Research Article |
Corresponding author: Andreas C. Dimitriou ( dimitriou.c.andreas@ucy.ac.cy ) Academic editor: Elisabeth Hornung
© 2018 Andreas C. Dimitriou, Stefano Taiti, Helmut Schmalfuss, Spyros Sfenthourakis.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Dimitriou AC, Taiti S, Schmalfuss H, Sfenthourakis S (2018) A molecular phylogeny of Porcellionidae (Isopoda, Oniscidea) reveals inconsistencies with present taxonomy. In: Hornung E, Taiti S, Szlavecz K (Eds) Isopods in a Changing World. ZooKeys 801: 163-176. https://doi.org/10.3897/zookeys.801.23566
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Porcellionidae is one of the richest families of Oniscidea globally distributed but we still lack a comprehensive and robust phylogeny of the taxa that are assigned to it. Employing five genetic markers (two mitochondrial and three nuclear) we inferred phylogenetic relationships among the majority of Porcellionidae genera. Phylogenetic analyses conducted via Maximum Likelihood and Bayesian Inference resulted in similar tree topologies. The mtDNA genes cytochrome oxidase I (COI) and 16s rRNA (16s) were used for clade dating using previously published mutation rates. Our results provide evidence against the monophyly of both Porcellionidae and the largest genus of the family Porcellio. These results are compared to previous published work based on morphological evidence. The genera Leptotrichus and Brevurus are not grouped with the rest of Porcellionidae whereas Agnaridae are grouped with part of Porcellionidae. Armadillidium and Schizidium (Armadillidiidae) occupy a basal position on the phylogenetic tree. Even though the African genera Tura and Uramba (distributed in East Africa) are grouped together there is no general geographical pattern in other sub-clades. Additional taxonomic issues that arise in this work such as the assignment of the recently described genus Levantoniscus, arealso discussed. The status of Porcellionidae should be further revised and morphological characters traditionally used in Oniscidea taxonomy should be reconsidered in view of molecular evidence. The origin of the monophyletic clade within Porcellionidae as indicated in the present work is dated back to the Oligocene (~32 mya).
Crinocheta , genetic markers, monophyly, node dating, taxonomic revision, woodlice
The Oniscidea family Porcellionidae is one of the richest in species, with 333 species, belonging to 19 genera, currently assigned to it (
Different authors have found Porcellionidae to be closely related with Oniscidae, Trachelipodidae, Cylisticidae, Agnaridae or Armadillidiidae (
Members of Porcellionidae were originally reported from the circum-Mediterranean region, Atlantic islands, Arabian Peninsula and East Africa. Nowadays they are known from all over the world, being introduced into many regions by human activities (
The present study aims to a more detailed investigation of phylogenetic relationships among genera of Porcellionidae, using two mitochondrial and three nuclear genes that allow estimation of divergence times among extant taxa.
Isopod specimens belonging to five Porcellionidae genera, one to Trachelipodidae (Levantoniscus Cardoso, Taiti & Sfenthourakis, 2015) and two to Armadillidiidae (Armadillidium Brandt, 1831 and Schizidium Verhoeff, 1901) were collected on Cyprus between 2014 and 2016. Additional specimens came from the collection of the Istituto per lo Studio degli Ecosistemi, deposited in the Museum of Natural History of the University of Florence, and from the personal collection of one of the authors (H.S.). Members of the families Armadillidiidae, Agnaridae and Trachelipodidae that are assumed to be closely related to Porcellionidae were included in the analyses to test the monophyly of the latter, whilst specimens of the more distant families Scyphacidae (Actaecia euchroa Dana, 1853) and Philosciidae (Chaetophiloscia elongata (Dollfus, 1884) were included as outgroups. More details about specimens used are given in Table
We were not able to include specimens of five Porcellionidae genera, namely the monotypic Congocellio Arcangeli, 1950 and Tropicocellio Arcangeli, 1950, both distributed in the Democratic Republic of the Congo, Dorypoditius Verhoeff, 1942 from Mozambique, Atlantidium Arcangeli, 1936 form Madeira, and Pondo Barnard, 1937 from South Africa (Pondoland and Natal).
