Canadian Journal of Zoology
Integrative taxonomy reveals a new genus and new species of Philosciidae (Crustacea: Isopoda: Oniscidea) from Neotropical region
Journal: Canadian Journal of Zoology
Manuscript ID cjz-2017-0289.R1
Manuscript Type: Article
Date Submitted by the Author: 22-Jan-2018
Complete List of Authors: Zimmermann, Bianca; Universidade Federal de Santa Maria Soares Campos Filho, Ivanklin; Universidade Federal de Campina Grande Araujo, Paula;Draft Universidade Federal do Rio Grande do Sul
Atlantoscia, Brazilian Atlantic Forest, isopods phylogeny, Neotropics, Keyword: Paratlantoscia gen. nov., Paratlantoscia robusta sp. nov.
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Integrative taxonomy reveals a new genus and new species of Philosciidae (Crustacea:
Isopoda: Oniscidea) from Neotropical region
Bianca L. Zimmermann1, Ivanklin S. Campos Filho2, and Paula B. Araujo3
1 Centro de Ciências Naturais e Exatas, Departamento de Ecologia e Evolução, Universidade
Federal de Santa Maria, Av. Roraima, 1000, Cidade Universitária, Bairro Camobi, 97105 900,
Santa Maria, Brazil. Fone/Fax: +55 55 3220 8465.
2 Programa de Pós Graduação em Recursos Naturais, Universidade Federal de Campina Grande,
Av. Aprígio Veloso, 882, Bairro universitário, 58429 140, Campina Grande, Brazil. Email: [email protected] Draft 3 Instituto de Biociências, Departamento de Zoologia, Universidade Federal do Rio Grande do Sul,
Av. Bento Gonçalves 9500, Bairro Agronomia, 91501 970, Porto Alegre, Brazil. Email:
Corresponding author. B.L. Zimmermann (email: [email protected]).
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Integrative taxonomy reveals a new genus and new species of Philosciidae (Crustacea:
Isopoda: Oniscidea) from Neotropical region.
B.L. Zimmermann, I.S. Campos Filho, and P.B. Araujo.
Abstract: Although new methods and data are conquering space in the field of taxonomy, such as integrative taxonomy, most of the terrestrial isopod species are still described based only on morphology. Species of the genus Atlantoscia Ferrara & Taiti, 1981 were the first and are the unique terrestrial isopods from the Neotropics for which a molecular phylogeny was already conducted. Previous results indicated thatDraft this genus could be paraphyletic, and a more detailed analysis would be required. Our aim was to reconstruct the phylogeny of Atlantoscia using mitochondrial and nuclear markers and test its monophyly by integrating molecular and morphological data. We observed that, indeed, Atlantoscia is paraphyletic. Atlantoscia ituberasensis Campos Filho, Lisboa & Araujo, 2013 and Atlantoscia rubromarginata Araujo &
Leistikow, 1999 were placed in a new genus of terrestrial isopods, Paratlantoscia gen. nov., together with a new species described in the present study, Paratlantoscia robusta sp. nov. The new genus is defined by the presence of specialized respiratory areas in the pleopod exopods and its validity is highly corroborated by molecular analyses and by biogeographic information. This study highlights the importance of multiple and complementary perspectives as a way to improve the quality of species hypothesis and associated descriptions.
Keywords: Atlantoscia, Brazilian Atlantic Forest, isopods phylogeny, Neotropics, Paratlantoscia gen. nov., Paratlantoscia robusta sp. nov.,
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Introduction
Species are a cornerstone of biology, ecology, and conservation (Petit and Excoffier 2009). Hence,
identifying the boundaries between a set of species has been a largely discussed issue in the last
years in the field of systematic biology (e.g., De Queiroz 2007; Knowles and Carstens 2007;
Aguilar et al. 2013; Grummer et al. 2014). Due to practical and historical reasons most species were
primarily described based on morphology for most part of our history (Padial et al. 2010).
Nonetheless, new methods and data are conquering space in taxonomy. For example, assigning
groups of organisms to species is one of the most challenging aspects of phylogenetic studies
(Coyne and Orr 2004). One of these new approaches is integrativeDraft taxonomy. It integrates information from different sources (usually from morphological and molecular data) to delimit and describe taxa. The result is
improved rigor in species delimitation, which is its ultimate goal as well (Marshall et al. 2006;
Schilick Steiner et al. 2010; Castalanelli et al. 2017). Integrative taxonomy has gained many
supporters in recent years, especially because this approach has been proved to be effective in
solving systematic issues in a variety of groups (e.g., Castro Romero et al. 2016; Decker et al. 2016;
Ekimova et al. 2016; Hernandez Orts et al. 2017; Mazancourt et al. 2017), thus improving the
quality of species hypothesis and associated descriptions (Pante et al. 2015). In this context,
terrestrial isopods (Oniscidea) are an interesting group, since the majority of the species are still
described based only on morphology, even though in many cases morphological characters do not
provide a clear taxonomic resolution (Schmalfuss 2003). Terrestrial isopods comprise the most
successful colonizers of terrestrial environments among crustaceans and show morphological,
physiological, and behavioral adaptations to this lifestyle (Richardson and Araujo 2015). For
example, the development of the respiratory structures on the pleopod exopods was a key factor in
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the success of terrestrial colonization in woodlice. Variations in these structures represent both convergence and functional constraints. The complexity of respiratory structures is generally linked to different levels of environment adaptation (Richardson and Araujo 2015) and could potentially be used in the species delimitation..
