Key words: new species, net weaving mechanism, largest onychophoran, fossil and extant velvet worms, Peripatidae, taxonomy, Costa Rica, Peripatus.

Los onicóforos o "peripatos" son animales escasos y poco conocidos. Aquí informamos el descubrimiento del onicóforo más grandes conocido, una hembra de 22cm de longitud del bosque costero caribeño de Costa Rica. Analizamos los especímenes con microscopia electrónica de barrido. La nueva especie, Peripatus solorzanoi, sp. nov. se caracteriza así: papilas primarias convexas y cónicas, con bases redondeadas y más de 18 filas de escamas de alto. Sección apical grande y esférica, con diámetro basal de al menos 20 filas. Parte apical con 6-7 filas de escamas de alto. Fórmula de lámina mandibular externa 1/1/1, lámina interna: 1/1/1/9-10. Diente accesorio romo en ambas láminas. Cuatro almohadillas en cuarto y quinto oncopodios; cuarta almohadilla arqueada. Describimos el mecanismo, previamente desconocido, mediante el cual tejen su red los onicóforos, usando fotografías para mostrar que es de naturaleza muscular. Como todos los demás onicóforos, P. solorzanoi es una especie escasa: consideramos urgente la protección del hábitat del onicóforo más grande del mundo.

Palabras clave: especie nueva, mecanismo de tejido de red, onicóforo más grande, onicóforos fósiles y vivientes.

Onychophorans, or velvet worms, are small invertebrates that most biologists study in theory but due to their rarity, never see in the real life (Bouvier 1905, 1907, New 1995). There are two living families: the egg-laying Peripatopsidae occuring in Chile, South Africa and Oceania, and the Peripatidae that bears live young and occurs in the Neotropics, and in isolated tropical areas of Africa and Asia (Bouvier 1905, 1907, Ruhberg 1985).

Onychophorans are predators that hunt for small invertebrate prey that they capture with an adhesive net mainly composed of water and protein (Bouvier 1905, Read & Hughes 1987, Mora et al. 1996a, b). The phylum has been considered a landmark of the evolutionary process, sharing important features with both the annelids and the arthropods (Bouvier 1905, Ballard et al. 1992). In addition, the onychophorans are an ancient group that is known in fossil records from the mid Cambrian (Dzik & Krumbiegel 1989, Hou & Bergstrom 1995) and are regarded as the first animals that could raise their bodies from the substrate and walk (Monge-Nájera & Hou 2000). They originally were a marine taxon with varied body shapes, often protected by spiculae and armor, but the nearly 180 named extant species (Reid 1996, Trewick 1998, 2000) are all terrestrial, showing no spiculae or armor and presenting the same simple body plan (Monge-Nájera & Hou 2000).

Previously known onychophorans range in body length from 10mm through 15cm (Read 1988a, b; Ruhberg 1985) and it has been suggested that growth is limited by their tracheal respiratory system and by their lack of a hard skeleton (Monge-Nájera & Lourenço 1995). No formal studies were made, so far, dealing with size constraints caused by the hydraulic skeleton of Onychophora.

The scientific information about most species is limited to the species description and minimal collection data; mating behavior in the wild has not been properly documented(Ruhberg 1985, Tait & Briscoe 1995) and there are many taxonomical problems (Read 1988a, 1988b and Reid 1996). Here we describe the uncommonly sized Peripatus solorzanoi sp. nov. from the Caribbean coastal forest of Costa Rica, and provide additional information regarding its taxonomy, behaviour and conservation.

Scanning Electron Microscopy: Specimens were prepared for SEM according to standard procedures (Morera-Brenes & Monge- Nájera 1990).

DNA isolation and sequencing: DNA was extracted from tissue samples using the Wizard genomic DNA purification kit (Promega) according to the manufacturer’s instructions. DNA concentration in aqueous solutions was measured spectrophotometrically and adjusted to 50ng/μl with TE. Polymerase chain reaction (PCR) amplification and sequencing conditions were previously described by Podsiadlowski et al. (2008). A 658 bp fragment (region) of the cytochrome c oxidase subunit I (COX1/ COI) gene was amplified using the primer pair LCO1490/HCO2198 (Folmer et al. 1994). Sequences were obtained from two specimens and confirmed by double-check.

Sequences were analyzed using Geneious software (Biomatters). Default parameters settings for DNA global alignment with free end gaps (Blosum 62 cost matrix, gap opening 12, extension penalties 3) were used.

