First modern morphological and molecular description of Saccocoelium cephali larvae stages (Digenea: Haploporidae) from the Black Sea

doi:10.21203/rs.3.rs-4338263/v1

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Research Article

First modern morphological and molecular description of Saccocoelium cephali larvae stages (Digenea: Haploporidae) from the Black Sea

https://doi.org/10.21203/rs.3.rs-4338263/v1

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published 26 Oct, 2024

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Purpose

Knowledge of haploporid trematode larvae is very poor. Until recently, only scattered reports from Black and Medditeranean Seas are known. The present research is the first report of haploporid cercariae S. cephali from the gastropod Hydrobia acuta inhabiting the Black Sea. Thus, the present study aimed to investigate the larvae of one species from Haploporidae family.

Methods

Hydrobia acuta molluscs were collected in the estuary of the Chernaya River ( Black Sea) during 2011-2013 and examined for trematode larvae. Found parthenitae were drawn and preserved for DNA extractions. Morphological features were compared to nominal species and genetic comparisons were made of the subunit ribosomal DNA with other Haploporidae species.

Results

The haploporid parthenitae were found in the gonads of Hydrobia acuta molluscs. Most of morphological characteristics of specimens investigated agree with those of Saccococoelium sp. and closest to S. cephali by shape and length of the body, ratio of suckers, ratio of tail length to body, shape and size of pharynx. Phylogenetic analysis from our study generated with Bayesian algorithm showed that studied cercariae specimens ex H. acuta from the Black Sea was within hapolporine clade and closely related with S. cephali from Spain. The process of encystation of 1 cercaria in same mollusc was observed (adolescaria).Cercariae and metacercariae have been described and named as Saccocoelium cephalibased on its morphological, morphometric and molecular characteristics.

Conclusion

Morphological characteristics of cercariae emerging from Hydrobia acuta from Black Sea, also the analysis of partial 28S rDNA sequences support conspecificity of parasite with S.cephali mature worms from Mediterranean Sea.

Trematoda

larva

Saccocoelium

Hydrobia acuta

Black Sea

Haploporidae is a small family of Digenea, the vast majority of which are specific parasites of marine or brackish-water mullet fishes. The family includes 15 genera, but only adult worms of four of them (Saccocoelium, Haploporus, Dicrogaster,Lecithobotrys) were registered from the Black Sea (Skrjabin, 1956; Dmitrieva, Gaevskaya, 2001; Popjuk, Pron’kina, 2011). In the Black Sea the life cycles of haploporid trematodes are still unknow. This is largely due to the fact that most of previous studies have focused on the parasites of marine fishes (Gaevskaya, Korniychuk. 2003; Popjuk, Pron’kina, 2011). For most earlier described adult worms of Haploporidae family, the species are morphologically closed. I creates difficulties for making an accurate their species identification (Blasco-Costa et al., 2009a). Therefore, molecular studies are required.

In this study, the first complex data, including morphological descriptions and molecular characteristic of larvae of Saccocoelim cephali from gastropod molluscs H.acuta in the Black Sea are presented.

Sample collection

A total 5188 specimens of H.acuta were collected in the estuary of the Chernaya River (44°27′49′′ N, 33°51′37′′ E) (Sevastopol, Black Sea) during 2011–2013. All snails were examined for helminthic infections using standard methods (Bykhovskaya-Pavlovskaya, 1985). Parthenitae of trematodes were studied as alive asstained using an Olympus CX41 microscope equipped with an CX50 camera with software Infinity Analyze 1 (Canada). Trematodes were fixed under a cover glass with slight pressure, stained with acetocarmine. The colour grade was differentiated by “iron water” (H₂O + Fe₂O₃) and acidified alcohol (70% ethanol + 3% HCl). After dehydration in ethanol of increased concentrations (70, 80, 90, and 100%) and clarification in clove oil, trematodes were mounted in Canada balsam(Roskin, Levinson, 1957).

Morphological data

All measurements were made on stained parasites. The abbreviations of metric features included in Table 1 are according to Blasco-Costa et. al (2009a). The excretory system of the larvae was investigated on living individuals when the larvae were stained with neutral red, as a result of which the flickering of the flame cells was observed. Drawings were made using a drawing software Inkscape 0.48.2.-1 (Scalable Vector Graphics, 2011).

