whales. A total of 176 striped dolphins Stenella coeruleoalba stranded along the Mediterranean coast of Spain during 1980–2016 were examined. The prevalence of S. aequus was 27.3% (95% CI: 20.9–34.3), and populations were sparse, with a mean number of whale lice per infecte…

¿Por qué conservamos la biodiversidad? Un breve recorrido sociológico, psicológico y moral

espanolEn este trabajo se muestran, de manera muy sucinta, algunos conceptos fundamentales sobre los valores que subyacen en la toma de las decisiones sobre la conservacion de la biodiversidad, asi como las bases psicologicas de la deliberacion en este contexto. Para ello, se describe, en primer lugar, un modelo sociologico clasico que ayuda a predecir la asignacion de recursos para la conservacion, basado en la construccion social y el poder “politico” oculto de las diferentes especies. En segundo lugar, se vincula dicho modelo con la taxonomia de valores subyacentes (no necesariamente excluyentes). Finalmente, se discute la base psicologica de la deliberacion valorativa a partir de la teo…

Consumption of pelagic tunicates by cetaceans calves in the Mediterranean Sea

Gelatinous zooplankton, including jellyfish, ctenophores and pelagic tunicates, constitutes fragile marine animals that live in the water column, and represent an important resource for marine food webs through their seasonal pulses. Although there is scarce evidence on the occurrence of gelatinous zooplankton in stomach contents of apex, endothermic predators such as cetaceans, the ecological significance of such observations requires consideration. In this study, we report on the occurrence of pelagic tunicates in the stomach of three individual calves of two cetacean species from the western Mediterranean, and collate all previous reports of gelatinous zooplankton in cetacean diets. We t…

Tetraphyllidean plerocercoids from Western Mediterranean cetaceans and other marine mammals around the world: a comprehensive morphological analysis.

Tetraphyllidean plerocercoids have occasionally been reported in marine mammals, but they have rarely been described in detail, and the ecological significance of these infections is unclear. We described plerocercoids collected from the mucosa of the terminal colon and rectum, the anal crypts, and the hepatopancreatic ducts of 7 striped dolphins Stenella coeruleoalba, 1 Cuvier's beaked whale Ziphius cavirostris, and 3 Risso's dolphins Grampus griseus from the Spanish Mediterranean. We also examined undescribed plerocercoids from 3 cetacean species from the Atlantic and the Pacific. All plerocercoids had a lanceolate body, and a scolex with an apical sucker and 4 sessile monolocular bothrid…

Gastrointestinal Helminths of Loggerhead Turtles (Caretta caretta) from the Western Mediterranean: Constraints on Community Structure

Richness and composition of gastrointestinal helminth communities of 54 loggerhead turtles, Caretta caretta, from the western Mediterranean were interpreted from patterns of helminth exchange at 2 host taxonomic scales: exchange between marine turtles and other marine hosts and exchange within turtles. We predicted exchange of the former to be unimportant ecologically and evolutionarily because of the host phylogenetic distance. The absence of records of successful exchange at this host taxonomic scale confirmed that host physiological barriers seem to prevent contemporary parasite transfer between marine turtles and other sympatric hosts. Marine turtles also seem to exhibit an evolutionary…

Life-history trade-offs in a generalist digenean from cetaceans: the role of host specificity and environmental factors

Background Adults and larvae of generalist parasites are exposed to diverse hosts and local environmental conditions throughout their life cycles, thus local adaptation is expected to occur through phenotypic plasticity and/or natural selection. We investigated how the combined effect of cryptic host specificity and local selective pressures could shape reproductive traits of a putative generalist parasite in the oceanic realm. Methods The LSU rDNA, ITS2 and the mt-COI of individuals of the digenean Pholeter gastrophilus (Kossack, 1910) Odhner, 1914 (Heterophyidae Leiper, 1909) from oceanic striped dolphins, Stenella coeruleoalba Meyen, and coastal bottlenose dolphins, Tursiops truncatus Mo…

