A new species of freshwater crab genus Fredius Pretzmann, 1967 (Crustacea: Brachyura: Pseudothelphusidae) from a naturally isolated orographic forest enclave within the semiarid Caatinga in Ceará, northeastern Brazil

A new species of freshwater crab, Fredius ibiapaba, is described and illustrated from a mid-altitude forested patch in Ipú (Ibiapaba plateau, Ceará, northeastern Brazil), between 635 to 782 m. The new species can be separated from its congeners by the morphology of its first gonopod: proximal half remarkably swollen, sloping abruptly downwards distally to a nearly right-angular shoulder; mesial lobe much smaller than cephalic spine; cephalic lobe moderately developed; auxiliary lobe lip, delimiting field of apical spines, protruded all the way to distal margin of auxiliary lobe. Comparative 16S rDNA sequencing used to infer the phylogenetic placement of Fredius ibiapaba n. sp. revealed that it is the sister taxon of F. reflexifrons, a species which occurs allopatrically in the Amazon and Atlantic basin’s lowlands (<100 m). Fredius ibiapaba n. sp. and F. reflexifrons are highly dependent upon humidity and most probably were once part of an ancestral population living in a wide humid territory. Shrinking humid forests during several dry periods of the Tertiary and Quaternary likely have resulted in the fragmentation of the ancestral humid area and hence of the ancestral crab population. Fredius reflexifrons evolved and spread in a lowland, humid river basin (Amazon and Atlantic basins), whilst F. ibiapaba n. sp. evolved isolated on the top of a humid plateau. The two species are now separated by a vast intervening area occupied by the semiarid Caatinga


INTRODUCTION
Cumulative evidence from many independent sources argue in favor of the mid-altitude forested patches in northeastern Brazil being remnants of a once much larger humid forest, connected to both the Amazonian and Atlantic rainforests during the moister periods (e.g., Andrade-Lima, 1982;Cartelle & Hartwig, 1996;De Vivo, 1997;Ab'Saber, 2000;Auler et al., 2004;Carnaval & Bates, 2007;Carmignotto, De Vivo & Langguth, 2012; and references therein). These humid forest refuges (Figs. 1A-1D), naturally isolated by the vast surrounding semiarid Caatinga (Figs. 1F,1G), are indeed known to harbor many woody plant and animal species (fossil and Recent) that are also found or are closely related to species occurring allopatrically in the Amazonian and Atlantic rainforests.
Here we describe and illustrate a new species of a freshwater pseudothelphusid crab, Fredius ibiapaba n. sp., from a humid forest refuge in Ipú (Ibiapaba plateau, Ceará, northeastern Brazil), between 665 to 782 m (Figs. 1A-1D). Evidence from a phylogenetic analysis using 16S rDNA is presented for a sister taxa relationship between Fredius ibiapaba n. sp. and F. reflexifrons (Ortmann, 1897), a species occurring allopatrically in the Amazonian humid lowlands. Previous hypothesis on the phylogenetic relationships of F. reflexifrons and the possible evolutionary scenario that led to the emergence of the sister taxa Fredius ibiapaba n. sp. and F. reflexifrons are discussed.

Procedures with material examined
The specimens were collected using license permission from the Sistema de Autorização e Informação em Biodiversidade (SISBIO #29615) of the Brazilian Ministry of Environment (MMA). The studied specimens are deposited in the collections of the INPA (Instituto Nacional de Pesquisas da Amazônia, Manaus), MZUSP (Museu de Zoologia, Universidade de São Paulo, Brazil) and LACRUSE (Laboratório de Crustáceos do Semiárido). Other acronyms: SMF (Naturmuseum Senckenberg) and CCDB (Coleção de Crustáceos do Departamento de Biologia da Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto). Measurements: cl (carapace length, taken along the carapace axis to the posterior median margin) and cw (carapace width, taken at the widest point), in millimeters (mm). Dates are written in the format day.month.year, with months in lower-case Roman numerals. Abbreviations are as follows: G1, G2, first and second gonopods, respectively. Mxp3, third maxilliped. The terminology used in the description of the G1 essentially follows (Rodríguez & Pereira, 1992;Rodríguez & Campos, 1998) (Fig. 2).