The protein-coding sequence of P. solorzanoi n.sp. was inferred by translation in silico using the invertebrate mitochondrial Genetic Code. The COI protein was compared to relevant taxa from the America’s: Peripatidae: Epiperipatus biolleyi (ACCESSION ABF93293, Podsiadlowski et al. 2008), Oroperipatus corradoi (ACCESSION AAC95414, Gleeson et al. 1998), and Peripatopsidae: Metaperipatus inae (Mayer 2007; ACCESSION ABQ95564, Braband et al. 2010).

Phylogenetic analysis: This analysis was performed with Geneious Tree Builder. A genetic distance matrix was constructed using the Jukes-Cantor model, and the tree build method was based on the Neighbor Joining Algorithm (Biomatters 2009). A tree was drawn using the Tree View program (Page 1998).

Following Ruhberg (1985) we call the legs "oncopods". Whenever we use a term that is not used by all authors, we include the equivalent.

Species account: Peripatus solorzanoi, n. sp.

Paratypes: Four young that were born to holotype shortly after capture and other collected by Miguel Solano, Norberto Solano and Alejandro Solórzano, 13 August 2000. Museo de Zoología, Universidad de Costa Rica, San José (UCRMZ-60-01).

Distribution: Guayacán and Liverpool de Siquirres and Barbilla National Park, Limón Province, Costa Rica. 

Etymology: Peripatus solorzanoi sp. nov. is dedicated to Costa Rican herpetologist Alejandro Solórzano, who discovered the species, in consideration of his extensive work on the Central American herpetofauna and for his frequent contribution of onychophoran specimens to the University of Costa Rica.

Diagnosis: The following combination of characters: Dorsal primary papillae: Convex and conical with rounded bases; more than 18 scale ranks. Basal piece separated from apical piece by a sligh constriction. Apical section dilated, spherical and symmetric, with a basal diameter of 20 ranks; 6 or 7 scale ranks on apical pieces. Sensory bristle central, thorn-shaped, straight or slightly curved with ornamented basis. Outer blade 1 tooth, 1 accessory tooth, 1 vestigial accessory tooth (formula: 1/1/1); inner blade 1 tooth, 1 accessory tooth, 1 rudimentary accessory tooth, 9 to 10 denticles (formula: 1/1/1/9-10). Accessory tooth blunt in both blades. Four pads in the fourth and fifth oncopods (oncopods="legs"). Nephridial tubercle free from third and fourth pads, in lateral posterior position. The 4th. pad is arched.

Oncopods: Males with 34 leg pairs (n=2) and females with 39-41 leg pairs (39, n=1; 40, n=2; 41, n=11). Total observed animals n=16.

Foot papillae: Three foot papillae, two anterior and one posterior (Fig. 1).

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Soles on foot (also known as creeping pads): Four complete creeping pads, without presence of the vestigal fifth one in all oncopods.

Nephridial tubercle: Present in the 4^th and 5th oncopod pairs, anteriorly displaced and opening between the 3^rd and 4^th creeping pads, free from the 3rd and indenting the proximal margin of 4^th pad, which is crescent-shaped around it. Fourth creeping pad not divided by the nephridial tubercle (Figs. 8, 9).

Integument: Structure of papillae. Dorsal primary papillae convex and conical with rounded bases; without grooves parallel to the main body axis between them. Primary papillae of dorsal surface all of one type, conical, usually 5 to 12 accessory papillae between two of the larger ones. Basal pieces height of >18 scale ranks (Fig. 2). Accessory papillae oriented both to ridge and borders of each fold (Fig. 3).

Sensory bristles. Thorn-shaped bristle, little developed, straight or slightly curved. Sensory bristles placed centrally on the apical pieces and bearing an ornamented basis (Fig. 2).

Plicae. Dorsal integument with 12 plicae per segment, arranged in rings separated by straight grooves perpendicular to the main body axis. Seven plicae pass to the ventral side between oncopods.

Mid-line (also known as dorsomedian furrow): The dorsomedian furrow is conspicuous, forming a channel which splits the folds anteroposteriorly (Fig. 3). Hyaline organs absent along the dorsomedian furrow.

Jaw: Outer jaw blade with one principal and one well developed accessory blunt tooth; presence of a vestigial second accessory tooth (Fig. 4A). Inner jaw blade with one principal and two accessory teeth, the first one well developed and blunt and the second less developed or vestigial (Fig. 4B), 9 to 10 denticles (Fig. 4C).