Among the main representatives of terrestrial isopods we can highlight the family Philosciidae
Kinahan, 1857, one of the most important groups in tropical and wetlands habitats (Leistikow 2001;
Schmalfuss 2003; Campos Filho et al. 2014). Within Philosciidae, the genus Atlantoscia Ferrara &
Taiti, 1981 consists of seven described species, mainly recorded in Brazil, except for the widely distributed species A. floridana Van Name, 1940 (Campos Filho et al. 2013). Members of this genus differ in few morphological characteristics and are defined based mainly on the shape of the male pleopods (Campos Filho et al. 2013).Draft Recently, Zimmermann et al. (2015) tried to overcome these taxonomic problems within Atlantoscia. The authors demonstrated that using integrative taxonomy by coupling morphology and DNA sequences is an efficient strategy for delimitation of
Atlantoscia species and to investigate their relationships. However, some issues still persist For instance, Atlantoscia rubromarginata Araujo & Leistikow, 1999 and Atlantoscia ituberasensis
Campos Filho, Lisboa & Araujo, 2013 are quite different from the other congeneric species and thus might not belong to this genus (Zimmermann et al. 2015). Specifically, they are genetically and geographically distant and possess unique and specialized morphologies, with well developed respiratory areas in the pleopods (Campos Filho et al. 2013).
Taking into account that populations that are considered to be candidate species will typically exhibit some evidence of genetic isolation, either in the form of phenotypic/genetic differences or geographic separation (Jackson et al. 2017), our aim was to reconstruct the phylogeny of the species in Atlantoscia using mitochondrial and nuclear markers and test its monophyly by integrating molecular, morphological and biogeographical data. We observed, in this study, that Atlantoscia is paraphyletic. Atlantoscia ituberasensis and A. rubromarginata were grouped with a new species
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described in the present study and all of them should be placed in a new genus of terrestrial isopods.
Information from morphology, genetic and biogeography corroborate these findings.
Material and Methods
Material studied
Terrestrial isopods used in this study come from samples deposited in scientific collections of
Museu de Zoologia (MZUSP), Universidade de São Paulo, São Paulo and Coleção de Carcinologia
(UFRGS), Universidade Federal do Rio Grande do Sul, Porto Alegre. Individuals from an
undescribed species sampled in the Brazilian state of Bahia and possibly related to the Atlantoscia genus (due to morphological similarities)Draft were also examined.
Morphological Analysis: description of the new genus and new species
The specimens were dissected and the appendages and pereonites were mounted on slides.
Drawings were prepared using a camera lucida. The noduli laterales were measured and illustrated
as in Vandel (1962). The terminology for the setae follows Campos Filho et al. (2013). The
descriptions are based on the paratypes mounted on slides. The material was deposited in the same
museums mentioned in the previous session.
Molecular Analysis: DNA extraction and PCR amplification
Dissections were conducted as described by Bouchon et al. (1998). Total DNA was extracted using
a PureLink Genomic DNA kit (Invitrogen/K1820 01). PCR amplifications were performed using
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the LCO / HCO primer set (Folmer et al. 1994), which amplifies an approximately 700 bp fragment of the mitochondrial COI gene (COI) and using primers SSU_04F / SSU_R22 (Blaxter et al. 1998) which amplifies an approximately 500 bp fragment of the nuclear 18S rRNA gene (18S). Standard polymerase chain reaction (PCR) was run and PCR products were checked by agarose gel electrophoresis (see Zimmermann et al. 2015). All DNA purification (performed with ExoSAP IT
PCR Product Cleanup Reagent) and sequencing was carried out by Macrogen (Seoul, South Korea), using BigDye technology, with each sample being sequenced in both the forward and reverse directions using the same primers as those used in amplification. All sequences generated in this study were deposited in the GenBank database (http://www.ncbi.nlm.nih.gov/). We also used COI mitochondrial haplotypes obtained by Zimmermann et al. (2015) for phylogenetic analysis. The analyzed terrestrial isopods and GenBankDraft accession numbers are shown in Table 1. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
Outgroup choice
Outgroup can affect reconstructed phylogenies due to heterogeneity of evolutionary rates and variation on base composition among taxa (Brinkmann and Philippe 2008; Caravas and Friedrich
2010). One way to overcome this problem is to choose species that are closely related to the in groups and include multiple outgroups (Brinkmann and Philippe 2008). We chose ten different outgroups for the mitochondrial tree and five for the nuclear tree. It is worth mentioning that terrestrial isopods, and in particular the family Philosciidae, are a group in which studies with molecular biology are still scarce. Thus, few species have DNA sequences available for constructing molecular phylogenies. The sequences of all specimens used as outgroup species were downloaded from GenBank.