Molecular data analysis
DNA extraction, amplification and sequencing: muscle tissue samples were obtained from the pereopods or pleon of Fredius ibiapaba n. sp., F. buritizatilis Mantellato in Magalhães et al., 2014, andPrionothelphusa eliasi Rodriguez, 1980. At the Laboratório de Biologia Molecular da Universidade Estadual do Sudoeste da Bahia-LBM/UESB a small region of the 16S rDNA gene was extracted with Wizard R Genomic DNA Purification Kit (Promega), amplified in a 12,5 µl final volume reaction with 2,5 mM de MgCl2  (Palumbi et al., 1991). PCR products were purified using a polietilenoglicol (PEG) 20% and sequenced in an ABI Prism 3100 Genetic Analyzer R (Applied Biosystems) at the   Hall, 2005). The identities of the final sequences were confirmed with a BLAST (Basic Local Alignment Search Tool) on GenBank database. Additional comparative sequences were retrieved from GenBank (Table 1). Phylogenetic analyses: substitution saturation in 16S rDNA was tested using the saturation index implemented in DAMBE 5 (Xia, 2013). The sequences were grouped and edit in BioEdit and aligned using the ClustalW interface (Thompson, Higgins & Gibson, 1994). Prionothelphusa eliasi (Pseudothelphusidae) and Trichodactylus dentatus H. Milne Edwards, 1853 (Trichodactylidae) were chosen as outgroups. The best-fit model HKY + G was selected using jModeltest 2.1.7 (Darriba et al., 2012). This model was used to generate Maximum Likelihood gene trees in MEGA 6.06 (Tamura et al., 2013). Branch support values were calculated using bootstrap analyses with 1,000 replicates (Felsenstein, 1985). Only nodes with bootstrap support greater than 50 are shown on the phylogenetic tree. Nucleotide divergence estimated from pairwise distance was calculated in MEGA 6.06 with the same best-fit model ( Table 2).

Registration of nomenclatural act
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: [urn:lsid:zoobank.org:pub:0925982D-7441-120 4256-9856-A553987956A6]. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS. 1 Fredius ibiapaba n. sp.
Chelipeds moderately heterochelous, right cheliped larger than left one (Figs. 3E, 3F). Major cheliped merus subtriangular in cross-section; lateral surface smooth, with irregular row of small tubercles of different sizes along dorsal surface; mesial surface smooth, slightly concave to fit lateral sides of carapace; mesial lower margin with row of conical teeth slightly increasing in size distally; lateral lower margin with row of small teeth. Carpus smooth dorsally; mesial margin with row of small, irregular teeth and strong, acute spine about midlength of margin. Palm moderately swollen, smooth on lateral and mesial surfaces, with minute granules on rounded dorsal and ventral faces. Dactylus in process of regeneration.
Cutting margin of dactylus and fixed finger both with larger teeth interspersed with smaller ones. Fingers not gaping when closed, tips not crossing. Minor cheliped similar in shape.
Fredius ibiapaba n. sp. closely resembles F. reflexifrons, but the following characters derived from G1 distinguish the new species from the latter species: (1) in having the auxiliary lobe lip, delimiting the field of apical spines, protruded all the way to the distal margin of the auxiliary lobe (Figs. 4A, 4B), whereas in F. reflexifrons the lip fades away well before reaching the distal margin of the lobe (Figs. 4D, 4E); (2) the subapical bulge markedly less swollen (Figs. 5A, 5C) and the G1 apex much less tilted so that the mesial lobe is not visible in sternal view (Fig. 5A), in contrast to F. reflexifrons (Fig. 5B, 5D, respectively). Also, in F. ibiapaba n. sp. the distal margin of the cephalic lobe is blunt (Figs. 4A, 6A), whereas in F. reflexifrons it tapers progressively to a distinct narrower tip (Figs.  4D, 6D).
Fredius ibiapaba n. sp. further differs from F. ykaa in that the G1 shoulder is high and robust (Figs. 4B, 4C), whilst in F. ykaa the G1 shoulder is remarkably lower; it can be easily further differentiated from F. denticulatus in that its G1 caudal lobe lacks a field of spines spirally twisted to a transverse position (viz., Rodríguez & Campos, 1998) and from F. fittkaui in having the G1 cephalic spine straight and sharply acuminate, whereas in F. fittkaui it is curved and round tipped. Morphological differentiation between female specimens is difficult.

