Multi-locus assessment of the phyletic status of Urolophidae (Elasmobranchii, Batoidea)

B.Sc. DISSERTATION

Mul1-locus assessment of the phyle1c status of

Urolophidae (Elasmobranchii, Batoidea)

Juan Sebas*an González

Advisor: Susana Caballero

Co-advisor: Andrés Del Risco

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Even though molecular phylogene*cs has mostly reshaped the previously morphological based taxonomy of the pre-molecular era, methodological limita*ons such as poor character sampling or superficial analyses are oFen overlooked. This has been evident for taxonomic groups with a complicated history of phylogene*c resolu*on such as batoids (skates and rays). Of par*cular interest is the family Urolophidae (Indo-Pacific round s*ngrays), which has been usually recovered as diphyle*c by recent molecular phylogene*c analysis despite the striking morphological resemblance of its two genera: Urolophus and Trygonoptera. In this study, the phyle*c status of the family urolophidae was assesed by re-evalua*ng their phylogeny with publicly available mitochondrial and nuclear loci (COI, NADH, RAG1 and POMC). My results show that the concatenated dataset and three out of four individual loci consistently recover a diphyle*c Urolophidae, while the nuclear RAG1 supports its monophyly. By removing the third codon posi*ons of mitochondrial loci, urolophid monophyly is also supported. Given these results, I speculate that the non-monophyly might be a result of the fast evolu*onary rates of mitochondrial loci, in contrast with the slower rate of the nuclear RAG1 locus, which may be more appropriate for deeply diverging lineages, which appear to be the case for urolophids. This should be further supported by the exclusion of fast evolving sites in mitochondrial datasets. As such, we refrain from sugges*ng taxonomic changes to this taxon as there is some molecular support for its monophyly in addi*on to their morpholocal synapomorphies, despite the preliminar findings of previous studies

Keywords: Long branch aXrac*on, monophyly, phylogene*cs, s*ngrays, Urolophidae,

INTRODUCTION

The widespread use of molecular phylogene*cs have challenged and re-shaped the previously morphological based taxonomy over all groups or organisms. While many well established groups have found support from molecules, several higher level taxa that were thought to represent natural groups are now known to be the outcome of extensive

homoplasy in morphological traits that ini*ally misled taxonomists of the pre-molecular cladis*c era. However, this over-reliance in molecular phylogene*cs has some*mes led researchers to make rushed taxonomic emenda*ons, even when results are conflic*ng. This has been especially evident for groups presen*ng deep and short internodes that are evaluated by studies that employ insufficient loci, inadequate data, or superficial methodological analyses (Betancur & Or^ 2014). The batoid order Mylioba*formes, which comprises the s*ngrays, is an excellent model group for illustra*ng an apparent need for taxonomic emenda*ons due to extensive non-monophyly at of some groups at various taxonomic levels (Last et al. 2016; Li et al. 2015). Instances in which new taxa have been unnecessarily erected based on insufficient data and shallow analyses have also been seen within this group, especially within the family Dasya*dae (i.e. Li et al. 2015).

Within mylioba*forms, an interes*ng case regarding this issue is the family Urolophidae, commonly called Indo-Pacific round s*ngrays. Urolophids are boXom-dwelling s*ngrays distributed over the coastal waters of the Indo-West Pacific. They consist in 28 species classified in two genera, Urolophus J. P. Müller & Henle, 1837 and Trygonoptera J. P. Müller & Henle, 1841. Although the monophyly of the family was previously evident by the overall similar appearance of its members and some alleged morphological synapomorphies (Figure 1) and by their occupa*on of the same oceanographic region, recent molecular analyses have found that urolophids represent a diphyle*c group, with each genera being distantly located from each other in the phylogene*c trees (Li et al. 2015; Naylor et al. 2013). If those analyses are effec*vely represen*ng the true evolu*onary history of urolophids, then the morphological similari*es between Urolophus and Trygonoptera species were independently acquired by convergent evolu*on, and the proposal of taxonomic changes should be considered to solve the non-monophyly of Urolophidae. As previously men*oned, this would not be an uncommon case given the ubiquitous nature of morphological homoplasies. However, the aforemen*oned studies employed single-locus analyses and recovered poor support for the deep internodes of the mylioba*form trees, calling into ques*on of whether such results should be considered reliable enough to proceed with taxonomic modifica*ons. In this study, I thoroughly evaluate the phyle*c status of the family Urolophidae by re-evalua*ng their phylogeny

with publicly available mitochondrial and nuclear loci and various par**ons and metholodogical procedures to evaluate if taxonomic changes are really necessary.