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Phylogenetic Analysis
Consensus sequences were aligned with Muscle (Edgar 2004), implemented in MEGA v. 6.0
(Tamura et al. 2013). Phylogenetic analyses were conducted for mitochondrial and nuclear
sequences data sets using Bayesian (BI) and maximum likelihood (ML) methods. The best fit
models of sequence evolution for each gene were selected with JModeltest 2.1.10 (Darriba et al.
2012). The GTR + I + G model was selected for both mitochondrial and nuclear sequences.
Bayesian analyses were performed using Monte Carlo Markov Chain (MCMC) implemented in
BEAST version 1.8.0 (Drummond et al. 2012). The Bayesian analysis was run for 50 million chains
and sampled every 1,000 generations. The species tree prior assumed a Yule process. Posterior probabilities were calculated with a burn inDraft of 5 million states and checked for convergence using Tracer version 1.6 (Rambaut et al. 2014). ML analyses were performed with raxmlGUI 1.5
(Silvestro and Michalak 2012) using a nodal support estimated by 1,000 bootstrap replicates.
Divergence times, i.e. time to the most recent common ancestor (TMRCA), among mitochondrial
haplotypes were also estimated with BEAST with four gamma categories and the strict molecular
clock. The COI substitution rate employed was 0.0164 ± 0.008 substitutions per site per Myr, an
estimative used for other terrestrial isopods (Poulakakis and Sfenthourakis 2008; Lee et al. 2014).
All results were visualized and checked with FigTree 1.4.2 (Rambaut 2014). Pairwise genetic
distances of COI and 18S sequences were calculated using p distance model in MEGA 6.0 (Tamura
et al. 2013). The average values of the intraspecific and interspecific distances were obtained with
1000 bootstrap replicates.
Testing species models hypotheses
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We attempted to test the monophyly of Atlantoscia by comparing species models hypotheses using a Bayes Factor (BF) approach for model selection. The tested models were: Model 1 = constrained tree considering Atlantoscia as monophyletic (i. e., one main clade with all species included); and
Model 2 = constrained tree considering Atlantoscia as paraphyletic (i. e., two distinct clades, according to the tree generated in this study). BF calculates the ratio of the marginal likelihood of two models, which has the advantage of taking into account priors used in Bayesian analysis (Xie et al. 2011). The marginal likelihood values of these competing models were estimated using stepping stone sampling (SS, Xie et al. 2011) in BEAST package and run for 10 million generations of 50 path steps. The better model was chosen when twice the natural logarithm of the Bayes factor testing statistic (2lnBf) was greater than 2 (Kass and Raftery 1995). A value greater than 10 was assumed to indicate decisive support forDraft distinguishing between competing species delimitation hypotheses (Grummer et al. 2014). All parameters were setup as described in the Section
Phylogenetic Analyses. This allows for the direct comparison of the two models considering both the topology and the branch lengths of species trees.
Results
Taxonomy
Paratlantoscia B. L. Zimmermann, I. S. Campos Filho & P. B. Araujo, gen. nov.
Type species: Paratlantoscia robusta, sp. nov. by present designation.
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Species included: Paratlantoscia ituberasensis (Campos Filho et al., 2013), Paratlantoscia robusta
sp. nov., and Paratlantoscia rubromarginata (Araujo & Leistikow, 1999).
Genus diagnosis
Body convex; cephalon with supra antenal line, without frontal line, lateral lobes not developed;
telson triangular with lateral margin sinuous; antennal flagellum of three articles; mandibles with
molar pecinil dichotomized; maxillula outer endite of 4+6 teeth; maxilla bilobate; dactylar organ
longer than outer claw; uropod endopod inserted proximally; pleopod 1 5 exopods with well
developed uncovered lungs.
Remarks Draft The genus Atlantoscia was erected by Ferrara and Taiti (1981) to allocate A. alceui from Ascension
Island (currently synonym of A. floridana). The authors distinguished the genus from the
Mediterranean genus Chaetophiloscia Verhoeff, 1908 by having: pleopod 1 exopod with uncovered
lung, and distinct noduli laterales coordinates. Araujo and Leistikow (1999) described the second
species of the genus, A. rubromarginata from the state of Sergipe. This species was distinguished
from A. floridana by the pleopod exopods (all of them) with respiratory areas partially covered.
