A new species of Parastacus Huxley , 1879 ( Crustacea , Decapoda , Parastacidae ) from a swamp forest in southern Brazil

In this contribution we describe a new species of burrowing crayfi sh of the genus Parastacus Huxley, 1879 from a swamp forest in southern Brazil and determine its conservation status. Th e distinction of the new species is based on morphology and the mitochondrial DNA marker 16S rRNA. Th e extinction risk was assessed according to the sub-criterion B1 of IUCN that estimates the Extent of Occurrence (EOO). Parastacus tuerkayi sp. nov. is morphologically distinguishable from all species of Parastacus by having three lines of verrucous tubercles on the dorsomesial margin of the cheliped propodus and a suborbital angle exceeding 90°. Th e EOO comprises 647,674 km2, and the species is classifi ed as “endangered”. Phylogenetic relationships indicate the distinct position of this new species in relation to the already described species. key words 16S, mtDNA sequence, burrowing crayfi sh, Neotropical region, taxonomy .


IntroductIon
The freshwater crayfish of the genus Parastacus Huxley, 1879 are currently represented by ten species, distributed in the southern regions of South America, specifically in Chile, Argentina, Uruguay and Brazil (for the latter in the states of Rio Grande do Sul and Santa Catarina) (Buckup and Rossi, 1980;1993;Ribeiro et al., 2016).According to previous phylogenetic studies, Parastacus forms a well supported monophyletic clade and is closely related to Samastacus Riek, 1971and Viralastacus Hobbs, 1991(Crandall et al., 2000;Toon et al., 2010).
Burrowing crayfish differ in both behaviour and type of burrows.Hobbs (1942) classified crayfish burrowing behaviour into three categories, taking into account the complexity of burrows, the connection or not to open waters, seasonality and reproductive period, and time individuals spend inside the burrows.Horwitz and Richardson (1986) classified crayfish burrows based on the relationship to the water availability: (1) located in permanent water bodies, (2) connected to the watertable, water from underground or surface run-off and (3) no connection to water-table, the water supply being the surface run-off.Specifically for Parastacus, Riek (1972) classified all species as strong burrowers, but Buckup and Rossi (1980) noted differences in burrowing abilities, depending on habitat.
Molecular tools to complement species descriptions in parastacids were increasingly adopted in the last years (Rudolph and Crandall, 2005;2007;2012), especially in the recognition of new species, when cryptic species are involved.The use of DNA sequencing can be very useful in uncovering genetic variation and increasing the speed of species description, thus acting as a stimulus to further conservation efforts (Burnham and Dawkins, 2013).
In this contribution, we describe a new burrowing species of the crayfish genus Parastacus, discovered in a small fragment of a swamp forest located inside a theme park in southern Brazil.In addition, the distinctive position of this new species is discussed in a phylogenetic context.Habitat characterization and conservation status of the species based on the IUCN Red List criteria are also discussed.

Sampling
Specimens were collected in one small section of a swamp forest, located inside the Beto Carreiro World Park, in the municipality of Penha, state of Santa Catarina, Brazil (26°48'10"S 48°37'2"W).The type material was deposited in the Museu de Zoologia da Universidade de São Paulo (MZUSP), São Paulo, Brazil, and in the Carcinological Collection of the Departamento de Zoologia, Instituto de Biociências, Universidade Federal do Rio do Grande do Sul (UFRGS), Porto Alegre, Brazil.For sampling, burrows were excavated manually in order to obtain crayfish specimens and to provide some information about the burrow system.In addition, a vacuum pump (7cm x 72 cm) was used to capture the individuals.

Morphological analysis
Drawings were prepared under a stereomicroscope fitted with a camara lucida and measurements were performed with vernier calipers with 0.1 mm accuracy and a millimetric ocular on a stereomicroscope.Morphological parameters used were defined by Buckup and Rossi (1980), Hopkins (1970), Morgan (1997) and Ribeiro et al. (2016).Measurements of all type specimens can be found in Tab. 1. Size and shape of the S2 pleura were defined according to Ribeiro et al. (2016).Sex was determined based on the morphology of the genital apertures, according to Rudolph (1997).Morphological descriptions follow Riek (1971), Buckup and Rossi (1980), Hobbs (1987), Morgan (1997), Holdich (2002) and Ribeiro et al. (2016).The taxonomic classification follows De Grave et al. (2009).Branchial count follows Huxley (1879).
Conditions for the polymerase chain reactions (PCR) were: initial denaturation at 94°C for 4 min, followed by 40 cycles of 95°C for 45 s, annealing at 48 or 50°C for 1 min, elongation at 72°C for 1 min,