Phylogenetic analysis
The mitochondrial loci 16S was successfully amplified and sequenced for Fredius buritizatilis, F. ibiapaba n. sp., and Prionothelphusa eliasi. Additional sequences used were retrieved from GenBank (Table 1). Bootstrap support values are shown on nodes of the phylogenetic tree (Fig. 8). The sister species relationships between Fredius reflexifrons and the new species is well supported by high bootstrap value. The close morphological similarity between the two species also supports such relationship.
The divergence rates between Fredius reflexifrons and F. ibiapaba n. sp. (4%) is higher than between F. estevisi x F. stenolobus, F. platyacanthus x F. stenolobus and F. platyacanthus x F. estevisi all with 2% of divergence (Table 2). Morphology and molecular data hence provide evidences for the differentiation between F. ibiapaba n. sp. and F. reflexifrons.
A survey of the pseudothelphusids described from 1840 to 2004 (Yeo et al., 2008) showed that the curve of described species is still far from being asymptotic. And indeed, new species are still being discovered either by collecting in new biomes (e.g., F. buritizatilis from a palm swamp known as ''buritizal''), or by revisiting the taxonomy of widely disjunct species for testing as to their conspecific identity, such as F. ibiapaba n. sp. and F. reflexifrons.
The distribution range of Fredius ibiapaba n. sp. is very narrow and currently restricted to a humid enclave, a small mid-altitude forested patch in Ipú (Ceará, northeastern Brazil, Figs. 1A-1E), nested within the vast semiarid Caatinga domain (Figs. 1F, 1G). The orographic forest enclaves, such as Ipú, are typically located along the slopes of plateaus, between 600 and 1,100 m, hence high enough to receive rainfall of more than 1,200 mm year −1 of Atlantic origin (Tabarelli & Santos, 2004 and references therein). These enclaves are regionally known as ''Brejos'' (or ''Brejos de altitude'' or even ''Brejos nordestinos'') (Andrade-Lima, 1982;Silva & Casteletti, 2003;Tabarelli & Santos, 2004). Fredius ibiapaba n. sp. inhabits the mid-highlands of the Ibiapaba plateau, between about 635 to 782 m, where it digs burrows among the leaf litter, alongside little streams and water ponds inside forest stands or directly on the humid forest floor (Fig. 1E). In contrast, F. reflexifrons is widely distributed in the Amazon basin's lowlands (<100 m) from as far west as Peru (Ampyiacu River, a tributary of the Amazonas River) to as far east as the Atlantic basin (French Guiana) (Magalhães, 2003). It is found in burrows alongside the ''igarapés'' (streams) or digs its burrows on the humid forest floor (Magalhães & Rodríguez, 2002). Magalhães et al. (2005) misidentified the specimens from the mid-highlands of the Ibiapaba plateau with F. reflexifrons and explained its presence in Ibiapaba by a migration ''. . . eastwards as far as Serra de Ibiapaba'' during the expansion of the humid tropical forest.
Fredius ibiapaba n. sp. and F. reflexifrons are highly dependent upon humidity and our view is that they most probably were once part of an ancestral population living in a wide humid territory. The shrinking humid forests during several dry periods of the Tertiary and Quaternary (Katzer, 1933;Andrade-Lima, 1953;Bigarella, Andrade-Lima & Riehs, 1975;Ab'Saber, 1977;Bigarella & Andrade-Lima, 1982;Andrade-Lima, 1982;Clapperton, 1993;Thomas, 2000;Haffer, 2001;Haffer & Prance, 2002) likely have resulted in the fragmentation of the ancestral humid area and hence of the ancestral crab population, which was split into two sister species. Fredius reflexifrons evolved and spread in a lowland, humid river basin and is now widely distributed, whilst F. ibiapaba n. sp. evolved isolated on the top of a humid plateau (Figs. 1A-1E). The two species are now separated by a vast intervening area occupied by the semiarid Caatinga (Figs. 1F, 1G).
He found two floristic components in these refuges on the top of hills, one whose species and genera have mostly originated from the southeastern flora, lies further inland in the states of Alagoas and Rio Grande do Norte; and a second one in the humid mid highlands closer to the coast, especially between Pernambuco and the border of Ceará and Piauí states (referred to as the Pernambuco Centre by Santos et al. (2007)), in which the Amazonian flora are better represented (Andrade-Lima, 1982). Santos et al. (2007) found strong bootstrap support for a close floristic relationship between the Pernambuco Centre and Amazonian localities.