Submitted 30 August 2017Accepted 22 November 2017Published 11 December 2017

Corresponding authorsAndreacutes Martiacutenez-Aquinomaandres_hotmailcomM Leopoldina Aguirre-Macedoleopoldinaaguirrecinvestavmxleopoldina2305gmailcom

Academic editorJames Reimer

Additional Information andDeclarations can be found onpage 11

DOI 107717peerj4158

Copyright2017 Martiacutenez-Aquino et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

A molecular phylogenetic appraisal ofthe acanthostomines Acanthostomumand Timoniella and their positionwithin Cryptogonimidae (TrematodaOpisthorchioidea)Andreacutes Martiacutenez-Aquino Victor M Vidal-Martiacutenez andM Leopoldina Aguirre-MacedoDepartamento de Recursos del Mar Centro de Investigacioacuten y de Estudios Avanzados del InstitutoPoliteacutecnico Nacional Unidad Meacuterida Meacuterida Yucataacuten Meacutexico

ABSTRACTThe phylogenetic position of three taxa from two trematode genera belonging tothe subfamily Acanthostominae (Opisthorchioidea Cryptogonimidae) were analysedusing partial 28S ribosomal DNA (Domains 1ndash2) and internal transcribed spacers(ITS1ndash58SndashITS2) Bayesian inference and Maximum likelihood analyses of combined28S rDNA and ITS1 + 58S + ITS2 sequences indicated the monophyly of the genusAcanthostomum (A cf americanum and A burminis) and paraphyly of the Acanthos-tominae These phylogenetic relationships were consistent in analyses of 28S aloneand concatenated 28S + ITS1 + 58S + ITS2 sequences analyses Based on molecularphylogenetic analyses the subfamily Acanthostominae is therefore a paraphyletic taxonin contrast with previous classifications based on morphological data Phylogeneticpatterns of host specificity inferred from adult stages of other cryptogonimid taxaare also well supported However analyses using additional genera and species arenecessary to support the phylogenetic inferences from this study Our molecularphylogenetic reconstruction linked two larval stages of A cf americanum cercariaeand metacercariae Here we present the evolutionary and ecological implications ofparasitic infections in freshwater and brackish environments

Subjects Biodiversity Evolutionary Studies Marine Biology Parasitology ZoologyKeywords Cichlasoma urophthalmus Evolutionary ecology of parasites ITS1ndash58SndashITS2 28SPyrgophorus coronatus Acanthostominae

INTRODUCTIONThe Cryptogonimidae Ward 1917 is a speciose family (ge370 species) consisting of 93genera associated with the intestine or pyloric caeca of marine and freshwater teleostsreptiles and occasionally amphibians around the world (Miller amp Cribb 2008a Milleramp Cribb 2013 Miller et al 2009 Miller et al 2010a Miller et al 2010b Cribb amp Gibson2017 Tkach amp Bush 2010 Fernandes et al 2013) Since taxonomic identification basedon morphological characters is complex (ie it is based on combinations of characters)the taxonomic classification of species within Cryptogonimidae (eg at the subfamily

How to cite this article Martiacutenez-Aquino et al (2017) A molecular phylogenetic appraisal of the acanthostomines Acanthostomum andTimoniella and their position within Cryptogonimidae (Trematoda Opisthorchioidea) PeerJ 5e4158 DOI 107717peerj4158

level) has been reworked several times (Miller amp Cribb 2008a) Taxonomic schemes ofsubfamilies can also be detected based on ecological factors and host preferences Forexample studies based on phylogenetic approaches infer hierarchical-taxonomic patternsbetween cryptogonimid species associated with specific marine fish hosts (eg Retrovariumspp that are associated with perciform marine fishes) or cryptogonimid genera associatedwith reptile taxa (eg the subfamily Acanthostominae Looss 1899) (Brooks 1980Miller ampCribb 2007aMiller amp Cribb 2008a) In particular theAcanthostominaewas inferred basedon morphology phylogeny and biogeographical and host-parasite association patterns(Brooks 1980 Brooks amp Holcman 1993) The criteria for the subfamily Acanthostominaeas recognized by Brooks amp Holcman (1993) was based on six characters (1) a terminaloral sucker (2) a body armed with single row of spines (3) a preacetabular pit (4) agenital pore not in preacetabular pit (5) a seminal vesicle coiled posteriorly and (6) asucker-like gonotyl Based on these criteria the acanthostomine trematodes include fivegenera Timoniella Rebecq 1960 Proctocaecum Baught 1957 Gymnatrema Morozov1955 Caimanicola Freitas and Lent 1938 and Acanthostomum Looss 1899 (Brooks2004) Nevertheless Miller amp Cribb (2008a) were not convinced by the morphologicalcharacteristics that were used to justify subfamily-level divisions in Cryptogonimidaebecause several subfamilies were separated by few and often trivial characters Miller ampCribb (2008a) also recognized that the phylogenetic analyses of acanthostomines by Brooks(1980) could be used to infer intergeneric relationships between cryptogonimids

To explore the diversity of helminth parasite fauna from aquatic invertebrate andvertebrate hosts in Mexico (Vidal-Martiacutenez et al 2001 Aguirre-Macedo et al 2017)molecular phylogenetic analyses based on nuclear gene fragments (partial 28S ribosomalDNA and the internal transcribed spacers (ITS1ndash58SndashITS2)) were carried out oncryptogonomids from Mexicorsquos Yucataacuten Peninsula The analyses were used to answerquestions regarding the phylogenetic position of acanthostomines within the familyCryptogonomidae and possible life-cycle links between cercariae and metacercariaewere additionally examined Based on the results of the molecular phylogenetic analysesthe systematic position of the acanthostomine genera Acanthosthomum and Timoniellawere evaluated with a brief discussion of the taxonomic implications for the subfamilyAcanthostominae and phylogenetic evidence to support the different intergenericrelationships among Cryptogonimidae is provided

MATERIAL AND METHODSCollection of hosts and trematode parasitesAs part of our ongoing study in the Celestun lagoon (Sosa-Medina Vidal-Martiacutenez ampAguirre-Macedo 2015) we collected specimens of cryptogonimid metacercariae presumedto be of the subfamily Acanthostominae Acanthostomum americanum (=Atrophecaecumastorquii) Peacuterez-Vigueras 1956 and Timoniella (=Pelaezia) loossi Peacuterez-Vigueras1956 from the Ria Celestun Biosphere Reserve Yucatan Peninsula Mexico (basedon Moravec 2001 Vidal-Martiacutenez et al 2001 Brooks 2004 Miller amp Cribb 2008a)These metacercariae were collected from the euryhaline fish Cichlasoma urophthalmus(Guumlnter 1862) (Perciformes Cichlidae) from the Yaxaaacute water spring (2053prime1257primeprimeN