New species of Parastacus
Nauplius, 25: 2017008 and a final extension step at 72°C for 5 min.PCR products were outsourced for sequencing to Macrogen Europe (Amsterdam, The Netherlands).The obtained chromatograms were proofread using Chromas Lite version 2.23 (Technelysium Pty Ltd., 2005).Resulting sequences were blasted in GenBank and compared with the available Parastacus assemble.The new sequences were deposited at GenBank under accession numbers KY192525 and KY192526.
The best nucleotide substitution model was selected using jMODELTEST 2.1.10with the Akaike Information Criterion (AIC) (95% confidence) (Darriba et al., 2012), suggesting HKI + G as evolutionary model.Phylogenetic relationships were estimated using Bayesian Inference implemented in BEAST 1.8.3 (Drummond et al., 2012).The gene tree search was run on computational resources provided by CIPRES portal (Miller et al., 2015) using the tool BEAST on XSEDE (Drummond and Rambaut, 2007;Suchard and Rambaut, 2009).We used 10 million generations with Markov Chain Monte Carlo (MCMC) sampling, saving trees every 1,000 steps.The efficiency of the chain was assessed in Tracer 1.6 (Rambaut et al., 2007), and the software TreeAnnotator (BEAST package) was used to summarize the trees, with 10% of initial trees discarded as burn-in.Genetic distances were also calculated by pairwise comparisons using uncorrected p-distances with the software Mega 7.0 (Kumar et al., 2013).

