Canadian Journal of Zoology

Integrative reveals a new and new of (Crustacea: : 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. CamposFilho2, 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, 97105900,

Santa Maria, . Fone/Fax: +55 55 3220 8465.

2 Programa de PósGraduação em Recursos Naturais, Universidade Federal de Campina Grande,

Av. Aprígio Veloso, 882, Bairro universitário, 58429140, 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, 91501970, Porto Alegre, Brazil. Email:

[email protected]

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. CamposFilho, 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 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 CamposFilho, 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;

SchilickSteiner 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., CastroRomero et al. 2016; Decker et al. 2016;

Ekimova et al. 2016; HernandezOrts 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 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; CamposFilho 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 (CamposFilho et al. 2013). Members of this genus differ in few morphological characteristics and are defined based mainly on the shape of the male pleopods (CamposFilho 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

CamposFilho, 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 (CamposFilho 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 CamposFilho 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/K182001). PCR amplifications were performed using

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the LCO / HCO primer set (Folmer et al. 1994), which amplifies an approximately 700bp fragment of the mitochondrial COI gene (COI) and using primers SSU_04F / SSU_R22 (Blaxter et al. 1998) which amplifies an approximately 500bp 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 ExoSAPIT

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 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 bestfit

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 speciestree prior assumed a Yule process. Posterior probabilities were calculated with a burninDraft 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 pdistance 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 steppingstone sampling (SS, Xie et al. 2011) in BEAST package and run for 10 million generations of 50 pathsteps. The better model was chosen when twice the natural logarithm of the Bayesfactor 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 speciesdelimitation 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. CamposFilho & P. B. Araujo, gen. nov.

Type species: Paratlantoscia robusta, sp. nov. by present designation.

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Species included: Paratlantoscia ituberasensis (CamposFilho et al., 2013), Paratlantoscia robusta

sp. nov., and Paratlantoscia rubromarginata (Araujo & Leistikow, 1999).

Genus diagnosis

Body convex; cephalon with supraantenal 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 15 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.

CamposFilho et al. (2012) described one new species from the State of Rio Grande do Sul,

Atlatoscia petronioi CamposFilho et al. 2012. Subsequently, CamposFilho et al. (2013) described

Atlantoscia sulcata CamposFilho 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 CamposFilho & Araujo, 2015 from the state of Rio Grande

do Sul, and Atlantoscia meloi CamposFilho & 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 welldeveloped respiratory areas on pleopod exopods (see also Araujo and Leistikow 1999; CamposFilho 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 CamposFilho 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 welldeveloped 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. CamposFilho & P. B. Araujo, gen. et sp. nov.

Figs. 1 4

Zoobank: urn:lsid:zoobank.org:pub:0E1E3881E48E412394586DBB6DB6C3A0.

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.

CamposFilho, 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. PintodaRocha, Bragagnolo & da Silva, 2426 Jun. 2009; 1

♂, 4 ♀♀ (MZUSP 24254), Camacan, Reserva Particular do Patrimônio Natural Serra Bonita, 2426

Jun. 2009, leg. R. PintodaRocha, 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 .

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 17 epimera strongly pigmented, unpigmented longitudinal spots on median and

paramedian areas, epimera 57 unpigmented on posterior corners; pleonites 2, 4 and 5 strongly

pigmented, pleonites 13 with median longitudinal unpigmented area; telson with three

unpigmented spots (Fig. 1A). Dorsum smooth covered with sparse short scalesetae. Cephalon

(Figs. 1BC) with supraantenal line slightly bent downwards in the middle; eyes of about 1113

ommatidia arranged in rows. Pleon (Fig. 1A) narrower than pereon; neopleurae 35 welldeveloped,

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posterior corners acute and directed backwards. Telson (Fig. 1D) triangular, distal portion concave.

Noduli laterales (Figs. 1EG) 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 scalesetae, cuticle scaled proximally and distal margin slightly prominent bearing slender fringe of thin setae; endite subrectangular covered with thin setae on distal portion, distal margin rounded bearing two hooklike setae, median seta not surpassing the distal margin.

Pereopods (Figs. 3AC) rather slender bearing sparse long setae along sternal margin of merus and carpus; carpus 1 with transverse antennalgrooming brush and distal seta with handlike 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 heartshaped, 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 wellsupported 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 (CamposFilho 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, , and Ascension and St. Helena Islands (CamposFilho 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; CamposFilho 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

(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 PliocenePleistocene 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).

https://mc06.manuscriptcentral.com/cjz-pubs Page 17 of 38 Canadian Journal of Zoology 17

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 , 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 rstrategist, 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; CamposFilho et al. 2013, 2014, 2015; Souza et al. 2015; López

Orozco et al. 2017). Unfortunately, few studies had focused in the BAF (i. e., CamposFilho 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 ISCF (2017137055) and BLZ, to Conselho Nacional de Desenvolvimento CientíficoDraft e Tecnológico CNPq for scholarships to ISCF 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 562202/20102, 470286/20113 and 204468/20140).

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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 DraftAJ287058 Philosciidae sp. KR424678

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Table 2. Pairwise genetic divergence (pdistance) 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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