Acanthocephalans from Marine Fishes from Patagonia, Argentina

In this study, 542 individual fish from 20 species from the Patagonian continental shelf of Argentina were examined for acanthocephalans. A total of 1,547 acanthocephalans belonging to 5 species were collected from 18 species of fish. Adult forms were represented by 2 species: Aspersentis johni (Baylis, 1929) (Heteracanthocephalidae) from longtail southern cod, Patagonotothen ramsayi (Regan) (new host record), and Breizacanthus aznari Herna´ndez-Orts, Alama-Bermejo, Crespo, Garcı´a, Raga and Montero, 2012 (Arhythmacanthidae) from raneya, Raneya brasiliensis (Kaup). Immature worms of B. aznari were also collected from the intestine of pink cusk-eel, Genypterus blacodes (Forster) (new host re…

Emergence of the epibiotic barnacle Xenobalanus globicipitis in Guiana dolphins after a morbillivirus outbreak in Sepetiba Bay, Brazil

Abstract We report on the emergence of the epibiotic barnacle Xenobalanus globicipitis in Guiana dolphins (Sotalia guianensis) from Sepetiba Bay, Rio de Janeiro, Brazil, in September 2018, six months after a lethal morbillivirus epidemic. Small boat surveys were conducted at the entrance and in the interior of the bay from January 2018 to February 2021. Dolphins were photo-identified and classified according to overall spatial use of the Bay. We examined the potential influence of the period and habitat on the prevalence of X. globicipitis. The overall prevalence of the barnacle was 38.7% in 214 dolphins. X. globicipitis was not observed in 99 individually identified dolphins from January t…

Insight into the role of cetaceans in the life cycle of the tetraphyllideans (Platyhelminthes: Cestoda)

Abstract Four types of tetraphyllidean larvae infect cetaceans worldwide: two plerocercoids differing in size, ‘small’ (SP) and ‘large’ (LP), and two merocercoids referred to as Phyllobothrium delphini and Monorygma grimaldii . The latter merocercoid larvae parasitize marine mammals exclusively and exhibit a specialised cystic structure. Adult stages are unknown for any of the larvae and thus the role of cetaceans in the life cycle of these species has been a long-standing problem. The SP and LP forms are thought to be earlier stages of P. delphini and M. grimaldii that are presumed to infect large pelagic sharks that feed on cetaceans. A molecular analysis of the D2 variable region of the …

Girdles as the main infection site for Paradeontacylix kampachi (Sanguinicolidae) in the greater amberjack Seriola dumerili.

In this study, we provide new information about the habitats selected by the blood fluke Paradeontacylix kampachi in the greater amberjack Seriola dumerili based on an exhaustive anatomical examination. From May to October 1998, 21 fish of the 0+ age class were collected from tanks of the Spanish Institute of Oceanography in Puerto de Mazarrón, Spain, for parasitological analysis. Individuals of P. kampachi were found in 17 of the 21 fish analysed (mean intensity +/- SD: 13.6 +/- 16.6; median: 6). Worms occurred in the girdles, cephalic kidney, sinus venosus, kidney and branchial arteries. A Friedman test with a post-hoc contrast revealed a significantly higher number of worms in the girdle…

First evidence of avian pox-like outbreaks in Eurasian coots, Fulica atra, from Europe: A widespread phenomenon?

Avian pox is a disease caused by Avipoxvirus spp. that is known to produce self-limiting, proliferative epithelial lesions in over 275 bird species from 70 families. Although generally benign, this emerging disease can sometimes provoke significant outbreaks of mortality. In rallids, there is a single serological record of Avipoxvirus. In this study, we report on field evidence for two avian pox-like outbreaks in Eurasian coots, Fulica atra. In Utterslev Mose, Denmark, 17 birds were found in the winter and early spring of 2006 with putative signs of pox-like lesions on the frontal shield and bill. Similar lesions were detected in a population of c. 30 Eurasian coots from a small wetland in…