METHODS

In order to elucidate the phylogene*c posi*on and phyle*c status of the Urolophidae within Mylioba*formes, we obtained one representa*ve sequence of every urolophid species available in GenBank for at least one of four loci: the mitochondrial genes cytochrome oxidase subunit I (COI), NADH dehydrogensase subunit 2 (NADH2), the nuclear recombina*on ac*va*ng gene 1 (RAG1) and proopiomelanocor*n (POMC). To account for any poten*al affinity of a “urolophid” species to any other non-urolophid lineage, we also sampled a representa*ve species for every genus of the order

Mylioba*formes. Moreover, we included Zanobatus schoenleinii as the outgroup, because it is thought to be the sister group to the Mylioba*formes (Aschliman, et al., 2012). Detailed informa*on with accession numbers of sampled sequences is summarized in Table 1.

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Phylogene1c analyses

Sequences were ini*ally aligned with the MUSCLE algorithm implemented in GENEious (Kearse et al, 2012), with subsequent modifica*ons performed manually. Phylogene*c analyses were accomplished on each of the individual gene alignments, and on a concatena*on of the alignments of the four genes. We also conducted phylogene*c

analysis on the concatenated sequences of mitochondrial loci for two addi*onal par**ons: one with the nucleo*de sequences translated to amino acids, and another one excluding third codon posi*ons. The aforemen*oned procedures were performed by the soFware MEGA (Kumar et al. 2016).

Models of sequence evolu*on were es*mated for each of those alignments using JModelTest (Darriba et al, 2012) according to the Akaike Informa*on Criterion (AIC). The best finng models for each locus are indicated in Table 2. An addi*onal set of par**ons were performed in which Par**onFinder was employed to divide the sequences into

par**on blocks of subs*tu*on models for es*ma*ng models of sequence evolu*on, rather than assigning such models for each locus.

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Phylogene*c inference was then conducted for all par**ons with PhyML (Guindon et al. 2010) for maximum likelihood (ML) and *BEAST v1.8.1 (Drummond & Rambaut, 2007) for bayesian inference (BI). For the ML analysis, nodal support was assessed with 1000 bootstrap replicates. BI analysis was performed with unlinked par**on blocks for the concatenated alignment of four genes, choosing the Yule Tree prior as suggested for inter-species phylogenies (Ho, 2007). The individual gene alignments and the concatenated datasets were run for 10.000.000 genera*ons, sampling every 1000 genera*on. Chain convergence was then visually inspected in Tracer, discarding the first 10% of trees for each run and producing the maximum-clade credibility trees with TreeAnotator.

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RESULTS AND DISCUSSION

Our preliminary results with the concatenated dataset failed to find support for a monophyle*c Urolophidae, recovering the family as diphyle*c by separa*ng the genera Trygonoptera and Urolophus (Figure 3a). This is in agreement with results from previous studies (Naylor et al. 2013; Li et al. 2015), even though this was based on a concatena*on of four genes, in contrast with the single locus analyses of such studies. However, by par**oning the data in different ways, interes*ng results were found as summarized in Table 1. While conduc*ng the analysis with each of the four loci individually, we found that only the nuclear RAG1 gene supported the monophyly of Urolophidae with significant support from Bayesian and Maximum likelihood analyses. Addi*onally, when the analysis of the concatenated datasets was re-run excluding the 3rd _{codon posi*on of the protein}