Campos Filho et al. (2012) described one new species from the State of Rio Grande do Sul,
Atlatoscia petronioi Campos Filho et al. 2012. Subsequently, Campos Filho et al. (2013) described
Atlantoscia sulcata Campos Filho et al. 2013 from the state of São Paulo, and A. ituberasensis from
the state of Bahia. Based on author’s descriptions the last two species show well developed
respiratory areas on all pleopod exopods (especially A. ituberasensis), characteristic shared with A.
rubromarginata. Recently, Zimmermann et al. (2015) based on molecular and morphological
evidences described Atlantoscia inflata Campos Filho & Araujo, 2015 from the state of Rio Grande
do Sul, and Atlantoscia meloi Campos Filho & Araujo, 2015 from the state of Santa Catarina. The
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authors also found some molecular divergences within Atlantoscia. In one of those divergences A. ituberasensis and A. rubromarginata were recovered in a clade with close relationships and with high molecular divergence from the main clade of Atlantoscia (see Fig. 5 on Zimmermann et al.
2015). As mentioned by the authors, within Atlantoscia, only these species have well developed respiratory areas on pleopod exopods (see also Araujo and Leistikow 1999; Campos Filho et al.
2013) these structures were previously recognized and classified as uncovered lungs by Ferrara et al. (1994). The authors mentioned that the molecular divergence between the clades could have resulted of geographic isolation, and the presence of specialized respiratory organs could be related to an adaptation to high temperatures and/or dry conditions.
Atlantoscia ituberasensis and A. rubromarginata are recorded in the Brazilian Atlantic Forest of the states of Bahia and Sergipe, northeasternDraft Brazil (see distribution map in Campos Filho et al. 2013). This region is inserted in the Equatorial Rainforest (Af) or Equatorial Savannah climate classifications (Aw), characterized by high temperatures, dry and concentrated precipitations periods along of the year (Kottek et al. 2006; Peel et al. 2007).
Paratlantoscia gen. nov. most resembles the genus Atlantoscia in almost all morphological characteristics, but it can be distinguished by having well developed uncovered lungs on all pleopod exopods. Paratlantoscia gen. nov. is supported in the integrative view using molecular, morphological and biogeographical evidences.
Paratlantoscia robusta B. L. Zimmermann, I. S. Campos Filho & P. B. Araujo, gen. et sp. nov.
Figs. 1 4
Zoobank: urn:lsid:zoobank.org:pub:0E1E3881 E48E 4123 9458 6DBB6DB6C3A0.
Holotype: Brazil: Bahia: ♂ (MZUSP 24749), Ituberá, 13°44’S 39°09’W, Sep. 2009, leg. M.C.L.
Peres.
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Paratypes: Brazil: Bahia: 1 ♂, 2 ♀♀ (MZUSP 24750), same data as holotype; 2 ♂♂, 4 ♀♀ (UFRGS
4830), Ilhéus, Reserva Experimental CEPLAC (Comissão Executiva para o Plano da Lavoura
Cacaueira – Executive Comission to Cocoa Tillage Plan), 12 Nov. 2010, leg. J.T. Lisboa & I.S.
Campos Filho, in cocoa plantation; 1 ♂, 2 ♀♀ (MZUSP 24751), Ilhéus, Reserva Experimental
CEPLAC, 14°45’19”S 39°14’15”W, 30 May 2008, leg. J.T. Lisboa, in cocoa plantation; 3 ♂♂, 5
♀♀ (UFRGS 4597), Ilhéus, Reserva Experimental CEPLAC, 30 May 2008, leg. J.T. Lisboa, in
cocoa plantation; 1 ♂, 4 ♀♀ (MZUSP 24252), Camacan, Reserva Particular do Patrimônio Natural
Serra Bonita, 15°23’S 39°33’W, leg. R. Pinto da Rocha, Bragagnolo & da Silva, 24 26 Jun. 2009; 1
♂, 4 ♀♀ (MZUSP 24254), Camacan, Reserva Particular do Patrimônio Natural Serra Bonita, 24 26
Jun. 2009, leg. R. Pinto da Rocha, Bragagnolo & da Silva; 1 ♂, 10 ♀♀, 10 mancas (MZUSP 24238), Porto Seguro, Parque Nacional doDraft Pau Brasil, 16°24’S 39°12’W, 23 Jun. 2009, leg. not identified.
Etymology: The new species name refers the robust shape of the animal.
Description
Maximum body length: ♂ 5.2 mm, cephalon width 1.0 mm; ♀ 5.3 mm, cephalon width 1.2
mm. Color light brown; cephalon with irregular unpigmented spots; antennae with third article and
distal half portion of fifth article of peduncle and second article of flagellum unpigmented;
pereonites 1 7 epimera strongly pigmented, unpigmented longitudinal spots on median and
paramedian areas, epimera 5 7 unpigmented on posterior corners; pleonites 2, 4 and 5 strongly
pigmented, pleonites 1 3 with median longitudinal unpigmented area; telson with three
unpigmented spots (Fig. 1A). Dorsum smooth covered with sparse short scale setae. Cephalon
(Figs. 1B C) with supra antenal line slightly bent downwards in the middle; eyes of about 11 13
ommatidia arranged in rows. Pleon (Fig. 1A) narrower than pereon; neopleurae 3 5 well developed,
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posterior corners acute and directed backwards. Telson (Fig. 1D) triangular, distal portion concave.