Two decades of monitoring in marine debris ingestion in loggerhead sea turtle, Caretta caretta, from the western Mediterranean

Abstract Anthropogenic marine debris is one of the major worldwide threats to marine ecosystems. The EU Marine Strategy Framework Directive (MSFD) has established a protocol for data collection on marine debris from the gut contents of the loggerhead sea turtle (Caretta caretta), and for determining assessment values of plastics for Good Environmental Status (GES). GES values are calculated as percent turtles having more than average plastic weight per turtle. In the present study, we quantify marine debris ingestion in 155 loggerhead sea turtles collected in the period 1995–2016 in waters of western Mediterranean (North-east Spain). The study aims (1) to update and standardize debris inges…

Epidemiological pattern of tattoo skin disease: a potential general health indicator for cetaceans

The presence of tattoo skin disease (TSD) was examined in 1392 free-ranging and dead odontocetes comprising 17 species from the Americas, Europe, South Africa, New Zealand and Greenland. We investigated whether TSD prevalence varied with sex, age and health status. TSD was encountered in cetaceans from the Pacific and Atlantic Oceans as well as in those from the North, Mediterranean and Tasman Seas. No clear patterns related to geography and host phylogeny were detected, except that prevalence of TSD in juveniles and, in 2 species (dusky dolphin Lagenorhynchus obscurus and Burmeister’s porpoise Phocoena spinipinnis), in adults was remarkably high in samples from Peru. Environmental factors …

Aggressive interactions between juvenile swordfishes and blue sharks in the Western Mediterranean: a widespread phenomenon?

There are numerous reports of billfishes spearing objects, marine organisms, and even humans. Whether or not this behaviour is intentional and, if so, what is its functional meaning, are open questions. In 2016, an adult blue shark (Prionace glauca) was found to be killed by a juvenile swordfish (Xiphias gladius) in the western Mediterranean. Here we report on three more recent cases involving both species in the same area. In February 2017, an adult male blue shark was found stranded in Garrucha (Spain) with a fragment of a juvenile swordfish’s rostrum (18cm long x 2cm wide at proximal end) inserted in its cranium. In March 2017, an adult pregnant female blue shark was stranded alive on th…

Morphological and molecular characterization of tetraphyllidean merocercoids (Platyhelminthes: Cestoda) of striped dolphins (Stenella coeruleoalba) from the Western Mediterranean.

Two types of tetraphyllidean merocercoids, Phyllobothrium delphini and Monorygma grimaldii, are well known from most cetaceans world-wide. The role of cetaceans in the life-cycle of these merocercoids is unclear because their specific identity is as yet unknown. The problem is compounded by poor descriptions of both merocercoids. We used light and scanning electron microscopy, and histological techniques to provide a thorough description of merocercoids collected from 11 striped dolphins, Stenella coeruleoalba, from the Spanish Mediterranean. We also described, for the first time, specimens of P. delphini with immature proglottides. Our merocercoids were morphologically similar to those des…

Living on the Edge: Settlement Patterns by the Symbiotic Barnacle Xenobalanus globicipitis on Small Cetaceans

The highly specialized coronulid barnacle Xenobalanus globicipitis attaches exclusively on cetaceans worldwide, but little is known about the factors that drive the microhabitat patterns on its hosts. We investigate this issue based on data on occurrence, abundance, distribution, orientation, and size of X. globicipitis collected from 242 striped dolphins (Stenella coeruleoalba) that were stranded along the Mediterranean coast of Spain. Barnacles exclusively infested the fins, particularly along the trailing edge. Occurrence, abundance, and density of X. globicipitis were significantly higher, and barnacles were significantly larger, on the caudal fin than on the flippers and dorsal fin. Ba…

Parasitic outbreak of the copepod Balaenophilus manatorum in neonate loggerhead sea turtles (Caretta caretta) from a head-starting program