Species, locality of origin, available sequence data from targeted genes, and GenBank accession numbers of individuals used in the molecular phylogenetic analyses.
Species (code) | Locality | Genes | Acc. No | ||||
---|---|---|---|---|---|---|---|
COI | 16s | 18s | 28s | NAK | |||
Porcellionidae | |||||||
Proporcellio vulcanius (Verhoeff, 1908) (1) | Cyprus (Larnaca) | √ | √ | √ | √ | MG887933/MG887948/-/MG887988/MG887906 | |
Agabiformius excavatus Verhoeff, 1941 (2) | Cyprus (Paphos) | √ | √ | √ | √ | -/MG887955/MG887969/ MG888009/MG887921 | |
A. excavatus (3) | Cyprus (Paphos) | √ | √ | -/MG887956/-/-/MG887922 | |||
Porcellio laevis Latreille, 1804 (4) | Cyprus (Lemesos) | √ | √ | √ | √ | √ | MG887936/MG887957/MG887986/MG887993/MG887913 |
P. laevis (5) | Cyprus (Lemesos) | √ | √ | √ | √ | √ | MG887937/MG887958/MG887987/ MG887994/MG887914 |
Porcellionides pruinosus (Brandt, 1833) (6) | Cyprus (Larnaca) | √ | √ | √ | √ | MG887934/MG887949/-/MG888010/MG887907 | |
P. pruinosus (7) | Cyprus (Larnaca) | √ | √ | √ | √ | MG887935/ MG887950/-/ MG887989/MG887908 | |
Leptotrichus kosswigi Strouhal, 1960 (8) | Cyprus (Paphos) | √ | √ | -/-/-/MG888013/MG887915 | |||
L. kosswigi (9) | Cyprus (Paphos) | √ | √ | √ | √ | -/MG887963/MG887970/ MG888014/MG887916 | |
Porcellio nasutus Strouhal, 1936 (10) | Greece (Parnon) | √ | √ | √ | √ | MG887944/ MG887953/-/MG887998/MG887910 | |
P. nasutus (11) | Greece (Parnon) | √ | √ | √ | √ | -/MG887954/MG887980/ MG887999/MG887911 | |
Tura sp. (12) | Kenya (Mombasa) | √ | √ | √ | √ | √ | MG887946/ MG887966/ MG887983/MG888001/MG887920 |
Caeroplastes porphyrivagus (Verhoeff, 1918) (13) | France (Toulon) | √ | √ | √ | MG887932/-/ MG887981/ MG887990/ - | ||
Uramba triangulifera Budde-Lund, 1910 (14) | Kenya (Aberdare National Park) | √ | √ | √ | -/ MG887961/-/MG888002/MG887923 | ||
Thermocellio sp. (15) | Tanzania (Dar es Salaam) | √ | √ | -/ MG887962/-/ MG887995/- | |||
Lucasius pallidus (Budde-Lund, 1885) (16) | Italy (Sardinia) | √ | √ | √ | -/-/MG887974/ MG887992/MG887917 | ||
Mica tardus (Budde-Lund, 1885) (17) | Italy (Sardinia) | √ | √ | -/ MG887959/-/MG887996/- | |||
Acaeroplastes melanurus melanurus (Budde-Lund, 1885) (18) | Italy (Sardinia) | √ | √ | √ | √ | √ | MG887945/ G887960/MG887982/ MG887991/MG887912 |
Soteriscus laouensis Taiti & Rossano, 2015 (19) | Morocco (Tirinesse) | √ | √ | √ | √ | √ | MG887931/MG887964/MG887975/MG887997/MG887918 |
Brevurus masandaranus Schmalfuss, 1986 (20) | Iran | √ | √ | -/-/-/MG888008/MG887919 | |||
Porcellionides cilicius (Verhoeff, 1918) (21) | Cyprus (Nicosia) | √ | -/-/-/-/MG887909 | ||||
Trachelipodidae | |||||||
Levantoniscus bicostulatus Cardoso, Taiti & Sfenthourakis, 2015 (22) | Cyprus (Paphos) | √ | √ | √ | -/-/MG887976 /MG888000/MG887928 | ||
Trachelipus aegaeus (Verhoeff, 1907) (26) | Greece (Naxos) | √ | √ | √ | √ | EF659961/KF891440/ MG887984 /-/MG887925 | |
Agnaridae | |||||||
Hemilepistus klugii (Brandt, 1933 (23) | Iran (Isfahan) | √ | √ | √ | √ | √ | MG887938/MG887951/MG887978 /MG888011/MG887926 |
