Org Divers Evol (2017) 17:813–820 https://doi.org/10.1007/s13127-017-0346-2 ORIGINAL ARTICLE An evolutionary timescale for terrestrial isopods and a lack of molecular support for the monophyly of Oniscidea (Crustacea: Isopoda) Luana S. F. Lins1,2,3 & Simon Y. W. Ho1 & Nathan Lo1 Received: 25 July 2016 /Accepted: 7 October 2017 /Published online: 15 October 2017 # Gesellschaft für Biologische Systematik 2017 Abstract The marine metazoan fauna first diversified in the of the suborder Oniscidea was not supported in any of our early Cambrian, but terrestrial environments were not colo- analyses, conflicting with classical views based on morphology. nized until at least 100 million years later. Among the groups This draws attention to the need for further work on this group of organisms that successfully colonized land is the crustacean of isopods. order Isopoda. Of the 10,000 described isopod species, ~ 3,600 species from the suborder Oniscidea are terrestrial. Although it Keywords Isopods . Oniscids . Phylogeny . Molecular clock is widely thought that isopods colonized land only once, some studies have failed to confirm the monophyly of Oniscidea. To infer the evolutionary relationships among isopod lineages, we Introduction conducted phylogenetic analyses of nuclear 18S and 28S and mitochondrial COI genes using maximum-likelihood and The marine metazoan fauna first diversified in the early Bayesian methods. We also analyzed a second data set com- Cambrian around 540 million years (Myr) ago, but continental prising all of the mitochondrial protein-coding genes from a (terrestrial and freshwater) environments were not colonized smaller sample of isopod taxa. Based on our analyses using a by the descendants of these taxa until at least 100 Myr later relaxed molecular clock, we dated the origin of terrestrial iso- (Labandeira 2005;Wilson2010). The crustacean order pods at 289.5 million years ago (95% credibility interval 219.6– Isopoda is one of the groups of organisms that have success- 358.9 million years ago). These predate the known fossil record fully colonized multiple environments, including continental of these taxa and coincide with the formation of the supercon- environments. Modern isopods represent a wide range of mor- tinent Pangaea and with the diversification of vascular plants on phological forms and include more than 10,000 described land. Our results suggest that the terrestrial environment has species. They are found in virtually all environments, includ- been colonized more than once by isopods. The monophyly ing the deep sea, land, and freshwater (Wilson and Hessler 1987; Poulin 1995). About 57% of extant isopod species are found in marine environments, whereas about 34% of the Electronic supplementary material The online version of this article species (~ 3600 species in the suborder Oniscidea) are terres- (https://doi.org/10.1007/s13127-017-0346-2) contains supplementary trial (Schmalfuss 2003; Wilson 2008; Sfenthourakis and Taiti material, which is available to authorized users. 2015). Although oniscids typically rely on habitats with high moisture levels, some species are found in arid and semi-arid * Luana S. F. Lins regions (Warburg 1992, 1995). Other species, such as those in [email protected] the genus Ligia Fabricius, 1798, are amphibious, living in the supralittoral zone. 1 School of Life and Environmental Sciences, The University of Colonization of the terrestrial environment by oniscids Sydney, Camperdown, NSW, Australia was once thought to have taken place via freshwater, but 2 Australian Museum, Sydney, NSW, Australia some studies have argued that the colonization of these two 3 School of Biological Sciences, Washington State University, environments occurred independently from marine envi- Pullman, WA 99164-4236, USA ronments (Carefoot and Taylor 1995; Tabacaru and 814 Lins L.S.F. et al. Danielopol 1996;Schmidt2008). Species found in Materials and methods supralittoral environments are considered to represent tran- sitional forms because of their intermediate adaptations to Data sets water restrictions. However, not all amphibious species have their most recent ancestor in the oceans but are in- We assembled two data sets for phylogenetic analysis: a data stead secondarily amphibious, such as Haloniscus Chilton, set derived from complete or near-complete mitochondrial 1920 (Wilson 2008). Species of the genus Ligia are con- genomes (both at the nucleotide and amino acid levels) and sidered as the prototypes of land isopods because of fea- a mito-nuclear supermatrix consisting of nucleotide sequences tures such as undifferentiated sac-like pleopods for gas from one mitochondrial protein-coding gene (COI) and two exchange, an open marsupium for egg incubation nuclear rRNA genes (18S