coding sequences (COI and NADH2), urolophids were found to be monophyle*c with significant support from Maximum likelihood, although with low support from Bayesian inference. Based on these paXerns, I argue that the non-monophyly of urolophids as recovered by previous studies and some of the par**ons of the present study might be

explained by the fast evolu*onary rates of mitochondrial loci. It is well known that mitochondrial data are not well-suited for resolving phylogenies at deeper levels because of their rela*vely higher subs*tu*on rates, and this is further complicated when the internodes as such levels are short. In fact, that seems to be the case for urolophids, whose basalmost divergence seems to be deep into the mylioba*form phylogeny, with a very short internode close to the ini*al radia*on of the order. Such subs*tu*onal rates not only weaken phylogene*c signal, but may also led to bias in the form of long branch aXrac*on, which might play a role as well in clustering each genera of urolophids with non-urolophid lineages. Nuclear loci, having slower subs*tu*on rates, are oFen presumed to be more adequate for resolving the deeper nodes of a phylogeny. This is consistent with the fact that the nuclear gene RAG1 was the only loci employed in this study that found support for urolophid monophyly. Furthermore, the fact that urolophid monophyly is also recovered aFer removing 3rd _{codon posi*ons for the whole datasets also reinforces the}

idea that fastly evolving sequences are the core of the problem.

Although I acknowledge that the phyle*c Urolophidae is s*ll not an undisputed fact given all the “methodological tricks” needed to find support for its monophyly, it should be noted that in addi*on to the evidence from molecular data found in this study, it is also well backed by morphological data that were implicitly aXached to the taxonomic descrip*on of the family. As such, my results present a more parsimonious explana*on for the evolu*on of anatomical traits of their ocular and fin morphology, as well as their biogeography, as opposed to the diphyle*c hypothesis. With the results of this and other similar studies, I encourage a more careful examina*on of the data and overall higher methodological standars for phylogene*c studies that might lead to significant taxonomic modifica*ons.

REFERENCES

Aschliman N. C., Nishida M., Miya M., Inoue J.G., Rosana K.M., and Naylor G.J.P. (2012). “Body plan convergence in the evolu*on of skates and rays (Chondrichthyes: Batoidea), “Mol. Phylogenet. Evol., vol. 63, no. 1, pp. 28-48, 2012

Betancur-R R. & Or^ G. (2014). “Molecular evidence for the monophyly of atshes (Carangimorpharia: Pleuronec*formes)”. Molecular Phylogene*cs and Evolu*on, 73: 18-22.

Brown W.M., George M. & Wilson A.C. (1979). "Rapid evolu*on of animal mitochondrial DNA". Proceedings of the Na*onal Academy of Sciences. 76 (4): 1967–71.

Del Risco A., Angel L., Caballero S. (2016). “Taxonomic and Biogeographic implica*ons of a mul*-locus phylogeny of whiptail s*ngrays and allies (Mylioba*formes: Dasyatoidea)” (in prep.).

Darriba D., Taboada G.L., Doallo R., and Posada D. (2012). “jModelTest 2: more models, new heuris*cs and parallel compu*ng,” Nat. Methods, vol. 9, no. 8, p. 772, Aug.

Drummond A.J. and Rambaut A. (2007). “BEAST: Bayesian evolu*onary analysis by sampling trees.,” BMC Evol. Biol., vol. 7, p. 214.

Guindon S., Dufayard J.F., Lefort V., Anisimova M., Hordijk W., Gascuel O. (2010). "New Algorithms and Methods to Es*mate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0.” Systema*c Biology, 59(3):307-21, 2010.