H. schirazi Lincoln, 1970 (24) | Iran (Shahreza) | √ | √ | √ | √ | √ | MG887939/MG887952/MG887979 /MG888012/MG887927 |
Agnara madagascariensis (Budde-Lund, 1885) (25) | U.A.E. | √ | √ | √ | -/-/MG887977 /MG888003/MG887924 | ||
Armadillidiidae | |||||||
Armadillidium vulgare (Latrteille, 1904) (27) | Cyprus (Limassol) | √ | √ | √ | √ | KR424609/AJ419997/ MG887972/MG888006/- | |
Schizidium fissum (Budde-Lund, 1885) (28) | Cyprus (Paphos) | √ | √ | -/-/MG887973/MG888005/- | |||
Philosciidae | |||||||
Chaetophiloscia elongata (Dollfus, 1884) (29) | Italy (Sardinia) | √ | √ | √ | √ | √ | KJ668161/AJ388091/MG887971/MG888004/-/MG887929 |
Scyphacidae | |||||||
Actaecia euchroa Dana, 1853 (30) | New Zealand | √ | √ | √ | √ | √ | GQ302701/AJ388093/MG887985/MG888007/MG887930 |
Fresh specimens were placed in 96% alcohol immediately after collection and stored at -20 °C. The majority of samples from museums and private collections had been preserved in 70% alcohol. Whole animals or legs of larger specimens were used for extraction of total genomic DNA using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following manufacturer’s instructions. NanoDrop 2000/200c (Thermo Fisher Scientific Inc., USA) was used to determine the final concentration and purity (A260/A280nm absorption rate) of DNA extractions.
The following mitochondrial and nuclear genetic loci were targeted using common PCR procedures: partial mitochondrial cytochrome c oxidase subunit 1 (COI), ribosomal 16S rRNA (16s), the nuclear, non-coding 18S ribosomal RNA (18s) and 28S ribosomal RNA (28s), and the protein coding Sodium-Potassium Pump (NAK). Mitochondrial COI and 16s genes were successfully amplified using the universal LCO1490/HCO2198 (
All PCR reactions were performed in a Veriti thermal cycler (Applied Biosystems, USA) with the following common steps: a) initial denaturation for 5 min at 94 °C, followed by b) 5 cycles of 3 minutes equally separated at 94 °C/60 °C/72 °C, c) 5 cycles of 3 minutes equally separated at 94 °C/55 °C/72 °C, d) 10 cycles of 3 minutes equally separated at 94 °C/50 °C/72 °C, e) 10 cycles of 3 minutes equally separated at 94 °C/47 °C/72 °C, f) 10 cycles of 3 minutes equally separated at 94 °C/42 °C/72 °C, and g) a final extension step of 72 °C for 10 min. Beyond fresh specimens, this touchdown PCR approach with 50 cycles in total allowed us to successfully amplify genes from ill-preserved samples increasing specificity, sensitivity and yield, eliminating aspecific products (
The final reaction volume in all cases was 20 μL, and consisted of 0.1 µL of Kapa Taq DNA Polymerase (5U/μL), 1.2 μL of 25 mM MgCl2, 2 μL of Kapa PCR buffer A, 0.6 μL of 10 mM dNTP (Kapa) 0.6 μL of each primer (10 µM) and >10 ng of DNA template. PCR product purification was made using Qiaquick Purification Kit (Qiagen, Germany) under manufactures protocol instructions. Both DNA strands of purified products were sequenced at Macrogen facilities (Amsterdam, The Netherlands).