Martiacutenez-Aquino et al (2017) PeerJ DOI 107717peerj4158 218

Kilometers

10 0 10

NGULF OF MEXICO

Ria Celestun Biosphere Reserve

Yaxaaacute spring

9030ordm 9020ordm 9010ordm

9030ordm 9020ordm 9010ordm

2100ordm

2050ordm

2040ordm

2030ordm

Mexico

Yucatan

Celestun lagoon

Figure 1 Map of the study area Yaxaaacute spring Celestun coastal lagoon Yucatan MexicoFull-size DOI 107717peerj4158fig-1

9020prime5886primeprimeW) located in the Celestun tropical lagoon (Fig 1) We also collectedcercariae presumed to be of the Cryptogonimidae from the aquatic gastropod Pyrgophoruscoronatus (Pfeiffer 1840) (Hybrobiidae) (see Scholz et al 2000) at the same location totest for possible life-cycle links between the cercariae and metacercariae with moleculardata In March 2016 we collected 223 snails of P coronatus from two localities BaldioceraSpring (2054prime629primeprimeN 9020prime2646primeprimeW) (156 snails) and Yaxaaacute Spring (67 snails) (the twosprings are approximately 1400 m apart) Snails were collected using strainers placedseparately into glass tubes and maintained in artificial light in the laboratory to stimulatethe emergence of cercariae After 2ndash3 days portions of the snails were removed from theirshells by dissection under a stereomicroscope The only representatives of Cyptogonimidae(three cercariae) were collected from a single P coronatus from Yaxaaacute Spring Forrepresentatives of other families of the 156 P coronatus examined from Baldiocera Springwe observed two cercaria of Ascocotyle (Phagicola) nana Ransom 1920 (Heterophyidae)in each of two individual snails and one metacercaria of Crassicutis cichlasomae Manter1936 (Apocreadiidae) from one snail Both larvae have been previously recorded fromP coronatus (Scholz et al 2000) Of the 67 P coronatus examined from Yaxaaacute Spring theonly cercariae observed belonged to the aforementioned cryptogonimids We also sampledspecimens of other adult cryptogonimids eg Oligogonotylus mayae (Razo-MendivilRosas-Valdez amp Peacuterez-Ponce de Leoacuten 2008) from the cichlid fish C urophthalmusThe protocols for host dissection examination collection and preservation and the

Martiacutenez-Aquino et al (2017) PeerJ DOI 107717peerj4158 318

morphological study of parasitic specimens followed Vidal-Martiacutenez et al (2001) Wealso collected adult specimens of the apocreadiid species Crassicutis cichlasomae from thesame fish host Crassicutis cichlasomae was used as an outgroup taxon for the phylogeneticanalyses in this study based on its previously established sister group relationship ofOphisthorchioidae (Bray et al 2009 Fraija-Fernaacutendez et al 2015) Trematodes wereidentified based on morphological criteria suggested by Vidal-Martiacutenez et al (2001)Milleramp Cribb (2008a) Razo-Mendivil Rosas-Valdez amp Peacuterez-Ponce de Leoacuten (2008) and Razo-Mendivil et al (2010) Reliable identification to genus level is possible for both Timoniellaand Acanthosthomum based on metacercariae morphology Microphotographs of bothtaxa can be found in Fig S1 However identification to species level may be questionabletherefore we hereafter refer to the species as T cf loossi and A cf americanum Severalmetacercariae and adult specimens collected for morphological analysis were deposited asvoucher specimens [T cf loossi (No 525)A cf americanum (No 526)C cichlasomae (No527) andO mayae (No 528)] in the ColeccioacutenHelmintoloacutegica del CINVESTAV (CHCM)Departamento de Recursos del Mar Centro de Investigacioacuten y de Estudios Avanzadosdel Instituto Politeacutecnico Nacional Unidad Meacuterida Yucataacuten Meacutexico Acanthostominecercariae were not deposited because each specimen was required for the molecular studyComisioacuten Nacional de Acuacultura y Pesca (PPFDGOPA-07016) issued the collectingpermits

DNA extraction PCR amplification and sequencingDNA was extracted from individual cercariae metacercariae and adult trematodes DNAextraction was performed using the DNAeasy blood and tissue extraction kit (QiagenValencia CA USA) following the manufacturerrsquos instructions For the four trematodetaxa the partial 28S ribosomal gene region was amplified by Polymerase Chain Reaction(PCR) (Saiki et al 1988) using 28sl forward (5prime-AAC AGT GCG TGA AAC CGC TC-3prime)(Palumbi 1996) and LO reverse (5prime-GCT ATC CTG AG(AG) GAA ACT TCG- 3prime) (TkachPawlowski amp Mariaux 2000) The primers BD1 forward (5prime-GTC GTA ACA AGG TTTCCG TA-3prime) and BD2 reverse (5prime-TAT GCT TAA ATT CAG CGG GT-3prime) (Bowles Blairamp McManus 1995) were used for ITS1ndash58SndashITS2 fragment The reactions were preparedusing the Green GoTaq Master Mix (Promega Madison WI USA) This procedure wascarried out using an Axygen Maxygen thermocycler PCR cycling conditions by bothmolecular markers were as follows an initial denaturing step of 5 min at 94 C followedby 35 cycles of 92 C for 30 s 55 C for 45 s and 72 C for 90 s and a final extension stepat 72 C for 10 min The PCR products were analysed by electrophoresis in 1 agarose gelusing TAE 1times buffer and observed under UV light using the QIAxcel Rcopy Advanced SystemThe purification and sequencing of the PCR products were carried out by Genewiz (SouthPlainfield NJ USA httpswwwgenewizcom)

Molecular data and phylogenetic reconstructionTo obtain the consensus sequences of the larvae and adults ofA cf americanum T cf loossiO mayae and C cichlasomae we assembled and edited the chromatograms of forward andreverse sequences using the Geneious Pro v517 platform (Drummond et al 2010) To

Martiacutenez-Aquino et al (2017) PeerJ DOI 107717peerj4158 418

investigate the monophyly of the taxa included in Cryptogonimidae at the subfamilylevel the 28S ITS1 58S and ITS2 sequences that were generated during this study werealigned with sequences of other cryptogonimids and their sister groups heterophyid andopisthorchiid taxa (based on Thaenkham et al 2011 Thaenkham et al 2012) obtainedfrom GenBank (see GenBank accession numbers in Table S1) using an interface availablewith MAFFT v7263 (Katoh amp Standley 2016) an lsquolsquoautorsquorsquo strategy and a gap-openingpenalty of 153 with Geneious Pro and a final edition by eye in the same platform The bestpartitioning scheme and substitution model for each molecular marker was selected byusing the lsquolsquogreedyrsquorsquo search strategy in Partition Finder v111 (Lanfear et al 2012 Lanfearet al 2014) and applying the Bayesian Information Criterion (BIC) (Schwarz 1978) Thenucleotide substitution model that best fit the 28S data was TVM + I + G (Posada 2003)for ITS1 and ITS2 it was TVMef + G (Posada 2003) and for 58S it was JC + G (Jukesamp Cantor 1969) Hypervariable regions of 28S ITS1 and ITS2 alignments were excludedusing the Gblocks Web Server (Castresana 2000 Talavera amp Castresana 2007)