Ho S. Y. M. (2007). “Calibra*ng molecular es*mates of subs*tu*on rates and divergence *mes in birds.,” J. Avian Biol., vol. 38, pp. 409–414

Kumar S., Stecher G., and Tamura K. (2016) MEGA7: Molecular Evolu*onary Gene*cs Analysis version 7.0 for bigger datasets. Molecular Biology and Evolu*on 33: 1870–1874. Last P.R., Naylor G.J. & Manjaji-Matsumoto B.M. (2016). "A revised classica*on of the family Dasya*dae (Chondrichthyes: Mylioba*formes) based on new mor-phological and molecular insights". Zootaxa. 4139 (3): 345–368

Lim P-E., Chong V.C. & Loh K-H. (2015). “Molecular and Morphological Analyses Reveal Phylogene*c Rela*onships of S*ngrays Focusing on the Family Dasya*dae

M. Kearse, R. Moir, A. Wilson, S. Stones-Havas, M. Cheung, S. Sturrock, S. Buxton, A. Cooper, S. Markowitz, C. Duran, T. Thierer, B. Ashton, P. Meintjes, and A. Drummond (2012). “Geneious Basic: An integrated and extendable desktop soFware plaxorm for the organiza*on and analysis of sequence data,” Bioinforma. , vol. 28 , no. 12 , pp. 1647–1649, Jun.

Naylor G.J.P., Caira J.N., Jensen K., Rosana K.A.M., Straube N. & Lakner C. (2012).

“Elasmobranch Phylogeny: A mitochondrial es*mate based on 595 species.” In Carrier J.C., Musak J.A. & Heithaus M.R. (editors), “The Biology of Sharks and Their Rela*ves”. p. 31-56. CRC Press, Taylor &Francis Group.

Nishida K. (1990). “Phylogeny of the suborder Mylioba*doidei”. Mem Fac Fish Hokkaido Univ 37: 1–108.

Yearsley G.K., Last P.R. & Gomon M.F. (2008). "Trygonoptera imitata sp. nov., a new s*ngaree (Myliobatoidei: Urolophidae) from southeastern Australia". In Last P.R., White W.T. & Pogonoski J.J. “Descrip*ons of new Australian Chondrichthyans”. CSIRO Marine and Atmospheric Research. pp. 261–267.

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Figure and Table Legends. Table 1.

List of GenBank accession numbers for taxa included in this study. Table 2.

Support for the monophyly of Urolophide from each of the methodological varia*ons and par**ons applied to the dataset. Cells filled with dashes indicate lack of evidence for monophyly. Analysis in which monophyly was recovered are represented by cells with numerical values deno*ng the nodal support for the clade.

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Figure 1.

Bayesian phylogene*c trees recovered by BEAST with posterior probabili*es annotates on branches. Inferred from the same concatenated dataset but excluding 3rd codon posi*ons, suppor*ng the monophyly of urolophids as well as the other mylioba*form families. Figure 2.

Bayesian phylogene*c trees recovered by BEAST with posterior probabili*es annotated on branches. Inferred from the concatenated dataset of four loci employing the complete nucleo*de sequences, illustra*ng urolophid polyphyly (as well as the polyphyly of some other families) as in previous studies.