Abstract Background Diseases associated to external parasitosis are scarcely reported in sea turtles. During the last decades several organism have been documented as a part of normal epibiont community connected to sea turtles. The copepod Balaenophilus manatorum has been cited as a part of epibiont fauna with some concern about its parasitic capacity. This study serves three purposes, i.e. (i) it sheds light on the type of life style that B. manatorum has developed with its hosts, particularly turtles; (ii) it makes a cautionary note of the potential health risks associated with B. manatorum in sea turtles under captivity conditions and in the wild, and (iii) it provides data on effective…

Evolutionary relationships between digeneans of the family Brachycladiidae Odhner, 1905 and their marine mammal hosts: A cophylogenetic study.

Cophylogenetic studies examine the congruence between host and parasite phylogenies. There are few studies that quantify the relative contribution of coevolutionary events, i.e. duplication, loss, failure-to-diverge, host-switching and spreading in trophically-transmitted parasites at the marine realm. We addressed this issue in the Brachycladiidae, a cosmopolitan digenean family specific to marine mammals. We used, for the first time, distance-based and event-based methods to explicitly test the coevolutionary events that have shaped the current brachycladiid-marine mammal associations. Parasite phylogeny was constructed using mtDNA ND3 sequences of nine brachycladiid species, and host phy…

Anisakis infections in lantern fish (Myctophidae) from the Arabian Sea: A dual role for lantern fish in the life cycle of Anisakis brevispiculata?

Abstract Data on geographic distribution and life cycles of parasitic nematodes of the genus Anisakis in tropical oceanic regions are extremely scarce. Myctophids (lantern fishes) are a key component of mesopelagic trophic webs and could play a significant role in the transmission of Anisakis spp. in these regions. We examined Anisakis infections in a sample of 160 myctophids collected in three localities from the Indian Exclusive Economic Zone (Arabian Sea) in 2015–16. Due to the poor condition of fish, a combination of morphological data and molecular barcoding was used to identify myctophids. Of the 104 specimens that could be identified at least to genus level, 75 (72.1%) were assigned …

POPULATION STRUCTURE OF ANISAKIS SIMPLEX (NEMATODA) IN HARBOR PORPOISES PHOCOENA PHOCOENA OFF DENMARK

The population structure and habitat selection of Anisakis simplex in 35 harbor porpoises off Denmark are described. The nematodes were collected from the stomach and duodenal ampulla and were categorized as third-stage larvae, fourth-stage larvae, subadults, and adults. The porpoises harbored 8,043 specimens of A. simplex. The proportion of adults and subadults increased with infrapopulation size. The number of development stages across infrapopulations covaried significantly (Kendall's test of concordance). Concordance was higher in hosts with the highest intensities than in those with low and medium intensities. All stages occurred mainly in the forestomach, but this trend was stronger f…

From mammals back to birds: Host-switch of the acanthocephalan Corynosoma australe from pinnipeds to the Magellanic penguin Spheniscus magellanicus.

Trophically-transmitted parasites are regularly exposed to potential new hosts through food web interactions. Successful colonization, or switching, to novel hosts, occur readily when ?donor? and ?target? hosts are phylogenetically related, whereas switching between distantly related hosts is rare and may result from stochastic factors (i.e. rare favourable mutations). This study investigates a host-switching event between a marine acanthocephalan specific to pinnipeds that is apparently able to reproduce in Magellanic penguins Spheniscus magellanicus from Brazil. Detailed analysis of morphological and morphometrical data from acanthocephalans from penguins indicates that they belong to Cor…

A new cystidicolid nematode from Mullus surmuletus (Perciformes: Mullidae) from the western Mediterranean.

Ascarophis valentina n. sp. is described from Mullus surmuletus off the Valencian coast of Spain on the basis of both light and scanning electron microscopy. It can be distinguished from the other members of the genus by the length of the left (long) spicule of the males and by egg morphology. An updated grouping of the species of Ascarophis considered valid is provided with respect to these characters. The new species resembles Ascarophis capelanus, belonging to the group of species possessing eggs with a single polar knob with filaments, but is distinguished by the size of the body, the length of the esophagus (especially in relation to body length), the position of the vulva, and the siz…

Taxonomic status and epidemiology of the mesoparasitic copepod Pennella balaenoptera in cetaceans from the western Mediterranean.