The datasets were analysed by Bayesian inference (BI) and Maximum likelihoodanalyses (ML) using the CIPRES Science Gateway v 33 (Miller Pfeiffer amp Schwartz2010) ML analyses were conducted in RaxML v 8 (Stamatakis 2014) using the GTRCATapproximation as a model of nucleotide substitution (Yang 1994 Yang 1996 Stamatakis2006) BI analyses were carried out with MrBayes v 321 (Ronquist et al 2012) TheBayesian phylogenetic trees were reconstructed for each gene separately using two parallelanalyses of Metropolis-Coupled Markov Chain Monte Carlo (MCMC) for 20 times 106

generations each Topologies were sampled every 1000 generations and the averagestandard deviation of split frequencies was observed until it reached lt001 as suggestedby Ronquist et al (2012) A majority consensus tree with branch lengths was reconstructedfor the two runs after discarding the first 5000 sampled trees For both ML and BIanalyses model parameters were independently optimized for each partition Nodesupport was evaluated by non-parametric bootstrapping (Felsenstein 1985) with 1000replicates performed with RAxML (ML) and BI by Posterior probabilities (PP) wherebootstrap values ge75 and PP ge 095 were considered strongly supported

RESULTSDNA sequences and dataset analysesIn total 36 bi-directional partial 28S (domains 1 and 2) and ITS1-58S-ITS2 sequenceswere obtained from three individual cercariae and three individual metacercariae fromA cf americanum as well as three individual metacercariae from T cf loossi O mayae(one adult specimen) and C cichlasomae (one adult specimen outgroup) (Table 1) Thepartial 28S rDNA sequence fragment consisted of 881 base-pairs (bp) for the cercariae andmetacercariae of A cf americanum 880 bp in T cf loossi 871 bp in O mayae and 870 bpin C cichlasomae The 28S sequences of cercariae and metacercariae of A cf americanumfrom P coronatus were identical while the sequences of T cf loossi showed a divergenceof 003 Nucleotide sequence variation in the 28S alignment from cryptogonimids(excluding the outgroup taxon) from 28S included 722 conserved sites 537 variable sites

Martiacutenez-Aquino et al (2017) PeerJ DOI 107717peerj4158 518

Table 1 GenBank accession numbers for cryptogonimid species sequences newly generated for thisstudy Codes used for each cryptogonimid sequenced are as shown in the terminal taxa names of Fig 1and Figs S2ndashS4

Name Code Life cycle stage GenBank accession

28S ITS1-58S-ITS2

Timoniella cf loossi 1 Metacercarie MG383502 MG383515Timoniella cf loossi 2 Metacercarie MG383503 MG383516Timoniella cf loossi 3 Metacercarie MG383504 MG383517Timoniella cf loossi 4 Metacercarie MG383505 MG383518Timoniella cf loossi 5 Metacercarie MG383506 MG383519Acanthostomum cf americanum 1c Cercarie MG383496 MG383509Acanthostomum cf americanum 2c Cercarie MG383497 MG383510Acanthostomum cf americanum 3c Cercarie MG383498 MG383511Acanthostomum cf americanum 1m Metacercarie MG383499 MG383512Acanthostomum cf americanum 2m Metacercarie MG383500 MG383513Acanthostomum cf americanum 3m Metacercarie MG383501 MG383514Oligogonotylus mayae Adult MG383507 MG383520Crassicutis cichlasomae Adult MG383508 MG383521

403 parsimony-informative sites and 134 singleton sites The sequence fragments for theITS1 nuclear marker were between 709 and 781 bp in length for A cf americanum andwere 805 bp in T cf loossi 613 bp in O mayae and 424 bp in C cichlasomae The 58Snuclear marker was composed of 160 bp in A cf americanum T cf loossi O mayae andC cichlasomae The length of the ITS2 nuclear marker ranged from 259 bp to 277 bp inA cf americanum and from 268 bp to 277 bp in T cf loossi 260 bp in O mayae and295 bp in C cichlasomae The ITS1 and ITS2 sequences of A cf americanum displayed 4and 07 divergence respectively and those from T cf loossi displayed 09 divergenceand 100 pairwise identity the 58S sequences were identical Nucleotide sequencevariation (excluding the outgroup taxa) for ITS1 58S and ITS2 were 626950 conserved40692212 variable 34136184 parsimony-informative and 655628 singleton sitesrespectively

Phylogenetic reconstructionsWe inferred the phylogenetic relationships of Cryptogonimidae based on the BI andML analyses from the following two datasets The partial 28S gene dataset contained92 terminals belonging to 81 species and the combined dataset (28S + ITS1 + 58S +ITS2) contained 294 sequences belonging to 81 taxa concatenated (all sequences availablefrom GenBank see Table S1) The phylogenetic trees constructed from the 28S and theconcatenated datasets (28S + ITS1 + 58S + ITS2) based on BI and ML analyses werebroadly congruent For example all clades with high nodal support values (PP ge 095 andbootstrap ge 75) and analysed with the concatenated and 28S datasets were recoveredwith both BI and ML (Fig 2 Figs S2ndashS4) Only three clades were recovered with highnodal support values (PP ge 095) using BI but not ML (ie (Gynichthys diadikidnusNeoparacryptogonimus ovatus) (Metagonimus takahashii M yokogawai) and (Haplorchis

Martiacutenez-Aquino et al (2017) PeerJ DOI 107717peerj4158 618

007

Siphoderina poulini

Retrovarium formosum

Siphoderina quasispina

Caecincola parvulus

Metagonimus miyatai

Euryakaina sp

Acanthostomum burminis

Acanthostomum cf americanum 1m

Timoniella cf loossi 3

Siphomutabilus gurukun

Siphoderina subuterus

Clonorchis sinensis

Siphoderina virga

Euryhelmis costaricensis

Metagonimoides oregonensis

Retrovarium amplorificium

Metagonimus takahashii

Ascocotyle sp

Acanthostomum cf americanum 1c

Stellantchasmus falcatus

Retrovarium snyderi

Metagonimus katsuradai

Centrocestus formosanusHeterophyes heterophyes

Siphoderina grunnitus

Procerovum varium

Oligogonotylus mayae

Acanthostomum cf americanum 3m

Oligogonotylus manteri

Stictodora sp isolate St2

Opisthorchis noverca

Haplorchis popelkae

Timoniella cf loossi 5

Acanthostomum burminis

Timoniella cf loossi 1

Retrovarium planum

Metagonimus hakubaensis

Acanthostomum burminis

Amphimerus ovalis

Siphoderina infirma

Ascocotyle pindoramensis

Pygidiopsis macrostomum

Procerovum cheni

Acanthostomum cf americanum 2c

Tabascotrema verai

Retrovarium valdeparvum

Haplorchis taichui

Siphoderina territans

Retrovarium exiguiformosum

Stictodora sp isolate St1

Adlardia novaecaledoniae

Galactosomum lacteum

Haplorchis pumilio

Haplorchoides sp

Latuterus tkachi

Siphoderina hirastricta

Retrovarium mariae

Crassicutis cichlasomae (Apocreadiidae)