Table 1

Comparing morphological features of haploporid cercariae in the Black Sea and Mediterranean Seas (in µm)
Features	Saccocoelium cephali, Present study	Saccocoelium tensum, Fares, Maillard, 1974	S .obesum,Fares, Maillard, 1974	Haploporus benedeni, Fares, Maillard, 1974
BL	315–514 (404 ± 41)	210	500	350
BW	162–249 (205 ± 21)	120	220	240
OSL	51–69 (60 ± 3)	53	90	100
OSW	84–94 (88 ± 2)			90
VSL	57–68 (63 ± 3 )	50	80
VSW	63–83 (73 ± 6)
PL	12–69 (37 ± 10)			30
PHL	35–48 (42 ± 2)	29	70	50
PHW	38–57 (48 ± 4)	25	60	60
OL	74–131 (106 ± 17)
Tail length	139–260 (200 ± 61)	330		380
FO	186–370 (244 ± 32)
CEND	109–174 (132 ± 15)
OSL/BL	0,2	0,25
VSL/BL	0,2	0,2
PL/BL	0,1
PHL/BL	0,1	0,1
OL/BL	0,3
FO/BL	0,6
TEND/BL
CEND/BL	0,3
0SW/BW	0,4
VSW/BW	0,4
PHW/BW	0,2	0,2
TW/BW
OSL/VSL	1.05	1.06		1

Scanning electron microscopy

Live sporocysts and spontaneously emitting cercariae were fixed in 2.5% (v/v) glutaraldehyde buffered with 0.1 M Sorensen phosphate for 24 hours at 5°C. After samples were dehydrated through an ethanol series (70–96°C).Dried in Leica EM CPD 300 critical point dryer using liquid carbon dioxide as a transitional medium. After drying, they were mounted on aluminium stubs and coated with gold in an ion-sputtering apparatus Leica EM ACE 200.

Statistical methods

After the parasites were identified, we assessed the infection indexes in the Hydrobia acuta, including invasion intensiveness (prevalence) (IE), invasion intensity (II), and abundance index (AI) (Bush et al., 1997). For each morphological parameter, the mean with standard error (mean ± SE) was calculated. To calculate the statistical parameters, the Statistica 6 software package for Windows (Statsoft) was used.

DNA extraction, amplification and sequencing

Two haploporid cercariae specimens fixed by 96% ethanol were used for molecular analysis (Table 2). Total DNA was extracted using QIAamp DNA Micro Kit (Qiagen, Germany). A worm was incubated in 180 µl of Genomic Digestion buffer with 20µl of Proteinase K at 55°C for one hour with following mix by vortex for 20 s. DNA extraction was carried out according to the manufacturer’s protocol. The elution volume was 25 µl. The DNA was stored at -20⁰ C.Fragment of 28S ribosomal DNA (rDNA) 1200 base pairs (bp) in lenght was amplified by a polymerase chain reaction (PCR) method using the 2x GoTaq Green Master mix (Promega, Madison, Wisconsin, USA) and the primers 28S-A (5′-TCG ATT CGA GCG TGA WTA CCC GC-3′) (Matejusova& Cunningham, 2004) and 1500R (5′-GCT ATC CTG AGG GAA ACT TCG-3′) (Tkach et al., 2003) with an annealing temperature of 55°C. Negative and positive controls using both primer pairs were included. PCR parameters began with a 1 min denaturation at 98ºC, followed by 35 cycles of 10 s at 98ºC, 5 s at 55ºC and 2 min 30 s at 72ºC, and concluded with a 7 min extension at 72ºC.