Color in life: No dorsal ornamentation but greater dorsal primary papillae may look like dark dots. Oncopods pale or light yellow, contrasting with the darker body. Holotype was light brown (Fig. 5), her newborns were red. Apparently there are two color morphotypes present: light brown or red wine color. We also observed deep brown onychophorans in the area but they might represent a different species.

Molecular analysis: The COI DNA sequences of the two P. solorzanoi’s specimens differ in 15 bases (2.13%) (Table 1). Identity= 644/658 (97%), gaps=0.

Nevertheless, all the differing bases at DNA level do not affect amino acid coding, so the translated aminoacid sequences of 219 length show identical COI proteins in both P. solorzanoi’s individuals (Table 2). Such length comparison show a pairwise identity of 97.7% (214 identical sites) between P. solorzanoi and E. biolleyi, both from the same family, but just 89.5% (196 identical sites) between P. solorzanoi and M. inae, species from different families. Coherently E. biolleyi and M. inae show a pairwise identity of 88.6% (194 identical sites).

Note that E. biolleyi presents a deletion of 1 amino acid (at shown position no. 50) with respect to the other studied species. Unfortunately the comparison between P. solorzanoi and O. corradoi is possible just in a length of 151 amino acids, because the studied sequenced fragment is shorter in the last species. They show a pairwise identity of 92.7% (140 identical sites).

The phylogenetic analysis at the full comparison 151 amino acids COI fragment is shown at Fig. 6. The Caribbean Peripatidae species Peripatus and Epiperipatus are close to each other in a node. The Peripatopsidae M. inae was used as outgroup.

Behavior and net weaving: Individuals of this new species actively forage for prey at night in rivulet banks. They expel adhesive when touched; the amount of adhesive is larger than in other Costa Rican species. They move away from a common flashlight beam. Some individuals were born in captivity, probably prematurely as a result of the mother’s postcollecting stress. A high speed film (Fig. 7) shows the animal touching prey with the antennae before expelling two streams of liquid adhesive. Muscular action produces a swinging movement of the adhesive-spelling organs; as a result, the streams cross in mid air, weaving the net.

Placement in the Caribbean Group, genus Peripatus: The new species, P. solorzanoi belongs to the Caribbean group, sensu Bouvier 1905, by presenting three foot papillae arranged laterally, two anterior and one posterior (Figs. 1, 8) and four complete creeping pads with nephridial tubercle opening between the 3^rd and 4^th pad (Figs. 8, 9). These features are shared currently by five neotropical genera: Peripatus Guilding 1826, Epiperipatus (Clark 1913), Macroperipatus (Clark 1913), Plicatoperipatus (Clark 1913) and Speleoperipatus (Peck 1975).

The presence of eyes and only twelve plicae per body segment exclude the possibility of such species being to Speleoperipatus or Plicatoperipatus, respectively. The rounded bases of dorsal primary papillae exclude also Macroperipatus, that bears papillae with quadrangular basis.

The distribution of dorsal papillae in the new species does not fit the old definition of Peripatus and Epiperipatus made by Peck (1975), but the type is similar to Epiperipatus (all of one type, Peck 1975). The new species is unique in having 5 to 12 accessory papillae between two of the larger ones. This is somehow similar to Peripatus (the primary papillary tubercles separated by rather broad intervals where the accessory papillae occur, Peck 1975). Read (1988a) believed that the distinction between Peripatus and Epiperipatus was invalid. According to Read’s classification (1988a), the number of scale ranks at apical piece in this new species classifies it in the genus Peripatus (sensu strictu).

DNA diversity and phylogenetics: The observed DNA sequence diversity (2.13%) in P. solorzanoi is a normal value for intraspecific variation, often sister species differ by >8%, while intraspecific variation is up to 5%. In contrast to the DNA variation, the inferred amino acid sequence is conserved in P. solorzanoi, suggesting that the majority of amino acid positions are constrained within this region of the COI gene.

Differences with similar species: As an aid to identification, we present here some information on how to distinguish the new species from others that occur in the same region. Some of these characteristics may be highly variable within the family, but are reliable within the geographic region considered.

The position of the nephridial tubercle on the fourth and fifth foot distinguishes P. solorzanoi from P. ruber and P. bouvieri because they have "the tubercle largely fused with the third arc" as stated by Bouvier (1905, 1907). Additionally, P. solorzanoi does not have the fifth pad that P. bouvieri has.

The other species recorded from Costa Rica also differ from P. solorzanoi. The new species differs from E. isthmicola, E. hilkae and E. nicaraguensis because its fourth arc is complete. It also differs from M. valerioi "where the fourth creeping pad is thin and twists around the urinary tubercle, which is completely free and outerly bound" (Morera- Brenes & León 1986). If the size is not considered, the new species resembles Epiperipatus biolleyi (Fig. 9), but it can be separated because the third pad is not indented in P. solorzanoi.