Conservation analysis.
The extinction risk of the new species was defined according to the B1 sub-criterion of the International Union for Conservation of Nature -IUCN (IUCN, 2012).This sub-criterion takes into consideration the estimated Extent of Occurrence (EOO) that was calculated in the Arcview 9.3 program (ESRI, 2009).The definition of the hydrographic basins follows the Otto Bacias shape method (level 4) (ANA, 2006).Type material.Holotype: male, Brazil, Santa Catarina, Penha, Beto Carreiro World (26°48'10"S 48°37'02"W), 04/IX/2013, leg.K.M. Gomes and F.B. Ribeiro (MZUSP 34940) Diagnosis.Narrow front with short triangular rostrum.Rostral apex shaped as inverted "U", with an upward blunt spine.Suborbital angle >90°.Postorbital carinae weakly prominent.Cervical groove V-shaped.Areola narrow and barely discernible.Telson subrectangular with sharp spines on lateral margins.Mandible with caudal molar process bicuspidate with one cephalodistal cusp and one small distoproximal cusp.S2 pleurae high and long with deep groove parallel to margin.Internal ventral border of basal article of antennule without sharp spine in males.
Abdomen: lacking spines or tubercles, long and narrow (AL 78.2% of CL; AW 83.6% of CW), smooth, covered with small setae on pleural margins (Fig. 1A).Pleural somites with rounded posterior margins.S1 pleurae with a large distal lobe not overlapped by S2 pleurae.S2 pleurae high and short with deep groove parallel to margin (Fig. 1D).
Tailfan: telson uniformly calcified, subrectangular, longer than wide (TeW 76.6% of TeL), with sharp spines on lateral margins; rounded distal margin with abundant long plumose setae and short simple setae.Dorsal surface with tufts of short setae and inconspicuous dorsomedian longitudinal groove (Fig. 1E).
Uropod protopod bilobed, with rounded and unarmed margins; proximal lobe largest.Exopod lateral margin bears a small and sharp spine, mid-dorsal carina weakly prominent, ending in a very sharp spine.Transverse suture (diaeresis) straight, with ten dorsolateral spines (outer) and nine dorsolateral spines (inner) on right exopod and ten dorsolateral spines (outer) and eight dorsolateral spines (inner) on the left exopod.Endopod with mid-dorsal carina weakly prominent, ending in a very sharp spine; lateral margin with one sharp spine at level of exopod transverse suture (Fig. 1E).
Epistome: anterolateral section with conical projection.Posterolateral section smooth and with deep lateral grooves converging to the basis of the anteromedian lobe and reduced median circular concavity.Anteromedian lobe pentagonal, 1.2x longer than wide, apex acute and straight with some serrated setae, reaching median part of antepenultimate article of antennal peduncle; dorsal surface straight, and basis with a shallow groove (Fig. 2A).
Thoracic sternites: SLP4 smallest and close to each other, median keel present and not inflated; SLP5 small and very close to each other, median keel present and not inflated; SLP6 larger than SLP4, SLP5 and SLP8 and with a slightly concave surface, median keel inflated; SLP7 largest and with surface slightly concave, median keel inflated, bullar lobes absent; SLP8 small and slightly concave, median keel absent, vertical arms of paired sternopleural bridges close to each other, bullar lobes separated and clearly visible (Fig. 2B, C).
Mandible: cephalic molar process molariform, caudal molar process bicuspidate with one cephalodistal cusp and one distoproximal cusp.Incisive lobe with nine teeth.Third tooth from the anterior margin largest (Fig. 2E).
Third maxilliped: ischium bearing few setiferous punctuations, but with some long smooth simple setae on outer margin (Fig. 2F); dorsal surface without setae
Second pair of pereiopods: ventral and dorsal surface of carpus, propodus and dactylus with sparse cover of simple long setae (Fig. 2J).
Gonopores: presence of both genital apertures on coxae of third and fifth pairs of pereiopods.Female gonopores semi-ellipsoidal (maximum diameter 1.56 mm) with well-calcified membrane.Male gonopores rounded, opening onto apical end of a small, fixed, calcified and truncated phallic papilla, close to inner border of ventral surface of coxae of fifth pair of pereiopods.Male cuticle partition present (Fig. 4B).
Description of the female paratype: Differs from the holotype in the following morphological characters: rostrum less sharp at apex, RW 81.9% of RL (Fig. 3A).Post orbital carinae shorter (RCL 65.8% of POCL) (Fig. 3A).Areola 2.4x as long as wide, constituting 27% of CL (Fig. 3A).S2 pleurae high and long (Fig. 3C).Transverse suture (diaresis) with seven dorsolateral spines (outer) and five dorsolateral spines (inner) on right exopod and five dorsolateral spines (outer) and six dorsolateral spines (inner) on left exopod.Anteromedian lobe of epistome 1.1x longer than wide.Internal ventral border of basal article of antenulle with a sharp spine (Fig. 3B).Antennal flagellum reaching S2.Crista dentata bearing 24 and 28 teeth on the right and left ischium, respectively.Chelipeds shorter than in male.Merus of chelipeds with up to two spines in the midventral region.Carpal spine present in both chelipeds, right cheliped bears two spines (Fig. 3A).Female gonopores ellipsoidal (maximum diameter 1.21 mm) covered by a thin and non-calcified membrane.