Table 2

List of taxa used for molecular analysis. N - number of sequences of 28S rDNA
Species	N	Definitive host	Authors	Accession number in the NCBI
Haploporidae
Waretrematinae
Paraunisaccoides elegans n. sp.	4	Planiliza subviridis	Atopkin et al., 2022	KY501639-KY501641
Unisaccus halongi n. sp.	3	Crenimugil seheli	Atopkin et al., 2022	OK644190-OK644192
Unisaccus tonkini	1	Moolgarda cunnesius	Besprozvannykh et al., 2018	MF176843
Skrjabinolecithum pyriforme	1	Planiliza haematocheila	Besprozvannykh et al., 2017	HE806359
Skrjabinolecithum spinosum	1	Planiliza haematocheila	Besprozvannykh et al., 2018	MF176834
Skrjabinolecithum. spasskii	3	Planiliza haematocheila	Atopkin et al., 2015	LN614538, HG530210, HG530207
Parasaccocoelium mugili	1	Planiliza haematocheila	Atopkin et al., 2021	MW813991
Parasaccocoelium armatum	2	Mugil cephalus	Atopkin et al., 2020b	MT298950 - MT298950
Parasaccocoelium haematochelium	2	Liza haematocheila	Besprozvannykh et al., 2015	HF548466 - HF548467
Parasaccocoelium polyovum	2	Liza haematocheila	Besprozvannykh et al., 2015	HF548477 - HF548478
Spiritestis herveyensis	1	Moolgarda seheli	Pulis et Overstreet, 2013	KC206500
Capitimitta costata	1	Selenotoca multifasciata	Pulis et Overstreet, 2013	KC206497
Capitimitta darwinensis	1	Selenotoca multifasciata	Pulis et Overstreet, 2013	KC206498
Carassotrema koreanum	3	Carassius gibelio	Atopkin et al., 2019	MH763763- MH763765
Elonginurus mugilis	2	Mugil cephalus	Atopkin et al., 2019	MH763766 - MH763766
Pseudohaploporinae
Parahaploporus elegantus	2	Moolgarda seheli	Atopkin et al., 2020a	MN639712-MN639713
Pseudohaploporus vietnamensis	2	Osteomugil engeli	Atopkin et al., 2018	MF774420- MF774421
Pseudohaploporus vietnamensis	1	Moolgarda seheli	Atopkin et al., 2018	MF774422
Pseudohaploporus planilizum	3	Planiliza subviridis	Atopkin et al., 2018	MF774417- MF774419
Pseudohaploporus pusitestis	2	Moolgarda seheli	Atopkin et al., 2020a	MH986037, MH986038
Haploporinae
Saccocoelium brayi	1	Liza saliens	Blasco-Costa et al., 2009b	FJ211234
Saccocoalium cephali	2	Hydrobia acuta	Present study	Not available
Saccocoelium cephali	1	Mugil cephalus	Blasco-Costa et al., 2009b	FJ211233
Saccocoeliumo besum	2	Liza ramada	Blasco-Costa et al., 2009b	FJ211259- FJ211260
Saccocoelium tensum	2	Liza ramada	Blasco-Costa et al., 2009b	FJ211257- FJ211258
Dicrogaster contracta	1	Liza aurata	Blasco-Costa et al., 2009b	FJ211261
Dicrogaster perpusilla	1	Liza ramada	Blasco-Costa et al., 2009b	FJ211238
Lecithobotrys putrescen	1	Liza saliens	Blasco-Costa et al., 2009b	FJ211236
Litosaccusbrisbanensis	1	Mugil сephalus	Andres et al., 2014	KM253765
Haploporus benedeni	1	Liza ramado	Blasco-Costa et al., 2009b	FJ211237
Ragaia lizae	1	Liza aurata	Blasco-Costa et al., 2009b	FJ211235
Forticulcitinae
Forticulcita isabelae	1	Mugil curema	Andrade-Gómez et García Valera, 2021	MT957787
Forticulcita macropharyngis	1	Mugil curema	Andrade-Gómez et García Valera, 2021	MW796513
Forticulcita minuta	1	Mugil cephalus	Andrade-Gómez et García Valera, 2021	MT957822
Forticulcita venezuelensis	1	Mugil curema	Andrade-Gómez et García Valera, 2021	MW796522
Forticulcita gibsoni	1	Mugil cephalus	Blasco-Costa et al., 2009b	FJ211239
F. apiensis	1	Mugil cephalus	Andres et al., 2015	KP761087
F. platana	1	Mugil liza	Andres et al., 2015	KP761086
Xiha fastigata	1	Mugil cephalus	Andres et al., 2015	KP761088
Overstreetoidespacificus	2	Mugil curema	Andrade-Gómez et García Valera, 2021	MT957753- MT957754
Ekuarhuni papillatum	2	Mugil sp.	Andrade-Gómez et García Valera, 2021	MT957687- MT957688
Ekuarhuni mexicanum	2	Mugil sp.	Andrade-Gómez et García Valera, 2021	MW796487- MW796488
Chalcinotrematinae
Saccocoelioides sp.	1	Unidentified molly(Poecilidae)	Curran et al., 2006	EF032696
Saccocoelioides beauforti	1	Mugil cephalus	Curran et al., 2018	MG925104
Saccocoelioides elongatus	1	Prochilodus lineatus	Curran et al., 2018	MG925108
Saccocoelioides magnus	1	Cyphocharax voga	Curran et al., 2018	MG925112
Saccocoelioides nanii	1	Prochilodus lineatus	Curran et al., 2018	MG925114
Saccocoelioides orosiensis	1	Poecilia gillii	Curran et al., 2018	MG925118
Saccocoelioides tkachi	1	Astyanax aeneus	Curran et al., 2018	MG925122
Intromugil mugilicolus	1	Mugil cephalus	Pulis et al., 2013	KC430096
Intromugil alachuaensis	1	Mugil cephalus	Pulis et al., 2013	KC430095
Hapladeninae
Hapladena acanthuri	1	Acanthurus chirurgus	Andres et al., 2018	MH244119
Hapladena cf. varia	1	Acanthurus chirurgus	Andres et al., 2018	MH244120
Megasoleninae
Megasolena hysterospina	1	Archosargus rhomboidalis	Andres et al., 2018	MH244121
Megasolena sp.	1	Holacanthus ciliaris	Andres et al., 2018	MH244122
Cadenatelinae
Cadenatella americana	1	Kyphosussectatrix	Andres et al., 2018	MH244117
Cadenatella floridae	1	Kyphosus incisor	Andres et al., 2018	MH244118
Atractotrematidae
Isorchis anomalus	1	Chanos chanos	Andres et al., 2016	KU873018
Isorchis currani	1	Selenotoca multifasciata	Huston et al., 2018	KU873017
Isorchis megas	1	Selenotoca multifasciata	Andres et al., 2016	KU873015
Brachycladiidae
Brachycladium goliath	1	Balaenoptera acutorostrata	Briscoe et al., 2016	KR703279
Monorchiidae
Hurleytrematoides chaetodoni	1	Chaetodon striatus	Andres et al., 2018	MH244116