Pennella balaenoptera is a mesoparasitic copepod that has been reported in at least 17 cetacean species. Subtle morphological differences in the first antennae of adult females have been used to discriminate this species from P. filosa, a species infecting fishes. Other morphological traits are unreliable because of their high plasticity, and no molecular data are available to confirm the taxonomic status of P. balaenoptera as an independent species. We found no consistent morphological differences of the first antennae between P. balaenoptera and P. filosa collected from cetaceans and fish in the western Mediterranean. Molecular data on the mitochondrial cytochrome oxidase subunit I failed…

The Function of Stones in Nest Building: The Case of Black Wheatear (Oenanthe Leucura) revisited

Black Wheatears ( Oenanthe leucura) transport to breeding territories, including the nest, a large number of stones. Currently, there is negative evidence that stones serve for nest support, thermoregulation, weather protection or anti-predation, but in males, this behaviour appears to represent a form of post-mating display. This ‘sexual selection’ hypothesis, however, does not explain why females also carry stones, and why many stones are associated with the nest. We examined stone distribution and morphology in 277 abandoned nests of Black Wheatears from an arid area of southeastern Spain. Nests were built on the ground (14.4%), or in cracks (7.2%), cavities (54.6%) and ledges (23.8%) a…

Patterns of trunk spine growth in two congeneric species of acanthocephalan: investment in attachment may differ between sexes and species.

SUMMARYAcanthocephalans have evolved a hooked proboscis and some taxa have trunk spines to attach to their definitive hosts. These structures are generated before being used, thus a key question is how investment in attachment could optimally be allocated through the ontogeny. The number and arrangement of hooks and spines are never modified in the definitive host, but it is unclear whether these structures grow during adult development. A comparison of the size of trunk spines between cystacanths and adults of Corynosoma cetaceum and C. australe indicated that spines grow in both species, but only in females, which also had significantly larger spines than males. This sexual dimorphism did…

Nyctiphanes couchii as intermediate host for Rhadinorhynchus sp. (Acanthocephala, Echinorhynchidae) from NW Iberian Peninsula waters

12 páginas, 3 figuras, 3 tablas

Epibiont communities of loggerhead marine turtles (Caretta caretta) in the western Mediterranean: influence of geographic and ecological factors

This study reports for the first time on the whole epibiont fauna of loggerhead marine turtles,Caretta caretta, in the western Mediterranean, analysing the factors that account for the predictability and composition of the assemblage. A total of 104 loggerhead turtles stranded along the coasts of eastern Spain during 1995–2006 were surveyed for epibionts. A total of 39 epibiont taxa were identified, three of them being new records for loggerhead turtles:Bittiumsp.,Idotea metallicaandJassasp. The assemblage was composed of a group of 27 facultative taxa that use turtles as any inanimate buoyant substrate, and 12 taxa that have developed more specific associations to marine turtles, including…

Deadly impalement of a blue shark Prionace glauca by a swordfish Xiphias gladius

In September, 2016, an adult female blue shark (Prionace glauca) 247 cm long stranded alive on the coast of Valencia (Spain, Western Mediterranean) but died shortly afterwards. The necropsy revealed ongoing pregnancy, with 65 embryos in early stage of development, and a healthy condition with no signs of starvation. Two fishing hooks surrounded by scarred tissue were detected in the mandible, indicating past interaction with fisheries. In addition, a fragment of the tip of a swordfish (Xiphias gladius) rostrum (length: 18 cm long, width: 0.5 cm (distal) and 3 cm (proximal)) was removed from the animal. The fragment had pierced the head producing an incision of 3.5 cm close to the left eye, …