Neoparacryptogonimus ovatus

Cryptocotyle lingua

Retrovarium sablae

Acanthostomum cf americanum 2m

Metadena lutiani

Retrovarium manteri

Timoniella cf loossi 2

Metagonimus otsurui

Retrovarium gardneri

Latuterus maldivensis

Haplorchis yokogawai

Acanthostomum cf americanum 3c

Metagonimus yokogawai

Lobosorchis tibaldiae

Retrovarium brooksi

Siphomutabilus raritas

Opisthorchis viverrini

Caulanus thomasi

Lobosorchis polygongylus

Euryakaina manilensis

Gynichthys diakidnus

Euryakaina marina

Timoniella cf loossi 4

Chelediadema marjoriae

I-P

Cla

de I

CR

YP

TOG

ON

IMID

AE

EA

DIIH

CR

OHT

SIP

O E

ADI

YH

PO

RET

EH

AEDI

OIH

CR

OHTSI P

O

Acanthostominae

IW-P

IW-P

Haemulidae

Siphoderina jactus

Beluesca littlewoodiBeluesca longicolla

Varialvus charadrusVarialvus jenae

Varialvus lacertus

Lutjanidae

Cichlidae CA

NACentrarchidae

I-P

IW-P

Siphodera vinaledwardsii GMHaemulidae

Lutjanidae

Nemipteridae

LutjanidaeHaemulidae

Mitotrema anthostomatum

Centrovarium lobotes NAAphalloides coelomicola EA

I-P

SE-A amp SL

IH CA DH CAIH CA

28S + ITS1 + 58S + ITS2IBML

Figure 2 Phylogenetic tree obtained from Bayesian inference analysis of the concatenated data (28S+ ITS1+ 58S+ ITS2) of species of the Cryptogonimidae The scale bar represents the number of nu-cleotide substitutions per site Codes following taxon names are cross-referenced in Table 1 and Table S1Filled black circles above and white circles below the branches represent Bayesian posterior probability ge095 and Maximum likelihood bootstrap support values ge 75 respectively Diffused green Freshwaterenvironment Diffused green-yellow Brackish environment Diffused blue Marine environment IH In-termediate host DH Definitive host Intermediate host unknown (continued on next page )

Full-size DOI 107717peerj4158fig-2

Martiacutenez-Aquino et al (2017) PeerJ DOI 107717peerj4158 718

Figure 2 ( continued)I-P Indo-Pacific IW-P Indo-west Pacific CA Central America GM Gulf of Mexico NA NorthAmerica EA Eastern Atlantic Se-A amp SL South-eastern Asia and Sri Lanka The black snail outlinecorresponds to Pyrgophorus coronatus The black fish outline corresponds to Cichlasoma urophthalmusThe black crocodile outline corresponds to Crocodylus moreletii The black fishes outline on the remainingCryptogonomidae refer to host specificity at family (ies) recording to species species groups or genus(black line) of cryptogonomids The animalsrsquo silhouettes were modified from Ditrich et al (1997)(snail) Gray (1830) (snake) Nelson (2006) (fishes) and Saacutenchez Herrera et al (2011) (crocodile) Thecryptogonomid taxa without black fish outline are not specific to one host See text for more details

yokogawai (Haplorchis popelkae Haplorchis pumilio))) while only one clade receivedhigh nodal support value (bootstrap ge 75) with ML and not BI (ie (Haplorchoidessp (Stictodora sp isolate St1 Stictodora sp isolate St2))) (Fig 2) Conversely onlyone difference was observed between the topology of the phylogenetic trees obtainedfrom the 28S and concatenated datasets with BI and ML Namely the phylogenetic treeobtained from the ML analysis of the 28S sequence dataset contained a polyphyleticgroup (without nodal support value) ie Siphodera vinaledwardsii Gynichthys diakidnusChelediadema marjoriae Caecincola parvulus and Tabascotrema verai (Fig S3) In alltrees acanthostomines were paraphyletic with high nodal support values (PP ge 095 andbootstrap ge 75) Based on all trees the family Cryptogonimidae appears to have arisenfrom a paraphyletic HeterophyidaeOpisthorchiidae group As well all trees clearly showedthat the generated sequences in this study of T cf loossi and A cf americanum form amonophyletic group with high nodal support values (PP ge 095 and bootstrap ge 75)respectively These acanthostomine genera are sister to the remaining cryptogonomidsFurthermore the genus Acanthostomum is a monophyletic group with high nodal supportvalues (PP ge 095 and bootstrap ge 75) Lastly the 28S and ITS1-58S-ITS2 fragmentsequences of acanthostomine metacercaria from C urophthalmus were identical to thoseof cercarie from P coronatus and therefore both trematode stages correspond to the sametaxa A cf americanum

The phylogenetic relationships among Cryptogonimidae at the generic level had highsupport (PPge 095) and the genera Siphoderina BeluscaVarialvusCaulanus and Latuterusform a monophyletic group (Clade I) (Fig 2) distributed in the Indo-Pacific region (I-P) Of these the genera Belusca and Varialvus Caulanus and Latuterus have been foundparasitizing themarine fish familiesHaemulidae and Lutjanidae FurthermoreRetrovariumspp was found parasitizing Lutjanidae andHaemulidae from the Indo-West Pacific (IW-P)(Fig 2)

DISCUSSIONThe phylogenetic trees obtained from BI and ML analyses inferred from the 28S andconcatenated dataset identified the phylogenetic position of the acanthostominesA cf americanum and T cf loossi and illustrate different intergeneric relationshipsamong cryptogonimids Phylogenetic analyses show that the Heterophyidae andOpisthorchiidae are paraphyletic as previously reported (Thaenkham et al 2011Thaenkham et al 2012 Fraija-Fernaacutendez et al 2015 Stoyanov et al 2015 Borges et al

Martiacutenez-Aquino et al (2017) PeerJ DOI 107717peerj4158 818

2016) and that the family Cryptogonimidae appears to have arisen from the paraphyleticHeterophyidaeOpistorchiidae This phylogenetic inference is based on a dataset of 51 taxaof Cryptogonimidae that included 24 genera At present the family Cryptogonimidaeincludes 93 genera (Cribb amp Gibson 2017) and we analysed almost 40 (3875)of recorded genera of Cryptogonimidae Therefore the phylogenetic inference ofCryptogonimidae has an appropriate taxonomical representation but it is still necessary tocomplete this work with more sampling and sequencing of the remaining non-investigatedgenera

Based on the phylogenetic position of A cf americanus A burminis (which formeda single clade) and T cf loossi (independent lineage) we find that the subfamilyAcanthostominae is paraphyletic Therefore the monophyly proposed for the subfamilyAcanthostominae based on morphological analyses (ie Brooks 1980 Brooks 2004Brooks amp Caira 1982 Brooks amp Holcman 1993) does not appear to be valid These datasupport the proposed invalidity of the subfamily-level division of Acanthostominaeinto Cryptogonimidae as previously suggested by Miller amp Cribb (2008a) Thereforeit is necessary to include more acanthostomine taxa (ie Proctocaecum GymnatremaCaimanicola) in future studies to determine their phylogenetic positions and test theirmonophyly