PCR products were directly sequenced using an ABI Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, USA) as recommended by the manufacturer. The internal sequencing primers for 28S rDNA are described in Tkach et al. (2003PCR product sequences were analysed using an ABI 3500 genetic analyser at the FSC of Biodiversity FEB RAS. Sequences were submitted to the GenBank database (Table 2).

Alignments and phylogenetic analysis

Ribosomal DNA sequences were assembled using the SeqScapev. 2.6 software provided by Applied Biosystems. Alignments and estimations of the number of variable sites and sequence differences were performed using the MEGA 7.1 software (Kumar et al., 2016).

Phylogenetic analysis was performed with Bayesian Inference (BI) using MrBayes 3.2.6 program (Ronquist et al., 2012).The best nucleotide substitution models, the TVM + I + G (Posada, 2003) was estimated with jModeltest v. 2.1.5 software (Darriba et al., 2012) using BIC criterion (Huelsenbecket al., 2001).

Monte Carlo Markov chains algorithm was performed with 1000000 generations during two independent runs, sampling each 1000 generation and burnin of first 25% of all generations. Significance of phylogenetic relationships was estimated with posterior probabilities (Huelsenbeck et al., 2001).

Sequences of 28S rDNA of Brachycladium goliath (van Beneden, 1858) Fraija-Fernández, Aznar, Raga, Gibson &Fernández, 2014 and Hurleytrematoides chaetodonti (Manter, 1942) Yamaguti, 1954 from GenBank were used as the outgroup (Table 2).

Saccocoelium cephali Blasco-Costa, Montero, Gibson, Balbuena, Raga &Kostadinova, 2009

Host:Hydrobia acuta(Draparnaud, 1805)

Locality

Black Sea, near Sevastopol (44°27′49′′ N, 33°51′37′′ E)

Site

gonads

Intensity of infection IE = 1%, II = 1–30

Description

Sporocyst long, 238–356 (302 ± 24) × 73–133 (95 ± 11) µm, fill gonads of their host.

Cercariae (Based on 10 specimens, Table 1, Figs. 1 and 2).