Running against time: conservation of the remaining hawksbill turtle (Eretmochelys imbricata) nesting population in the Dominican Republic

Saona Island hosts the last hawksbill turtle (Eretmochelys imbricata) nesting population in the Dominican Republic, which has experienced a severe decline in the last decades, mostly due to illegal egg take. Here we present the results of an artificial incubation programme started in 2007 to protect the clutches from human predation. A preliminary survey in 2006 showed that about 60% of clutches laid were taken by humans. Over the study period (2007–2010) we recorded 400 clutches, of which 38.2% were predated by humans, 40.7% were artificially incubated and 21% were incubated in situ. Overall, the artificial incubation programme allowed the release of 12,340 hatchlings. No differences were …

Feeding on clean food? Potential effects of electric organ discharges by Torpedo spp. (Torpediniformes: Torpedinidae) on their trophically transmitted parasites.

Members of the Torpedinidae (torpedoes) and Hypnidae (coffin ray) use electric organ discharges (EOD) to stun or kill their prey before consumption. We investigated whether EOD could also negatively affect the helminth larvae infecting these preys through a surrogate model: we applied electric discharges to individuals of blue whiting, Micromesistius poutassou, that harbored live larvae of Anisakis. Larval mortality throughout a 6-h period was significantly higher in the treatment group, suggesting that EODs could significantly hamper helminth recruitment. We then tested whether torpedinids and hypnids ("strong-EOD" families) harbored species-poor helminth (cestode) assemblages compared wit…

Reproductive inequalities in the acanthocephalan Corynosoma cetaceum: looking beyond 'crowding' effects.

Background: At present, much research effort has been devoted to investigate overall (average) responses of parasite populations to specific factors, e.g., density-dependence in fecundity or mortality. However, studies on parasite populations usually pay little attention to individual variation (inequality) in reproductive success. A previous study on the acanthocephalan Corynosoma cetaceum in franciscana dolphins, Pontoporia blainvillei, revealed no overall intensity-dependent, or microhabitat effects, on mass and fecundity of worms. In this study, we investigated whether the same factors could influence mass inequalities for this species of acanthocephalan.Methods: A total of 10,138 speci…

Microtriches of tetraphyllidean metacestodes from Western Mediterranean striped dolphins (Stenella coeruleoalba)

The tegumental structures of two types of tetraphyllidean plerocercoids and two types of merocercoids (Phyllobothrium delphini and Monorygma grimaldii) from Mediterranean striped dolphins, Stenella coeruleoalba, are described for the first time using scanning electron microscopy. The tegument of all of the specimens was fully covered with microtriches. Four basic types were found: filitriches, blade-like spinitriches of different sizes and shapes, cone-shaped spinitriches (with two parallel small projections of equal length at the apex), and crowned cylindrical spinitriches (with 6–7 small papillae forming a crown at the apex); the two latter types are newly described. The two types of pler…

Dolphin morbillivirus epizootic resurgence, Mediterranean Sea

In July 2007, > 100 striped dolphins, Stenella coeruleoalba, were found dead along the coast of the Spanish Mediterranean. Of 10 dolphins tested, 7 were positive for a virus strain closely related to the dolphin morbillivirus that was isolated during a previous epizootic in 1990.

The origin of Lecithodesmus (Digenea: Campulidae) based on ND3 gene comparison

Species of Lecithodesmus (Campulidae) occur almost exclusively in baleen whales throughout a wide geographical distribution. Other campulids occur only in odontocetes and, secondarily, in pinnipeds and the sea otter. Therefore, the ancestor of Lecithodesmus might have either cospeciated with mysticetes during the early divergence of mysticete and odontocete cetaceans or originated later via host switching. We evaluate both possibilities based on a phylogenetic analysis. The ND3 mitochondrial gene sequence of a species of Lecithodesmus was included in a previous partial molecular phylogeny of the Campulidae. Fasciola hepatica and Dicrocoelium dendriticum were used as outgroups. Maximum parsi…