Sequence chromatograms were manually edited and assembled with CodonCode Aligner (v. 3.7.1; CodonCode Corp., USA). Separate multiple alignments for each gene/data set were performed using MAFFT v.7 (
Construction of phylogenetic trees was conducted using Bayesian inference (BI) and Maximum Likelihood (ML) methods. The analysis of BI was implemented in MRBAYES v. 3.2.6 (
RAxML (v. 8.1.21) (
Molecular dating of clades was inferred using BEAST v. 2.3.0 (
Four independent runs were performed for 100 million generations, each sampling every 5,000th generation. An uncorrelated lognormal relaxed clock under a Yule tree prior and the default options for all other prior and operator settings, were used in each case. Trace plots were inspected in order to compare the divergence estimates across runs and ensure the convergence of Markov Chain Monte Carlo chains using TRACER v. 1.5 (
At least four out of five targeted genes were successfully amplified and sequenced for the great majority of available individuals, with final DNA extraction yield over 20 ng/μl and A260/A280 purity rate over 1.5.Since some important samples were old (collected more than two decades ago, mainly from Africa) or ill-preserved for a long time (i.e., in 70% alcohol) we didn’t manage to retrieve sequences from all targeted genes. However, specimens not represented by all gene fragments were also included in the analyses. The final concatenated alignment obtained consisted of 3,841 base pairs (bp). More details about the aligned sequences length, conserved, variable and parsimony-informative sites for each gene are given in Table
Aligned bases length, conserved, variable, and parsimony-informative sites for each gene used in the present analysis.
Gene | Alignment length (bp) | Conserved sites | Variable sites | Parsimony informative sites |
---|---|---|---|---|
COI | 655 | 214 | 434 | 302 |
16S | 454 | 151 | 277 | 211 |
18S | 863 | 417 | 332 | 177 |
28S | 1167 | 314 | 827 | 567 |
NAK | 702 | 512 | 188 | 109 |
Available sequences were separated in different groups at the genus level except for Porcellio species which were treated as different groups due to the alleged non-monophyly of the genus. Between groups p-genetic distances for each gene are given in Suppl. material
The best-fit nucleotide substitution models for each partition/gene selected under the BIC criterion were (for both MRBAYES and BEAST) the HKY+G+X, HKY+G+X, TRNEF+G, TRN+G, TRN+G+X and GTR+G+X for COI, 16s, 18s, 28s and NAK genes, respectively. The selected model under --raxml commandline option at PartionFinder was the GTR+G (-ln =26511.0556641) for all genes.
Maximum Likelihood and Bayesian Inference analyses (implemented both in BEAST and MRBAYES) resulted into phylogenetic trees with similar, well-supported topologies. Given the congruence among the results of the two methods, only the Bayesian tree is presented herein (Figure
Dated phylogram based on concatenated data set including five genes (COI, 16s, 18s, 28s, NAK), generated using a relaxed lognormal clock in BEAST. BI posterior probabilities (>0.9) and ML bootstrap values (>60) are presented above the nodes. Estimated mean divergence time is given below the nodes only where nodes are statistically supported or the topology was identical between BI, ML and BEAST analyses. Subclades including individuals from more than one species have been collapsed to genus level, since all (except Porcellio) were monophyletic. Abbreviations: P. Porcellionidae, T. Trachelipodidae, A. Agnaridae, R. Armadillidiidae. Numbers in parentheses after each taxon name refer to numbering of taxa in Table
Our results provide evidence against the monophyly of both the family Porcellionidae and the genus Porcellio. Brevurus appears to belong to a supported distant clade, external to that formed by the remaining Porcellionidae+Trachelipodidae+Agnaridae. Leptotrichus is an external branch to Agnaridae + part of Porcellionidae. Monophyly of Agnaridae is supported. Levantoniscus forms the sister clade of all monophyletic Porcellionidae. Finally Armadillidiidae branches early in the tree, not showing any close relationship to Porcellionidae.