Noduli laterales (Figs. 1E G) flagelliform, d/c coordinates reaching maximum on pereonite 4 relative to lateral margin of pereonites, b/c coordinates gradually decreasing relative to posterior margin of pereonites. Antennula (Fig. 1H) with distal article longest, eight aesthetascs arranged in three rows plus apical pair. Antenna (Fig. 1I) when extended posteriorly reaches posterior margin of fourth pereonite; flagellum with distal article longest; apical organ short, about half of length of distal article of flagellum. Mandibles with molar penicil of about five branches and dense cushion of setae, left mandible (Fig. 2A) with 2+1 penicils, right mandible (Fig. 2B) with 1+1 penicils.
Maxillula (Fig. 2C) inner endite with two slender penicils, distal margin rounded; outer set of outer endite with slender seta and accessory tooth, inner set with five teeth cleft at apex, one of them trifid. Maxilla (Fig. 2D) outer lobe twiceDraft as wide as inner endite, distal margin rounded covered with thin seate; inner lobe rounded covered with thick setae. Maxilliped (Fig. 2E) base rectangular bearing sparse piliform scale setae, cuticle scaled proximally and distal margin slightly prominent bearing slender fringe of thin setae; endite sub rectangular covered with thin setae on distal portion, distal margin rounded bearing two hook like setae, median seta not surpassing the distal margin.
Pereopods (Figs. 3A C) rather slender bearing sparse long setae along sternal margin of merus and carpus; carpus 1 with transverse antennal grooming brush and distal seta with hand like apex; dactylus with inner claw reaching distal margin of outer claw, ungual seta simple not surpassing distal margin of outer claw, dactylar organ with lanceolate apex. Uropod (Fig. 4A) protopod grooved on outer margin, exopod and endopod with sparse setae along inner and outer margins; exopod longer than endopod, grooved on outer margin and bearing five setae on apex, endopod with three setae on apex.
Male: Genital papilla with triangular ventral shield and subapical orifices with thin and short setae (Fig. 4B). Pleopod 1 (Fig. 4C) exopod heart shaped, outer margin sinuous with three setae; endopod 1 robust, slightly bent outwards, distal part pointed with slightly inflated apex. Pleopod 2
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(Fig. 4D) exopod triangular, outer margin concave with two setae, distal portion elongated; endopod
2 slender, reaching fourth pleopod. Pleopod 3 triangular, outer margin with 5 setae, distal portion
elongated (Fig, 4E). Pleopod 4 subtriangular, outer margin with with 4 setae near the distal portion
(Fig. 4F).. Pleopod 5 exopod (Fig. 4G) triangular with transverse plumose fringe, outer margin
slightly sinuous bearing two setae (one on tip), distal portion elongated and acute.
Molecular Analyses
All the specimens utilized in the phylogenetic analysis are described in Table 1. We obtained COI
sequences for two individuals of Paratlantoscia robusta sp. nov. (640 bp unambiguous alignment), and nuclear 18S sequences for 30 individualsDraft belonging to nine species (434 bp aligned, including gaps). No intraspecific variation was observed in the 18S sequences. The number of parsimony
informative sites was 162 for COI and 20 for 18S sequences. Tree topologies were congruent
between maximum likelihood and Bayesian analyses. Besides that, the phylogenies generated with
both mitochondrial (Fig. 5) and nuclear (Fig. 6) sequences were congruent regarding the taxa of
interest; that is, both markers support the monophyly of Paratlantoscia. The results observed
confirm the paraphyly of Atlantoscia, justifying the new genus Paratlantoscia with the inclusion of
the new species described P. robusta plus P. ituberasensis and P. rubromarginata, positioned in a
distinct and well supported clade. The other species of Atlantoscia, A. floridana, A. inflata, A.
meloi, A. petronioi and A. sulcata also comprise a well supported clade and can be considered a
monophyletic group.
The average mtDNA genetic distance among species of Atlantoscia was 15.7%, ranging from
13.6 to 18.3%, and among species of Paratlantoscia was 16.5%, ranging from 13.1 to 18.4% (Table
S1). The mean mtDNA genetic divergence between genera was 20.8%. In relation to the nuclear
sequences, the average genetic distance between genera was 5.4%, a considerably higher value than
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that observed among species of Atlantoscia (0.7%) and Paratlantoscia (1.7%) (Table 2).
Interestingly, Atlantoscia species appear to be more related to the specimen of Balloniscus sellowii
(Brandt, 1833) than to the Paratlantocia species (Figs. 5 and 6). However, despite this supposed closeness, B. sellowii is also genetically distant from the Atlantoscia species (e.g., they show a genetic divergence of 4.1% among the nuclear gene sequences).
Bayes Factor model selection results show that SS marginal likelihood estimators prefer Model
2 over Model 1 ( 5,668.73 versus 5,681.05; 2lnBF = 24.64), suggesting the paraphyly of
Atlantoscia. Estimates of divergence times show that Atlantoscia and Paratlantoscia seem to have diverged at about 22.93 Mya. The TMRCA for Atlantoscia and Paratlantoscia haplotypes was estimated at 14.02 and 16.09 Mya, respectively. Divergence times among congeneric species are more recent, most of them dating from PleistoceneDraft (between 11 and 8 Mya).