Based on the phylogenetic positions ofAcanthostomum spp andT loossi in this study wepostulate a probable host-specificity pattern at a supra-specific level The adult trematodesA burminis A americanum and T loossi are associated with freshwater diapsid sauropsidsie Xenochrophis piscator (Schneider 1799) (snake) (Reptilia Colubridae) and Crocodylusmoreletii Dumeacuteril amp Bibron 1851 (crocodile) (Reptilia Crocodylidae) (Moravec 2001Jayawardena et al 2013 Sosa-Medina Vidal-Martiacutenez amp Aguirre-Macedo 2015) Themolecular evidence that links the two larval stages of A americanum to the freshwaterenvironment (from their intermediate hosts snails and fish) and their later developmentas adults in freshwater crocodiles may reflect an ecological preference to a freshwaterenvironments More specifically the first larval stage (ie cercaria) of A cf americanumis restricted to freshwater environments due to the intermediate host snailrsquos intolerance tobrackish water (Scholz et al 2000) The trematodersquos intermediate and definitive vertebratehosts (Cichlasoma urophthalmus and Crocodylus moreletii) are both tolerant to brackishwater and can move between the two aquatic environments (Platt Sigler amp Rainwater2010 Miller et al 2009) however the freshwater environment is essential to completingthe trematodersquos life cycle This assertion is supported by taxonomic records ofmetacercariaeof A cf americanum being from freshwater fishes of the families Characidae CichlidaeClupeidae and Poeciliidae (Salgado-Maldonado 2006 Sosa-Medina Vidal-Martiacutenez ampAguirre-Macedo 2015)

Our phylogenetic trees indicated that the Acanthostominae were sister to theremaining marine cryptogonimids (supporting the sister-group relation found byStoyanov et al 2015) (Fig 2) If the acanthostomine taxa are truly sister to the remainingCryptogonomidae there would be a strong argument for the hypothesis that thecryptogonimids originated in a freshwater environment and later diversified and colonizedbrackish and marine environments The transition from the freshwater environment to

Martiacutenez-Aquino et al (2017) PeerJ DOI 107717peerj4158 918

the brackish and marine environments is an evolutionary process also inferred for otherplatyhelminth groups (egTorchin Lafferty amp Kuris 2002Boeger Kritsky amp Pie 2003VanSteenkiste et al 2013) Future studies may test this hypothesis regarding the colonizationfrom freshwater tomarine environments (egWaters amp Wallis 2001Grosholz 2002 Lee ampGelembiuk 2008) The identification of the link between the cercariae and metacercariae ofA cf americanummay represent a step in the understanding of the evolutionary strategiesemployed within different aquatic environments and the potential repercussions on foodwebs (eg Shoop 1988 Dobson Lafferty amp Kuris 2006 Poulin 2006)

It is noteworthy that the hydrobiid snail P coronatus is highly susceptible to trematodeinfection as it has been reported to harbour 12 trematode species ie Genarchellaastyanactis Watson 1976 Echinochasmus leopoldinae Scholz et al 1996 Echinochasmusmacrocaudatus Ditrich et al 1996 Saccocoelioides cf sogandaresi Lumsden 1963 Crassicutiscichlasomae Manter 1936 Homalometridae gen sp Oligogonotylus manteri Watson 1976A (Phagicola) nana Ransom 1920 Ascocotyle (Ascocotyle) sp Xiphidiocercaria type1 Xiphidiocercaria type 2 and Xiphidiocercaria type 3 (Scholz et al 2000) The recordof A cf americanum in P coronatus is a new cercaria record for this snail Howeverunfortunately we did not collect sufficient cercariae of A cf americanum to describe theirmorphology

Our analyses recovered a monophyletic group (Clade I) that includes Belusca CaulanusLatuterus Siphoderina and Varialvus distributed in the Indo-Pacific (Miller amp Cribb2007a Miller amp Cribb 2008b Miller et al 2010b) (Fig 2) Based on the diversity of generain this clade possible taxonomic implications include the erection of a new taxonomichierarchy at the subfamily level Future studies based on morphological evidence maysupport or reject this taxonomic inference Presently more than 50 cryptogonimid taxahave been recorded from fishes belonging to the Lutjanidae and Haemulidae of the IW-P(Miller amp Cribb 2007b Cribb et al 2016) reflected in the phylogenetic topology revealedin this study eg the genera Beluesca Varialvus Caulanus Latuterus SiphomutabilusMetadena Chelediadema and Gynichthys (Fig 2) (Miller amp Cribb 2007c Miller amp Cribb2009 Miller amp Cribb 2013 Miller et al 2010a Miller et al 2010b Miller Bray amp Cribb2011 Overstreet Cook amp Heard 2009) Furthermore Adlardia novaecaledoniae has beenfound in Nemipteridae from the Indo-West Pacific (Miller et al 2009) On the other handprevious records of Euryakaina spp and Retrovarium spp have been found in the familiesLutjanidae andHaemulidae families from the Indo-West Pacific and were attributed by theauthors to a host specificity pattern at the supra-specific level (Miller amp Cribb 2007bMilleret al 2010aMiller Bray amp Cribb 2011) Similarly cases of monophyletic groups from thisstudy originating from specific families could indicate cases of host specificity (probablyresulting from co-divergence Page 2003Martiacutenez-Aquino 2016) although we cannot ruleout the possibility of these cases are an artefact of sampling bias Future taxonomical studiesof cyryptogonomid trematodes from marine fishes from other parts of the world will shedmore light on host-specificity patterns (eg Barger 2010 Montoya-Mendoza et al 2014)

Additionally several non-acanthostomine cryptogonimid clades associated with thefreshwater environment are specialist parasites of particular families of freshwater fishesfromNorth and Central America eg Caecincola parvulus is associated with Centrarchidae

Martiacutenez-Aquino et al (2017) PeerJ DOI 107717peerj4158 1018

fromNorth America (NA) andTabascotrema veraiO mayae andO manteri are associatedwith Cichlidae from Central America (CA) (Choudhury et al 2016) Even though thesegroups did not have valid nodal support in this study (Fig 2) it is important to mentionthree points First the freshwater cryptogonimids appear to have arisen from amongthe marine taxa Second C parvulus and Oligogonotylus spp occur in freshwater fishesas both adults and metacercariae (Stoyanov et al 2015 Choudhury et al 2016) Thirdconsidering that centrarchids and cichlids are both members of Percomorpha and havemarine affinities Choudhury et al (2016) suggested that a close relationship exists betweenMiddle-American cryptogonimids of cichlids and cryptogonimids of North Americancentrarchids The phylogenetic relationship we found between cryptogonimids of cichlidsand centrarchids supports this hypothesis However recent records of C parvulus fromother freshwater fish families must also be considered before final conclusions are made(McAllister et al 2015McAllister et al 2016)