and 28S). For both data sets, we (Bamphibious type^), and an open-type water-conducting obtained sequence data from GenBank (Supplemental system (Carefoot et al. 2000). Table 1). Most sequences were obtained from studies by The colonization of continental environments by other ar- Michel-Salzat and Bouchon (2000), Dreyer and Wägele thropods, such as insects and arachnids, is relatively well un- (2001), and Raupach et al. (2004, 2009). derstood (Friedrich and Tautz 1995; Grimaldi and Engel 2005; The mitogenome data set included sequences from 17 iso- Labandeira 2006), but the timing of the transition to land in pod species and one outgroup species (the amphipod isopods is uncertain. This is partly because of the paucity of Gammarus duebeni Lilljeborj, 1852). To minimize the impact fossil data for the group, which is a consequence of terrestrial of indels and mutational saturation, we limited our analyses to isopods lacking a waxy epicuticle and thus having low pres- the amino acid sequences and the first and second nucleotide ervation probability (Broly et al. 2013). codon positions of the 13 protein-coding genes. Preliminary Until recently, oniscids were thought to be a recently analyses of the mitochondrial ribosomal RNA genes revealed evolved group because of the lack of fossils older than the an abundance of indels and a high degree of mutational satu- Eocene (Schmidt 2008; Broly et al. 2013). The oldest oniscid ration, leading to their exclusion from our analyses. Thirteen fossil, preserved in amber, has been dated to the Albian isopod taxa had complete sequences, but Janira maculosa (Lower Cretaceous; Neraudeau 2008; Broly et al. 2015). The Leach, 1814, was missing four protein-coding genes, first formal description of a Cretaceous terrestrial isopod was Oniscus asellus Linnaeus, 1758, was missing two, and published only recently (Broly et al. 2015). Bathynomus sp. A. Milne-Edwards, 1879, and Porcellio The phylogenetic relationships within Isopoda have been dilatatus Brandt, 1831, were each missing one protein- discussed extensively (see Brandt and Poore 2003) but have coding gene. An alignment of the amino acid sequences was not been resolved with confidence. In particular, the suborder first performed in the Muscle v3.8.31 (Edgar 2004), and the Oniscidea has a complex taxonomic history (summarized by result was used to align the corresponding nucleotide se- Schmidt 2008). The evolutionary relationships within the quences in Pal2Nal (Suyama et al. 2006). The resulting data group have mainly been defined by morphological evidence. sets comprised a total of 4136 amino acids and 8272 nucleo- The monophyly of oniscids is supported by several tide sites (with the third codon positions removed). A chi- apomorphies, including the complex water-conducting system squared test for compositional heterogeneity, conducted using present in all members of this group (Wägele 1989; Brusca PAUP* 4.0 (Swofford 1998), revealed no significant variation and Wilson 1991; Tabacaru and Danielopol 1996;Erhard in nucleotide composition among taxa. 1997; Schmidt 2008). Other studies, however, have suggested The mito-nuclear data set included sequences from 192 that different lineages of terrestrial isopods evolved indepen- isopods and an outgroup species from the tanaid genus dently, which would render Oniscidea polyphyletic (Vandel Parapseudes Sars, 1882. Owing to the potential problem of 1943, 1964). Although some molecular phylogenetic studies mutational saturation, we did not include more distant have included oniscids (Michel-Salzat and Bouchon 2000; outgroup taxa even though they would have allowed us to Wetzer 2002), the monophyly of the suborder has not yet been implement a larger number of calibration constraints for mo- tested. lecular dating. All species had sequences for 18S, whereas In this study, we examined the evolution of Isopoda using sequences of 28S and COI were available only for a subset all available genetic data for the order. Our phylogenetic anal- of taxa. Taxon sampling was aimed at including most of the yses were based on a data supermatrix of one mitochondrial families of Isopoda. When sequences were available for mul- and two nuclear genes and on a second data set comprising tiple species from the same genus, we included only the spe- sequences of all of the protein-coding genes from mitochon- cies for which the largest number of markers was available. drial genomes. We specifically tested the placement of This approach was taken in order to minimize the amount of oniscids within the isopod tree. Using a molecular clock, we missing data. The sequences of Paragnathia formica (Hesse, estimated the timing of terrestrial colonization by these 1864) and Paranthura japonica (Richardson, 1909) were not organisms.