Discussion
A new genus of terrestrial isopods described by means of integrative taxonomy
This study demonstrated that Atlantoscia constitute a paraphyletic assemblage. Additionally, a new genus and a new species of terrestrial isopod were described, Paratlantoscia robusta gen. nov et sp. nov., and other two species of Atlantoscia were placed within this new genus. The validity of this new taxonomic entity was supported both by morphological and molecular data, reinforcing the merits of integrative taxonomy to delimit species and other taxa boundaries.
As pointed out previously, Atlantoscia species are morphologically similar and the diagnostic characteristics traditionally used to delimit species are few and subtle (Campos Filho et al. 2013;
Zimmermann et al. 2015). Despite this similarity, the respiratory areas on the pleopod exopods of
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Parantlantoscia are considerably more developed than in Atlantoscia. The various stages of the
evolution of respiratory areas in Oniscidea are considered a key factor for their terrestrial
colonization (for a more comprehensive overview see Hornung 2011; Richardson and Araujo
2015). Specifically, specialized respiratory areas are observed typically in derived taxa presumably
as an adaptation to life in xeric environments (Paoli et al. 2002). Furthermore, all representatives of
Paratlantoscia are found only in northeastern Brazil, a region characterized by high temperatures
and dry climate (Kottek et al. 2006). Therefore, since the presence of well develop respiratory area
is one of the main diagnostic features of Paratlantoscia, the inclusion of A. ituberasensis and A.
rubromarginata into the new genus is justified: they present more developed respiratory areas than
what is expected for Atlantoscia. In addition to the morphological evidence,Draft molecular analysis with both mitochondrial and nuclear genes also showed that the Atlantoscia and Paratlantoscia comprise distinct and well
supported clades with a high genetic divergence between them. Levels of sequence divergence
found in this study are consistent with other studies conducted for congeneric species of terrestrial
isopods (e.g., Rivera et al. 2002; Cooper et al. 2008; Sicard et al. 2014; Javidkar et al. 2016;
Karasawa 2016), corroborating results of the morphological analysis and the effectiveness of an
integrated approach to solve taxonomic issues.
We should mention that while the development of new concepts and approaches are
indispensable, more taxonomists and more funding for taxonomy are also urgently needed. A
stronger focus on actually publishing systematic revisions and species descriptions should be part of
a strategy to help overcoming the taxonomic impediment (Evenhuis 2007). In most cases, when
new species are discovered, they are not systematically described (Pante et al. 2015). Taxonomic
studies are the foundation of biological research and naming newly delimited species should be
strongly stimulated.
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Molecular clock and biogeography aspects
Geographic distribution and divergence times of Atlantoscia and Paratlantoscia provide additional evidence for the existence of these two genera. Atlantoscia species are mainly distributed in the southeast and southern Brazil (i. e., they are included in the southern component of the Brazilian
Atlantic Forest BAF), except the widespread A. floridana, recorded from USA, Brazil, Argentina, and Ascension and St. Helena Islands (Campos Filho et al. 2013). On the other hand,
Paratlantoscia gen. nov. occurs only in northeastern Brazil, the northern component of the BAF
(Araujo and Leistikow 1999; Campos Filho et al. 2012, 2013; Hoffmesiter and Ferrari 2016).
One of the main geographic barriers associated to the general historical patterns of taxa distribution in the BAF is the Doce River,Draft which separates the Northern and Central components from the Southern component (DaSilva et al. 2015; Hoffmesiter and Ferrari 2016). Considering that the divergence between Atlantoscia and Paratlantoscia was estimated in the Late Miocene (ca. 23
Mya), and based on their distribution patterns, past events as marine transgressions and/or tectonic activities, Doce River probably have drove the split of Atlantoscia and Paratlantoscia gen. nov.
(see also Almeida and Carneiro 1998; Mohriak 2003; DaSilva et al. 2015; Thomaz et al. 2015).
Large rivers are clear geographic barriers for the terrestrial invertebrate fauna not adapted to fly
(Dantas et al. 2011), and similar results were reported for other groups in the BAF during the
Miocene (e. g., Pellegrino et al. 2005; Fouquet et al. 2012; Roxo et al. 2014). On the other hand, climate fluctuations and tectonic events during the Pliocene Pleistocene would have led to the radiation of Atlantoscia and Paratlantoscia species. It is interesting to highlight that representatives of Paratlantoscia have larger sizes than those of Atlantoscia (data not shown). In an evaluation of biogeography of body size in terrestrial isopods, the authors concluded that climatic factors alone do not define the differential response of body size among isopod species. This variation, on the other hand, seems to be best predicted by generic affiliation (Karagkouni et al. 2016).