Studies of cryptogonimids (and trematodes in general) are negatively impacted bythe lack of taxonomical records of helminth parasites of freshwater and marine fishes ofdifferent regions (Scholz amp Choudhury 2014 Cribb et al 2016 Vidal-Martiacutenez Torres-Irineo amp Aguirre-Macedo 2016) as well as the lack of knowledge concerning intermediateand definitive host life cycles (Cribb amp Bray 2011 Blasco-Acosta amp Poulin 2017) This hasled to a reduction in postulated evolutionary hypotheses on the diversification patternsof parasites However the development of phylogenetic hypotheses as presented herecan provide a modern framework in parasite evolutionary ecology (eg Littlewood 2011Goacutemez amp Nichols 2013 Poulin Blasco-Costa amp Randhawa 2016)

ACKNOWLEDGEMENTSThanks are due to staff of the laboratory of Patologiacutea Acuaacutetica Clara Vivas RodriacuteguezGregoryArjona-Torres Ana LMay-Tec Francisco Puc ItzaacuteNadiaHerreraCastillo JhonnyG Garciacutea-Teh Arturo Centeno-Chale Germaacuten Loacutepez-Guerra Daniel Aguirre-Ayala andEfraiacuten Sarabia from CINVESTAV-IPN Unidad Meacuterida Meacutexico We are grateful to AbrilGamboa-Muntildeoz and Joseacute Garciacutea Maldonado for their technical assistance in the molecularlab We also thank Dr Fadia Sara Ceccarelli who reviewed the first draft of this manuscriptand made very useful suggestions of the phylogenetic analyses that we present in thiscontribution The manuscript greatly benefited from comments of Dr Terry Miller andtwo anonymous referees

ADDITIONAL INFORMATION AND DECLARATIONS

FundingFinancial support of Andreacutes Martiacutenez-Aquino at the laboratory of Aquatic Pathology atCINVESTAVndashIPN came from Grant No 201441 financed by the Sectorial HidrocarbonFund CONACYT-SENER to the CIGoM Consortium The funders had no role in studydesign data collection and analysis decision to publish or preparation of the manuscript

Martiacutenez-Aquino et al (2017) PeerJ DOI 107717peerj4158 1118

Grant DisclosuresThe following grant information was disclosed by the authorsSectorial Hidrocarbon Fund CONACYT-SENER 201441

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull Andreacutes Martiacutenez-Aquino conceived and designed the experiments performed theexperiments analyzed the data wrote the paper prepared figures andor tables revieweddrafts of the paperbull VictorM Vidal-Martiacutenez contributed reagentsmaterialsanalysis tools wrote the paperreviewed drafts of the paperbull M Leopoldina Aguirre-Macedo conceived and designed the experiments contributedreagentsmaterialsanalysis tools wrote the paper reviewed drafts of the paper

Field Study PermissionsThe following information was supplied relating to field study approvals (ie approvingbody and any reference numbers)

Comisioacuten Nacional de Acuacultura y Pesca (PPFDGOPA-07016) issued the collectingpermits

Data AvailabilityThe following information was supplied regarding data availability

The sequences are provided as a Supplemental File

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj4158supplemental-information

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Barger MA 2010 A new species of Caecincola (Trematoda Cryptogonimidae) fromspotted bass (Micropterus punctulatus) in the Big Thicket National Preserve TexasUSA Comparative Parasitology 776ndash8 DOI 10165444171

Blasco-Acosta I Poulin R 2017 Parasite life-cycle studies a plea to resurrect an oldparasitological tradition Journal of Helminthology 6647ndash656DOI 101017S0022149X16000924

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Martiacutenez-Aquino et al (2017) PeerJ DOI 107717peerj4158 1218

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Bowles J Blair D McManus DP 1995 A molecular phylogeny of the human schisto-somesMolecular Phylogenetics and Evolution 4103ndash109DOI 101006mpev19951011

Bray RAWaeschenbach A Cribb THWeedall GD Dyal P Littlewood DTJ 2009The phylogeny of the Lepocreadioidea (Platyhelminthes Digenea) inferred fromnuclear and mitochondrial genes implications for their systematics and evolutionActa Parasitologica 54310ndash329 DOI 102478s11686-009-0045-z

Brooks DR 1980 Revision of the Acanthostominae Poche 1926 (Digenea Cryptogo-nimidae) Zoological Journal of the Linnean Society 70313ndash382DOI 101111j1096-36421980tb00855x

Brooks DR 2004 Comments on the gonotyl of Proctocaecum macroclemidis (Tkach andSnyder 2003) n comb (Digenea Acanthostomidae Acanthostominae) with a keyto the genera of Acanthostominae and a new phylogenetic for Proctocaecum Baugh1957 Journal of Parasitology 90594ndash597 DOI 101645GE-133R

Brooks DR Caira JN 1982 Atrophecaecum lobacetabulare n sp (Digenea Cryptogonim-idae Acanthostominae) with discussion of the generic status of ParacanthosthomumFischthal and Kuntz 1965 and Ateuchocephala Coil and Kuntz 1960 Proceedings ofthe Biological Society of Washington 95223ndash231

Brooks DR Holcman B 1993 Revised classification and phylogenetic hypothesis forthe Acanthostominae Looss 1899 (Digenea Opisthorchiformes Cryptogonimidae)Proceedings of the Biological Society of Washington 106207ndash220

Castresana J 2000 Selection of conserved blocks from multiple alignments fortheir use in phylogenetic analysisMolecular Biology and Evolution 17540ndash552DOI 101093oxfordjournalsmolbeva026334

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Cribb TH Bray RA 2011 Trematode families and genera have we found them allTrends in Parasitology 27149ndash154 DOI 101016jpt201012008

Cribb TH Bray RA Diaz PE Huston DC Kudlai O Martin SB Yong RQ-Y CutmoreSC 2016 Trematodes of fishes of the Indo-west Pacific told and untold richnessSystematic Parasitology 93237ndash247 DOI 101007s11230-016-9625-0

Cribb TH Gibson D 2017 Cryptogonimidae Ward 1917 World register of marinespecies Available at httpwwwmarinespeciesorgaphiaphpp=taxdetailsampid=108440

Ditrich O Scholz T Aguirre-Macedo L Vargas-Vaacutezquez J 1997 Larval stages oftrematodes from freshwater molluscs of the Yucatan Peninsula Mexico FoliaParasitolica 44109ndash127

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Drummond AJ Ashton B Buxton S CheungM Cooper A Duran C Field M HeledJ Kearse M Markowitz S Moir R Stones-Havas S Sturrock S Thierer TWilsonA 2010 Geneious v504 Available at httpwwwgeneiouscom (accessed on 01December 2016)

Felsenstein J 1985 Confidence limits on phylogenies an approach using the bootstrapEvolution 39783ndash791 DOI 1023072408678