Body oval. Tegument without spines. Oral sucker subterminal, almost the same size as ventral sucker. Ventral sucker in middle of body. Two global eye spots behind oral sucker. Oral to ventral sucker distance (493 ± 54) slightly less than ventral sucker to end of body distance (651 ± 114). Prepharynx present. Pharynx round, muscular, well defined. Oesophagus long. Intestinal bifurcation slightly posterior to ventral sucker in two sacciform branches. Two digestive branches reach the posterior 1/3 of body. Excretory bladder Y-shaped, both branches approach level of ventral sucker. Stenostome excretory system. Formula of excretory system is 2 [(2 + 2 + 2) + (2 + 2 + 2)] = 24.Tail slender, unadorned, almost twice as short as body length.

Adolescaria

The process of encystation of 1 cercaria in same mollusc was observed (adolescaria).Cyst oval, elongate, 180 ×116 µm (Fig. 3).

Molecular data

The alignment consisted of 1022bp for the 28S rRNA gene of two investigated haploporid cercariae specimens collected from Hydrobia acuta from the Black Sea basin.. The nucleotide composition of the 28S rDNA fragment was 25.6% for T(U), 21.9% for C, 21.0% for A, and 31.5% for G bases, which was typical for Saccocoelium species. A 1022bp 28S rDNA sequences from this study was highly similar to that for Saccocoelium cephali ex Mugil cephalus from Spain (FJ211233).These sequences differ from each other with single T/C transition. However, we cannot identify is it substitution fixed because of single sequence of adult S. cephalic is available. Genetic p-distances between our specimens and S. cephali were 0.098 ± 0.093%, whereas between our samples and other Saccocoelium species ranged from 2.75 ± 0.5% (S. tensum) to 4.8 ± 0.6% (S. brayi), demonstrating typical interspecific differentiation level for Haploporidae by 28S rDNA sequence data(Table 3).

Table 3

Interspecific genetic p-distance values (%, below diagonal) and standard errors (above diagonal) for Haploporinae
		1	2	3	4	5	6	7	8	9	10
1	Cercaria ex H. acuta #1		0	0.093	0.621	0.632	0.508	0.828	0.832	0.884	0.791
2	Cercaria ex H. acuta #2	0		0.093	0.621	0.632	0.508	0.828	0.832	0.884	0.791
3	Saccocoeliumcephali	0.098	0.098		0.61	0.62	0.495	0.817	0.823	0.872	0.783
4	Saccocoeliumobesum	4.617	4.617	4.519		0.293	0.558	0.764	0.798	0.878	0.777
5	Saccocoeliumbrayi	4.813	4.813	4.715	0.884		0.593	0.777	0.84	0.88	0.799
6	Saccocoeliumtensum	2.75	2.75	2.652	3.733	4.126		0.72	0.797	0.829	0.761
7	Dicrogastercontracta	6.883	6.883	6.785	6.293	6.686	5.801		0.606	0.809	0.823
8	Dicrogasterperpusilla	8.358	8.358	8.26	8.063	8.456	7.473	4.523		0.881	0.863
9	Haploporusbenedeni	8.644	8.644	8.546	8.35	8.743	7.859	6.785	8.063		0.844
10	Litosaccusbrisbanensis	7.389	7.389	7.291	7.685	8.374	6.7	7.594	9.172	8.473

Phylogenetic analysis from our study generatedwith Bayesian algorithm showed that studied cercariae specimens ex H. acuta from the Black Sea was within hapolporine clade and closely related with S. cephali from Spain (Fig. 4).

Adult warms of Saccocoelium cephali was first described by Isabel Blasco-Costa with co-authors in 2009 (Blasco-Costa et al., 2009a) from the Mediterranean Sea (Spain). The Black Sea cercariae and metacercariae of S.cephali trematodes have not been described before.

In the Black Sea parthenithae of haploporid trematode firstly were noted by Sinitzin (1911). The author reported two morphologically similar haploporid cercariae: Cercaria metentera (Sinitzin, 1911) from gastropod Iravadia quadrasi (O. Boettger, 1893) (= Rissoa venusta) and C. mesentera (Sinitzin, 1911) from mollusc Ecrobia ventrosa (Montagu, 1803) (= Hydrobia ventrosa) in the water off Sevastopol. According to Sinitzin (1911), C. mesentera differs from C. metentera in the sizes of the body, oesophagus, caeca, excretory bladder form and absence of prepharynx. Afterwards the trematode fauna of molluscs in the Black Sea was widely studied by Dolgikh (1965). Author reported one type of haploporid parthenitae C. metentera (Sinitzin, 1911) from mollusc Rissoa splendida (Eichwald, 1830) and discussed their biology and the identity of the adult form, suggesting that it is a species of Saccocoelium tensum Looss, 1902.