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GENES RAYAS

MYLIOBATIFORMES Mitocondrial Nuclear

Family Scientific Name ND2/NADH2 (1,044bp) COI (655bp) RAG1 (948bp) POMC

UROLOPHIDAE

Trygonoptera galba - -

-Trygonoptera imitata JQ518936.1 EU399075.1 KT187533.1 KT187430.1

Trygonoptera mucosa - EU399063.1 - KT187431.1

Trygonoptera ovalis JQ519029.1 EU399064.1 KT187534.1 KT187432.1

Trygonoptera personata JQ519030.1 EU399066.1 -

-Trygonoptera testacea JQ518935.1 EU399089.1 JN184258.1

-Urolophus armatus - - -

-Urolophus aurantiacus KF927988.1 EU339355.1 -

-Urolophus bucculentus JQ518992.1 EU399099.1 -

-Urolophus circularis - - -

-Urolophus cruciatus JQ518937.1 EU399102.1 JN184129.1 KT187426.1

Urolophus deforgesi - - -

-Urolophus expansus JQ519040.1 EU399106.1 -

-Urolophus flavomosaicus JQ518991.1 EU399107.1 -

-Urolophus gigas - - KT187535.1 KT187433.1

Urolophus halleri JQ518939.1 KF930522.1 JN544201.1 KT187425.1

Urolophus javanicus - - -

-Urolophus kaianus - - -

-Urolophus kapalensis JQ519018.1 EU399121.1 -

-Urolophus lobatus JQ519026.1 EU399123.1 -

-Urolophus maculatus JQ518940.1 - -

-Urolophus mitosis - - -

-Urolophus neocaledoniensis - - -

-Urolophus orarius - - -

-Urolophus papilio - - -

-Urolophus paucimaculatus JQ518938.1 EU399131.1 KT187536.1 KT187434.1

Urolophus piperatus - - -

-Urolophus sufflavus - EU399136.1 -

-Urolophus viridis JQ518994.1 EU399139.1 KT187537.1 KT187435.1

Urolophus westraliensis JQ519004.1 EU399140.1 JN184260.1

-HEXATRYGONIDAE _{Hexatrygon bickelli} JQ518835.1 JN184061.1 JN184120.1 KT187420.1

PLESIOBATIDAE Plesiobatis daviesi KF927936.1 KF899649.1 JN184125.1 KT187424.1

GYMNURIDAE _{Gymnura australis JQ518828.1 EU398798.1 KT187547.1 KT187456.1}

MYLIOBATIDAE

Aetobatus narinari JQ518988.1 KR003775.1 JN184121.1 KT187462.1

Aetomylaeus nichofii KM396928.1 EU398510.1 JN184246.1

-Manta birostris JQ519062.1 EU398904.1 JN184248.1 KT187440.1

Mobula japanica JQ519163.1 KP175660.1 JN184122.1 KT187421.1

Myliobatis californica JQ519159.1 GU440417.1 KT187538.1 KT187437.1

Rhinoptera steindachneri JQ518918.1 JN184076.1 JN184124.1 KT187423.1

UROTRYGONIDAE Urobatis jamaicensis JQ518941.1 GU225504.1 -

-Urotrygon rogersi JQ519162.1 - KT187546.1 KT187455.1

POTAMOTRYGONIDAE

Heliotrygon sp - - -

-Paratrygon aiereba JN184309.1 - JN184257.1

-Plesiotrygon iwamae - EF532668.1 -

-Potamotrygon hystrix JN184071.1 JN184071.1 JN184127.1

-DASYATIDAE

Dasyatis brevis JN184058.1 JN184058.1 JN184114.1 KT187416.1

Dasyatis microps JQ518779.1 KU936199.1 -

-Himantura bleekeri - KC508511.1 -

-Himantura schmardae JN184062.1 JN184062.1 JN184126.1

-Neotrygon kuhlii KR019777.1 KR019777.1 KC249797.1 KT187417.1

Pastinachus atrus JQ518815.1 EU398973.1 KT187550.1 KT187472.1

ZANOBATIDAE Zanobatus schoenleinii JN184086.1 JN184086.1 JN184113.1

Tables Table 2.

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Subs. model by loci Subs. model by PartitionFinder

Subs. model by codon

Partition Bayes PP Likelihoo d BS

Bayes PP Likelihoo d BS

Bayes PP Likelihoo d BS

POMC - - - - N/A N/A

RAG-1 0.97 86 0.95 86 N/A N/A

COI - - -

-NADH2 - - -

-Concatenated

(All loci) - - - - N/A N/A

COI (aa) - - - N/A N/A

NADH2 (aa) - - - - N/A N/A

Concatenaed

(aa) - - N/A N/A N/A N/A

Concatenated (w/o 3rd codon positions for mitochondrial loci)

Graphics Figure 1.

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Zanobatidae

Hexatrygonidae

Urolophidae

Plesiobatidae

Gymnuridae

Myliobatidae

Dasyatidae

Urotrigonidae Potamotrygonidae

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Figure 2.

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Zanobatidae

Hexatrygonidae

“Urolophidae”

“Dasyatidae”

“Myliobatidae”

Gymnuridae

“Dasyatidae”

Potamotrygonidae

“Myliobatidae”

“Urolophidae”

Plesiobatidae

“Myliobatidae” “Dasyatidae” “Dasyatidae” Urotrygonidae