Fernandes BMM Cohen SC Mendonca HS Justo MCN 2013 Annakohniella travassosin gen n sp (Digenea Cryptogonimidae) parasite of Rhaphiodon vulpinus (PiscesCynodontidae) from Brazil Comparative Parasitology 8017ndash21 DOI 10165445821

Fraija-Fernaacutendez N Olson PD Crespo EA Raga JA Aznar FJ FernaacutendezM2015 Independent host switching events by digenean parasites of cetaceansinferred from ribosomal DNA International Journal for Parasitology 45167ndash173DOI 101016jijpara201410004

Goacutemez A Nichols E 2013 Neglected wild life parasitic biodiversity as a conservationtarget International Journal for Parasitology Parasites and Wildlife 2222ndash227DOI 101016jijppaw201307002

Gray JE 1830 Illustrations of Indian zoology chiefly selected from the collection of major-general hardwicke FRS Volumen II London Treuttel Wurtz Treuttel Jun andRichter

Grosholz E 2002 Ecological and evolutionary consequences of coastal invasions Trendsin Ecology and Evolution 1722ndash27 DOI 101016S0169-5347(01)02358-8

Jayawardena UA Tkach VV Navaratne AN Amerasinghe PH Rajakaruna RS 2013Malformations and mortality in the Asian Common Toad induced by exposureto Pleurolophocercous cercariae (Trematoda Cryptogonimidae) ParasitologyInternational 62246ndash252 DOI 101016jparint201301003

Jukes TH Cantor CR 1969 Evolution of protein molecules In Munro HM edMammalian protein metabolism New York Academic Press 21ndash132

Katoh K Standley DM 2016 A simple method to control over-alignment in theMAFFT multiple sequence alignment program Bioinformatics 321933ndash1942DOI 101093bioinformaticsbtw108

Lanfear R Calcott B Ho SY Guindon S 2012 Partition Finder combined selection ofpartitioning schemes and substitution models for phylogenetic analysesMolecularBiology and Evolution 291695ndash1701 DOI 101093molbevmss020

Lanfear R Calcott B Kainer D Mayer C Stamatakis A 2014 Selecting optimalpartitioning schemes for phylogenomic datasets BMC Evolutionary Biology 1482DOI 1011861471-2148-14-82

Lee CE Gelembiuk GW 2008 Evolutionary origins of invasive populations EvolutionaryApplications 1427ndash448 DOI 101111j1752-4571200800039x

Littlewood DTJ 2011 Systematics as a cornerstone of parasitology overview andpreface Parasitology 1381633ndash1637 DOI 101017S0031182011001533

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Martiacutenez-Aquino A 2016 Phylogenetic framework for coevolutionary studies acompass for exploring jungles of tangled trees Current Zoology 62393ndash403DOI 101093czzow018

McAllister CT Bursey CR Robison HW Connior MB Trauth SE 2016 New recordsof helminth parasites (Trematoda Cestoda Nematoda) from fishes in the Arkansasand Red River Drainages Oklahoma Proceedings of the Oklahoma Academy of Science9683ndash92 DOI 10165447141

McAllister CT FontWF Connior MB Robison HW Fayton TJ Stokes NG CriscioneCD 2015 Trematode parasites (Digenea) of the Slender Madtom Noturus exilis andBlack River Madtom Noturus maydeni (Siluriformes Ictaluridae) from ArkansasUSA Comparative Parasitology 82137ndash143 DOI 10165447141

Miller TL Adlard RD Bray RA Justine J-L Cribb TH 2010a Cryptic species ofEuryakaina n g (Digenea Cryptogonimidae) from sympatric lutjanids in the Indo-West Pacific Systematic Parasitology 77185ndash204 DOI 101007s11230-010-9266-7

Miller TL Bray RA Cribb TH 2011 Taxonomic approaches to and interpretationof host specificity of trematodes of fishes lessons from the Great Barrier ReefParasitology 1381710ndash1722 DOI 101017S0031182011000576

Miller TL Bray RA Goiran C Justine J-L Cribb TH 2009 Adlardia novaecaledoniaen g n sp (Digenea Cryptogonimidae) from the fork-tailed threadfin bream Ne-mipterus furcosus (Val) (Perciformes Nemipteridae) off New Caledonia SystematicParasitology 73151ndash160 DOI 101007s11230-009-9187-5

Miller TL Bray RA Justine J-L Cribb TH 2010b Varialvus gen nov (DigeneaCryptogonimidae) from species of Lutjanidae (Perciformes) off the Great BarrierReef New Caledonia and the Maldives Acta Parasitologica 55327ndash339DOI 102478s11686-010-0045-z

Miller TL Cribb TH 2007a Coevolution of Retrovarium n g (Digenea Cryptogonimi-dae) in Lutjanidae and Haemulidae (Perciformes) in the Indo-West Pacific Interna-tional Journal for Parasitology 371023ndash1045 DOI 101016jijpara200701006

Miller TL Cribb TH 2007b Two new cryptogonimid genera Beluesca n gen andChelediadema n gen (Digenea Cryptogonimidae) from tropical Indo-West PacificHaemulidae (Perciformes) Zootaxa 154345ndash60 DOI 105281zenodo177912

Miller TL Cribb TH 2007c Two new cryptogonimid genera (Digenea Cryptogo-nimidae) from Lutjanus bohar (Perciformes Lutjanidae) analyses of ribosomalDNA reveals wide geographic distribution and presence of cryptic species ActaParasitologica 52104ndash113 DOI 102478s11686-007-0019-y

Miller TL Cribb TH 2008a Family Cryptogonimidae Ward 1917 In Bray RA GibsonDI Jones A eds Keys to the Trematoda Vol 3 Wallingford CAB International51ndash112

Miller TL Cribb HT 2008b Eight new species of SiphoderinaManter 1934 (DigeneaCryptogonimidae) infecting Lutjanidae and Haemulidae (Perciformes) off AustraliaActa Parasitologica 53344ndash364 DOI 102478s11686-008-0053-4

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Miller TL Cribb TH 2009 Gynichthys diakidnus n g n sp (Digenea Cryptogonimi-dae) from the grunt Plectorhinchus gibbosus (Laceacutepegravede 1802) (Perciformes Haemuli-dae) off the Great Barrier Reef Australia Systematic Parasitology 74103ndash112DOI 101007s11230-009-9194-6

Miller TL Cribb TH 2013 Dramatic phenotypic plasticity within species of Sipho-mutabilus n g (Digenea Cryptogonimidae) from Indo-Pacific caesionines (Perci-formes Lutjanidae) Systematic Parasitology 86101ndash112DOI 101007s11230-013-9436-5

Miller MA PfeifferW Schwartz T 2010 Creating the CIPRES Science Gateway forinference of large phylogenetic treesrsquo In Proceedings of the gateway computingenvironments workshop (GCE) New Orleans IEEE Computer Society 1ndash8