Color of living specimens.Rostrum reddish brown.Cephalothorax anterior and lateral regions greenish brown to reddish brown.First pair of pereiopods reddish brown with dark reddish brown fingers.Pereiopod pairs 2-5 light brown to reddish brown.Dorsal abdomen light brown to dark reddish brown.Tailfan light brown to reddish brown (Fig. 4E-G).Phylogenetic position.The phylogenetic relationships based on 512bp of the 16S rRNA gene provide clear evidence for the separation of P. tuerkayi sp.nov.from other species of the genus Parastacus with high values of posterior probability (Fig. 6).Genetic distances estimated between P. tuerkayi sp.nov.and other Parastacus species range from 6.2% (P.defossus) to 13.1% (P.nicoleti) for the16S gene (Tab.2).Intraspecific genetic distance was not more than 0.03%.
Habitat and ecology.Parastacus tuerkayi sp.nov.was collected in a small fragment (approximately 500 m²) of a swamp forest located inside the theme park "Beto Carreiro World" in the coastal region of the state of Santa Catarina.This physiographic region belongs to the Atlantic Forest Biome and the vegetation is composed predominantly by Myrtaceae, Poaceae, Piperaceae (genus Piper) and some pterydophyta of the family Blechnaceae (genus Blechnum) (P.Brack pers.comm.).Soil is mainly composed by clay and temporarily flooded with a large amount of organic matter derived from leaf decomposition (F.B. Ribeiro pers.obs.).Found in a flooded area, burrows of P. tuerkayi sp.nov.can be identified as type 2 according to Horwitz and Richardson's (1986) classification.
Based on Hobbs' (1942) classification, P. tuerkayi sp.nov.can be considered a primary burrower, in which the individuals spend almost their entire life underground and build deep and relatively complex burrows.
Burrows can reach a depth of up to one meter, but with few branches and with long (up to 15 cm) and large (up to 12 cm) chimneys.
This burrow structure is very similar to the one of P. caeruleodactylus that is also found in swamp forests in the state of Rio Grande do Sul, near the foothills of the Serra Geral mountains and in the coastal region, and P. pugnax, found in small valleys or depressions between mountains or topographic depressions, usually associated with perennial forests in Chile (Rudolph, 2013;Ribeiro et al., 2016).Parastacus tuerkayi sp.nov. is ecologically similar to P. pugnax, P. caeruleodactylus, P. defossus and P. nicoleti.These species share some morphological adaptations to the burrowing life style, as the narrow areola, which is indicative of one extended branchial chamber; carapace, abdomen and appendages covered by setae in some regions, reduced eyes and the abdomen narrower than the cephalothorax (Horwitz and Richardson 1986;Richardson, 2007).
Regarding reproductive biology, the ovigerous female (paratype UFRGS 6376) bears approximately 20 eggs (average maximum diameter 2.4 mm) attached to its pleopods.The low fecundity is also a characteristic shared by strong burrowing species (Richardson, 2007).
Distribution.Parastacus tuerkayi sp.nov.appears to have an extremely limited distribution, being found only in the municipality of Penha, state of Santa Catarina, southern Brazil (Fig. 5).
Conservation status.The EOO was estimated as comprising approximately 647.674 km² based on the   Otto Bacia shape level 4 (ANA, 2006), indicating that this species can be included in the Endangered -EN category, in which the EOO is less than 5,000 km² (IUCN, 2012).The species is categorized as EN under subitem "a": for an EOO, which is severely fragmented; and subitem "b" (iii): continuing decline in quality of habitat.Both subitems are appropriate, due to the threats existing in the species occurrence area.Urbanization may be the main cause of habitat loss and fragmentation, since P. tuerkayi sp.nov.was AbbreviationsSLP = Thoracic Sternite Lateral Processes S1 = Abdominal Somite 1 S2 = Abdominal Somite 2 TL = Total Length CL = Carapace Length CW = Carapace Width CD = Carapace Depth CeL = Cephalon Length RL = Rostral Length RW = Rostral Width RCL = Rostral Carina Length CMW = Cornea Maximum Width OW = Orbital Width POCL = Post Orbital Carina Length FW = Frontal Width ASL = Antennal Scale Length ASW = Antennal Scale Width AreL = Areola Length AreW = Areola Width RPrT/LPrT = Right/Left Propodus Thickness RPrL/LPrL = Right/Left Propodus Length RPrW/LPrW = Right/Left Propodus Width RDL/LDL = Right/Left Dactylus Length RML/LML = Right/Left Merus Length AL = Abdomen Length AW = Abdomen Width TeL = Telson Length TeW = Telson Width

Figure 3 .
Figure 3. Parastacus tuerkayi sp.nov., female paratype (UFRGS 6376).A, habitus, dorsal view; B, epistome; C, first and second abdominal pleura.Scale bars: A = 1 cm; B, C = 5 mm.Red arrows indicate the spine on the carpus and on the internal ventral border of basal article of antenulle respectively in A and B.ellipsoidal (average maximum diameter 1.18 mm) covered by a calcified membrane.Male gonopores are very similar in male and female paratypes.Parastacus tuerkayi sp.nov. is morphologically similar to P. caeruleodactylus, P. defossus, P. nicoleti and P. pugnax in having the post orbital carinae weakly prominent, the areola narrow and barely discernible and the abdomen narrower than the cephalothorax.Parastacus tuerkayi sp.nov. is also similar to P. nicoleti in having the dorsal surface of dactylus with tubercles in the proximal portion.Parastacus tuerkayi sp.nov.differs from all other Parastacus species in having three well defined lines of verrucous tubercles in the dorsomesial margin of the palm of chelipeds and the

Figure 5 .Figure 6 .
Figure 5. Distribution of Parastacus tuerkayi sp.nov. in the state of Santa Catarina, southern Brazil.The type locality is represented by a red triangle.