The mandible’s outer blade formula in P. solorzanoi (1.1.1) is different from all confirmed and unconfirmed species of Peripatus (as detailed by Read 1988a, b); and it is also different from the other Costa Rican onychophorans (Morera-Brenes & León 1986). Only Plicatoperipatus from Jamaica has a similar outer blade formula. The outer blade shape resembles that of Macroperipatus torquatus from Trinidad, except that the accessory teeth have a more pointing shape in M. torquatus (Read 1988a, b).

The inner blade formula of P. solorzanoi (1/1/1(9-10) is similar to that of Macroperipatus valerioi: (1/1/1/11) (Morera-Brenes & León 1986). The shape of the inner blade also resembles that of M. valerioi, except that in M. valerioi has more denticles and the accessory tooth is more acute (Morera-Brenes & León 1986). It distantly resembles the accessory tooth in the inner blade of "E. brasiliensis" from Venezuela (in quotation marks because we believe this is a misidentification). However, both M. valerioi and "E. brasiliensis" have a well developed  second accessory tooth that is lacking in the new species. The bilobular shape of the inner blade diastema resembles those of P. sedgwicki from Venezuela and P. swainsonae from Jamaica.

Both males and females of the new species have more oncopods than other Neotropical Peripatus (sensu stricto) and other Costa Rican onychophorans (Epiperipatus and Macroperipatus).

Geographic distribution: These are the species of Peripatus that have been corroborated with SEM: P. juliformis Guilding from St. Vincent, P. d. dominicae Pollard from Dominica, P. antiguensis Bouv. from Antigua and P. antiguensis from Montserrat, P. d. lachauxensis Brues from Haiti, P. swainsonae Cockerel from Jamaica, P. sedgwicki Bouv. from Venezuela, and the new P. solorzanoi from Costa Rica (Fig. 10). When only these species are considered, Peripatus has a circum-Caribbean distribution, reaching northward from South America into the Central American bridge and Antillean arc. All the Antillean species occur on continental islands, so we hypothesize that the genus colonized the current islands in times of a lower sea level, as suggested by Monge-Nájera (1996). The exception to this hypothesis was Barbados, an oceanic island reported to have a Peripatus species, but Read (1988a, b) concluded that it was misidentified and relocated the species in another genus (as Epiperipatus barbadensis).

The unconfirmed continental species of Peripatus (P. ruber from Costa Rica, P. bouvieri from Colombia, P. brolemani from Venezuela, P. torrealbai from Venezuela (Scorza 1953), P. bavaysi from Guadalupe, P. daniscus from St. Tomas, P. daniscus juanensis from Porto Rico, P. manni from Haiti and P. haitienisis Brues from Haiti) fit our hypothesis. If they were removed from Peripatus in the future, it would not affect our hypothesis.

In contrast with Peripatus, the genus Epiperipatus seems to be more widely distributed in South America. Nevertheless, both genera have the same circum-Caribbean distribution, suggesting they originated from a common ancestor before their parallel expansion (Monge- Nájera 1995). We must note, however, that maybe Peripatus is not a monophyletic genus.

Body size: The body size has been regarded as of little taxonomic use due to the contraction capacity of Onychophora. Thus, it is difficult to obtain a precise measurement of body length, mainly after fixation, when several degrees of contraction may occur (Read 1988a). Despite Read’s statement (1988a), the body size of P. solorzanoi is quite uncommon among extant and also extinct onychophorans and should be regarded as a diagnostic feature in addition with other morphological characters. The size has been already used to help the diagnosis of Metaperipatus inae Mayer 2007 and is very useful to characterize adults of P. solorzanoi.

We appreciate the assistance and support of Alejandro Solórzano, who brought the new species to our attention and provided the photograph in Fig. 5-B, José A. Vargas Z. for the photograph in Fig. 5-A, and the British Broadcasting Corporation, through Tim Haynes, for allowing us to reproduce frames from the high speed video of P. solorzanoi (Fig. 7). We are indebted to Lars Podsiadlowski for his great support with DNA sequencing and critical reading of the paper. Francisco Hernández helped greatly with the SEM. We also thank Hilke Ruhberg, Randall Rubí Chacón, Robert Mesibov, V.M.S.J. Read and three anonymous reviewers for suggestions to improve an earlier draft, as well as Sergio Aguilar for preparing the final version of figures.

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