Montoya-Mendoza J Jimeacutenez-Badillo L Salgado-Maldonado G Mendoza-Franco EF2014Helminth parasites of the Red Snapper Lutjanus campechanus (PerciformesLutjanidae) from the Reef Santiaguillo Veracruz Mexico Journal of Parasitology100868ndash872 DOI 10164513-4291

Moravec F 2001 Some helminth parasites from Moreletrsquos crocodile Crocodyle moreletiifrom Yucatan Mexico Folia Parasitologica 4847ndash62 DOI 1014411fp2001008

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Overstreet RM Cook JC Heard RW 2009 Trematoda (Platyhelminthes) of the Gulfof Mexico In Felder DL Camp DK eds Gulf of Mexicomdashorigins waters and biotaBiodiversity College Station Texas AampM University Press 419ndash486

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Palumbi SR 1996 Nucleic acids II The polymerase chain reaction In Hillis D MoritzC Mable BK edsMolecular systematics Sunderland Sinauer 321ndash383

Platt SG Sigler L Rainwater TR 2010 Moreletrsquos crocodile Crocodylus moreletii InManolis SC Stevenson C eds Crocodiles Status survey and conservation action planDarwin Crocodile Specialist Grouo SSCIUCN 79ndash83

Posada D 2003 Using MODELTEST and PAUP to select a model of nucleotidesubstitution In Baxevanis AD Davison DB Page RDM Petsko GA Stein LDStormo GD eds Current protocols in bioinformatics New York John Wiley amp Sons651ndash6514

Poulin R 2006 Evolutionary ecology of parasites Second Edition New Jersey PrincetonUniversity Press

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Ronquist F TeslenkoM Van der Mark P Ayres DL Darling A Houmlhna S Larget B LiuL SuchardMA Huelsenbeck JP 2012MrBayes 32 efficient Bayesian phylogeneticinference and model choice across large model space Systematic Biology 61539ndash542DOI 101093sysbiosys029

Saiki RK Gelfand DH Stoffel S Scharf SJ Higuchi R Horn GT Mullis KB Erlich HA1988 Primer-directed enzymatic amplification of DNA with a thermostable DNApolymerase Science 239487ndash491 DOI 101126science2448875

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Saacutenchez Herrera O Loacutepez Segurajaacuteuregui G Garciacutea Naranjo Ortiz de la Huerta ABeniacutetez Diacuteaz H 2011 Programa de monitoreo del cocodrilo de pantano (Crocodylusmoreletii) Meacutexico-Belice-Guatemala Mexico City Comisioacuten Nacional para elConocimiento y Uso de la Biodiversidad

Scholz T Aguirre-MacedoML Flores Diacuteas de Leacuteon ATS Ditrich O 2000 Larvalstages of trematodes in Mexican freshwater molluscs a review of present state andmethodology for future research In Salgado-Maldonado G Garciacutea-Aldrete ANVidal-Martiacutenez VM edsMetazoan parasites in the neotropics a systematic andecological perspective Meacutexico Instituto de Biologiacutea UNAM 77ndash100

Scholz T Choudhury A 2014 Parasites of freshwater fishes in North America why soneglected Journal of Parasitology 10026ndash45 DOI 10164513-3941

Schwarz G 1978 Estimating the dimension of a model Annals of Statistics 6461ndash464DOI 101214aos1176344136

ShoopWL 1988 Trematode transmission patterns Journal of Parasitology 7446ndash59DOI 1023073282478

Sosa-Medina T Vidal-Martiacutenez VM Aguirre-MacedoML 2015Metazoan parasites offishes from the Celestun coastal lagoon Yucatan Mexico Zootaxa 4007529ndash544DOI 1011646zootaxa400744

Stamatakis A 2006 Phylogenetic models of rate heterogeneity a high performancecomputing perspective In Parallel and distributed processing symposium 2006Rhodes Island IEEE

Stamatakis A 2014 RAxML version 8 a tool for phylogenetic analysis and post-analysisof large phylogenies Bioinformatics 301312ndash1313 DOI 101093bioinformaticsbtu033

Stoyanov B Neov B Pankov P Radoslavov G Hristov P Georgiev BB 2015 Re-description of Aphalloides coelomicola Dollfus Chabaud amp Golvan 1957 (DigeneaOpisthorchioidea) based on specimens from Knipowitschia caucasica (Berg)(Actinopterygii Gobionellidae) from a Black Sea lagoon with comments on thesystematic position of the genus Systematic Parasitology 911ndash12DOI 101007s11230-015-9559-y

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ThaenkhamU Nawa Y Blair D PakdeeW 2011 Confirmation of the paraphyleticrelationship between families Opisthorchiidae and Heterophyidae using small andlarge subunit ribosomal DNA sequences Parasitology International 60521ndash523DOI 101016jparint201107015

Tkach VJ Bush SE 2010 Serpentoanisocladium sinense n g n sp (Digenea Cryp-togonimidae) from the eastern water snake Sinonatrix percarinata (Boulenger)(Serpentes Colubridae) in Guizhou Province China Systematic Parasitology76205ndash210 DOI 101007s11230-010-9246-y

Tkach V Pawlowski J Mariaux J 2000 Phylogenetic analysis of the suborder Plagiorchi-ata (Platyhelminthes Digenea) based on partial lsrDNA sequences InternationalJournal for Parasitology 3083ndash93 DOI 101016S0020-7519(99)00163-0

TorchinME Lafferty KD Kuris AM 2002 Parasites and marine invasions Parasitology124S137ndashS151 DOI 101017S0031182002001506

Van Steenkiste N Tessens BWillemsW Backeljau T Jondelius U Artois T 2013 Acomprehensive molecular phylogeny of Dalytyphloplanida (Platyhelminthes Rhab-docoela) reveals multiple escapes from the marine environment and origins of sym-biotic relationships PLOS ONE 8(3)e59917 DOI 101371journalpone0059917

Vidal-Martiacutenez VM Scholz T Aguirre-MacedoML Gonzaacutelez-Soliacutes D Mendoza-Franco EF 2001 Atlas of the helminth parasites of cichlid fishes of Mexico PragueAcademia

Vidal-Martiacutenez VM Torres-Irineo E Aguirre-MacedoML 2016 A century (1914ndash2014) of studies on marine fish parasites published in The Journal of ParasitologyIn Janovy J Esch GW eds A century of Parasitology discoveries ideas and lessonslearned by scientists who published in the Journal of Parasitology 1914ndash2014 Chich-ester John Wiley and Sons Ltd 57ndash76

Waters JMWallis GP 2001 Cladogenesis and loss of the marine life-history phasein freshwater galaxiid fishes (Osmeriformes Galaxiidae) Evolution 55587ndash597DOI 1015540014-3820(2001)055[0587CALOTM]20CO2

Yang Z 1994Maximum likelihood phylogenetic estimation from DNA sequenceswith variable rates over sites Journal of Molecular Evolution 39306ndash314DOI 101007BF00160154

Yang Z 1996 Among-site rate variation and its impact on phylogenetic analyses Trendsin Ecology amp Evolution 11367ndash372 DOI 1010160169-5347(96)10041-0

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