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Contrary to other species of Atlantoscia, the biogeographic history of the widely distributed A.
floridana is quite hard to trace. As previously mentioned, A. floridana is recorded from coastal
regions of Florida, USA until north of Argentina, and Ascension and St. Helena islands. Along this
wide distribution range many historical barriers are well known (i. e., Nihei and Carvalho 2007;
Morrone 2014; Silva and Noll 2014; DaSilva et al. 2015). Thus, the distribution of A. floridana is
probably the result of human introductions (see also Ferrara and Taiti 1981). This argument is
supported by molecular comparisons between sequences of A. floridana specimens from Florida
and south Brazil, which demonstrated high levels of similarity (based on DNA Barcoding
http://www.boldsystems.org/, data not shown). In addition, A. floridana is referred as an abundant
species (Lopes et al. 2005; Bugs et al. 2014) and is classified as r strategist, which explains the high success to adapt and colonize distinct environmentsDraft along its distribution range (Quadros and Araujo 2007; Quadros et al. 2009).
Recently, many efforts have been made to describe the diversity of the terrestrial isopods from
Neotropics (e. g., Schmidt, 2007; Campos Filho et al. 2013, 2014, 2015; Souza et al. 2015; López
Orozco et al. 2017). Unfortunately, few studies had focused in the BAF (i. e., Campos Filho et al.
2013, 2015; Zimmermann et al. 2015). Many surveys were made in the last years and many
specimens still wait for recognition and formal descriptions (i. e., Fernandes et al. 2015; Silva and
Ferreira 2015). The identification of this hidden diversity provides very useful information to
conduct evolutionary and conservation works, giving a better understanding about the species
distribution patterns in the Atlantic forest.
In summary, in this study we used evidence from different sources to demonstrate that
Atlantoscia is a paraphyletic group. Previously, morphological variations in the complexity of
respiratory structures in Atlantoscia species were not considered a strong enough argument to
challenge the validity of this genus. However, molecular and biogeographic data reinforced and
corroborated the hypothesis that A. rubromarginata and A. ituberasensis (with their specialized
https://mc06.manuscriptcentral.com/cjz-pubs Canadian Journal of Zoology Page 18 of 38 18
morphologies) compose a new genus, Paratlantoscia. In addition, a new species of Paratlantoscia was described, which also exhibits well developed respiratory areas in the pleopods. We hope that the current popularity of integrative taxonomy will instigate researchers to use this approach, thus enhancing the quality of taxonomic descriptions and facilitating taxa delimitation.
Acknowledgments
We are grateful to Profs. Marcos Tavares and Maria José from MZUSP for the assistance in material deposit and collection analysis process; to Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior - CAPES for PNPD scholarship to ISC F (201713705 5) and BLZ, to Conselho Nacional de Desenvolvimento CientíficoDraft e Tecnológico CNPq for scholarships to ISC F during his Ph.D. and postdoctoral, and productivity fellowship to PBA. Special thanks to Alexandre V.
Palaoro for providing helpful comments and suggestions on the manuscript. This work was supported by CNPq (grant numbers 562202/2010 2, 470286/2011 3 and 204468/2014 0).
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Zimmermann, B.L., Campos Filho, I.S., Deprá, M., and Araujo, P.B. (2015). Taxonomy and
molecular phylogeny of the Neotropical genus Atlantoscia (Oniscidea, Philosciidae): DNA
barcoding and description of two new species. Zool. J. Linn. Soc. 174(4): 702–717. doi:
10.1111/zoj.12256.
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Table 1. Data on specimens used for this study. Terrestrial isopods species and GenBank accession
numbers. Accession numbers in bold represent sequences obtained in this study.
Acession numbers
Species COI 18S rRNA
Atlantoscia floridana KM200831 KY706091
Atlantoscia floridana KM200833
Atlantoscia floridana KM200839
Atlantoscia inflata KM200841 KY706092
Atlantoscia inflata KM200842
Atlantoscia inflata KM200843Draft
Atlantoscia meloi KM200848 KY706093
Atlantoscia petronioi KM200851 KY706094
Atlantoscia petronioi KM200853
Atlantoscia petronioi KM200855
Atlantoscia sulcata KM200863
Paratlantoscia ituberasensis KM200846 KY706095
Paratlantoscia robusta KY706089 KY706096
Paratlantoscia robusta KY706090
Paratlantoscia
rubromarginata KM200862 KY706097
Outgroup
Anchiphiloscia pilosa AF191112
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Australophiloscia societatis AF191111
Balloniscus sellowii KJ814235 KY706099
Benthana taeniata KY706098
Burmoniscus meeusei KM200865 AB889805
Chaetophiloscia elongata KJ668161
Laevophiloscia yalgooensis EU364629
Littorophiloscia culebrae AF191115
Littorophiloscia hawaiiensis AF191120
Papuaphiloscia laevis AF191113
Philoscia affinis FN824100 Philoscia muscorum DraftAJ287058 Philosciidae sp. KR424678
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Table 2. Pairwise genetic divergence (p distance) among terrestrial isopods species (outgroup in
bold) using nuclear (18S rRNA) gene sequences.
