A new giant species of placented worm and the mechanism by which onychophorans weave their nets (Onychophora: Peripatidae)

Onychophorans, or velvet worms, are poorly known and rare animals. Here we report the discovery of a new species that is also the largest onychophoran found so far, a 22cm long female from the Caribbean coastal forest of Costa Rica. Specimens were examined with Scanning Electron Microscopy; Peripatus solorzanoi sp. nov., is diagnosed as follows: primary papillae convex and conical with rounded bases, with more than 18 scale ranks. Apical section large, spherical, with a basal diameter of at least 20 ranks. Apical piece with 6-7 scale ranks. Outer blade 1 principal tooth, 1 accessory tooth, 1 vestigial accessory tooth (formula: 1/1/1); inner blade 1 principal 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; 4th. pad arched. The previously unknown mechanism by which onychophorans weave their adhesive is simple: muscular action produces a swinging movement of the adhesive-spelling organs; as a result, the streams cross in mid air, weaving the net. Like all onychophorans, P. solorzanoi is a rare species: active protection of the habitat of the largest onychophoran ever described, is considered urgent.

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. 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 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, 1988band 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.

MATERIALS AND METHODS
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.
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. 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.
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 5 th oncopod pairs, anteriorly displaced and opening between the 3 rd and 4 th creeping pads, free from the 3 rd 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.    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%) ( 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). Phylogenetic analysis: The available information on the COI protein sequence of the species: P. solorzanoi, E. biolleyi, O. corradoi and M. inae was used to estimate the genetic distances between such taxa. Genetic distance matrix (substitutions per site) is shown at Table 3.
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.
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.   (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).  Fig. 7. Prey capture and feeding behavior of the new species: the onychophoran walks at night looking for prey; (A) touches the potential prey with the antennae, and (B) expels two streams of liquid adhesive from specialized organs that (C) oscillate so that the streams cross in mid air and produce the net. D. Finally, when the prey is secured with the adhesive, it is processed with external digestion. 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.

DNA diversity and phylogenetics:
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 (1905Bouvier ( , 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. Read (1988a) has previously commented: "The largest onychophorans include the large species of Oroperipatus (section 1 of Peripates Andicoles in Bouvier 1905), and Macroperipatus torquatus. Certain Epiperipatus, including E. lewisi, reach a large size (Arnett 1961)". Of these, Macroperipatus torquatus was, so far, the largest living species with a total length of 15cm (Read 1985). The new species, P. solorzanoi, is even longer with 22cm and its size can be only compared with extinct taxa, namely as the fossil Xenusion Pompeckj, 1927 from the Baltic, with 20cm (Dzik & Krumbiegel 1989) and the not fully identified fossil Jianshanopodia decora that may reach an equal size if confirmed as being an onychophoran (Liu et al. 2006 of the animal's length, but the correlation has important exceptions. The Onychophora's ancestors were a few millimeters long, lived under water and had few but long oncopods (Monge-Nájera 1994, so we suspect that at the extreme size of 22cm, P. solorzanoi is close to the practical limit for a functional onychophoran because they lack a hard skeleton and cannot excavate their own burrows (Monge-Nájera et al. 1993).

Conservation:
The Onychophora's conservation has been previously discussed (Mesibov 1990, New 1995 and as all other species of Onychophora, P. solorzanoi is rare. Despite additional effort to find the species during fieldwork in the Caribbean of Costa Rica, it has never been found outside the limited area where the type was collected. Furthermore, much of the original tropical rainforest in that area has been deforested for farming and housing development. So far, the only known population is limited to a few kilometers of riparian vegetation. So the species is at least under the Vulnerable IUCN category (IUCN 2000). Under these circumstances, we believe that active protection of the habitat of the largest onychophoran ever described, is urgent.