The African genera Tura and Uramba are sister taxa sharing a common ancestor at around 22.2 mya (95% HPD 12.1 – 33.5 mya) and are grouped with Agabiformius. On the other hand, Thermocellio, also distributed in Kenya and the neighboring Tanzania, appears to be more closely related to Porcellio laevis, native to Europe and North Africa. Another African/Atlantic genus, Soteriscus, forms a well-supported clade with Lucasius and Mica that are distributed in Africa and on some Mediterranean islands. The Mediterranean genera Acaeroplastes, Caeroplastes, Porcellionides and Proporcellio, together with part of Porcellio, are grouped in the most derived clade that diverged at around 27 mya.
The genus Porcellio as currently perceived is represented in two well-supported separate clades. P. laevis groups with Thermocellio while P. nasutus with Acaeroplastes in a clade also including Caeroplastes.
Genetic distances between Porcellionidae genera (or species in the case of the non-monophyletic Porcellio) varied significantly among genes. The range of variation per gene is: COI: 16.9–50.3 %; 16s: 16.9–36.5 %; 18s:3.6–28.5 %; 28s:0.4–44.2%; NAK: 2.3–9.1%. The p-distances between Trachelipus and Agnara for NAK, and P. laevis and Lucasius for 18s, could be artifacts due to the comparatively shorter sequence length in Agnara and P. laevis, respectively (see Suppl. material
It is worth noticing also that minimum and maximum distances are not exhibited by the same taxa for all genes. More specifically, highest / lowest genetic divergence is found between the following groups: Tura - Porcellio nasutus / Soteriscus – Leptotrichus (16s), Porcellio laevis – Lucasius / Proporcellio - Porcellionides (COI), Agabiformius - Porcellio nasutus / Caeroplastes - Acaeroplastes (18s), Brevurus - Thermocellio / Porcellio laevis - Thermocelio (28s) and Uramba – Brevurus / Proporcellio - Porcellionides (NAK). The allegedly congeneric Porcellio species never exhibit a minimum genetic distance.
This is the first comprehensive study aiming to resolve phylogenetic relationships among Porcellionidae genera using a multi-locus approach, thus increasing reliability of results. Our findings undermine the monophyly of both the family Porcellionidae and the genus Porcellio, in line with suggestions by previous authors (
The extremely high genetic distances, which reached up to 50.3 in mtDNA and 44.2 in nDNA, are confirming the vast divergence among taxa within Porcellionidae. Observed inconsistencies of group distances among different genes highlight the usefulness of the multi-locus approach followed herein for a reliable phylogenetic reconstruction of the taxa examined.
In view of the herein estimated phylogeny, a monophyletic Porcellionidae should exclude Brevurus and Leptotrichus. Moreover, the supposedly subtle morphological differences between Leptotrichus and Agabiformius that had led to a presumed sister-group relationship between these genera, are misleading, since they are found to be very distant (
The genus Levantoniscus, tentatively assigned to Trachelipodidae (
As indicated by the tree topology, Porcellionidae is more closely related to Trachelipodidae and Agnaridae rather than Armadillidiidae. A similar result has been found by
In conclusion, the monophyly of Porcellionidae as currently perceived cannot be supported by molecular evidence. Of course, we still need to identify phenotypic synapomorphies defining the family, since the characters used so far cannot be considered as valid. In addition, the genus Porcellio needs to be revised, as it appears to be polyphyletic, comprising of at least two separate groups.
The monophyletic subgroup of Porcellionidae seems to have an African origin, diverging at the end of the Palaeogene (Oligocene) and then differentiating further during the Miocene. Based on the cladochronology estimated herein, more basal cladogenetic events, leading to the branching of other related families, happened in the Eocene. This chronology is compatible with the very old (Mesozoic) origin of Oniscidea suggested by Broly et al. (2013,
We are grateful to Prof. Nikos Poulakakis and Emmanouela Karameta for their support with data processing.
Percentage sequence divergence among the main clades of Porcellionidae and Maximum Likelihood phylogenetic tree.