Species 1 2 3 4 5 6 7 8 9 10 11
1. Atlantoscia floridana
2. Atlantoscia inflata 0.012
3. Atlantoscia meloi 0.009 0.002
4. Atlantoscia petronioi 0.009 0.002 0.000
5. Paratlantoscia ituberasensis 0.059 0.059 0.056 0.056
6. Paratlantoscia robusta 0.052 0.052 0.049 0.049 0.021
7. Paratlantoscia rubromarginata 0.054 0.054 0.052 0.052 0.016 0.014
8. Balloniscus sellowii 0.037 0.044 0.042Draft 0.042 0.047 0.050 0.047
9. Benthana taeniata 0.084 0.096 0.094 0.094 0.095 0.085 0.090 0.092
10. Burmoniscus meeusei 0.119 0.119 0.117 0.117 0.106 0.104 0.106 0.113 0.120
11. Philoscia muscorum 0.092 0.095 0.095 0.095 0.098 0.093 0.098 0.098 0.119 0.117
12. Philosciidae sp. 0.078 0.080 0.080 0.080 0.081 0.078 0.081 0.083 0.112 0.134 0.094
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Figure captions
Figure 1. Paratlantoscia robusta sp. nov., male paratype MZUSP 24750. (A) habitus; (B) cephalon, dorsal view; (C) cephalon, frontal view; (D) Telson; (E) d/c noduli laterals coordinates; (F) b/c noduli laterales coordinates; (G) epimeron 1; (H) antennule; (I) antenna. Scale bars: (A) = 1 mm; (B
– I) = 0.1 mm
Figure 2. Paratlantoscia robusta sp. nov., male paratype MZUSP 24750. (A) Left mandible; (B) right mandible; (C) maxillula; (D) maxilla; (E) maxilliped. Scale bars = 0.1 mm
Figure 3. Paratlantoscia robusta sp. nov.,Draft male paratype MZUSP 24750. (A) pereopod 1; (B) pereopod 7; (C) dactylus of pereopod 1. Scale bars = 0.1 mm
Figure 4. Paratlantoscia robusta sp. nov., male paratype MZUSP 24750. (A) uropod; (B) genital papilla; (C) pleopod 1; (D) pleopod 2; (E) pleopod 3 exopod; (F) pleopod 4 exopod; (G) pleopod 5 exopod. Scale bars = 0.1 mm; (C), detail of endopod apex = 0.05 mm
Figure 5. ML tree based on COI mitochondrial sequences of terrestrial isopods. Numbers above branches represent Bayesian posterior probabilities (bold) and bootstrap support, respectively. Only posterior probabilities ≥ 0.95 and bootstrap values ≥ 70% are shown
Figure 6. ML tree based on 18S rRNA nuclear sequences of terrestrial isopods. Numbers above branches represent Bayesian posterior probabilities (bold) and bootstrap support, respectively. Only posterior probabilities ≥ 0.95 and bootstrap values ≥ 70% are shown
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Draft
Figure 1. Paratlantoscia robusta sp. nov., male paratype MZUSP 24750. (A) habitus; (B) cephalon, dorsal view; (C) cephalon, frontal view; (D) Telson; (E) d/c noduli laterals coordinates; (F) b/c noduli laterales coordinates; (G) epimeron 1; (H) antennule; (I) antenna. Scale bars: (A) = 1 mm; (B – I) = 0.1 mm
193x178mm (300 x 300 DPI)
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Draft
Figure 2. Paratlantoscia robusta sp. nov., male paratype MZUSP 24750. (A) Left mandible; (B) right mandible; (C) maxillula; (D) maxilla; (E) maxilliped. Scale bars = 0.1 mm
279x394mm (300 x 300 DPI)
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Draft
Figure 3. Paratlantoscia robusta sp. nov., male paratype MZUSP 24750. (A) pereopod 1; (B) pereopod 7; (C) dactylus of pereopod 1. Scale bars = 0.1 mm
261x369mm (300 x 300 DPI)
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Draft
Figure 4. Paratlantoscia robusta sp. nov., male paratype MZUSP 24750. (A) uropod; (B) genital papilla; (C) pleopod 1; (D) pleopod 2; (E) pleopod 3 exopod; (F) pleopod 4 exopod; (G) pleopod 5 exopod. Scale bars = 0.1 mm; (C), detail of endopod apex = 0.05 mm
217x237mm (300 x 300 DPI)
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Draft
Figure 5. ML tree based on COI mitochondrial sequences of terrestrial isopods. Numbers above branches represent Bayesian posterior probabilities (bold) and bootstrap support, respectively. Only posterior probabilities ≥ 0.95 and bootstrap values ≥ 70% are shown
208x200mm (300 x 300 DPI)
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Draft
Figure 6. ML tree based on 18S rRNA nuclear sequences of terrestrial isopods. Numbers above branches represent Bayesian posterior probabilities (bold) and bootstrap support, respectively. Only posterior probabilities ≥ 0.95 and bootstrap values ≥ 70% are shown
308x426mm (300 x 300 DPI)
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