Based on morphological data, our samples belong to family Haploporidae. Most of morphological characteristics of specimens investigated agree with those of Saccococoelium sp. and closest to S. cephali by shape and length of the body, ratio of suckers, ratio of tail length to body, shape and size of pharynx.The found samples of S.cephali are differ from the all other species of Saccocoelium in its distinctly bigger size (size of body and other organs see in Table 1). Thus, morphological data supports the identification of haploporid cercaria from mollusc H.acuta in the Black Sea as Saccocoelium cephali.

The body surface, suckers and tail structure are described in present SEM research. But they were not reported for Saccocoelium trematode larvae until now. Thus, the new characters are provided in this article. The entire surface of the cercaria was not armed with spines. On the figures some authors noted the armed protein coagulation on body surface of Saccocoelium cercaria from Mediterranean (Fares, Maillard, 1974) and Azov Seas (Kudlai, 2011). Our SEM study reflects the absence of formations on the body surface (Fig. 2).

Based on molecular data, cercariae ex Hydrobia acuta from our study belong to species Saccocoelium cephali unambiguously, despite of single substitution between 28S rDNA sequences of our specimens and S. cephali from Ebro Delta, Spain (GenBank #?). We do not exclude that this substitution will be characterized as fixed after additional 28S rDNA sequence data of adult S. cephali will be available. In any case our taxonomical conclusion about cercaria from Black Sea will stand firm. Besides, fixed substitutions between 28S rDNA sequences within same species are known for Haploporidae (Besprozvannykh et al., 2015; Atopkin et al., 2015).

In the life cycle of haploporid trematode, marine gastropods of the family Hydrobiidae and Rissoidae serve as the first intermediate hosts (Fares, Maillard, 1974) and mullet fish as definitive hosts (Dmitrieva, Gaevskaya, 2001; Popjuk, Pron’kina, 2011).

Thus, we can expect that Saccocoelium cephali trematode completes its life cycle in the Black Sea, using molluscs Hydrobia acuta as first intermediate host and the definitive host can be mullet fish.

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgements

Authors are grateful to researcher of Institute of Biology of the Southern Seas. PhD Makarov V. Mikhail for collection and identification of Black Sea molluscs and Lishaev Vyacheslav for carried out SEM research.

This work was supported by budget funding of RAS (project №124022400148-4) “Biodiversity as the basis for the sustainable functioning of marine ecosystems, criteria and scientific principles for its conservation”.

Declarations

Funding:This work was carried out within the framework of IBSS state research assignment “Biodiversity as the basis for the sustainable functioning of marine ecosystems, criteria and scientific principles for its conservation” (No. 124022400148-4).

Conflict of interest: The authors declare that there is no conflict of interest.

Availability of data and material: DNA sequence data was deposited in GenBank.

Author Contributions Statement

Y.V.B. provided support for the fieldwork portion of the study, including transport, supplies,

and catching of molluscs. Y.V.B. carried out dissections and parasitological examinations. When parasites were encountered, they were photographed by Y.V.B. Figure 1, Figure 2 and Figure 3 was made by Y.V.B. All molecular biology work and analysis was carried out by D.M.A. Figure 4 was created by A.D.M. Table 1 was created by Y.V.B and Table 2 was made by D.M.A. All authors reviewed the manuscript and provided edits on the final version.

Code Availability: Not applicable.

Animal Handling and Use: All applicable institutional, national and international guidelines for the care and use of animals were followed.

Ethical standards: All applicable institutional, national and international guidelines for the care and use of animals were followed, protocol № 2(7)/24 of March 28, 2024

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First modern morphological and molecular description of Saccocoelium cephali larvae stages (Digenea: Haploporidae) from the Black Sea

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Abstract

Figures

Introduction

Materials and Methods

Sample collection

Morphological data

Scanning electron microscopy

Statistical methods

DNA extraction, amplification and sequencing

Alignments and phylogenetic analysis

Results

Description

Adolescaria

Molecular data

Discussion

Declarations

References

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